MASARYK UNIVERSITY
FACULTY OF SCIENCE
DEPARTMENT OF BIOCHEMISTRY
Advanced Immunochemical
Biosensors and Assays: From LabelFree
to Single-Molecule Detection
Habilitation Thesis
Zdeněk Farka
Brno 2022
Abstract
The ability of rapid detection of low analyte concentrations, in particular of biomarkers,
microorganisms and their products, or pharmaceuticals, is of fundamental importance in many
fields, including clinical diagnostics, food control, and environmental screening.
Immunochemical biosensors and assays combine the excellent selectivity provided by
antibodies with highly sensitive detection based on various readout techniques. This habitation
thesis presents a commented summary of 22 scientific papers focused on advanced
immunoanalytical techniques, to which I have contributed as a corresponding author, first
author, or co-author. After introducing the field of immunosensing, the thesis starts with labelfree
biosensors and continues through catalytic and luminescent labels to the detection by laserinduced
breakdown spectroscopy. Numerous assays were developed for a wide range of
analytes, starting from small molecules (pharmaceuticals, mycotoxins), through proteins
(disease biomarkers), to bacteria (Salmonella, honeybee pathogens). The research was focused
not only on testing new methodologies but also on the practical applicability of the sensors, as
represented by a large focus on the analysis of representative real samples.
Acknowledgments
First of all, I would like to express my sincere thanks to all my students, colleagues, and
collaborators for any contribution to our research summarized in this thesis.
I want to thank all of my past supervisors and mentors who contributed to my scientific
education. In particular, I thank Petr Skládal, who allowed me to join his research group in the
first year of my bachelor studies, which started my scientific career. Throughout my studies,
he shared his knowledge and provided all the instrumentation, allowing me to become
proficient in the field of biosensing. I thank Hans-Heiner Gorris, who always willingly hosted
me on my numerous stays at the University of Regensburg and who introduced me to the
application of photon-upconversion nanoparticles. I also thank Niko Hildebrandt for hosting
me at the University of Rouen and allowing me to gain experience in the detection of nucleic
acids.
I wish to thank all my students and colleagues from the Masaryk University. Special
thanks go to Matěj Pastucha, to whom I could always rely on. I am grateful to David Kovář,
Veronika Poláchová, Eliška Macháčová, Radka Obořilová, Dorota Sklenárová, Ekaterina
Makhneva, Pavlína Botíková, Květa Mertová, Zuzana Mikušová, Tomáš Juřík, Karel Lacina,
Veronika Křešťáková, Karel Novotný, Libuše Trnková, Lenka Zajíčková, Hana Šimečková,
Ivana Mašlaňová, and Roman Pantůček for their inspiring thoughts, great effort during our
common work, and for creative and friendly environment that they created.
I am grateful for the perfect operation of core-facilities within CEITEC Masaryk
University, especially Nanobiotechnology (Jan Přibyl, Šimon Klimovič), Cryo-Electron
Microscopy and Tomography (Vít Vykoukal, Miroslav Peterek), and Proteomics (Zbyněk
Zdráhal, Ondrej Šedo, Hana Konečná, and Kamil Mikulášek).
I would also like to thank the colleagues and collaborators from other institutes,
especially the Institute of Analytical Chemistry of the AS CR (Antonín Hlaváček, František
Foret), Brno University of Technology (Pavel Pořízka, Pavlína Modlitbová), University of
Regensburg (Julian C. Brandmeier, Simone Rink), University of Turku (Riikka Petlomaa, Tero
Soukka), Institute of Macromolecular Chemistry of the AS CR (Uliana Kostiv, Daniel Horák),
University of Rouen (Mariia Dekaliuk), Leibniz Institute for Plasma Science and Technology
(Katja Fricke), Institute of Physics of the AS CR (Ivana Víšová, Hana Lísalová), and Adam
Mickiewicz University in Poznań (Natalia Jurga).
A great deal of thanks goes to Matthias J. Mickert, my colleague, friend, and an
industrial partner. Together, we have done an incredible amount of scientific work, connected
with an even more incredible amount of fun, which I will never forget.
Finally, I am immensely grateful to my parents for their limitless support.
Table of Contents
1 Commentary to Habilitation Thesis...................................................................................6
2 Introduction (Papers I and II)...........................................................................................11
3 Label-Free Biosensing .....................................................................................................14
3.1 Electrochemical Impedance Spectroscopy Biosensing of Salmonella (Paper III)....14
3.2 Quartz Crystal Microbalance Biosensor for Aerosolized Bacteria (Paper IV) .........16
3.3 Plasma-Polymerized Surfaces for SPR Biosensing (Papers V–VII).........................19
4 Catalytic Labels ...............................................................................................................23
4.1 Amperometric Detection of M. plutonius (Paper VIII).............................................23
4.2 Enzymatic Precipitation-Enhanced SPR Detection of Salmonella (Paper IX) .........24
4.3 Nanozyme-Linked Immunosorbent Assay (Paper X)...............................................27
5 Photon-Upconversion Nanoparticles ...............................................................................30
5.1 Competitive Upconversion-Linked Immunosorbent Assays ....................................30
5.1.1 Competitive ULISA for Diclofenac (Papers XI and XII)..................................30
5.1.2 Competitive ULISA for Zearalenone (Paper XIII)............................................32
5.2 Sandwich Upconversion-Linked Immunosorbent Assays ........................................34
5.2.1 Detection of M. plutonius with BSA-Modified UCNPs (Paper XIV) ...............34
5.2.2 Preparation of PEG-Modified UCNPs and Analysis of HSA (Paper XV) ........35
5.2.3 Detection of P. larvae with PEG-Modified UCNPs (Paper XVI).....................37
5.3 Single-Molecule Upconversion-Linked Immunosorbent Assays .............................38
5.3.1 Digital ULISA for PSA (Papers XVII and XVIII) ............................................38
5.3.2 Digital ULISA for Cardiac Troponin (Paper XIX)............................................41
5.4 UCNP-Based Immunocytochemistry (Paper XX) ....................................................43
6 Laser-Induced Breakdown Spectroscopy (Papers XXI and XXII)..................................45
7 Conclusions and Outlook.................................................................................................47
References................................................................................................................................48
List of Abbreviations ...............................................................................................................60
Appendix..................................................................................................................................62
List of Publications ..............................................................................................................62
Paper I..................................................................................................................................66
Paper II...............................................................................................................................137
Paper III .............................................................................................................................166
Paper IV .............................................................................................................................174
Paper V...............................................................................................................................182
Paper VI .............................................................................................................................192
Paper VII............................................................................................................................202
Paper VIII...........................................................................................................................216
Paper IX .............................................................................................................................225
Paper X...............................................................................................................................233
Paper XI .............................................................................................................................241
Paper XII............................................................................................................................249
Paper XIII...........................................................................................................................257
Paper XIV ..........................................................................................................................267
Paper XV............................................................................................................................277
Paper XVI ..........................................................................................................................290
Paper XVII.........................................................................................................................302
Paper XVIII........................................................................................................................309
Paper XIX ..........................................................................................................................317
Paper XX............................................................................................................................327
Paper XXI ..........................................................................................................................339
Paper XXII.........................................................................................................................350
6
1 Commentary to Habilitation Thesis
This habitation thesis presents a commented summary of 22 scientific papers published
between 2014 and 2021, to which I have contributed as a corresponding author, first author, or
co-author. All these publications are focused on immunochemical biosensors and assays;
however, they are based on different sensing schemes and the detection of various analytes.
After introducing the field of immunosensing, the thesis starts with label-free biosensors and
continues through catalytic and luminescent labels to the detection by laser-induced breakdown
spectroscopy. The research was focused not only on testing new methodologies but also on the
practical applicability of the sensors, as represented by a large focus on the analysis of
representative real samples.
The label-free sensors especially provide rapid and straightforward analysis, making
them suitable for in-field detection, especially of larger analytes. The thesis discusses the
development and performance of biosensor based on electrochemical impedance spectroscopy
for Salmonella and quartz crystal microbalance biosensor for aerosolized biological warfare
agents. We have also focused on biosensor surface modifications by plasma-polymerized films
and their application in surface plasmon resonance biosensing.
The catalytic labels are beneficial due to their ability of signal enhancement. Apart from
the conventional use of horseradish peroxidase in a sandwich immunoassay for European
foulbrood diagnosis, the thesis demonstrates advanced approaches based on enzymaticallycatalyzed
precipitation for signal enhancement in surface plasmon resonance and the catalytic
Prussian blue nanoparticles as a promising alternative to enzymes.
The luminescence detection was done with photon-upconversion nanoparticles, which
overcome the optical background interference by the ability to be excited in the near IR region,
followed by the emission in the visible range. The methods of their surface modification and
conjugation with biomolecules were thoroughly studied. The conjugates were used for
immunochemical detection of a wide range of analytes from small molecules, through proteins,
to bacteria, demonstrating even the capabilities of single-molecule detection.
Finally, laser-induced breakdown spectroscopy was introduced as a novel way of signal
readout, which is not dependent on the catalytic or luminescent properties of the labels. This
approach was used in the microtiter plate-based immunoassay but also as the readout method
in immunocytochemical imaging.
Roman numerals will be used to address the individual publications in the following text. Full
articles have been reproduced in the appendix with permissions from the respective copyright
holders. Asterisk denotes corresponding author.
I. Farka, Z.; Juřík, T.; Kovář, D.; Trnková, L.; Skládal, P., Nanoparticle-Based
Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev. 2017,
117 (15), 9973–10042.
Contribution: Literature research, manuscript writing
(Supervision 10%, Manuscript 30%, Research direction 30%)
7
II. Farka, Z.; Mickert, M. J.; Pastucha, M.; Mikušová, Z.; Skládal, P.; Gorris, H. H., Advances
in Optical Single-Molecule Detection: En Route to Super-Sensitive Bioaffinity Assays. Angew.
Chem. Int. Ed. 2020, 59 (27), 10746–10773. (Z.F. and M.J.M. contributed equally)
Contribution: Outline of review, literature research, manuscript writing
(Supervision 50%, Manuscript 30%, Research direction 40%)
III. Farka, Z.; Juřík, T.; Pastucha, M.; Kovář, D.; Lacina, K.; Skládal, P., Rapid immunosensing
of Salmonella Typhimurium using electrochemical impedance spectroscopy: the effect of
sample treatment. Electroanalysis 2016, 28 (8), 1803–1809. (Z.F. and T.J. contributed equally)
Contribution: Design of experiments, development and optimization of EIS immunosensor, characterization of
sensing surface by AFM, data evaluation, manuscript writing
(Experimental work 30%, Supervision 30%, Manuscript 50%, Research direction 40%)
IV. Kovář, D.; Farka, Z.; Skládal, P., Detection of aerosolized biological agents using the
piezoelectric immunosensor. Anal. Chem. 2014, 86 (17), 8680–8686. (D.K. and Z.F.
contributed equally)
Contribution: Development and optimization of QCM immunosensor, data evaluation, manuscript writing
(Experimental work 50%, Supervision 10%, Manuscript 50%, Research direction 30%)
V. Makhneva, E.; Farka, Z.; Skládal, P.; Zajíčková, L., Cyclopropylamine plasma polymer
surfaces for label-free SPR and QCM immunosensing of Salmonella. Sens. Actuators B Chem.
2018, 276, 447–455.
Contribution: Development and optimization of SPR and QCM immunosensors, characterization of sensing
surface by AFM, data evaluation, participation in manuscript writing
(Experimental work 30%, Supervision 10%, Manuscript 30%, Research direction 20%)
VI. Makhneva, E.; Farka, Z.*; Pastucha, M.; Obrusník, A.; Horáčková, V.; Skládal, P.;
Zajíčková, L., Maleic anhydride and acetylene plasma copolymer surfaces for SPR
immunosensing. Anal. Bioanal. Chem. 2019, 411 (29), 7689–7697.
Contribution: Design of experiments, development and optimization of SPR immunosensor, characterization of
sensing surface by AFM, data evaluation, manuscript writing
(Experimental work 20%, Supervision 50%, Manuscript 50%, Research direction 40%)
VII. Makhneva, E.; Barillas, L.; Farka, Z.; Pastucha, M.; Skládal, P.; Weltmann, K. D.; Fricke,
K., Functional Plasma Polymerized Surfaces for Biosensing. ACS Appl. Mater. Interfaces
2020, 20 (14), 17100–17112.
Contribution: Development and optimization of SPR immunosensor, data evaluation, participation in manuscript
writing
(Experimental work 20%, Supervision 10%, Manuscript 20%, Research direction 20%)
VIII. Mikušová, Z.; Farka, Z.*; Pastucha, M.; Poláchová, V.; Obořilová, R.; Skládal, P.,
Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus
plutonius. Electroanalysis 2019, 31 (10), 1969–1976.
8
Contribution: Design of experiments, preparation of immunization antigen and antibody, optimization of
electrochemical immunosensor, data evaluation, manuscript writing
(Experimental work 30%, Supervision 80%, Manuscript 50%, Research direction 80%)
IX. Farka, Z.; Juřík, T.; Pastucha, M.; Skládal, P. Enzymatic Precipitation Enhanced Surface
Plasmon Resonance Immunosensor for the Detection of Salmonella in Powdered Milk. Anal.
Chem. 2016, 88 (23), 11830–11836.
Contribution: Design of experiments, development and optimization of precipitation-enhanced SPR assay,
characterization of precipitation reaction by AFM, data evaluation, manuscript writing
(Experimental work 40%, Supervision 50%, Manuscript 50%, Research direction 50%)
X. Farka, Z.*; Čunderlová, V.; Horáčková, V.; Pastucha, M.; Mikušová, Z.; Hlaváček, A.;
Skládal, P., Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich Nanozyme-Linked
Immunosorbent Assay. Anal. Chem. 2018, 90 (3), 2348–2354. (Z.F. and V.Č. contributed
equally)
Contribution: Design of experiments, bioconjugation and characterization of PBNPs, development and
optimization of sandwich assay, data evaluation, manuscript writing
(Experimental work 40%, Supervision 60%, Manuscript 60%, Research direction 60%)
XI. Hlaváček, A.; Farka, Z.; Hübner, M.; Horňáková, V.; Němeček, D.; Skládal, P.; Knopp,
D.; Gorris, H. H., Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive
Detection of Diclofenac. Anal. Chem. 2016, 88 (11), 6011–6017.
Contribution: Design of experiments, bioconjugation and characterization of UCNPs, development and
optimization of competitive immunoassay, data evaluation, participation in manuscript writing
(Experimental work 30%, Supervision 10%, Manuscript 30%, Research direction 20%)
XII. Hlaváček, A.; Peterek, M.; Farka, Z.; Mickert, M. J.; Prechtl, L.; Knopp D.; Gorris, H. H.,
Rapid single-step upconversion-linked immunosorbent assay for diclofenac. Microchim. Acta
2017, 184 (10), 4159–4165.
Contribution: Development and optimization of competitive immunoassay, data evaluation, participation in
manuscript writing
(Experimental work 20%, Supervision 10%, Manuscript 20%, Research direction 10%)
XIII. Peltomaa, R.; Farka, Z.; Mickert, M. J.; Brandmeier, J. C.; Pastucha, M.; Hlaváček, A.;
Martínez-Orts, M.; Canales, Á.; Skládal, P.; Benito-Peña, E.; Moreno-Bondi, M. C.; Gorris, H.
H., Competitive upconversion-linked immunoassay using peptide mimetics for the detection
of the mycotoxin zearalenone. Biosens. Bioelectron. 2020, 170, 112683.
Contribution: Design of experiments, bioconjugation and characterization of UCNPs, development and
optimization of competitive immunoassay, data evaluation, participation in manuscript writing
(Experimental work 30%, Supervision 30%, Manuscript 30%, Research direction 30%)
XIV. Poláchová, V.; Pastucha, M.; Mikušová, Z.; Mickert, M. J.; Hlaváček, A.; Gorris, H. H.;
Skládal, P.; Farka, Z.*, Click-conjugated photon-upconversion nanoparticles in an
immunoassay for honeybee pathogen Melissococcus plutonius. Nanoscale 2019, 11 (17),
8343–8351.
9
Contribution: Design of experiments, preparation of immunization antigen and antibody, bioconjugation and
characterization of UCNPs, development and optimization of sandwich immunoassay, data evaluation,
manuscript writing
(Experimental work 20%, Supervision 80%, Manuscript 50%, Research direction 80%)
XV. Kostiv, U.; Farka, Z.; Mickert, M. J.; Gorris, H. H.; Velychkivska, N.; Pop-Georgievski,
O.; Pastucha, M.; Odstrčilíková, E.; Skládal, P.; Horák, D., Versatile bioconjugation strategies
of PEG-modified upconversion nanoparticles for bioanalytical applications.
Biomacromolecules 2020, 21 (11), 4502–4513. (U.K. and Z.F. contributed equally)
Contribution: Design of experiments, bioconjugation of UCNPs, development and optimization of sandwich
immunoassay, data evaluation, participation in manuscript writing
(Experimental work 30%, Supervision 40%, Manuscript 40%, Research direction 40%)
XVI. Pastucha, M.; Odstrčilíková, E.; Hlaváček, A.; Brandmeier, J. C.; Vykoukal, V.; Weisová,
J.; Gorris, H. H.; Skládal, P.; Farka Z.*, Upconversion-linked Immunoassay for the Diagnosis
of Honeybee Disease American Foulbrood. IEEE J. Sel. Top. Quantum Electron. 2021, 27 (5),
6900311.
Contribution: Design of experiments, preparation of immunization antigen and antibody, bioconjugation and
characterization of UCNPs, development and optimization of sandwich immunoassay, data evaluation,
manuscript writing
(Experimental work 20%, Supervision 80%, Manuscript 50%, Research direction 80%)
XVII. Farka, Z.; Mickert, M. J.; Hlaváček, A.; Skládal P.; Gorris, H. H., Single Molecule
Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive
Detection of Diagnostic Biomarkers. Anal. Chem. 2017, 89 (21), 11825–11830. (Z.F. and
M.J.M. contributed equally)
Contribution: Design of experiments, optimization of single-particle microscope setup, bioconjugation and
characterization of UCNPs, development and optimization of sandwich immunoassay, data evaluation,
manuscript writing
(Experimental work 40%, Supervision 20%, Manuscript 40%, Research direction 20%)
XVIII. Mickert, M. J.; Farka, Z.; Kostiv, U.; Hlaváček, A.; Horák, D.; Skládal, P.; Gorris, H.
H., Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through
Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay. Anal. Chem.
2019, 91 (15), 9435–9441. (M.J.M and Z.F. contributed equally)
Contribution: Design of experiments, bioconjugation and characterization of UCNPs, development and
optimization of sandwich immunoassay, data evaluation, manuscript writing
(Experimental work 30%, Supervision 20%, Manuscript 30%, Research direction 30%)
XIX. Brandmeier, J. C.; Raiko, K.; Farka, Z.*; Peltomaa, R.; Mickert, M. J.; Hlaváček, A.;
Skládal, P.; Soukka, T.; Gorris, H. H., Effect of Particle Size and Surface Chemistry of PhotonUpconversion
Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin. Adv.
Healthc. Mater. 2021, 10 (18), 2100506.
Contribution: Design of experiments, bioconjugation and characterization of UCNPs, development and
optimization of sandwich immunoassay, data evaluation, manuscript writing
10
(Experimental work 20%, Supervision 30%, Manuscript 30%, Research direction 30%)
XX. Farka, Z.*; Mickert, M. J.; Mikušová, Z.; Hlaváček, A.; Bouchalová, P.; Xu, W.; Bouchal,
P.; Skládal, P.; Gorris, H. H., Surface design of photon-upconversion nanoparticles for highcontrast
immunocytochemistry. Nanoscale 2020, 12 (15), 8303–8313. (Z.F. and M.J.M.
contributed equally)
Contribution: Design of experiments, optimization of microscope setup, bioconjugation and characterization of
UCNPs, development and optimization of ICC assay, data evaluation, manuscript writing
(Experimental work 40%, Supervision 40%, Manuscript 50%, Research direction 40%)
XXI. Modlitbová, P.; Farka, Z.; Pastucha, M.; Pořízka, P.; Novotný, K.; Skládal, P.; Kaiser, J.,
Laser-induced breakdown spectroscopy as a novel readout method for nanoparticle-based
immunoassays. Microchim. Acta 2019, 186, 629.
Contribution: Design of experiments, development and optimization of sandwich immunoassay, data evaluation,
participation in manuscript writing
(Experimental work 40%, Supervision 30%, Manuscript 30%, Research direction 30%)
XXII. Pořízka, P.; Vytisková, K.; Obořilová, R.; Pastucha, M.; Gábriš, I.; Brandmeier, J. C.;
Modlitbová, P.; Gorris, H. H.; Novotný, K.; Skládal, P.; Kaiser, J.; Farka, Z., Laser-Induced
Breakdown Spectroscopy as a Readout Method for Immunocytochemistry with Upconversion
Nanoparticles. Microchim. Acta 2021, 188, 147.
Contribution: Design of experiments, bioconjugation and characterization of UCNPs, development and
optimization of ICC assay, data evaluation, manuscript writing
(Experimental work 20%, Supervision 40%, Manuscript 50%, Research direction 50%)
11
2 Introduction (Papers I and II)
The capability to rapidly detect small analyte concentrations, particularly of low-abundance
biomarkers, is critical for diagnosing diseases in their early stages. The majority of bioaffinity
methods are employing antibodies;1, 2
however, also aptamers3
and molecularly imprinted
polymers (MIPs)4
can be used for specific capture of the target analyte. Antibodies with high
affinity can be prepared against generally any analyte molecule. The limit of affinity,
represented by a binding constant, is approximately 1010
M−1
,5
which is worse than 1014
M−1
in the case of (strept)avidin-biotin interaction.6
Due to the relatively large size of antibodies,
single binding site antibodies (camelids) are also attracting attention recently.7
Aptamers are
beneficial due to their easier large-scale production, and MIPs excel in chemical stability. MIPs
are particularly suitable for detecting small molecules with a rigid structure. However, they are
less suitable for the detection of bigger, more flexible analytes, as proteins.
Two approaches can be used for the detection of binding events: (i) Label-free assays
exploit the possibility to generate a signal directly upon analyte binding to the detection
element. (ii) The so-called sandwich format is based on binding a second affinity reagent
bearing a label that provides signal generation. Both approaches can be carried out in a
competitive (or inhibition) mode, based on competition of immunoreagents for a limited
amount of binding sites, resulting in lower signals for higher analyte concentrations. The first
immunoassays were based on radioactive labels;8
however, these were soon replaced by
enzymes, which allow higher safety. Furthermore, a single enzyme molecule can generate a
high number of measurable product molecules (signal amplification step). The enzyme-linked
immunosorbent assay (ELISA) is nowadays considered as a method of choice for quantitative
analysis of various analytes, from clinical diagnosis, through food control, up to environmental
protection.
Throughout the past 60 years, the progress in immunoassays was primarily focused on
enhancing sensitivity, specificity, and reproducibility. Even though ELISA can detect
picomolar analyte concentrations, even higher sensitivities are necessary. Only a few toxin
molecules can be harmful,3
individual infectious viruses or bacteria can initiate a disease,9
and
trace cancer biomarker quantities indicate the onset of a malignant transformation.10
Furthermore, developing immunoassays with higher sensitivity is critical to allow discovering
new biomarkers, which cannot be analyzed using the current methodology.11, 12
The conventional ELISA is carried out the laboratory conditions and is based on
relatively long incubation times and several washing steps. Therefore, the recent development
in the field aims also at faster analysis, with higher throughput and smaller sample
consumption. Such assays allow on-line analysis, e.g., at the bedside for clinical tests,13
or in
the field for environmental or military applications. Such methods are often referred to as pointof-care
(PoC) tests.14
It is preferred to use samples that require minimal invasiveness during
their collection, such as urine or saliva. Furthermore, assays without washing steps are desired.
The most famous PoC test based on antibodies is the home pregnancy test, the representative
of lateral flow immunoassays (LFIAs), which were developed in the 1980s.15
The user-friendly
operation, along with the possibility to provide reliable results, is necessary to allow the PoC
12
test to be used in predictive, preventive, personalized, and participatory medicine, commonly
termed P4 medicine.16
The group of label-based bioaffinity assays can be further divided according to the used
detection label (Figure 1). (i) Enzymes represent the most widespread approach, whereas
(ii) fluorescent molecular labels are generally easier, without the requirement of the product
generation step. However, the fluorescence immunoassays (FIAs) are typically limited by the
fluorescence of the background. Furthermore, fluorescence readout was adapted in
homogeneous assays based on fluorescence polarization and methods based on signal
amplification (e.g., based on Immuno PCR). Significant progress regarding the limitation of
background fluorescence was achieved by the development of time-resolved (TR) approaches
that exploit lanthanide-based labels with long lifetimes (μs) compared to small fluorophores
(ns).17
The time-gated approach is based on the luminescence excitation, which is not directly
followed by the signal acquisition, but the measurement is delayed by a few μs, so the
autofluorescence signal decays, and only specific lanthanide signals are measured. The
dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is currently the most
widespread commercially available TR approach.18
Figure 1: History of the development of label-based immunoassays with optical readout. Radioisotopes
were replaced by enzymes, fluorescent molecules, and nanoparticles. By choosing a suitable readout
method, all these labels can be used for the measurement at the single‐molecule level. Reprinted from
Paper II under the permission of Creative Commons Attribution-NonCommercial 4.0 International
License.
13
As an alternative label type (iii), various kinds of nanoparticles (NPs) are gaining
increasing popularity (Figure 1).2, 19
Gold nanoparticles (Au NPs) are widely used for the
readout of LFIAs. Because of their plasmonic properties, Au NPs exhibit strong absorption and
scattering of light, which makes them easily visible by the eye, and the color-based readout is
possible without the need for sophisticated instrumentation. Apart from the plasmonic NPs,
many other types of NPs and nanocomposites are being used for optical detection. Quantum
dots (QDs) represent an alternative to organic fluorophores due to their better photostability
and higher brightness, which is an essential aspect in immunoassay readout. Photonupconversion
nanoparticles (UCNPs) are another kind of luminescent labels, which allow
excitation by near-infrared light, followed by the emission of light with a shorter wavelength.
This anti-Stokes emission avoids autofluorescence and light scattering, leading to detection
without optical background interference.20
Nanocontainers (e.g., liposomes) can be packed by
many fluorescent molecules to generate strong signals. Compared to the enzyme-based labels,
which produce the fluorophores in situ from the non-fluorescent substrate, the fluorophores
encapsulated in nanocontainers can be released on demand, limiting the self-quenching inside
the confined environment.21
Furthermore, various mixed detection schemes, e.g.,
electrochemiluminescence, can be employed to generate strong signals without background.
Overall, the various detection schemes present different advantages and disadvantages
in terms of sensitivity, analysis time, miniaturization potential, etc. Therefore, a suitable
method has to be chosen not only concerning the target analyte but also for the intended
application and user base.
14
3 Label-Free Biosensing
3.1 Electrochemical Impedance Spectroscopy Biosensing of Salmonella
(Paper III)
Electrochemical immunosensors are receiving increasing focus because they can combine
highly sensitive measurements with portability and low cost. Electrochemical impedance
spectroscopy (EIS) is a technique, which allows the measurement of small changes in the
interface between the electrode and solution. EIS provides a fast response in combination with
high sensitivity and potential for real-time measurement and miniaturization.22
When used in
biosensors, EIS provides insight into the individual immobilized layers and coating on the
electrode in general. The EIS measurement is based on applying a low-amplitude sinusoidal
potential (or current) through the electrochemical cell with the electrolyte solution, typically
ferro/ferricyanide. The output current (or potential) is then measured over a range of
frequencies by a potentiostat, allowing the calculation of the impedance parameters. Compared
to the other electrochemical techniques, including cyclic voltammetry, the applied potential is
smaller, preventing the undesired influence on biomolecular layers and binding processes.23
The biosensors based on EIS typically employ antibodies immobilized in the electrode,
directly capturing the target analyte. The accumulated mass hinders the electron transfer; this
is evaluated as the increase of impedance. This allows operation in label-free mode, providing
robust and straightforward analysis. The label-free EIS can be used for rapid analysis of
pathogens within small sample amounts.
Our research focused on Salmonella enterica serovar Typhimurium, a non-typhoidal
strain, which is one of the leading causes of gastrointestinal diseases. Salmonella is a gramnegative
bacterium, which can cause diarrhea, fever, and abdominal spasm within 12 to 72 h
after infection. In the worst scenario, Salmonella can enter blood, bones, brain, or nervous
system, which can cause even lethal infections.24
The infection is typically caused by
consuming contaminated food.25
Salmonella can be present in raw animal food products,
including meat, eggs, and unpasteurized dairy products. There are globally 94 million cases of
gastroenteritis and 155,000 deaths attributed to Salmonella each year.26
According to the
Centers for Disease Control and Prevention (CDC), there are 1.2 million illnesses and 450
deaths per year in the United States caused by non-typhoidal Salmonella strains.26
This
highlights the danger of Salmonella to human health and the importance of developing devices
that can allow rapid and sensitive Salmonella detection.
The standard approaches allowing the detection of Salmonella include traditional
cultivation-based methods, ELISA, and polymerase chain reaction (PCR). The cultivationbased
approaches are considered a gold standard for Salmonella detection because of the high
sensitivity and selectivity. However, the long analysis times (5–7 days) with labor-intensive
procedures do not allow using cultivation for rapid screening purposes.27
The ELISA can
provide sensitive results generally within 24 h.28
Usually, a time-consuming pre-enrichment
step is necessary to increase the bacteria count in the samples.29
PCR overcomes the sensitivity
and analysis times on conventional methods; however, it requires expensive instrumentation
15
and trained personnel to carry out the
analysis.30
Furthermore, the abovementioned
methods are typically
limited only to laboratory conditions
and do not allow PoC operation.
We have developed an EIS
approach for the detection of
Salmonella Typhimurium, based on a
simple, easy to fabricate, and low-cost
immunosensor. The screen-printed
electrodes (SPEs) were modified by a
self-assembled monolayer of
cysteamine, followed by binding of
glutaraldehyde and specific antibody
(Figure 2). The increase of impedance
after incubation with the sample
revealed the presence of bacteria.
Different sample treatment methods
(viable bacteria and combinations of
heat-treatment and sonication) were tested to find the optimal way of sample preparation
regarding the specificity of the chosen antibody. The achieved results have shown that the
antibody did not exhibit the necessary affinity towards native Salmonella. After the heattreatment
(80 ºC, 40 min), the affinity of the antibody to the microbe increased significantly.
This allowed reaching a limit of detection (LOD) of 7×104
CFU/mL with a wide linear range
up to 108
CFU/mL. The treatment by heat does not present significant technical difficulty for
the real sample analyses. Furthermore, it can even be beneficial to work with the killed or
weakened bacteria due to the reduced level of pathogenicity.
To improve the sensitivity further, the Salmonella cells were disrupted by sonication.
The sonicated heat-treated sample has shown a higher level of specific binding than whole
cells, resulting in an LOD of 1×103
CFU/mL; the linear range was up to 108
CFU/mL. The total
analysis time (including the incubation of the sensor with the sample) was 20 min. The
treatment by sonication is also not a technical problem for practical analysis. Even though
additional instrumentation is required, sonication is beneficial because it is less timeconsuming
than heat-treatment. The presence of bacteria on the sensor surface was confirmed
by atomic force microscopy (AFM; Figure 3). It was shown that the sensor captured a large
number of cell fragments, with the size and structure corresponding to heat-treated and
sonicated bacteria adsorbed on the glass.
In the case of the cross-reactivity with E. coli K-12, only negligible increases of
impedance were observed, confirming the excellent selectivity of the method. Because both
Salmonella and E. coli are relatively similar gram-negative bacteria, a low level of crossreactivity
can also be expected in the case of more phylogenetically distant bacteria.31
Figure 2: Scheme of antibody immobilization (blocking
by BSA not shown) and the design of the SPE electrode.
Reprinted from Paper III with permission. Copyright 2016
Wiley-VCH.
16
The practical applicability of the sensor was demonstrated by the analysis of real
samples of milk spiked with Salmonella. The electrode had to be washed thoroughly after the
incubation with complex samples; insufficient washing was connected with the increase of
non-specific signals. The detection capabilities in the case of complex samples decreased
slightly compared to the detection in the buffer, resulting in the LOD of 9×103
CFU/mL and
linear range up to 107
CFU/mL. These results are comparable to the infection dose of
Salmonella32
and highlight the potential of the developed method.
Figure 3: (A) EIS response of the SPE immunosensor to different concentrations of heat-treated and
sonicated Salmonella; (B) AFM scan of the electrode after binding (error signal); (C) negative control
(no antibody on the surface); (D) AFM scan of the blank electrode. Reprinted from Paper III with
permission. Copyright 2016 Wiley-VCH.
3.2 Quartz Crystal Microbalance Biosensor for Aerosolized Bacteria
(Paper IV)
In 1959, Sauerbrey described the dependence between the resonant frequency of quartz sensor
and the mass accumulated on the sensor surface, which led to the development of the
microgravimetric biosensing approach – quartz crystal microbalance (QCM). QCM allows
detecting binding events based on the measurement of changes of frequency of the quartz
17
resonator.33
The great advantage of QCM is the ability to provide the results in real-time,
finding applications in monitoring of surface modifications, adsorption/desorption effects, and
binding interactions. When the analyte is captured on the surface, it increases the loaded mass,
which can be directly estimated from the decrease of the resonance frequency.
We employed the QCM biosensor for the detection of bioaerosols. Airborne
microorganisms (bacteria, viruses, fungi, etc.) are an integral part of the environment. Bacteria
can be spread to the air by natural as well as anthropogenic sources; the misuse of pathogens
can result in targeted biological attacks.34, 35
Modern history has shown that the potential of
abuse of biological warfare agents is very high.36
The contamination of outdoor and indoor air by bacteria can infect a large number of
people within a short timeframe.37
The detection of bioaerosols is difficult due to the low levels
of target bacteria, combined with a potentially complex sample matrix containing pollen grains,
mold, fungi, dust, and ubiquitous microbial organisms.38
Further interferences might be caused
by industrial products, which are emitted into the atmosphere in large quantities. The low
visibility and lack of odor present additional challenges for sampling and analysis. In contrast
to chemical agents, also the delayed effect of biological agents has to be considered, leading to
a potentially large number of casualties before protective steps are taken.36
The bioaerosols can
contribute to the spread of epidemics and pandemics in places with high population densities.
In such cases, rapid detection and identification of the pathogen are critical to allow early
treatment. Therefore, the specific detection of bacteria in the air is of particular importance also
during peacetime.
The bioaerosol analysis consists of two critical parts: collection of the bioaerosol and
detection of the bacteria. The systems for the collection of bioaerosols (samplers) have been
extensively developed in recent years;39
their function can be based on various physical
principles.40
The two main approaches for the detection are based on the analysis of general
biological compounds in the air or specific detection of target bacteria. The non-specific
detection is typically based on optical methods, exploiting the fluorescence of the molecules
present in biological systems (ATP, NADH, tryptophan, etc.).41, 42
These systems, however,
cannot differentiate between pathogenic and non-pathogenic bacteria, and the presence of
atmospheric pollutants with fluorescence properties can interfere with the analysis.43
Furthermore, this principle is only useful for living bacteria, and it does to allow detection of
spores, as they exhibit only low ATP levels.44
The specific detection of bacteria typically
exploits culture-based techniques, PCR,45, 46
Raman spectroscopy, or mass spectrometry.47
The
culture-based methods are time-consuming and usually require collection of samples for
subsequent laboratory analysis. The PCR is highly sensitive; however, various interfering
substances can be present in the complex samples, which hinders the analysis.48
Therefore,
PCR is often combined with other methods to prevent false-positive results.49
In the case of
MS, the widespread use for in-field detection is limited mainly by the requirement of vacuum
and extensive instrumentation in general.
We have employed the immunosensor technology to overcome these disadvantages,
aiming at quick-response, real-time, and on-site detection. First, a bioaerosol chamber was
18
constructed for safe and controlled dissemination of biological agents and applied for
experiments with model bacterial aerosols of E. coli (Figure 4A). The samples were
disseminated using a piezoelectric humidifier, distribution of bioaerosol inside the chamber
was achieved using three 12-cm fans. The disseminated bacteria were collected and
preconcentrated using the wetted-wall cyclone SASS 2300; the analysis was done using the
on-line linked QCM immunosensor. The measurement was fully automated; the flow system
was used for the on-line delivery of the samples from the cyclone to the QCM, allowing to
perform one detection cycle within 16 min. The achieved LOD of E. coli in the bioaerosol was
104
CFU/L of air, based on the amounts of the disseminated microbe (Figure 4B). The whole
experiment, including sample collection, detection, sensor surface regeneration, and bioaerosol
chamber ventilation, took 40 min. The reference experiments based on cultivation showed that
the disseminated amount of E. coli was reduced, probably because of the surface adsorption,
desiccation, and mechanical stress caused by the cyclone. The great benefit of the developed
detection system is the possibility of entirely remote operation; the users do not come into
contact with potentially dangerous microorganisms during the experiments. Furthermore, the
system is fully portable (desk-top size) and requires only power and ethernet connections.
The achieved results proved the suitability of the developed QCM immunosensor
combined with cyclone sampling to detect aerosolized bacteria. In the future, multiplexed
detection based on monoclonal antibodies specific to different pathogens can allow
comprehensive screening of the air quality in a reasonably short time. The system based on
single-step analysis provides the necessary simplicity, robustness, and reliability, making it a
suitable option for in-field applications.
Figure 4: (A) The constructed bioaerosol chamber. (B) Calibration curve for the QCM detection of
E. coli in bioaerosol. The inset shows the binding curves of samples captured by the cyclone. Adapted
from Paper IV with permission. Copyright 2014 American Chemical Society.
19
3.3 Plasma-Polymerized Surfaces for SPR Biosensing (Papers V–VII)
Surface plasmon resonance (SPR) biosensors are based on the oscillations of electrons in
conduction bands of metal films (typically gold) upon the excitation by light. This effect
strongly depends on the dielectric constant of the environment50
and can be exploited in
immunosensing because the biological interactions lead to the changes in oscillation frequency.
The measurement can be based on changes of intensity, angle, refractive index, or phase of the
reflected light.51
The SPR biosensors can be divided into two main groups: (i) propagating SPRs (PSPRs;
also simply referred to as SPRs) and localized SPRs (LSPRs).52
The excitation of PSPR is
typically done on continuous metal thin films using a prism or grating. The resonance then
spreads along the metal/dielectric surface to a distance up to hundreds of micrometers.53
In the
case of the LSPR, the plasmon resonance is not propagating and is excited on nanostructured
metal surfaces. The properties can be adjusted by the size, shape, or composition of the
nanostructures or nanoparticles.54
The SPR experiments are typically done in a direct,55
sandwich,56
or inhibition format.57
The direct assays are useful for bigger analytes that provide a sufficient response upon binding
(Figure 5). The sandwich assay is based on a two-step procedure. The antibody first binds the
analyte as in the direct format, followed by the capture of secondary antibodies (potentially
labeled with enzymes or nanoparticles) to enhance the signal.58
The inhibition assay is based
on mixing the analyte with respective antibodies, followed by the injection to the flow cell
containing a chip with a known amount of immobilized analyte. The binding of free antibodies
from the solution is evaluated to determine the analyte concentration.
To efficiently immobilize the capture molecules to the sensor surface, coatings bearing
chemical groups that allow the formation of covalent bonds are typically used.59, 60
Apart from
the amount of the functional groups, sufficient layer stability under various conditions is
necessary. Typically, the biomolecules are immobilized via primary amines or carboxyl
groups; therefore, there should be either
carboxyls or primary amines available on
the surface to provide immobilization via
the common carbodiimide / Nhydroxysuccinimide
zero-length coupling
reactions providing amide-based bonds.
Numerous methodologies were
investigated for surface modification with
the desired chemical groups. Typically,
wet-chemical procedures are being used,
including binding of thiols on the surfaces
of noble metals.61
However, these
methods are time-consuming and often
require the use of aggressive chemicals.
Alternatively, plasma
polymerization can be used to prepare thin
Figure 5: Scheme of SPR immunoassay in direct
format. Adapted from Paper VII with permission.
Copyright 2020 American Chemical Society.
20
polymer films on various surfaces in a fast and eco-friendly procedure without the requirement
of washing steps or reagent additions. Physical plasma is (partially) ionized gas consisting of
photons, electrons, positive and negative ions, atoms, free radicals, and excited or non-excited
molecules. Generation of plasma can be done by applying energy at low-, atmospheric-, or
high-pressure conditions. Non-thermal plasmas are gaining increasing attention in many
applications due to the possibility to provide enhanced gas-phase chemistry with high
concentrations of chemically active species without the need for increased gas temperatures.
The prepared plasma-polymerized (PP) coatings typically exhibit high branching and crosslinking,
excellent adhesion to practically any surfaces, and high stability.
The amine-rich PP coatings are the most commonly used and studied to allow
biomolecule immobilization and cell binding.62, 63
The use of amine-rich PP films was
demonstrated in biosensing, cell proliferation, and other biological applications.64, 65
Compared
with the conventional layers, e.g., carboxymethylated dextran (CMD), faster and more costeffective
layer preparation can be achieved by employing plasma processes. PP films with
carboxyl groups are usually prepared by plasma polymerization of various acrylates.66
Alternative approaches can be based on gas mixtures of CO2 and ethylene67
or deposition from
maleic anhydride (MA). The MA-based coating provided a highly reactive surface; however,
the polymerization required fine-tuning as the layer was initially not sufficiently stable.68
We have explored different ways of surface modification by PP films for biosensing
applications. First, we explored amine-based PP films composed of cyclopropylamine (CPA).
The pulsed plasma polymerization of CPA can be used to prepare reactive nitrogen-containing
films in a fast and eco-friendly way. As the layer stability is one of the critical aspects, we have
first investigated the behavior of CPA PP films in aqueous media. The immersion in the buffer
for 18 h before the glutaraldehyde activation turned out to be a critical step in maintaining longterm
layer stability. The FT-IR and ellipsometry showed that the number of amine groups
decreased, which was connected by the decreases of thickness by up to 17% after the immersion
in the buffer. The results were explained by the hydrolysis of enamines or imines in the CPA
PP; the chemical changes without thickness losses were caused by the hydrolysis of nitriles.
The activation by glutaraldehyde led to the growth of a 5–7 nm thick film of glutaraldehyde
and its oligomers. This surface was used to immobilize antibodies against human serum
albumin (HSA) or Salmonella for the biosensing experiments. Furthermore, regeneration with
10 mM NaOH allowed multiple measurements with a single sensor.
Since the commercial SPR sensors are most commonly based on carboxyl-containing
layers,69
we have also examined the field of carboxyl-rich PP films. In our preliminary study,
we have demonstrated the potential of PP films in SPR biosensing.70
We compared two kinds
of PP films prepared under different plasma conditions; the first was based on polymerization
from gas mixtures of maleic anhydride, acetylene, and argon (MA/C2H2/Ar; Figure 6A), the
other on the mixture of CO2, ethylene, and argon (CO2/C2H4/Ar). The capacity of both surfaces
to bind anti-HSA antibody was demonstrated, and the specific binding of HSA showed the
biosensing potential. The layer based on CO2/C2H4 exhibited lower stability and smaller
binding capacity, which resulted in the drift of the baseline and small response upon HSA
injection. In contrast, the film based on MA/C2H2 allowed efficient immobilization of the
21
antibody and provided a stable baseline signal, which resulted in a higher response to HSA
compared to the immunosensor based on CO2/C2H4.
Based on these results and with the aim to develop a robust biosensing layer, we
continued studying the preparation and properties of films based on MA/C2H2. We carried out
a systematic comparison of MA/C2H2 PP films with sensors based on a mixed self-assembled
monolayer of mercaptoundecanoic acid with mercaptohexanol (MUA/MCH) and with
carboxymethylated dextrans (2D and 3D CMD) in term of the performance in the detection of
HSA (Figure 6B). Compared with MUA/MCH and 2D CMD layers, the MA/C2H2 PP films
showed better performance, demonstrated by about two times higher signals. On the other
hand, the sensor based on 3D CMD still exhibited higher performance, especially providing a
wider working range. This, however, can be explained by the significantly smaller surface area
of the planar PP film compared to the 3D CMD.
Figure 6: (A) Scheme of sensor surface modification by MA/C2H2 PP film with subsequent
immobilization of the antibody via EDC/NHS chemistry. (B) SPR binding curves of HSA on antibodymodified
MA/C2H2 PP film. Adapted from Paper VI with permission. Copyright 2019 Springer.
In our next contribution, we used a high frequency-driven atmospheric-pressure plasma
jet (APPJ; Figure 7) to prepare PP coatings based on 1,2,4-trivinylcyclohexane (TVC),
tetrahydrofurfuryl methacrylate (THFMA), and a mixture of thereof. THFMA was selected
because of the presence of the vinylidene group that can form polymers chains and
tetrahydrofuran (THF) group, which served as a protective group against the breakdown of the
THFMA. TVC contains three vinyl groups, which makes it effective as a monomer, but TVC
also served as the source of carbon functionalities to adjust the carbon content and stability of
the resulting PP films. Under plasma-induced polymerization conditions, the THF or
cyclohexane ring-opening can happen, which results in further polymer chain cross-linking.
Both the TVC and the THFMA are non-toxic, which fits the eco-friendly procedure of plasma
polymerization.
22
The behavior of the films in an
aqueous environment was studied. The
highest stability was observed in the case
of ppTVC, which contained the lowest
amount of oxygen. In the case of
ppTHFMA and ppTHFMA-co-TVC,
more significant thickness losses occurred
during the initial storage in water,
however, with no impact on the chemical
composition. After the stabilization for
24 h in liquid, all films have shown a high
level of stability. The initial losses of film
thickness can be explained by the removal
of loosely bound oligomers from the film
surface. AFM has demonstrated that the
thickness losses were connected with the
formation of characteristic morphological
features. These led to an increase in the surface area and can be beneficial for the
immobilization of a higher amount of antibody, resulting in higher sensor sensitivity.
After the immobilization of the specific antibody, the PP-modified chips were used for
the SPR detection of HSA. We have demonstrated that not only the number of functional
groups affect the sensitivity of the measurement, but also the layer morphology is an essential
factor. The ppTHFMA-co-TVC layer with the highest surface roughness provided the largest
binding capacity. The sensors exhibited an excellent level of stability; the regeneration allowed
to perform up to 9 measurements with a single sensor. The achieved LOD of 50 ng/mL is
comparable with the performance of the 3D CMD chip, which confirms that the PP films are a
promising alternative to the conventional surface modification techniques.
Figure 7: Scheme of the atmospheric-pressure plasma
jet (APPJ; left) and photograph of the operating APPJ
(right). Reprinted from Paper VII with Permission.
Copyright 2020 American Chemical Society.
23
4 Catalytic Labels
4.1 Amperometric Detection of M. plutonius (Paper VIII)
Amperometric biosensors are devices based on the measurement of the current, corresponding
to the analyte concentration, as a function of electrode potential or time. The amperometric
methods can be used to determine the redox potential of the analyte, its electrochemical activity
(adsorption, interaction with modified layers, electrocatalysis), but they are also sensitive to
the changes of the electrode surface. In the case of amperometric catalytic biosensors, a suitable
enzyme (or multi-enzyme system) is typically immobilized on the electrode, which catalyzes
the transformation of the analyte; the concentration is determined from the measured current.
In the case of amperometric immunosensors, the most common approach is based on labeling
the analyte with a tracer (antibody conjugated with suitable enzyme); the analyte concentration
is determined from the current measured upon the addition of the substrate solution.
The most common potential-controlled (potentiostatic) measurement techniques
include (i) chronoamperometry based on the measurement of a current at a fixed potential in
time; (ii) single-potential amperometry based on the measurement of direct current as a
function of the potential difference between two electrodes, and (iii) multiple-potential
amperometry based on sweeping the potential in time and recording the corresponding current
in the whole potential window.2
We have developed a chronoamperometric biosensor for the detection of Melissococcus
plutonius, the causative agent of honeybee disease European foulbrood (EFB). EFB can
typically be found in honeybee larvae up to five days of age, which get infected by the ingestion
of food contaminated with M. plutonius. Upon the infection, the larvae color changes from
white to yellow or brown, and larvae usually die displaced in the brood cells instead of normal
coiled position.71
Because the infection can affect a large percentage of the brood, it can
severely weaken the colony or even cause its collapse.
It is crucial to prevent the uncontrolled spreading of the EFB to limit the economic and
environmental consequences of the honeybee colony losses. Therefore, there is a high demand
for methods that can detect M. plutonius in the stages of EFB infections, ideally in the PoC
format. The typical detection approach is based on the microscopic evaluation of smears
stained by carbol fuchsin. However, the sensitivity of this approach is not high enough to reveal
the EFB in its early stages. Cultivation-based methods traditionally exhibit very high sensitivity
at the cost of high time demands. However, in the case of EFB, the analysis is complicated by
the low cultivation recoveries of M. plutonius and overgrowing by secondary invaders.71
The
sensitivity, time-requirements, and throughput of the conventional methods can be overcome
by using molecular detection methods based on either DNA or antibodies. Nowadays, realtime
PCR is considered the gold standard for laboratory confirmation of M. plutonius.71
Even
though antibody-based methods are widely used to detect various pathogens,72
they are not yet
commonly used in the EFB diagnosis. There are no antibodies against M. plutonius
commercially available; the need to prepare the antibodies in-house is clearly one of the factors
limiting the faster growth of antibody-based methods for EFB. There is only a single report on
the ELISA for the laboratory detection of M. plutonius.73
Furthermore, an LFIA assay for EFB
24
was recently reported.74
However, it allows only qualitative disease confirmation, suggesting
room for more sensitive approaches.
To start working in the field of EFB diagnosis, we have first prepared a rabbit
polyclonal anti-M. plutonius antibody, and tested it in an ELISA assay.75
To develop an
amperometric immunosensor based on a sandwich assay (Figure 8A), the antibody was
immobilized to the gold working electrode via a self-assembled monolayer of cysteamine and
glutaraldehyde. As the tracer, antibody conjugated with horseradish peroxidase (HRP) was
used to provide electrochemical readout based on reducing the enzymatically oxidized
3,3´,5,5´-tetramethylbenzidine (TMB). Due to the use of the sandwich format, the effect of the
complex sample matrix was suppressed compared to the label-free procedure based on EIS.
Therefore, the amperometric approach is preferred for the analysis of complex samples of bees
and larvae. The specific capture of M. plutonius on the sensor was verified by AFM. Even
though the electrode exhibited substantial roughness, the bacteria were clearly visible.
The achieved LOD was 6.6×104
CFU/mL for the pure bacterial sample in the buffer,
and the sensor provided a working range up to 109
CFU/mL (Figure 8B). In the case of the
real sample analysis, LODs of 2.4×105
and 7.0×105
CFU/mL were achieved for homogenized
bees and larvae, respectively. The experiments with P. alvei as a negative control confirmed
the high selectivity of the assay. The achieved sensitivity, together with a short analysis time
of 2 h, confirm the suitability of the developed sensor in PoC diagnosis of EFB.
Figure 8: (A) Scheme of an amperometric immunoassay for the detection of M. plutonius.
(B) Amperometric response traces after the addition of TMB in the M. plutonius detection. Adapted
from Paper VIII with permission. Copyright 2019 Wiley-VCH.
4.2 Enzymatic Precipitation-Enhanced SPR Detection of Salmonella
(Paper IX)
In recent years, various kinds of nanoparticles are being employed to enhance the performance
of SPR immunosensors. For example, the application of gold nanoparticles in a sandwich
format allowed to significantly increase the refractive index in the Salmonella detection.76
Magnetic nanoparticles can provide signal amplification due to the increased refractive index
and the immunomagnetic preconcentration.77
However, the nanoparticle-based labels often
suffer from a higher level of non-specific interactions compared to smaller molecules. To
25
overcome this limitation, a method of amplification based on enzyme-catalyzed precipitation
of solid product on the sensor surface was developed.78, 79
The approach found application
mainly in electrochemical sensing, with reports on the detection of prostate-specific antigen
(PSA),80, 81
carcinoembryonic antigen (CEA),82
as well as E. coli.83
We introduced a method for the detection of Salmonella using SPR immunosensor
enhanced by biocatalyzed precipitation. Our strategy aimed to develop a highly sensitive,
robust, and straightforward assay while maintaining a reasonably short analysis time. The assay
was based on the formation of sandwich immunocomplex of capture antibody, Salmonella, and
HRP-conjugated detection antibody (Ab2-HRP). The HRP then catalyzed the conversion of
4-chloro-1-naphthol (4-CN) to insoluble benzo-4-chlorocyclohexadienone, which served as the
signal enhancement step (Figure 9A).
Figure 9: (A) Scheme of biocatalyzed precipitation-enhanced SPR detection of Salmonella. (B) SPR
sensorgrams of the final step of biocatalyzed precipitation enhancement for different Salmonella
concentrations. (C) Calibration curve. The inset shows the measuring chip after the precipitation
reaction (antibody-modified channel – left, reference – right). Adapted from Paper IX with permission.
Copyright 2016 American Chemical Society.
26
At the concentration of 107
CFU/mL, the signal change after precipitation enhancement
was 40× higher than the signal with the bare bacterium. A closer look at the SPR signal changes
in binding and reference channels revealed that even though some response was observed for
the non-specific binding of Salmonella, there was practically no signal change upon injection
of Ab2-HRP conjugate. This led to the increase of the ratio between the binding and reference
channel from 3.5 (after Salmonella binding) to 8.6 (after biocatalyzed precipitation). Even
though the injection of Salmonella in a very low concentration of 100 CFU/mL led to signal
change comparable to the level of noise, the following precipitation reaction allowed to
increase the signal to clearly distinguishable levels, allowing to reliably determine even very
low Salmonella concentrations (Figure 9B).
With the increasing concentration of bacteria, the measured signal increased
exponentially. This suggests that several Ab2-HRP conjugates can be bound on a single
Salmonella cells, leading to the precipitation of a large number of 4-CN molecules. This is a
significant advantage to nanoparticle-based signal amplification, which leads only to linear
enhancement of the signal.84
The obtained dependence of log(ΔR) on log(c) was linear from
102
to 106
CFU/mL, and the LOD was evaluated to be 100 CFU/mL (Figure 9C).
The total assay time was 60 min, which is substantially shorter than the conventional
methods for the detection of bacteria, including cultivation (~ days),85
ELISA (~ 10 h),86
and
PCR (~ hours).87, 88
The analysis time is also shorter than in other reports on the amplification
of SPR response with nanoparticles while achieving similar or better LOD.76, 84
Furthermore,
the real-time operation of SPR can reveal higher bacteria concentrations in a short time upon
sample injection (~ 10 min), allowing a rapid reaction even before the signal amplification is
finished.
After the measurement, the SPR chip was removed from the system and analyzed by
the AFM. It was visible already by the naked eye that there was more precipitate formed in the
measuring channel compared to the reference. AFM revealed the presence of bacteria and a
large number of precipitate particles (~ 22,000 particles on the area of 20×20 µm2
). Even
though the reference channel also contained some precipitate particles, the number was
significantly lower (~ 3,000 particles on 20×20 µm2
). The 6.9-fold difference in the number of
precipitate particles found by AFM corresponds to the 8.6-fold difference in SPR response for
the same concentration. A closer look at the individual Salmonella before and after the
precipitation revealed the presence of precipitate particles, leading to a three times increase of
the height upon the precipitation (Figure 10).
To demonstrate the applicability of the developed method for analyzing real samples,
Salmonella was detected in powdered milk. Although immunosensors used specific antibodies
to ensure selective detection, components of complex samples can still exhibit non-specific
binding towards the sensor surface. However, as the Ab2-HRP conjugate used in our method
is specific towards Salmonella, the potential non-specific binding is not transferred to the signal
amplification step, contributing to the high selectivity of the method. In powdered milk, the
achieved LOD was 103
CFU/mL, which is deterioration by one order of magnitude compared
to the analysis in the buffer. This was probably caused by the non-specific adsorption of milk
components, which can block some of the antibodies in the sensor surface or conceal some
27
epitopes on Salmonella. The ID50 (the number of bacteria that have to be ingested to result in
50% infection probability) of Salmonella is considered to be > 104
CFU.89
Furthermore, it was
shown that ingestion of low Salmonella levels below 102
CFU/g does not pose a risk to human
health.90
Therefore, the performance of the developed SPR immunosensor is suitable for the
practical analysis of Salmonella in contaminated food samples.
Figure 10: 3D representation of the AFM scan of (A) native and (B) precipitate-covered individual
Salmonella cells. (C) Cross-sections of the bacteria evaluated as perpendicular lines in the center.
Reprinted from Paper IX with permission. Copyright 2016 American Chemical Society.
4.3 Nanozyme-Linked Immunosorbent Assay (Paper X)
The typical catalytical labels used in immunoassays are represented by enzymes, especially
HRP. However, the enzymes suffer from several disadvantages, including the high cost of their
production, limited stability, and activity reduction upon conjugation with immunoreagents.
The properties of conventional enzymes can be overcome by using catalytic nanomaterials –
nanozymes.91, 92
Compared to the biomolecules, the inorganic nanomaterials provide very high
thermal and chemical stability.93
In particular, nanozymes with high peroxidase-like activity
are preferred for immunoassays due to the compatible assay procedure with conventional
ELISA.
Nanozyme production can be done using aqueous solutions and benign precursors,
making the procedure eco-friendly.94
The peroxidase-like activity was first discovered for
magnetite nanoparticles (Fe3O4), followed by many other nanomaterials, including CeO2, CuO,
Co3O4, and MnO2 nanoparticles, graphene oxide nanoplates, or Prussian blue nanoparticles
(PBNPs).95, 96
We have introduced a method for the conjugation of PBNPs with antibodies and applied
the conjugates in a nanozyme-linked immunosorbent assay (NLISA). The conjugation was
based on the modification of the PBNP surface by reductively denatured bovine serum albumin
(BSA), followed by the oxidation of antibodies by sodium periodate and binding them to the
amino group of BSA (Figure 11). We have developed two sandwich NLISA assays, first for
the detection of HSA in urine and the other for the detection of Salmonella in powdered milk.
Because the oxidation of TMB to the blue product was utilized in the assay, the readout could
be done using a standard colorimetric reader without special requirements on instrumentation.
For the analysis of HSA in urine, the possible trace amounts of HSA present in the urine
of healthy donors were first removed using centrifugal ultrafiltration on a 10-kDa membrane,
followed by spiking known HSA concentrations. Even though the urine contains various ions,
28
which can cause undesired oxidation of
TMB, the heterogeneous format with
several washing steps allowed to
overcome this limitation. As a result,
only small differences were observed
between HSA analysis in the buffer and
urine. Microalbuminuria, which
happens due to diabetic nephropathy, is
connected with HSA concentrations
from 20 to 200 μg/mL in the 24 h
specimens.97
The optimized NLISA
provided an LOD of 1.2 ng/mL of HSA
in urine and a working range up to 1
μg/mL (Figure 12). This confirms that
the NLISA is suitable for the practical
diagnosis of microalbuminuria.
Comparing the performance of
NLISA with ELISA based on the same
immunoreagents has shown only a
small difference in LODs (1.2 ng/mL for NLISA and 3.7 ng/mL for ELISA). The comparable
results suggest that the primary limiting step of the assay is not the detection step but rather the
antibody affinity. Nevertheless, PBNPs offer several practical advantages, including higher
stability, simple and cheap synthesis, and the possibility of catalyzing higher concentrations of
TMB. As the shelf-life of PBNPs is practically unlimited (several years at 4 °C), antibodies are
becoming the main limiting element. Therefore, the overall stability of the detection label could
be, in the future, improved by replacing antibodies with MIPs or aptamers.
Figure 12: Scheme of sandwich NLISA and calibration curve for HSA detection in spiked
urine. Reprinted from Paper X with permission. Copyright 2018 American Chemical Society.
Figure 11: Scheme of PBNP-Ab conjugate synthesis. The
PBNPs are modified by denatured BSA to introduce amino
groups on their surface, and the oxidized antibody is
conjugated via the aldehyde groups. Reprinted from
Paper X with permission. Copyright 2018 American
Chemical Society.
29
The universal applicability of PBNPs as immunoassay labels was also demonstrated by
detecting Salmonella in powdered milk. The incubation with milk served as an additional
blocking step and had a minimum effect on the LOD. The optimized assay provided LOD of
6×103
CFU/mL with a working range up to 106
CFU/mL. This performance is better than the
published ELISA assays that provide LODs between 104
and 106
CFU/mL.28, 98
The
comparison with ELISA based on the same immunoreagents (LOD 3×103
CFU/mL) confirmed
that the choice of antibodies affects the assay performance more significantly than the label.
Overall, PBNPs proved to be a suitable alternative to conventionally used HRP; the detection
of peroxidase-like activity is compatible with standard instrumentation and methodologies.
Furthermore, the nanoparticle-based labels provide higher stability than biomolecules with the
possibilities for cheap and large-scale production.
30
5 Photon-Upconversion Nanoparticles
Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanocrystals, which exhibit
anti-Stokes emission. The energy transfer upconversion belongs to the non-linear optical
processes and is based on the absorption of two or more photons, resulting in the emission of
a single photon with higher energy (shorter wavelength).99
Compared with other anti-Stokes
processes, including two-photon excitation and second harmonic generation, the excitation of
upconversion can be done at lower energy densities. Even though the upconversion process
was discovered already in the 1960s,100
it was only used in the form of bulk crystalline or glass
materials.101
The composition of inorganic upconversion phosphor is based on a crystalline
host matrix (typically NaYF4) with a dopant included at a low concentration (typically Yb3+
and Er3+
or Tm3+
). The dopant ensures luminescence, while the crystal structure of the host
lattice provides a matrix to bring the dopant ions into the optimal position.102
Due to the remarkable progress in nanotechnology, methods for the synthesis of
upconversion nanomaterials were discovered, leading to UCNPs with high luminescence
efficiency. Compared to conventional luminescence labels, such as organic fluorophores or
QDs, UCNPs can be detected without autofluorescence background, they provide large antiStokes
shifts allowing easy separation of the excitation and detection channels, exhibit
excellent photostability, and the emission wavelength is tunable to enable multiplexed
detection.102
The synthesis of UCNPs is typically done in hydrophobic solvents, such as oleic acid
and octadecene. Therefore, their surface has to be modified for biological applications.103
One
of the most widespread surface modification techniques is silanization. It is based on the
hydrolysis and condensation of siloxane precursors, typically tetraethyl orthosilicate104
with
other derivatives of silane, to provide functional groups for further bioconjugations.105
Other
modification techniques are based on exchanging the hydrophobic surface ligands by ligands
with hydrophilic properties. Ligands bearing phosphonate or carboxylate groups can
coordinate to the lanthanide ions on the UCNP surface; the functional groups on the other end
of the ligand are then used for the bioconjugation.106
Throughout our extensive work in the UCNP field, we have explored different ways of
UCNPs surface modifications and employed the conjugates to develop immunoassays for a
wide range of analytes, from small molecules to bacteria.
5.1 Competitive Upconversion-Linked Immunosorbent Assays
5.1.1 Competitive ULISA for Diclofenac (Papers XI and XII)
Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug. The widespread use
of DCF for cattle treatment in the Indian subcontinent has led to significant vulture population
losses in the 1990s because DCF caused renal failure in vultures feeding on contaminated
carcasses.107
DCF is one of the most frequently analyzed pharmaceuticals in the water-cycle in
Europe because it cannot be easily degraded in water treatment plants. It was detected in the
amounts of low μg/L wastewater effluents and amounts of ng/L in surface waters,108
groundwater, and drinking water.109
The sensitive detection of DCF is typically done by LCTOF-MS
or high-resolution mass spectrometers.110
However, these methods require expensive
31
instrumentation with trained personnel, and the analysis is lengthy. On the other hand, ELISA
assays are highly suitable for analyzing a large number of samples, even in smaller, lessequipped
laboratories.111
In our pioneering work on the immunoassay based on UCNPs – the upconversion linked
immunosorbent assay (ULISA) – we have synthesized conjugates of UCNPs with detection
anti-mouse antibody and applied them in an indirect competitive assay for DCF (Figure 13A).
We have synthesized oleic acid-capped UCNPs and coated them with a silica shell bearing
carboxyl groups on the surface. This modification was used to improve the water dispersibility
and conjugate antibodies using EDC/sulfo-NHS chemistry.112
The coating antigen in a competitive immunoassay has to be, on hand, in low-enough
concentration to allow efficient competition for the binding sites of the detection antibodies,
but, on the other hand, its concentration still has to provide strong-enough signals. To achieve
the optimal assay performance, we have prepared and characterized two different coating
conjugates by modifying bovine serum albumin (BSA) with DCF. The MALDI-TOF mass
spectrometry analysis revealed that the conjugates carried either 5.7 or 10 DCF molecules per
BSA. Even though the conjugate with the higher degree of derivatization provided about twice
as high signals, larger signal fluctuations and hook effect were observed. On the other hand,
the conjugate with 5.7 DCF molecules per BSA provided better signal stability, slightly lower
IC50 value (1.2 ng/mL compared to 1.5 ng/mL), and lower LOD. The optimized ULISA assay
provided an LOD of 0.05 ng/mL, which was five times higher than the LOD of a conventional
ELISA (0.01 ng/mL; Figure 13B). However, the ULISA allowed for a faster and easier signal
generation. However, it was most notably a first step in the further development of ULISA
assays that were eventually going to reach a single-molecule sensitivity.
Figure 13: (A) Scheme of indirect competitive ULISA for the detection of DCF. A microtiter plate is
coated with a BSA-DCF conjugate, dilution series of DCF are prepared in the microtiter plate followed
by the addition of anti-DCF mouse antibody, and the attachment of anti-DCF antibody is detected by a
conjugate of secondary antibody with UCNP. (B) Normalized calibration curves of ULISA and ELISA.
Adapted from Paper XI with permission. Copyright 2016 American Chemical Society.
In the follow-up work, we focused on improving the LOD and reducing the analysis
time by designing a single-step ULISA assay (Figure 14A). For the synthesis of the DCF
tracer, we have prepared a conjugate to DCF with bovine γ-globulin (BGG) and conjugated it
on the surface of UCNPs with a carboxylated silica shell. The DCF-BGG conjugate was used
32
because it provided structural flexibility between the DCF and the UCNPs, and it prevented
non-specific binding of the tracer to the microtiter plate. Because the UCNP-DCF tracer was
used to directly compete with the analyte DCF for the binding on the immobilized anti-DCF
antibody, the assay was done in a single step. Furthermore, we have optimized a method for
the lyophilization of the tracer, which did not negatively affect its performance even after
prolonged storage at room temperature. The single-step analysis and the possibility of storing
the tracer in a dry state without the necessity of cooling makes the ULISA an excellent option
for environmental analysis in low-resource settings.113
The optimized assay provided an LOD of 0.02 ng/mL with a signal-to-background (S/B)
ratio of 82 (Figure 14B). The high value of S/B was enabled by (i) the high brightness of the
used UCNPs with a diameter of 90 nm, (ii) the low level of non-specific interaction due to the
coating with BGG, and (iii) the presence of multiple DCF molecules per single UCNP ensures
efficient competition even when the molar concentration of tracer is significantly lower than
the concentration of DCF. Finally, we have demonstrated the practical potential of the method
on the successful analysis of real samples of drinking and river water.
Figure 14: (A) Scheme of single-step competitive ULISA for the detection of DCF and (B) calibration
curve. Adapted from Paper XII with permission. Copyright 2017 Springer.
5.1.2 Competitive ULISA for Zearalenone (Paper XIII)
We have also developed an assay for the analysis of mycotoxin zearalenone, based on the
competition with epitope mimicking peptide. Microbial toxins are produced by many
pathogenic microorganisms (bacteria and fungi) and act as their virulence agents. They are
represented by a heterogeneous group of compounds that interfere with biochemical processes,
including the function of membranes, transport of ions, release of transmitters, and synthesis
of macromolecules. Exposure to the toxins either in food or in the environment can cause
significant health problems; the individual symptoms vary significantly between the different
toxins.114
Unlike in the case of viable bacteria, toxins are typically not affected by the heat
processing of the product.
Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin produced by several fungi
species of the Fusarium genus.115
Even though the acute toxicity of ZEA is relatively low, it is
chronically toxic and has been connected with reproduction disorders of farm animals, mainly
pigs.116
ZEA exhibits estrogenic, genotoxic, haematotoxic, and anabolic effects.117
Along with
33
other mycotoxins, ZEA is often found in agricultural products, including maize, wheat, barley,
rice, and oats.118
Epitope mimicking peptides, also referred to as mimotopes, are used as an alternative
to conventional analyte-conjugates in competitive immunosensing. Such peptides mimic the
epitope of the analyte and allow competition with the native analyte for binding to the antibody.
Even though antibodies with high affinity are required in all immunoassay, competing peptides
with lower affinity can be beneficial for competitive assays. They shift the equilibrium towards
analyte binding, making a smaller amount of analyte produce the same response level.119
Based on the previously identified amino acid sequence,120
we have synthesized the
peptide mimetic of ZEA, introduced biotin on its C-terminus, and used it in an ULISA assay
for ZEA detection (Figure 15). The specific anti-ZEA antibody was bound on the surface of
the microtiter plate, allowing competition between analyte ZEA and biotinylated peptide
mimetic. The detection was carried out using the conjugate of UCNPs with streptavidin. The
optimized assay provided an LOD of 20 pg/mL with a working range up to 0.5 ng/mL,
representing a 200-fold improvement of LOD and 3-fold improvement of working range
compared to the previously reported bioluminescence immunoassay with the same peptide
mimetic.120
Figure 15: Scheme of competitive ULISA for ZEA. In the first step, a microtiter plate is coated with
an anti-ZEA antibody, and ZEA in the sample competes with the biotinylated peptide mimetic for a
limited amount of antibody binding sites. In the second step, the conjugates of UCNPs with streptavidin
bind to the biotinylated peptide. Reprinted from Paper XIII with permission. Copyright 2020 Elsevier.
34
To confirm the potential of ULISA for the analysis of complex samples, analysis of
ZEA-free maize samples (as confirmed by UPLC-MS/MS) spiked with ZEA was done. The
recoveries between 77% and 105% demonstrate the suitable accuracy for quantitative real
sample analysis. Furthermore, in comparison with UPLC-MS/MS, ULISA is based only on a
simple extraction in methanol and does not require extensive sample pre-treatment. The
achieved performance fulfills the requirements given by the European legislation, which
confirms the suitability of ULISA as a tool for simple analysis of food samples contaminated
by mycotoxins.
5.2 Sandwich Upconversion-Linked Immunosorbent Assays
5.2.1 Detection of M. plutonius with BSA-Modified UCNPs (Paper XIV)
UCNPs are also highly useful as labels in sandwich immunoassays. We have introduced a
method to conjugate UCNPs with streptavidin based on a copper-free click reaction and used
this conjugate to detect M. plutonius, the causative agent of European foulbrood (Figure 16).
The conjugation was based on strain-promoted cycloaddition between bicyclo[6.1.0]nonyne
(BCN) groups bound on the UCNP surface via BSA and azide-modified streptavidin.121
Apart
from serving as an intermediate to bind the BCN, BSA also contributes to the reduction of nonspecific
binding of the UCNP conjugates.
Figure 16: Scheme of the conjugation of UCNP with streptavidin based on functionalization of UCNP
surface with alkyne-modified BSA and click reaction with azide-modified streptavidin. The conjugates
are then employed as a label in a sandwich ULISA for the detection of M. plutonius. Reprinted from
paper XIV with permission. Copyright 2019 Royal Society of Chemistry.
The bioconjugation reaction was followed using agarose gel electrophoresis based on
fluorescence labeling of BSA-BCN and streptavidin-azide. The overlap of the fluorescence
signals, together with the mobility shift, confirmed the successful progress of the conjugation
reaction. Furthermore, the presence of BSA and streptavidin on the UCNP surface was
confirmed by mass spectrometry. After the digestion of proteins on the UCNP conjugates by
trypsin, the UCNP cores were removed by centrifugation, and the samples were analyzed by
35
LC-MS/MS. Both BSA and
streptavidin were successfully
identified; according to the integrated
signal intensities, BSA was detected as
the most abundant protein in the
sample. The amount of streptavidin
was approximately one order of
magnitude lower than the amount of
BSA.
First, we have optimized
ULISA on the detection of M. plutonius
in the buffer. The assay provided an
LOD of 340 CFU/mL and a wide
working range up to 109
CFU/mL. This
LOD is 400 times better in comparison
with ELISA (Figure 17). Since the
same antibodies were used in both
assays, the most significant impact on
enhancement can be accounted to the high label performance, providing a highly sensitive
readout of anti-Stokes emission and the low non-specific binding. To demonstrate the practical
applicability of the assay, real samples of bees, larvae, and bottom hive debris were analyzed,
representing the typical matrices where M. plutonius has to be detected during the infection by
EFB. The achieved LODs were 540 CFU/mL for bee extract, 8.5×103
CFU/mL for larvae
extract, and 570 CFU/mL for bottom hive debris. The level of M. plutonius in infected apiaries
with clinical symptoms is typically around 105
CFU/mL,122
demonstrating the suitability of the
developed ULISA for the practical EFB diagnosis in the early stages of the infection.
5.2.2 Preparation of PEG-Modified UCNPs and Analysis of HSA (Paper XV)
In our next work, we have introduced a different strategy for UCNP surface modification based
on coating the particles with a PEG linker and applying the conjugates in a sandwich
immunoassay for the detection of albuminuria marker HSA. For the surface modifications, we
have chosen heterobifunctional PEG with neridronate, and alkyne or maleimide functional endgroups
based on these considerations; (i) PEG can sterically stabilize the particles and resist
the non-specific interactions with surfaces and biomolecules;123
(ii) neridronate shows strong
coordination towards lanthanide ions of UCNPs;124
and (iii) the alkyne or maleimide groups
can be used for the subsequent conjugation of biomolecules.125
The first conjugation approach
was based on attaching azide-modified antibody or streptavidin to the alkyne groups via click
reaction (Figure 18). Alternatively, the disulfide bonds in the antibody were reduced by TCEP,
and the generated thiol moieties were bound to the maleimide groups.
Figure 17: Comparison of sandwich ULISA and ELISA
assays for the detection of M. plutonius. The normalized
signals were calculated by dividing the data by yMAX value
from the logistic fit. Reprinted from Paper XIV with
permission. Copyright 2019 Royal Society of Chemistry.
36
Figure 18: Scheme of the preparation of PEG-based conjugates of UCNPs with streptavidin. The oleic
acid on the surface of as-synthesized UCNPs was removed by a ligand exchange reaction with
nitrosonium tetrafluoroborate to prepare water-dispersible nanoparticles. The particles were then coated
with an alkyne-PEG-neridronate linker, and streptavidin-azide was coupled via copper-catalyzed click
reaction.
The prepared bioconjugates were used in the sandwich ULISA assay for HSA detection.
To allow efficient use in immunoassay, the nanoparticle-based labels must provide not only a
high level of modification with biorecognition molecule to allow specific binding, but the
conjugates must also show high uniformity with a small number of aggregates to reduce the
signal fluctuations.
First, we have tested the UCNPs modified by antibody via alkyne-azide click reaction.
Two different ratios between the antibody and NHS-PEG-N3 were tested. Even though higher
signals were observed in the case of conjugate based on antibody with a higher number of azide
molecules, there was no positive effect on the LOD. This can be explained by the higher
number of aggregated conjugates, resulting in higher signal fluctuation. The optimized ULISA
based on these particles provided an LOD of 3.5 ng/mL and a working range up to 1 μg/mL.
In contrast, the antibody conjugates based on maleimide coupling provided higher signals and
slightly lower background, resulting in the improvement of the LOD to 0.24 ng/mL and an
unchanged working range up to
1 μg/mL. The conjugates with
streptavidin reached an even lower LOD
of 0.17 ng/mL (Figure 19). This can be
explained by the more efficient binding
of the streptavidin-coated UCNPs to the
biotinylated antibody, caused mainly by
the flexibility of the additional antibody
present in the immunocomplex
compared to the binding directly to the
antigen. The comparison with ELISA
(LOD 0.56 ng/mL) and fluorescence
immunoassay (LOD of 0.59 ng/mL)
based on the same immunoreagents
demonstrated that the use of UCNPs is
advantageous in term of assay
performance compared to the
conventional labels.
Figure 19: ULISA for the detection of HSA with UCNPPEG-SA
label. Adapted from Paper XV with permission.
Copyright 2020 American Chemical Society.
37
The optimized assay based on streptavidin-conjugated UCNPs was then used for the
analysis of HSA in spiked urine. A slightly higher baseline was observed for the urine
compared to the buffer, which was probably caused by the presence of some HSA levels, even
in the samples from healthy donors. However, this did not affect the ability to specifically
detect the HSA, demonstrating the potential of the method for practical applications.
5.2.3 Detection of P. larvae with PEG-Modified UCNPs (Paper XVI)
Afterward, we have employed the PEG-based UCNP conjugates to detect spore-forming
bacterium Paenibacillus larvae, the causative agent of American foulbrood (AFB). AFB
represents the most dangerous honeybee brood disease and causes significant economic losses
throughout the world.126
The honeybee larvae are infected by ingesting the fee contaminated
by spores. The spores then germinate and colonize the midgut of the larvae, which is followed
by spreading the bacteria over the midgut epithelium and the body cavity of the larva.127
The
dead larvae are found as a glue-like mass sticking to the side of the honeycomb cell, which is
used as the typical sign for the AFB diagnosis. Afterward, the bacteria sporulate, and the spores
are spread around the hive by the adult honeybees, which results in the infection of more larvae
by the ingestion of contaminated food reserves. The spores can be found not only in the
diseased larvae and the resulting dry scales but also in adult worker bees, honey, bottom hive
debris, beehive surfaces, and beekeeping equipment.128
Because the spores are highly resilient, the discovery of infection is usually connected
with burning down the honeybee colonies, as well as the contaminated equipment.129
Therefore, sensitive diagnostic approaches are required to allow early diagnosis, preventing the
infection from spreading further.130
The traditional diagnosis of AFB is based on observing the
clinical signs within the hive. Microscopic evaluation of stained smears from diseased larvae
can be used for fast detection; however, the sensitivity of this approach is not high enough to
diagnose the infection in its early stages.131
On the other hand, culture-based methods provide
excellent sensitivity, but the cultivation takes several days, making this approach not suitable
for screening purposes.132
Currently, PCR is considered the gold standard for AFB diagnosis,
as it combines high sensitivity with fast analysis.132, 133
However, PCR-inhibitors and other
contaminants in the honeybee material can complicate the analysis of real samples.134
The
development of immunochemical methods for AFB diagnosis is generally hampered by the
lack of commercially available antibodies against P. larvae. Even though there was a single
report on the ELISA for the AFB diagnosis,135
its sensitivity did not allow detecting sub-clinical
P. larvae levels.
We have prepared a rabbit polyclonal anti-P. larvae antibody and used it in ULISA
assay to allow early diagnosis of AFB. Cell wall fraction of P. larvae was used for the
immunization of two rabbits. However, only one serum was used for further experiments
because the other showed high cross-reactivity with M. plutonius. Even though affinity
purification is generally used to suppress the cross-reactivity of generated antibodies, it was
not possible here because of the complex nature of the used antigen.136
Therefore, all IgGs
present in the antiserum were isolated by the purification on protein G affinity column.
First, an ELISA assay was used for testing the antibody specificity. To allow performing
a sandwich assay, the antibody was conjugated with biotin, and the conjugate of streptavidin
38
with HRP (SA-HRP) was used as a tracer. The assay provided an LOD of 6.5×104
CFU/mL
and a S/B ratio of 34. This performance is comparable with the LOD of 1×105
CFU/mL
published by Olsen et al.;135
however, it is not sufficient to analyze sub-clinical levels of the
bacterium.
Therefore, the SA-HRP conjugate was replaced by the streptavidin-coated UCNPs
(Figure 20A) in the ULISA assay (Figure 20B). The S/B value of 128 was achieved in the
optimized assay, representing a 4-fold improvement compared to the ELISA. In the case of
negative controls of P. alvei, M. plutonius, and B. laterosporus, only small signal changes were
observed compared to the target bacterium P. larvae. The assay provided an LOD of
2.9×103
CFU/mL (Figure 20C), which is 22 times better than the ELISA with the same
antibody, clearly demonstrating the advantage of the labels based on UCNPs. Finally, the
successful analysis of real samples of bees, larvae, and bottom hive debris showed the potential
of ULISA in the practical diagnosis of AFB.
Figure 20: (A) Structure of UCNP-PEG-SA conjugate. (B) Scheme of sandwich ULISA for the
detection of P. larvae. (C) Cross-reactivity testing of ULISA with P. larvae as a specific target and
P. alvei, M. plutonius, and B. laterosporus as negative controls. Adapted from Paper XVI with
permission. Copyright 2021 IEEE.
5.3 Single-Molecule Upconversion-Linked Immunosorbent Assays
5.3.1 Digital ULISA for PSA (Papers XVII and XVIII)
Due to the very low optical background, it is even possible to detect individual UCNPs. We
have developed an approach allowing visualization of single UCNPs under a conventional
epiluminescence microscope and used it in a single-molecule assay of cancer biomarker
prostate-specific antigen (PSA). Prostate cancer is globally the fifth leading cause of death from
cancer and the most often diagnosed cancer type among men.137
PSA is secreted by the
epithelial cells of the prostate in a typical concentration in healthy men below 4 ng/mL; the
increase above this level can be used in the prostate cancer diagnosis.138
When the carcinoma
is removed by the radical prostatectomy, the PSA levels decrease significantly.139
However,
39
repeated monitoring of PSA is still necessary
because the increase from levels below 0.1
ng/mL to consistently above 0.2 ng/mL is
connected with the biochemical
recurrence,140
which occurs in up to 40% of
cases after the surgery.141
This highlights the
need for sensitive assays to detect the
recurrence of cancer as early as possible.
In the first step, we have modified an
epifluorescence microscope Nikon Eclipse
Ti-E for the imaging of UCNPs (Figure 21).
The microscope was equipped with a 980-nm
excitation laser, 100× heat resistant
objective, and suitable filter sets to detect
upconversion luminescence of Er- and Tmdoped
UCNPs. For optimizing the setup,
carboxylated UCNPs with sizes from 37 to
90 nm were immobilized on a glass slide
modified by cationized BSA. The excitation
by 980-laser resulted in a very low
background that – when there were no
UCNPs present on the surface – depended
only on the signal noise of the camera. All tested UCNP types were visible as individual
diffraction-limited spots with a diameter of ~ 400 nm; the number of detected UCNPs was
directly proportional to their concentration.
Microtiter plates with a thin foil (190 µm) at the bottom of each well were used in an
immunoassay because of the short working distance of the objective with the high numerical
aperture. The assay was based on the sandwich immunocomplex of the capture antibody, PSA,
and the conjugate of silica-coated UCNPs with detection antibody. First, the upconversion
luminescence was read out by a conventional microtiter plate reader (analog ULISA), followed
by counting the individual immunocomplexes under the microscope (digital ULISA). There
was a small number of upconversion spots visible, in the case of the blank with no PSA in the
samples, which corresponds to the non-specific binding of UCNPs to the surface of the
microtiter plate and defines the LOD similarly as in the conventional immunoassays. However,
compared to the analog readout, digital detection offers several advantages: Because the signal
of an individual label can be reliably distinguished from the background noise, the background
fluctuations do not influence the measurement. Therefore, the LOD is limited only by the
affinity and non-specific binding of the immunoreagents used in the assay. Furthermore, in
contrast to the intensity-based readout, where a few big aggregates can strongly affect the
overall intensity, the digital approach counts the aggregates as single binding events, reducing
their effect on the measured signal.142
This enhances the robustness of the measurement and
indirectly allows for achieving lower LODs.
Figure 21: Scheme of upconversion microscope.
The inset shows individual UCNPs as diffractionlimited
spots. Adapted from Paper XVII with
permission. Copyright 2017 American Chemical
Society.
40
Figure 22: Upconversion microscopy images of microtiter plate after binding of serial dilutions of PSA
in 25% serum. The calibration curves yield LOD of 1.2 pg/mL in the digital and 20.3 pg/mL in the
analog mode. Adapted from Paper XVII with permission. Copyright 2017 American Chemical Society.
The analog readout of the immunoassay for PSA in 25% serum provided an LOD of
20.3 pg/mL with a working range from 100 pg/mL to 10 ng/mL. This is comparable with the
commercial ELISA assays for the PSA.143
The digital readout of the same microtiter plate
(Figure 22) allowed to lower the LOD by more than one order of magnitude down to
1.2 pg/mL, with a working range from 10 pg/mL to 1 ng/mL. It was not possible to analyze
higher PSA concentrations because the point-spread functions of the UCNPs started to overlap
and did not allow reliable counting. It is also possible to combine both readout options,
extending the overall working range to three orders of magnitude from 10 pg/mL to 10 ng/mL.
The PSA detection in the buffer provided practically identical results, confirming that the 25%
serum has a negligible matrix effect.
In our follow-up work, we have further improved the single-molecule assay scheme by
replacing the conjugates of silica-coated UCNPs with antibody by PEG-coated UCNPs
conjugated with streptavidin (Figure 23). Such conjugate was expected to provide better
performance because (i) the PEG provides resistance of UCNPs against aggregation and
ensures high dispersibility in water; (ii) the steric hindrance of the PEG reduces the level of
non-specific interactions;144, 145
and
(iii) the subsequent addition of
biotinylated detection antibody and
streptavidin-coated UCNPs allows
using a relatively high detection
antibody concentration to efficiently
label all PSA molecules while being
able to reduce the UCNP
concentration due to the high affinity
between streptavidin and biotin,146
which leads to a lower amount of
non-specifically adsorbed UCNPs.
Figure 23: Upconversion microscopy image of a microtiter
plate after the specific capture of PSA and a scheme of
individual sandwich immunocomplex. Reprinted from Paper
XVIII with permission. Copyright 2019 American Chemical
Society.
41
Compared to our previous study,147
the new assay design lowered the LOD by 50 times.
The comparison of an assay based on Er-doped (LOD 23 fg/mL, 800 aM) and Tm-doped
UCNPs LOD 24 fg/mL, 840 aM; Figure 24A) demonstrated that the advantages of the digital
readout are not dependent on the label type. The three times lower value of IC50 further
confirms that the two-step label design provides improved binding kinetics. For the real sample
analysis, random samples of human serum were collected in the hospital and analyzed by
electrochemiluminescence immunoassay as a reference method. The dilution linearity
experiments have shown that human serum has a low matrix effect on the assay, as represented
by the recovery rate fluctuations below 20%. The results from electrochemiluminescence assay
and ULISAs (both analog and digital) were in great agreement, confirming the potential of
UCNPs to be used as a label in assays for the diagnosis of prostate cancer (Figure 24B).
Figure 24: (A) Calibration curves of the ULISA in the digital (red) and analog (black) mode. The
logarithmic scale of the y-axis highlights the signals in the low PSA concentration range. (B) Correlation
between the PSA concentrations in human serum samples determined by the digital (red) or analog
(black) ULISA and an electroluminescence immunoassay. Reprinted from Paper XVIII with
permission. Copyright 2019 American Chemical Society.
5.3.2 Digital ULISA for Cardiac Troponin (Paper XIX)
The digital ULISA can be adapted for the detection of other biomarkers by simply exchanging
the immunoreagents. Thus, to demonstrate the universal nature of the approach, we focused on
detecting cardiac troponin, a biomarker of acute myocardial infarction (AMI). Heart diseases
represent the leading cause of death worldwide. There is typically only a short time available
since the symptoms start before the treatment is necessary, creating demand for rapid and
reliable diagnostic assays.148
Cardiac troponin is one of the recommended biomarkers for the
diagnosis of AMI. Because it is located only in myocardial tissue in healthy individuals, its
elevated concentration in blood can indicate the onset of AMI.149
Cardiac troponin is a
heterotrimeric complex, which consists of three distinct subunits – cTnI, cTnT, and TnC.150
The subunits cTnI and cTnT are present only in the myocardium (heart muscle), and during the
AMI, they are released into the bloodstream.151, 152
The diagnosis of AMI can be made based
on measuring the changes in the cTnI levels.153
42
However, cTnI is a challenging analyte for the detection by immunochemical methods
as its recognition by antibodies can be affected by several factors.154, 155
The N- and C-terminal
parts of cTnI are susceptible to proteolytic degradation,156
favoring the use of antibodies that
target epitopes in the central region.157
In addition, cTnI is typically present in blood in the
form of a binary cTnI-TnC complex,158
thus, the antibodies should recognize free as well as
complex form cTnI. Furthermore, the epitopes can be phosphorylated or blocked by
autoantibodies or heterophile antibodies, hindering immunochemical recognition.159
For this
reason, assays for cTnI are often based on the combination of two capture or two detection
antibodies.154
In our work, we studied the impact of size and surface modification of UCNP labels on
analog and digital ULISA for the detection of cTnI. The size of UCNP-based detection labels
is one of the critical factors affecting assay performance. On the one hand, the particles should
be as small as possible to (i) provide stable dispersions, (ii) minimize the level of non-specific
binding, and (iii) limit the influence of the UCNPs and the immunochemical interaction. On
the other hand, larger size leads to higher brightness of the UCNPs, making them more easily
detectable. Furthermore, we tested two ways of surface modification, based on (i) alkyne-PEGneridronate
linker and streptavidin and (ii) poly(acrylic acid) (PAA) and antibody; the
corresponding assay schemes are shown in Figure 25.
Figure 25: ULISA configurations for the detection of cTnI with labels based on (A) UCNP-PEG-SA
and (B) UCNP-PAA-Ab. Adapted from Paper XIX under the permission of Creative Commons
Attribution-NonCommercial 4.0 International License.
We found out that the size and surface modification of UCNPs affect the assay
performance more than the difference between analog and digital readout modes. Varying the
UCNPs size affected especially the assays in human plasma; the increasing size resulted in a
higher level of non-specific binding; however, the smaller UCNPs exhibited a slightly higher
degree of aggregation. The highest sensitivity was achieved when using PAA-based UCNPs
with a diameter of 48 nm. Surprisingly, the LODs in human plasma provided by analog
(8.6 pg/mL) and digital (9.8 pg/mL) readout were very similar. This contradicts with the results
43
achieved when detecting PSA where digital readout showed significantly higher sensitivity
than the analog one. This discrepancy might be caused by the different affinity of the antibodyantigen
pairs for PSA and cTnI. The analog readout provided a 10-fold lower LOD for PSA
compared to cTnI, and the difference increased to 200-fold in the digital mode. These results
thus suggest that a large enough antibody affinity is required to allow the digital readout to
further increase the sensitivity of the ULISA.
5.4 UCNP-Based Immunocytochemistry (Paper XX)
We have also demonstrated that UCNPs can be advantageously used to label breast cancer
biomarkers in immunocytochemistry (ICC) and immunohistochemistry (IHC). With around
2.1 million new cases reported every year, breast cancer is the second most common type of
cancer worldwide.160
Even though mammographic screening and advances in adjuvant
systemic therapy help fighting the disease, its incidence continues growing.161
Human
epidermal growth factor receptors (HER or ErbB) belong to membrane receptors, which play
essential roles in biological processes, including apoptosis and migration, differentiation, and
proliferation of cells. The overexpression of the HER2 receptor on cancer cells happens in 10–
30% of all patients with breast cancer. Due to the association with an increased rate of cell
proliferation, which results in rapid cancer growth and poor prognosis, HER2 is often used as
a biomarker in cancer diagnostics.162
IHC allows detecting and localizing antigens within histological tissues, which can be
used to identify cancerous cells.163
The optimization of protocols and testing of new staining
and labeling methods can be done in ICC, which targets
cultivated cells prepared similarly as the tissue samples. The
most common counterstaining approach is based on the
combination of hematoxylin and eosin (H&E).164
However, to
allow specific detection of cancer biomarkers, conjugates of
antibodies with enzymes,165
fluorophores,166
or
nanoparticles167
have to be used. Typically, HRP is employed
to oxidize 3,3’-diaminobenzidine to a brown precipitation
product, which is evaluated by light microscopy.168
The
evaluation of the tissue sections is typically done by a timeconsuming
visual inspection by the trained pathologists. To
increase the throughput, the current research focuses on the
automation of imaging and evaluation aided by artificial
intelligence in so-called digital pathology.169
However, the
automation requires labels providing high specific signals and
low non-specific binding.170
Due to their high brightness and low non-specific
adsorption, we have explored the capabilities of BSA-based75
and PEG-based143
conjugates of UCNPs with streptavidin for
labeling of HER2 biomarker on cancer cells (Figure 26). The
conjugates based on PEG provided a higher S/B ratio,
Figure 26: Scheme of the ICC
assay. The primary antibody
binds to the HER2 receptor on the
cell surface, followed by a
biotinylated secondary antibody,
and detection UCNP-streptavidin
conjugate. Adapted from Paper
XX with permission. Copyright
2020 Royal Society of Chemistry.
44
probably due to the non-specific adsorption of BSA to the cell surface. The efficiency of
labeling was strongly dependent on the blocking conditions. The S/B ratios were calculated as
the ratio of signals between HER2-positive BT-474 cells incubated with and without primary
antibody, as evaluated by upconversion scanning. Even though the assay buffer based on BSA
and BGG allowed to reach an acceptable S/B ratio of 23, the use of commercial SuperBlock
solution reduced the non-specific binding more efficiently while even slightly increasing the
specific signals (Figure 27). This resulted in the improvement of the S/B ratio by more than
one order of magnitude to 319. This finding agrees with the previous results, suggesting that
the presence of serum proteins is not optimal for achieving low backgrounds in ICC. The
comparison of HER2-positive BT-474 cells with HER2-negative MDA-MB-231 cells under
the same experimental conditions also confirmed a high level of specific binding, producing
40 times higher signals in the case of BT-474. We have also shown that upconversion-based
labeling is compatible with the H&E counterstaining, suggesting good applicability in IHC,
where the H&E is the typical counterstaining method.
The performance of UCNP-based labels was compared with conventional fluorescence
labeling using a conjugate of streptavidin with carboxyfluorescein (SA-FAM). The
fluorescence labeling resulted in S/B value of only 6.1, which is connected with relatively high
background signals due to the cellular autofluorescence and cross-talk between the detection
channels (fluorescein and DAPI). Due to the 50-fold wider dynamic range, the upconversion
labeling allows a much finer distinction of HER2 expression within different cell lines.
Furthermore, the high S/B ratio can enable the application of UCNP labeling for IHC with
automated data evaluation in digital pathology.
Figure 27: Labeling of HER2-positive FFPE BT-474 cells using UCNP-PEG-SA conjugates:
(A) DAPI, (B) upconversion, (C) overlay. Negative control (without primary antibody): (D) DAPI,
(E) upconversion, (F) overlay. Adapted from Paper XX with permission. Copyright 2020 Royal Society
of Chemistry.
45
6 Laser-Induced Breakdown Spectroscopy (Papers XXI and
XXII)
The direct nanoparticle-based immunoassay readout (i.e., not employing catalytic
transformation of the substrate) is typically based on luminescence detection, which combines
high sensitivity and simple instrumentation. However, the requirement of luminescence
properties limits the range of potential labels. Therefore, there is a demand for alternative
readout techniques that would allow universal detection independent of the luminescent or
catalytic properties of the label.
Laser-induced breakdown spectroscopy (LIBS) is an optical emission technique
complementary to the conventional methods in bioimaging.171
LIBS combines high sensitivity,
rapid analysis, and the possibility to detect halogens and light elements. However, its main
advantage is the possibility of multi-elemental imaging on a large scale (few cm) and with a
high resolution (units of μm).172
LIBS can be used to detect different kinds of nanoparticles on
various matrices, from the analysis of QDs on a filter paper173
to UCNPs in model organisms.174
Furthermore, LIBS can be used for surface mapping, providing information about the 2D or
even 3D element distribution within the sample.175
We have developed a method for the detection of Ag NPs and Au NPs by LIBS from
the bottom of the conventional 96-well microtiter plate (Figure 28). The optimized setup was
then applied for the readout of a sandwich immunoassay to detect HSA based on streptavidinconjugated
Ag NPs. The performance of the LIBS-based assay was compared with a
conventional fluorescence readout based on the conjugate of detection antibody with
fluorescein isothiocyanate (FITC). Even though slightly higher sensitivity was observed in the
case of fluorescence, the great advantage of LIBS was the wider dynamic range. Furthermore,
LIBS allows detecting labels without luminescence properties and presents the possibility of
multi-elemental analysis without the necessity to consider spectral overlaps of the conventional
luminescence labels.
In the following work, we pioneered
the application of LIBS for the readout of
nanoparticle-labeled ICC sections. The cell
pellets were labeled with UCNPs according
to our previous report,176
and the
characteristic signal of the Y II 437.49 nm
emission line was used to construct the 2D
map of the sample surface with a resolution
of 100 μm. The results demonstrated the
ability of LIBS to map the yttrium
distribution and showed a clear difference
between HER2-positive and HER2-negative
cells. The results from LIBS were then
compared with upconversion optical
microscopy and upconversion luminescence
Figure 28: Scheme of LIBS immunoassay with
label based on conjugate of Ag NPs with
streptavidin. Reprinted from Paper XXI with
permission. Copyright 2019 Springer.
46
scanning. The main advantage of microscopy compared to scanning-based approaches is the
high resolution, which allows studying the target distribution within cellular structures.
However, it is not possible to use conventional optical microscopy to quantitatively determine
the amount of label (and therefore indirectly also of the target antigen) within the whole cell
pellet. The S/B ratio of LIBS was 5, whereas the upconversion scanning of the identical pellets
provided S/B of 159 (Figure 29). Because there was the same amount of UCNPs, the worse
S/B of LIBS was probably given by the lower measurement sensitivity. Despite the successful
results of the preliminary work, further improvements of LIBS are necessary to meet the
practical requirement of IHC, especially in terms of sensitivity and lateral resolution. In the
future, LIBS can significantly improve the multiplexing capabilities in IHC due to the
possibility of using multiple nanoparticle labels without having to deal with spectral overlaps,
as in the case of optical readout.
Figure 29: Comparison of (A) LIBS and (B) upconversion scanning of BT-474 and MDA-MB-231
cells with HER2 biomarker labeled with UCNP-PEG-SA conjugate. (C) The average intensities
evaluated by the two methods. Error bars correspond to standard deviations of intensities within the cell
pellet. Reprinted from Paper XXII. Copyright 2021 Springer.
47
7 Conclusions and Outlook
This thesis has summarized the recent progress in the rapidly developing field of
immunochemical biosensors and assays. This research topic represents an interdisciplinary
effort to combine various kinds of physicochemical transducers with appropriate assay
strategies to achieve highly sensitive and specific detection. Furthermore, the use of
nanoparticles, in particular with catalytic or luminescent properties, can further enhance the
performance of such assays.
The label-free biosensing was represented by EIS biosensor for Salmonella in milk,
QCM biosensor for aerosolized biological warfare agents, and application of plasmapolymerized
layers in SPR biosensing of HSA and Salmonella. Overall, the main advantage of
these approaches is the rapid analysis and simple procedure. However, the sensitivity can be
limited by the lack of the signal-amplification step.
On the other hand, the use of catalytic labels is typically connected with higher
sensitivity and reduced effect of the complex sample matrix, but the analysis requires the
substrate conversion step and, therefore, longer time. Enzyme-based labels were employed in
an amperometric biosensor for the diagnosis of EFB and in precipitation-based SPR assay for
Salmonella. As an alternative to enzymes, we have also used catalytic PBNPs in an NLISA for
HSA and Salmonella. Even though the sensitivity was similar to the conventional ELISA,
PBNPs provide several practical advantages, particularly higher stability and the possibility of
easy synthesis from cheap precursors.
UCNPs allow highly sensitive detection due to the anti-Stokes luminescence and lack
of optical background. We have thoroughly studied the different ways of UCNP surface
modification and conjugation with biorecognition molecules. The conjugates were then
employed in ULISA assays for a wide range of analytes, from small molecules (DCF, ZEA),
through proteins (HSA, PSA, troponin), to bacteria (M. plutonius and P. larvae). UCNPs also
proved useful for labeling HER2 biomarker on the surface of breast cancer cells in ICC
imaging.
Finally, we have explored the possibilities of LIBS as an alternative way of signal
readout not dependent on the catalytic or luminescent properties of the labels. We have
employed it in microtiter plate-based immunoassay for HSA and in ICC detection of HER2
biomarker.
Even though reaching ever lower LODs is one of the main challenges from the scientific
point of view, it is equally important to address the simplicity and robustness of the assay
procedure. An ideal assay should provide results within a few minutes and be based either on
a fully automated system or a cheap disposable sensor that requires minimum manipulation.
The ongoing advances in transducer technology and labels, especially based on nanoparticles,
promise that the performance of immunochemical biosensors and assays will keep improving
in the future. This will be beneficial for the biochemical and biological research field, and for
the large community of immunoassay users in clinical diagnosis, detection of biological agents,
food safety, and environmental protection.
48
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of pulmonary micrometastases. Nanoscale 2017, 9 (3), 1110–1119.
168. Fan, L.; Tian, Y. Y.; Yin, R.; Lou, D. D.; Zhang, X. Z.; Wang, M.; Ma, M.; Luo, S. H.;
Li, S. Y.; Gu, N.; Zhang, Y., Enzyme catalysis enhanced dark-field imaging as a novel
immunohistochemical method. Nanoscale 2016, 8 (16), 8553–8558.
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intelligence in digital pathology – new tools for diagnosis and precision oncology. Nat.
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promising tool in the elemental bioimaging of plant tissues. Trends Anal. Chem. 2020,
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advices. Spectrochim. Acta Part B Atom. Spectr. 2014, 101, 171–182.
173. Škarková, P.; Novotný, K.; Lubal, P.; Jebavá, A.; Pořízka, P.; Klus, J.; Farka, Z.;
Hrdlička, A.; Kaiser, J., 2D distribution mapping of quantum dots injected onto filtration
paper by laser-induced breakdown spectroscopy. Spectrochim. Acta Part B Atom. Spectr.
2017, 131, 107–114.
174. Modlitbová, P.; Hlaváček, A.; Švestková, T.; Pořízka, P.; Šimoníková, L.; Novotný, K.;
Kaiser, J., The effects of photon-upconversion nanoparticles on the growth of radish and
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176. Farka, Z.; Mickert, M. J.; Mikusova, Z.; Hlavacek, A.; Bouchalova, P.; Xu, W. S.;
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nanoparticles for high-contrast immunocytochemistry. Nanoscale 2020, 12 (15), 8303–
8313.
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List of Abbreviations
4-CN 4-chloro-1-naphthol
Ab antibody
AFB American foulbrood
AFM atomic force microscopy
AMI acute myocardial infarction
BSA bovine serum albumin
CFU colony-forming unit
CMD carboxymethylated dextran
CPA cyclopropylamine
DCF diclofenac
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
EFB European foulbrood
EIS electrochemical impedance spectroscopy
ELISA enzyme-linked immunosorbent assay
HRP horseradish peroxidase
HSA human serum albumin
ICC immunocytochemistry
IHC immunohistochemistry
LFIA lateral flow immunoassay
LIBS laser-induced breakdown spectroscopy
LOD limit of detection
MIP molecularly imprinted polymer
NHS N-hydroxysuccinimide
NLISA nanozyme-linked immunosorbent assay
PAA poly(acrylic acid)
PBNP Prussian blue nanoparticle
PCR polymerase chain reaction
PoC point-of-care
PP plasma polymerization
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PSA prostate-specific antigen
QCM quartz crystal microbalance
QD quantum dot
SPE screen-printed electrode
SPR surface plasmon resonance
THFMA tetrahydrofurfuryl methacrylate
TVC 1,2,4-trivinylcyclohexane
UCNP photon-upconversion nanoparticle
ULISA upconversion-linked immunosorbent assay
ZEA zearalenone
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Appendix
List of Publications
1. Hlaváček, A.; Farka, Z.*; Mickert, M. J.; Kostiv, U.; Brandmeier, J. C.; Horák, D.;
Skládal, P.; Foret, F.; Gorris, H. H., Bioconjugates of photon-upconversion nanoparticles
for cancer biomarker detection and imaging. Nat. Protoc. Accepted.
2. Obořilová, R.; Šimečková, H.; Pastucha, M.; Klimovič, Š.; Víšová, I.; Přibyl, J.;
Vaisocherová-Lísalová, H.; Pantůček, R.; Skládal, P.; Mašlaňová, I.; Farka Z.*, Atomic
force microscopy and surface plasmon resonance for real-time single-cell monitoring of
bacteriophage-mediated lysis of bacteria. Nanoscale 2021, 13 (31), 13538–13549.
3. Brandmeier, J. C.; Raiko, K.; Farka, Z.*; Peltomaa, R.; Mickert, M. J.; Hlaváček, A.;
Skládal, P.; Soukka, T.; Gorris, H. H., Effect of Particle Size and Surface Chemistry of
Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac
Troponin. Adv. Healthc. Mater. 2021, 10 (18), 2100506.
4. Pastucha, M.; Odstrčilíková, E.; Hlaváček, A.; Brandmeier, J. C.; Vykoukal, V.;
Weisová, J.; Gorris, H. H.; Skládal, P.; Farka Z.*, Upconversion-linked Immunoassay
for the Diagnosis of Honeybee Disease American Foulbrood. IEEE J. Sel. Top. Quantum
Electron. 2021, 27 (5), 6900311.
5. Pořízka, P.; Vytisková, K.; Obořilová, R.; Pastucha, M.; Gábriš, I.; Brandmeier, J. C.;
Modlitbová, P.; Gorris, H. H.; Novotný, K.; Skládal, P.; Kaiser, J.; Farka, Z., LaserInduced
Breakdown Spectroscopy as a Readout Method for Immunocytochemistry with
Upconversion Nanoparticles. Microchim. Acta 2021, 188, 147.
6. Petruš, O.; Macko, J.; Oriňaková, R.; Oriňak, A.; Múdra, E.; Kupková, M.; Pastucha, M.;
Farka, Z.; Socha, V., Detection of organic dyes by surface-enhanced Raman spectroscopy
using plasmonic NiAg nanocavity films. Spectrochim. Acta A 2020, 249, 119322.
7. Trnková, L.; Třísková, I.; Čechal, J.; Farka, Z., Polymer pencil leads as a porous
nanocomposite graphite material for electrochemical applications: The impact of
chemical and thermal treatments. Electrochem. Commun. 2021, 126, 107018.
8. Farka Z.*, Nanoparticle-Based Biosensing: En Route to Ultimate Sensitivity (Meet the
Board). Anal. Sens. 2021, 1 (4), 136–137.
9. Farka, Z.; Mickert, M. J.; Pastucha, M.; Mikušová, Z.; Skládal, P.; Gorris, H. H.,
Advances in Optical Single-Molecule Detection: En Route to Super-Sensitive Bioaffinity
Assays. Angew. Chem. Int. Ed. 2020, 59 (27), 10746–10773. (Z.F. and M.J.M.
contributed equally)
10. Farka, Z.*; Mickert, M. J.; Mikušová, Z.; Hlaváček, A.; Bouchalová, P.; Xu, W.;
Bouchal, P.; Skládal, P.; Gorris, H. H., Surface design of photon-upconversion
nanoparticles for high-contrast immunocytochemistry. Nanoscale 2020, 12 (15), 8303–
8313. (Z.F. and M.J.M. contributed equally)
11. Peltomaa, R.; Farka, Z.; Mickert, M. J.; Brandmeier, J. C.; Pastucha, M.; Hlaváček, A.;
Martínez-Orts, M.; Canales, Á.; Skládal, P.; Benito-Peña, E.; Moreno-Bondi, M. C.;
Gorris, H. H., Competitive upconversion-linked immunoassay using peptide mimetics
for the detection of the mycotoxin zearalenone. Biosens. Bioelectron. 2020, 170, 112683.
12. Makhneva, E.; Barillas, L.; Farka, Z.; Pastucha, M.; Skládal, P.; Weltmann, K. D.; Fricke,
K., Functional Plasma Polymerized Surfaces for Biosensing. ACS Appl. Mater. Interfaces
2020, 20 (14), 17100–17112.
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13. Kostiv, U.; Farka, Z.; Mickert, M. J.; Gorris, H. H.; Velychkivska, N.; Pop-Georgievski,
O.; Pastucha, M.; Odstrčilíková, E.; Skládal, P.; Horák, D., Versatile bioconjugation
strategies of PEG-modified upconversion nanoparticles for bioanalytical applications.
Biomacromolecules 2020, 21 (11), 4502–4513. (U.K. and Z.F. contributed equally)
14. Víšová, I.; Smolková, B.; Uzhytchak, M.; Vrabcová, M.; Chafai, D. E.; Houska, M.;
Pastucha, M.; Skládal, P.; Farka, Z.*; Dejneka, A.; Vaisocherová-Lísalová, H.,
Functionalizable Antifouling Coatings as Tunable Platforms for the Stress-Driven
Manipulation of Living Cell Machinery. Biomolecules 2020, 10 (8), 1146.
15. Šišoláková, I.; Hovancová, J.; Oriňaková, R.; Oriňak, A.; Trnková, L.; Třísková, I.;
Farka, Z.; Pastucha, M.; Radoňák, J., Electrochemical determination of insulin on
CuNPs/chitosan-MWCNTs and CoNPs/chitosan-MWCNTs modified screen printed
carbon electrodes. J. Electroanal. Chem. 2020, 860, 113881.
16. Poláchová, V.; Pastucha, M.; Mikušová, Z.; Mickert, M. J.; Hlaváček, A.; Gorris, H. H.;
Skládal, P.; Farka, Z.*, Click-conjugated photon-upconversion nanoparticles in an
immunoassay for honeybee pathogen Melissococcus plutonius. Nanoscale 2019, 11 (17),
8343–8351.
17. Mickert, M. J.; Farka, Z.; Kostiv, U.; Hlaváček, A.; Horák, D.; Skládal, P.; Gorris, H. H.,
Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through
Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay. Anal.
Chem. 2019, 91 (15), 9435–9441. (M.J.M and Z.F. contributed equally)
18. Pastucha, M.; Farka, Z.; Lacina, K.; Mikušová, Z.; Skládal, P., Magnetic nanoparticles
for smart electrochemical immunoassays: a review on recent developments. Microchim.
Acta 2019, 186, 312.
19. Modlitbová, P.; Farka, Z.; Pastucha, M.; Pořízka, P.; Novotný, K.; Skládal, P.; Kaiser, J.,
Laser-induced breakdown spectroscopy as a novel readout method for nanoparticlebased
immunoassays. Microchim. Acta 2019, 186, 629.
20. Mikušová, Z.; Farka, Z.*; Pastucha, M.; Poláchová, V.; Obořilová, R.; Skládal, P.,
Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus
plutonius. Electroanalysis 2019, 31 (10), 1969–1976.
21. Makhneva, E.; Farka, Z.*; Pastucha, M.; Obrusník, A.; Horáčková, V.; Skládal, P.;
Zajíčková, L., Maleic anhydride and acetylene plasma copolymer surfaces for SPR
immunosensing. Anal. Bioanal. Chem. 2019, 411 (29), 7689–7697.
22. Farka, Z.*; Čunderlová, V.; Horáčková, V.; Pastucha, M.; Mikušová, Z.; Hlaváček, A.;
Skládal, P., Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich NanozymeLinked
Immunosorbent Assay. Anal. Chem. 2018, 90 (3), 2348–2354. (Z.F. and V.Č.
contributed equally)
23. Makhneva, E.; Farka, Z.; Skládal, P.; Zajíčková, L., Cyclopropylamine plasma polymer
surfaces for label-free SPR and QCM immunosensing of Salmonella. Sens. Actuators B
Chem. 2018, 276, 447–455.
24. Hegemann, D.; Indutnyi, I.; Zajíčková, L.; Makhneva, E.; Farka, Z.; Ushenin, Y.;
Vandenbossche, M., Stable, nanometer‐thick oxygen‐containing plasma polymer films
suited for enhanced biosensing. Plasma Process. Polym. 2018, 15 (11), 1800090.
25. Modlitbová, P.; Pořízka, P.; Novotný, K.; Drbohlavová, J.; Chamradová, I.; Farka, Z.;
Zlámalová-Gargošová, H.; Romih, T.; Kaiser, J., Short-term assessment of cadmium
toxicity and uptake from different types of Cd-based Quantum Dots in the model plant
Allium cepa L. Ecotoxicol. Environ. Saf. 2018, 153, 23–31.
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26. Makhneva, E.; Obrusník, A.; Farka, Z.; Skládal, P.; Vandenbossche, M.; Hegemann, D.;
Zajíčková, L., Carboxyl-rich plasma polymer surfaces in surface plasmon resonance
immunosensing. Jpn. J. Appl. Phys. 2018, 57 (01AG06), 1–5.
27. Modlitbová, P.; Klepárník, K.; Farka, Z.; Pořízka, P.; Skládal, P.; Novotný, K.; Kaiser,
J., Time-Dependent Growth of Silica Shells on CdTe Quantum Dots. Nanomaterials
2018, 8 (6), 439.
28. Farka, Z.; Juřík, T.; Kovář, D.; Trnková, L.; Skládal, P., Nanoparticle-Based
Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev.
2017, 117 (15), 9973–10042.
29. Farka, Z.; Mickert, M. J.; Hlaváček, A.; Skládal P.; Gorris, H. H., Single Molecule
Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the
Sensitive Detection of Diagnostic Biomarkers. Anal. Chem. 2017, 89 (21), 11825–11830.
(Z.F. and M.J.M. contributed equally)
30. Hlaváček, A.; Peterek, M.; Farka, Z.; Mickert, M. J.; Prechtl, L.; Knopp D.; Gorris, H.
H., Rapid single-step upconversion-linked immunosorbent assay for diclofenac.
Microchim. Acta 2017, 184 (10), 4159–4165.
31. Škarková, P.; Novotný, K.; Lubal, P.; Jebavá, A.; Pořízka, P.; Klus, J.; Farka, Z.;
Hrdlička, A.; Kaiser, J., 2D distribution mapping of quantum dots injected onto filtration
paper by laser-induced breakdown spectroscopy. Spectrochim. Acta B. At. Spectrosc.
2017, 131, 107–114.
32. Kubesa, O.; Horáčková, V.; Moravec, Z.; Farka, Z.; Skládal, P., Graphene and graphene
oxide for biosensing. Monatsh. Chem. 2017, 148 (11), 1937–1944.
33. Kovář, D.; Malá, A.; Mlčochová, J.; Kalina, M.; Fohlerová, Z.; Hlaváček, A.; Farka, Z.;
Skládal, P.; Starčuk, Z.; Jiřík, R.; Slabý, O.; Hubálek, J., Preparation and Characterisation
of Highly Stable Iron Oxide Nanoparticles for Magnetic Resonance Imaging. J.
Nanomater. 2017, 2017 (7859289), 1–8.
34. Trnková, L.; Farka, Z., Advanced nano- and biomaterials in biophysical chemistry
(Editorial). Monatsh. Chem. 2017, 148 (11), 1899–1900.
35. Farka, Z.; Juřík, T.; Pastucha, M.; Skládal, P. Enzymatic Precipitation Enhanced Surface
Plasmon Resonance Immunosensor for the Detection of Salmonella in Powdered Milk.
Anal. Chem. 2016, 88 (23), 11830–11836.
36. Farka, Z.; Juřík, T.; Pastucha, M.; Kovář, D.; Lacina, K.; Skládal, P., Rapid
immunosensing of Salmonella Typhimurium using electrochemical impedance
spectroscopy: the effect of sample treatment. Electroanalysis 2016, 28 (8), 1803–1809.
(Z.F. and T.J. contributed equally)
37. Hlaváček, A.; Farka, Z.; Hübner, M.; Horňáková, V.; Němeček, D.; Skládal, P.; Knopp,
D.; Gorris, H. H., Competitive Upconversion-Linked Immunosorbent Assay for the
Sensitive Detection of Diclofenac. Anal. Chem. 2016, 88 (11), 6011–6017.
38. Juřík, T.; Podešva, P.; Farka, Z.; Kovář, D.; Skládal, P.; Foret, F., Nanostructured gold
deposited in gelatin template applied for electrochemical assay of glucose in serum.
Electrochim. Acta 2016, 188, 277–285.
39. Trnková, L.; Farka, Z., Physical and electrochemical aspects of bio- and nanomaterials
(Editorial). Monatsh. Chem. 2016, 147 (5), 845.
40. Farka, Z.; Kovář, D.; Skládal, P., Rapid detection of microorganisms based on active and
passive modes of QCM. Sensors 2015, 15 (1), 79–92. (Z.F. and D.K. contributed equally)
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41. Kovář, D.; Farka, Z.; Skládal, P., Detection of aerosolized biological agents using the
piezoelectric immunosensor. Anal. Chem. 2014, 86 (17), 8680–8686. (D.K. and Z.F.
contributed equally)
42. Farka, Z.; Kovář, D.; Přibyl, J.; Skládal, P., Piezoelectric and surface plasmon resonance
biosensors for Bacillus atrophaeus spores. Int. J. Electrochem. Sci. 2013, 8 (1), 100–112.
43. Farka, Z.; Kovář, D.; Skládal, P., Piezoelectric biosensor coupled to cyclone air sampler
for detection of microorganisms. Chem. Listy 2013, 107 (S3), s302–s304.
* corresponding author
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Paper I
Nanoparticle-Based Immunochemical Biosensors and Assays:
Recent Advances and Challenges
Farka, Z.; Juřík, T.; Kovář, D.; Trnková, L.; Skládal, P.
Chem. Rev. 2017, 117 (15), 9973–10042
DOI: 10.1021/acs.chemrev.7b00037
Contribution:
Literature research, manuscript writing
Copyright 2017 American Chemical Society. Reprinted with permission.
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Paper II
Advances in Optical Single-Molecule Detection: En Route to
Super-Sensitive Bioaffinity Assays
Farka, Z.; Mickert, M. J.; Pastucha, M.; Mikušová, Z.; Skládal, P.; Gorris, H. H.
(Z.F. and M.J.M. contributed equally)
Angew. Chem. Int. Ed. 2020, 59 (27), 10746–10773
DOI: 10.1002/anie.201913924
Contribution:
Outline of review, literature research, manuscript writing
Copyright 2019 the authors. Reprinted under the permission of Creative
Commons Attribution-NonCommercial 4.0 International License.
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Paper III
Rapid immunosensing of Salmonella Typhimurium using
electrochemical impedance spectroscopy: the effect of sample
treatment
Farka, Z.; Juřík, T.; Pastucha, M.; Kovář, D.; Lacina, K.; Skládal, P.
(Z.F. and T.J. contributed equally)
Electroanalysis 2016, 28 (8) 1803–1809
DOI: 10.1002/elan.201600093
Contribution:
Design of experiments, development and optimization of EIS immunosensor,
characterization of sensing surface by AFM, data evaluation, manuscript writing
Copyright 2016 Wiley-VCH. Reprinted with permission.
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Paper IV
Detection of aerosolized biological agents using the piezoelectric
immunosensor
Kovář, D.; Farka, Z.; Skládal, P.
(D.K. and Z.F. contributed equally)
Anal. Chem. 2014, 86 (17), 8680–8686
DOI: 10.1021/ac501623m
Contribution:
Development and optimization of QCM immunosensor, data evaluation,
manuscript writing
Copyright 2014 American Chemical Society. Reprinted with permission.
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Paper V
Cyclopropylamine plasma polymer surfaces for label-free SPR and
QCM immunosensing of Salmonella
Makhneva, E.; Farka, Z.; Skládal, P.; Zajíčková, L.
Sens. Actuators B Chem. 2018, 276, 447–455
DOI: 10.1016/j.snb.2018.08.055
Contribution:
Development and optimization of SPR and QCM immunosensors,
characterization of sensing surface by AFM, data evaluation, participation in
manuscript writing
Copyright 2018 Elsevier. Reprinted with permission.
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Paper VI
Maleic anhydride and acetylene plasma copolymer surfaces for
SPR immunosensing
Makhneva, E.; Farka, Z.*; Pastucha, M.; Obrusník, A.; Horáčková, V.; Skládal,
P.; Zajíčková, L.
Anal. Bioanal. Chem. 2019, 411 (29), 7689–7697
DOI: 10.1007/s00216-019-01979-9
Contribution:
Design of experiments, development and optimization of SPR immunosensor,
characterization of sensing surface by AFM, data evaluation, manuscript writing
Copyright 2019 Springer. Reprinted with permission.
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Paper VII
Functional Plasma Polymerized Surfaces for Biosensing
Makhneva, E.; Barillas, L.; Farka, Z.; Pastucha, M.; Skládal, P.; Weltmann, K.
D.; Fricke, K.
ACS Appl. Mater. Interfaces 2020, 20 (14), 17100–17112
DOI: 10.1021/acsami.0c01443
Contribution:
Development and optimization of SPR immunosensor, data evaluation,
participation in manuscript writing
Copyright 2020 American Chemical Society. Reprinted with permission.
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Paper VIII
Amperometric Immunosensor for Rapid Detection of Honeybee
Pathogen Melissococcus plutonius
Mikušová, Z.; Farka, Z.*; Pastucha, M.; Poláchová, V.; Obořilová, R.; Skládal, P.
Electroanalysis 2019, 31 (10), 1969–1976
DOI: 10.1002/elan.201900252
Contribution:
Design of experiments, preparation of immunization antigen and antibody,
optimization of electrochemical immunosensor, data evaluation, manuscript
writing
Copyright 2019 Wiley-VCH. Reprinted with permission.
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Paper IX
Enzymatic Precipitation Enhanced Surface Plasmon Resonance
Immunosensor for the Detection of Salmonella in Powdered Milk
Farka, Z.; Juřík, T.; Pastucha, M.; Skládal, P.
Anal. Chem. 2016, 88 (23), 11830–11836
DOI: 10.1021/acs.analchem.6b03511
Contribution:
Design of experiments, development and optimization of precipitation-enhanced
SPR assay, characterization of precipitation reaction by AFM, data evaluation,
manuscript writing
Copyright 2016 American Chemical Society. Reprinted with permission.
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Paper X
Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich
Nanozyme-Linked Immunosorbent Assay
Farka, Z.*; Čunderlová, V.; Horáčková, V.; Pastucha, M.; Mikušová, Z.;
Hlaváček, A.; Skládal, P.
(Z.F. and V.Č. contributed equally)
Anal. Chem. 2018, 90 (3), 2348–2354
DOI: 10.1021/acs.analchem.7b04883
Contribution:
Design of experiments, bioconjugation and characterization of PBNPs,
development and optimization of sandwich assay, data evaluation, manuscript
writing
Copyright 2018 American Chemical Society. Reprinted with permission.
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Paper XI
Competitive Upconversion-Linked Immunosorbent Assay for the
Sensitive Detection of Diclofenac
Hlaváček, A.; Farka, Z.; Hübner, M.; Horňáková, V.; Němeček, D.; Skládal, P.;
Knopp, D.; Gorris, H. H.
Anal. Chem. 2016, 88 (11), 6011–6017
DOI: 10.1021/acs.analchem.6b01083
Contribution:
Design of experiments, bioconjugation and characterization of UCNPs,
development and optimization of competitive immunoassay, data evaluation,
participation in manuscript writing
Copyright 2016 American Chemical Society. Reprinted with permission.
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Paper XII
Rapid single-step upconversion-linked immunosorbent assay for
diclofenac
Hlaváček, A.; Peterek, M.; Farka, Z.; Mickert, M. J.; Prechtl, L.; Knopp D.;
Gorris, H. H.
Microchim. Acta 2017, 184 (10), 4159–4165
DOI: 10.1007/s00604-017-2456-0
Contribution:
Development and optimization of competitive immunoassay, data evaluation,
participation in manuscript writing
Copyright 2017 Springer. Reprinted with permission.
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Paper XIII
Competitive upconversion-linked immunoassay using peptide
mimetics for the detection of the mycotoxin zearalenone
Peltomaa, R.; Farka, Z.; Mickert, M. J.; Brandmeier, J. C.; Pastucha, M.;
Hlaváček, A.; Martínez-Orts, M.; Canales, Á.; Skládal, P.; Benito-Peña, E.;
Moreno-Bondi, M. C.; Gorris, H. H.
Biosens. Bioelectron. 2020, 170, 112683
DOI: 10.1016/j.bios.2020.112683
Contribution:
Design of experiments, bioconjugation and characterization of UCNPs,
development and optimization of competitive immunoassay, data evaluation,
participation in manuscript writing
Copyright 2020 Elsevier. Reprinted with permission.
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Paper XIV
Click-conjugated photon-upconversion nanoparticles in an
immunoassay for honeybee pathogen Melissococcus plutonius
Poláchová, V.; Pastucha, M.; Mikušová, Z.; Mickert, M. J.; Hlaváček, A.; Gorris,
H. H.; Skládal, P.; Farka, Z.*
Nanoscale 2019, 11 (17), 8343–8351
DOI: 10.1039/C9NR01246J
Contribution:
Design of experiments, preparation of immunization antigen and antibody,
bioconjugation and characterization of UCNPs, development and optimization of
sandwich immunoassay, data evaluation, manuscript writing
Copyright 2019 Royal Society of Chemistry. Reprinted with permission.
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Paper XV
Versatile bioconjugation strategies of PEG-modified upconversion
nanoparticles for bioanalytical applications
Kostiv, U.; Farka, Z.; Mickert, M. J.; Gorris, H. H.; Velychkivska, N.; PopGeorgievski,
O.; Pastucha, M.; Odstrčilíková, E.; Skládal, P.; Horák, D.
(U.K. and Z.F. contributed equally)
Biomacromolecules 2020, 21 (11), 4502–4513
DOI: 10.1021/acs.biomac.0c00459
Contribution:
Design of experiments, bioconjugation of UCNPs, development and optimization
of sandwich immunoassay, data evaluation, participation in manuscript writing
Copyright 2020 American Chemical Society. Reprinted with permission.
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Paper XVI
Upconversion-linked Immunoassay for the Diagnosis of Honeybee
Disease American Foulbrood
Pastucha, M.; Odstrčilíková, E.; Hlaváček, A.; Brandmeier, J. C.; Vykoukal, V.;
Weisová, J.; Gorris, H. H.; Skládal, P.; Farka Z.*
IEEE J. Sel. Top. Quantum Electron. 2021, 27 (5), 6900311.
DOI: 10.1109/JSTQE.2021.3049689
Contribution:
Design of experiments, preparation of immunization antigen and antibody,
bioconjugation and characterization of UCNPs, development and optimization of
sandwich immunoassay, data evaluation, manuscript writing
Copyright 2021 IEEE. Reprinted with permission.
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Paper XVII
Single Molecule Upconversion-Linked Immunosorbent Assay with
Extended Dynamic Range for the Sensitive Detection of Diagnostic
Biomarkers
Farka, Z.; Mickert, M. J.; Hlaváček, A.; Skládal P.; Gorris, H. H.
(Z.F. and M.J.M. contributed equally)
Anal. Chem. 2017, 89 (21), 11825–11830
DOI: 10.1021/acs.analchem.7b03542
Contribution:
Design of experiments, optimization of single-particle microscope setup,
bioconjugation and characterization of UCNPs, development and optimization of
sandwich immunoassay, data evaluation, manuscript writing
Copyright 2017 American Chemical Society. Reprinted with permission.
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Paper XVIII
Measurement of Sub-femtomolar Concentrations of ProstateSpecific
Antigen through Single-Molecule Counting with an
Upconversion-Linked Immunosorbent Assay
Mickert, M. J.; Farka, Z.; Kostiv, U.; Hlaváček, A.; Horák, D.; Skládal, P.; Gorris,
H. H.
(M.J.M and Z.F. contributed equally)
Anal. Chem. 2019, 91 (15), 9435–9441
DOI: 10.1021/acs.analchem.9b02872
Contribution:
Design of experiments, bioconjugation and characterization of UCNPs,
development and optimization of sandwich immunoassay, data evaluation,
manuscript writing
Copyright 2017 American Chemical Society. Reprinted with permission.
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Paper XIX
Effect of Particle Size and Surface Chemistry of PhotonUpconversion
Nanoparticles on Analog and Digital Immunoassays
for Cardiac Troponin
Brandmeier, J. C.; Raiko, K.; Farka, Z.*; Peltomaa, R.; Mickert, M. J.; Hlaváček,
A.; Skládal, P.; Soukka, T.; Gorris, H. H.
Adv. Healthc. Mater. 2021, 10 (18), 2100506
DOI: 10.1002/adhm.202100506
Contribution:
Design of experiments, bioconjugation and characterization of UCNPs,
development and optimization of sandwich immunoassay, data evaluation,
manuscript writing
Copyright 2021 the authors. Reprinted under the permission of Creative
Commons Attribution-NonCommercial 4.0 International License.
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Paper XX
Surface design of photon-upconversion nanoparticles for highcontrast
immunocytochemistry
Farka, Z.*; Mickert, M. J.; Mikušová, Z.; Hlaváček, A.; Bouchalová, P.; Xu, W.;
Bouchal, P.; Skládal, P.; Gorris, H. H.
(Z.F. and M.J.M. contributed equally)
Nanoscale 2020, 12 (15), 8303–8313
DOI: 10.1039/C9NR10568A
Contribution:
Design of experiments, optimization of microscope setup, bioconjugation and
characterization of UCNPs, development and optimization of ICC assay, data
evaluation, manuscript writing
Copyright 2020 Royal Society of Chemistry. Reprinted with permission.
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Paper XXI
Laser-induced breakdown spectroscopy as a novel readout method
for nanoparticle-based immunoassays
Modlitbová, P.; Farka, Z.; Pastucha, M.; Pořízka, P.; Novotný, K.; Skládal, P.;
Kaiser, J.
Microchim. Acta 2019, 186, 629
DOI: 10.1007/s00604-019-3742-9
Contribution:
Design of experiments, development and optimization of sandwich
immunoassay, data evaluation, participation in manuscript writing
Copyright 2019 Springer. Reprinted with permission.
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Paper XXII
Laser-Induced Breakdown Spectroscopy as a Readout Method for
Immunocytochemistry with Upconversion Nanoparticles
Pořízka, P.; Vytisková, K.; Obořilová, R.; Pastucha, M.; Gábriš, I.; Brandmeier,
J. C.; Modlitbová, P.; Gorris, H. H.; Novotný, K.; Skládal, P.; Kaiser, J.; Farka, Z.
Microchim. Acta 2021, 188, 147.
DOI: 10.1007/s00604-021-04816-y
Contribution:
Design of experiments, bioconjugation and characterization of UCNPs,
development and optimization of ICC assay, data evaluation, manuscript writing
Copyright 2021 Springer. Reprinted with permission.
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