Miniaturization (of analytical instrumentation) Introduction – history Microfluidics Applications for mass spectrometry Miniaturized MS … Capillary gel electrophoresis Separation of nerve cell proteins H. Hydén et al. Anal.Biochem, 17, 1-15, 1966. 1973 Instrumentation Miniaturization Terry, S.C., Jerman, J.H., Angell, J.B. IEEE Transactions on Electron Devices, 1979, 26, 1880-86. A Gas Chromatographic Air Analyzer Fabricated on a Silicon Wafer Picture by Wyatt Vreeland, NIST There really is nothing new under the Sun Prototype analog pneumatic computer to operate in a nuclear attack NBS (now NIST) 1950’s https://www.ncbi.nlm.nih.gov/pubmed?term=Microfluidic%5BTitle%5D Incidence of the word “MICROFLUIDIC” in PubMed (as of Octoberer 2017) Technology Products Consequences Microelectronics Microfluidics ? Microfluidics? Speed of analysis Space saving Cost cutting Mass production Control of electric current control of fluid flows Stephen Quake, Dept. Bioengineering, Stanford University, http://thebigone.stanford.edu/index.html Microfabrication technology Micromilling 10 mm Optical lithography 200 nm e/ion beam lithography Multiple exposure techniques 10 nm Etching (resist dependent) ~ nm Replication (mass production) 10’s nm Injection molding Hot embossing Casting 1. Expose 2. develop and etch mask photoresist protecting layer (Cr, Au) Substrate (glass, silicon, ...) 3. Remove the protection layer 4. Close the structure (thermal bonding) Photolithography SILICON - ANISOTROPIC ETCHING * Anisotropic etching – direction dependent etch rate * Etch rate slower perpendicularly to the crystalline planes with the highest density * Typical etches: KOH, Tetramethyl Ammonium Hydroxide (TmAH) Ethylene Diamine Pyrocatechol (EDP) • Substrates up to 100 x 100 mm2 • Structures down to 1 μm • Address grid down to 40 nm • Standard or UV laser source mPG 101 Tabletop Laser Pattern Generator System Integration Spatial flow focusing Diffusion limited mixing Capillary force filling www.gyros.com Exact volume metering on the nl level sample Defined volume (nl) Hydrophobic patch http://www.dolomite-microfluidics.com/ Droplet generation in nl-pl volumes Seth Fraden et al., J. AM. CHEM. SOC. 2007, 129, 8825-8835. Benefits and Issues Size - space saving Low reagent/sample consumption Smaller size – faster analysis Microchannel junctions without dead volume Parallel systems for high throughput Disposable parts - point-of-care devices BUT Scaling issues Fabrication limitations Surface chemistry Concentration limits of detection Phenomena unimportant on the macro scale may dominate Small volume problem 2.15 mm => 10 ml ~ 10-15 M 1 mm => 1 ml ~ 10-14 M 0.1 mm => 1 nl ~ 10-11 M 0.001 mm => 1 fl ~ 10-5 M Example: LOD = 1000 molecules MICROFABRICATED DEVICES * Sensors - accelerometers, glucose monitors, ... * Genomics - first commercial applications * Proteomics - sample processing separation Yining Shi, Peter C. Simpson, James R. Scherer, David Wexler, Christine Skibola, Martyn T. Smith, and Richard A. Mathies. Anal. Chem. 1999, 71, 5354-5361 Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis. Karger, B.L., Foret, F., Qifeng, X., Dunayevski, Y., Zavracki, P., McGruer, N. U.S. Patent # 5,872,010, 1999. What is claimed is: 1. A liquid handling system, comprising a microscale liquid handling substrate having one or more channels integrally formed therein, for conducting a liquid sample in said substrate, said one or more channels terminating in one or more exit ports in an outer surface of said substrate for transfer of a microscale quantity of a liquid sample off said substrate by droplet, spray or stream; Microscale Fluid Handling System Xue, Q., Foret, F., Dunayevskiy, Y.M., Zavracky, P.M., McGruer, N.E., Karger, B.L. Anal.Chem., 69, 1997, 426-430. Multichannel Microchip Electrospray Mass Spectrometry J. M. Ramsey, R. Ramsey Anal. Chem., 1997, 69, 1174-1178. Generating Electrospray from Microchip Devices Using Electroosmotic Pumping Electrospray Flat surface - droplet Capillary tip Etching, pulling, … Grinding Original capillary ESI tip (micro)fabrication ? ESI exit spray fluid BGE waste sample nebulizer gas 10 mm Zhang, B. Liu, H. Karger, B. L. Foret, F. Anal. Chem., 1999, 71, 3258-3264. CE Microdevice with a Pneumatic Nebulizer 0 2.0 4.0 Time (min) 0 20 40 60 80 100 RelativeAbundance m/z 400 1200 591.6 1181.7 Angiotensin I des-AspI 450.5 899.5 630.6 775.4 m/z 400 1200 m/z 400 1200 m/z 400 1200 Angiotensin III 1-7 Angiotensin I Bullfrog Angiotensin III 3-8 Base peak 441.1 880.8 m/z 400 1200 Angiotensinogen 1-14 Porcine 1 2 3 4 5 CE Microdevice with a Pneumatic Nebulizer 1. 2. 3. 4. 5. Zhang, B. Liu, H. Karger, B. L. Foret, F. Anal. Chem., 1999, 71, 3258-3264. 1 mm Si wafer SU-8 Le Gac S., Arscott S., Rolando C. ELECTROPHORESIS 2003 , 24, 3640-3647. Micro-nib electrospray source (SU-8 on a silicon wafer) Sjödahl, J., Melin, J., Griss, P., Emmer, A., Stemme, G., Roeraade, J. Rapid Commun. Mass Spectrom. 2003, 17, 337—341. ESI tips produced by DRIE in silicon Infusion ESI Tip Array www.advion.com 100 mm Integrated Au tips ESI tip fabrication www.advion.com DRIE in silicone Plasma etched in polyimide www.diagnoswiss.com www.phoenix-st.com www.agilent.com Injection molding in polypropylene Applications Mao, P; Gomez-Sjoberg, R; Wang, DJ. Anal. Chem. 2013, 85, 816-819. Multinozzle Emitter Array Chips for Small-Volume Proteomics Microfabricated Monolith Columns for Liquid Chromatography Sculpting Supports for Liquid Chromatography Fred E. Regnier J. High Resol. Chromatogr. 2000, 23, (1) 19–26 Gradient LC separation of albumin digest (4ml/min, 1 mg/ml, UV detection) De Malsche W., De Bruyne S., Op de Beeck J., Eeltink S., Detobel F., Gardeniers H., Desmet G. Journal of Separation Science 2012, 35, 2010-2017 A microfabricated micropillar liquid chromatographic chip Monolithically integrated with an electrospray ionization tip Lauri Sainiemi, Teemu Nissilä, Risto Kostiainen, Sami Franssila and Raimo A. Ketola Lab Chip, 2012, 12, 325 Design of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography S. Eeltink, et al., J. Sep. Sci. 2015, 38,1123-1129. Mellors, JS; Black, WA; Chambers, AG; Starkey, JA; Lacher, NA; Ramsey, JM. Anal. Chem. 2013, 85, 4100-4106 Hybrid Capillary/Microfluidic System for Comprehensive Online Liquid Chromatography-Capillary Electrophoresis-ESI-MS Hybrid Capillary/Microfluidic System for Comprehensive Online Liquid Chromatography-Capillary Electrophoresis-ESI-MS Mellors, JS; Black, WA; Chambers, AG; Starkey, JA; Lacher, NA; Ramsey, JM. Anal. Chem. 2013, 85, 4100-4106 S. Fritzsche, S. Ohla, P. Glaser, D. S. Giera, M. Sickert, C. Schneider and D. Belder Angew. Chem., Int. Ed., 2011, 50, 9467-9470. Asymmetric Organocatalysis and Analysis on a Nanospray Chip J. Ji, L. Nie, L. Qiao, Y. Li, L. Guo, B. Liu, P. Yang and H. H. Girault, Lab Chip, 2012, 12, 2625-2629. Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry J. Ji, L. Nie, L. Qiao, Y. Li, L. Guo, B. Liu, P. Yang and H. H. Girault, Lab Chip, 2012, 12, 2625-2629. Commercialization Microfluidic Chip for Peptide Analysis with an Integrated HPLC Column, Sample Enrichment Column, and Nanoelectrospray Tip H. Yin, K. Killeen, R. Brennen, D. Sobek, M. Werlich, T. van de Goor Anal. Chem. 2005, 77, 527-533 Polyimide HPLC-chip, integrating an enrichment column, frits, a laser ablated ESI tip and trapazoidal separation column www.agilent.com Three LC columns – length 130 mm Each segment individually packed. BSA digest separated with a 30min gradient on a 2 column segmented chip, packed with 3.5μm particles Segmented column HPLC/chip www.agilent.com TRIZAIC nanoTile - Waters Enolase digest 70 fmol, 2 mm particles nanoACQUITY TRIZAIC nanoTile Green tape Al2O3–MgO–SiO2 glass particles mixed with organic binders and solvents to form glass ceramic Product Description 951 Green Tape is a low-temperature cofired ceramic tape. The 951 system comprises a complete cofireable family of Au and Ag metallizations, buried passives, and encapsulants. 951 is available in multiple thicknesses for use as an insulating layer in: •Multichip modules •Single chip packages •Ceramic printed wiring boards •RF modules http://www.dupont.com/mcm Cut Ceramic Material Fired Wafer Ceramic Microfluidic Fabrication Final Diced Part Packed glass LC chip Eksigent, part of AB SCIEX, www.eksigent.com Miniaturized (microfabricated) mass spectrometers? Applications of Miniaturized MS Instruments • trace explosive detection and airport security • space exploration • environmental monitoring • point-ofcare medical applications Miniature Mass Spectrometer Systems based on a Microengineered Quadrupole Filter A. Malcolm, S.Wright, R. R. A. Syms, N. Dash, M.-A. Schwab, A. Finlay Anal. Chem. 2010, 82, 1751–1758 Mass Analyzer ionchip® quadrupole mass spectrometer Mass Range m/z 50-800 with ionchip®150 Mass Accuracy ± m/z 0.3 in full scan Mass Resolution m/z 0.7±0.1 FWHM Sensitivity 10pg of reserpine in SIM mode S/N ratio of 10:1 (RMS) www.microsaic.com OI Analytical, www.oico.com Self-contained instrument Embedded PC Vacuum inlet flow 2 mL/min 24V input (battery operation) Advion expression Compact Mass Spectrometer www.advion.com http://908devices.com/ Microscale Ion Trap Internal glow discharge ionization Mass Range 15-450 amu Mass Resolution 1 amu Automated CE-MS on a glass chip What is a patent Invention disclosure Does it make sense to patent? Patent search Resources Patent? Patent! What Is a Patent? A patent for an invention is the grant of a property right to the inventor, issued by the United States Patent and Trademark Office. Generally, the term of a new patent is 20 years from the date on which the application for the patent was filed in the United States or, in special cases, from the date an earlier related application was filed, subject to the payment of maintenance fees. U.S. patent grants are effective only within the United States, U.S. territories, and U.S. possessions. Under certain circumstances, patent term extensions or adjustments may be available. What is granted is not the right to make, use, offer for sale, sell or import, but the right to exclude others from making, using, offering for sale, selling or importing the invention. Once a patent is issued, the patentee must enforce the patent without aid of the USPTO. There are three types of patents: 1) Utility patents may be granted to anyone who invents or discovers any new and useful process, machine, article of manufacture, or composition of matter, or any new and useful improvement thereof; 2) Design patents may be granted to anyone who invents a new, original, and ornamental design for an article of manufacture; and 3) Plant patents may be granted to anyone who invents or discovers and asexually reproduces any distinct and new variety of plant. Patentable subject 1. Does not fall under the laws of nature, natural phenomena or abstract ideas 2. Utility requirement - invention must be useful in association with machines, human-made products, compositions of matter or processing methods 3. Novelty the idea must not be presented to the public before the filing 4. Nonobviousness – it must be unrecognizable to a skilled person in the field of invention 5. Clarity of the description included in the application Patent je zákonná ochrana vynálezů zaručující vlastníkovi patentu výhradní právo k průmyslovému využití vynálezu. V České republice udělování patentů upravuje zákon 527/1990. Podle něj se patenty udělují na vynálezy, které jsou nové, jsou výsledkem vynálezecké činnosti a jsou průmyslově využitelné. Vynález se považuje za nový, jestliže není součástí stavu techniky. Stavem techniky je všechno, co bylo zveřejněno přede dnem přihlášení patentu, ať již v České republice nebo v zahraničí. Za vynálezy se naopak nepovažují zejména : objevy, vědecké teorie a matematické metody, pouhé vnější úpravy výrobků, plány, pravidla a způsoby vykonávání duševní činnosti, programy počítačů, pouhé uvedení informace Majitel patentu má výlučné právo vynález využívat (tj. výrobek vyrábět, uvádět do oběhu nebo upotřebit postup), dále poskytnout souhlas k využívání vynálezu jiným osobám (např. licenční smlouvou) a má právo převést patent na jinou osobu. Proto, aby patent zůstal v platnosti, je nutno platit tzv. udržovací poplatky, a to v každém státu zvlášť. Maximální možná délka patentové ochrany je 20 roků. http://cs.wikipedia.org/ United States Patent and Trademark Office www.uspto.gov European patent office www.epoline.org Úřad průmyslového vlastnictví www.upv.cz www.ctt.muni.cz Brno, No. 27 on the list of 52 places to visit in 2016 www.ce-ce.org CECE 2018 15th International Interdisciplinary Meeting on Bioanalysis Brno, October 15 - 17, 2018