Microfluidics Handling with microvolumes lacinak@chemi.muni.cz Microfluidics • Handling small volumes (µL, nL, pL) • Interdisciplinary discipline – Engineering – Physics – (Bio)chemistry – Nanotechnology – Biotechnology • Potential to influence subject areas from – Chemical synthesis – Biological analysis • to – Optics – Information technology Microfluidics • Firstly used in analysis – Small volume of sample – Small reagent consumption – High resolution – Low cost – Miniaturisation • New capabilities to control concentrations of molecules in space and time • Historical background – Defence purposes – NEED of miniaturaisation – Molecular biology – NEED of higher throughput, sensitivity and resolution – Microanalytical methods – ABILITIES of (HPLC, GC, CE) – Microelectronics – ABILITIES of Microfluidics • At small length scales (dimensions): – Transport phenomena can be precisely controlled: • mixing • thermal transfers • concentration • flows – surface effects are important and confinement plays role Microfluidics • Liquid behaves differently at microscale (and lower dimensions) – No turbulence – Only laminar flow – Two separated liquids mix only through diffusion – parallel flow alevelnotes.com www.veryst.com Soft (PDMS) lithography • Soft lithography using PDMS – polydimethylsiloxane http://www.elveflow.com/microfluidic-tutorials Soft (PDMS) lithography http://www.bioc.rice.edu http://bdml.stanford.edu Types of microfluidic flows • Pressure-driven flows • Capillary-induced flow • Droplet microfluidics • Evaporation-induced flows • Electroosmotic flow • Physical computations • Navier-Stokes equations • Dimensionless characteristic parameters – Reynolds number, Re Pressure-driven flows Jean-Baptiste Salmon Microfluidics for lab-on-chips www.lof.cnrs.fr Capillary-induced flow • Based on surface tension • Capillary forces • Paper based microfluidics • Diffusive spreading http://www.technologyreview.com Jean-Baptiste Salmon Microfluidics for lab-on-chips www.lof.cnrs.fr Droplets in microfluidics • Generation of monodisperse droplets when two immiscible fluids flow in microchannels • formation of droplets in microfluidics on the basis of surface tension • Microreactors http://www-microdroplets.ch.cam.ac.uk Jean-Baptiste Salmon Microfluidics for lab-on-chips www.lof.cnrs.fr Evaporation-induced flows Electroosmotic flow Wheeler and Stroock, Nature 2008 Jean-Baptiste Salmon Microfluidics for lab-on-chips www.lof.cnrs.fr Transport phenomena in microfluidics • Convection/diffusion in microfluidics • Hydrodynamic dispersion • Mixing strategies – diffusion is not sufficient • Droplet vs. co-flow for chemistry • Active concentration in µ-evaporators – crystallisation Jean-Baptiste Salmon Microfluidics for lab-on-chips www.lof.cnrs.fr Microfluidics applications • Screening for optimal protein crystallisation conditions – Screening of large number of conditions • Separation - coupled with MS • Screening in drug development – high throughput • Bioanalysis • Manipulation of samples consisting single cell or molecule • Organic synthesis of derivatives for emission tomography • Manipulation of multiphase flow (bubbles and droplets) • Combinatorial chemistry Whitesides, Nature 442 (2006) 368 Paper microfluidics • Photolithography or wax printing to define hydrophilic & hydrophobic zones in paper – channels • Capillary wicking of fluids • Low cost Martinez and Whitesides, Anal. Chem, 2008, PNAS 2009 Mcrofluidics enabling LoC • Microfabrication techniques enable – Microchannels = pipes – Valves – Mixers – Pumps • Enabling real Lab-on-Chip – factories on a chip Lab-on-Chip • Popular topic • Scientific journal “Miniaturisation for chemistry, physics, biology, materials science and bioengineering” • Point-of-care Lab-on-Chip (LoC) • Originated due to the microfluidics • Whole “laboratory” on single chip – All necessary procedures integrated on one single element http://lab-on-chip.gene-quantification.info/ http://www.azonano.com Lab-on-Chip (LoC) • Low volume of sample (droplet) • Faster analysis • Robustness of the system • Safe platform for handling with dangerous samples – Infectious or (bio)hazardous samples • Bringing down cost for one analysis – mass production • Not always ↑↑ – still only proof-of-concept stage • LoC for HIV diagnosis Point-of-care (PoC) • Generally – Delivery of healthcare products and services to patient at time of care • PoC testing – Transfer of analytical event to location of sample collection (location of patient) • Treatment can be adjusted before the patient leaves – Field applications, bed site (environmental analysis, blood test) Point of care testing (PoCT) • Simple – Experiment performance – Interpretation of results – Operation • Portable – small dimensions • Robust • Fast • Electronic/remote communication (data collection) • To ensure reliability of results! – Different, unskilled operators http://www.whitmiremedical.com Point-of-care testing (PoCT) • Sensing part (sample introduction) • Signal processing (electronics, microcomputer) • Evaluation (algorithms and programs) • Output (display) • Paper-based indicators – pH, urine test strips • Sophisticated instruments – small bench instruments Analytes performed by PoCT and devices • Glucose • Blood gases • Lactate • Co-oximetry • Hemoglobin A1c • Abbott Precision Xceed Pro Glucose Meter • IL Gem Blood Gas analyser • Siemens DCA 2000 Haemoglobin A1C Analyser Cell sorting • Sorting of cells on the basis of their properties • Physical principles (mainly) • Chemical properties • Preconcentration purposes for – Diagnosis – circulating tumour cells – Therapeutics – stem cells • Theranostics and personalised medicine – Treatments are tailored to the prognoses of patients Shields IV, Reyes, Lopez, Lab Chip 5 (2015) 1230 Cell sorting • Introduced in 1969 by Herzenberg et al. • Fluorescence-activated cell sorting (FACS) • Current instruments based on FACS – Up to 50 000 cells/s – automated, robust, and capable of exceptional specificity – using multiple morphological and fluorescent cell signatures (e.g. cell surface labels, cell size, and granularity) • Magnetic-activated cell sorting devices – Separating cells with magnetic labels by permanent magnet http://www.bio.davidson.edu Physical principles of cell sorting Size determined distribution Size determined filtering Size determined displacement Size determined guiding Wu, Hjort, Micro Nanosyst 1 (2009) 181 Physical principles of cell sorting Wu, Hjort, Micro Nanosyst 1 (2009) 181 slanted ribs slanted groovescombination staggered-herringbone grooves Size determined displacement particle separation Continuous wall based hydrodynamic separation Streamline following separation Physical principles of cell sorting Soft inertial separation Wu, Hjort, Micro Nanosyst 1 (2009) 181 Shields IV, Reyes, Lopez, Lab Chip 5 (2015) 1230 Physical principles of cell sorting • Centrifugal forces Shields IV, Reyes, Lopez, Lab Chip 5 (2015) 1230 “Active” cell sorting • After inspection cells are sorted by external operator Electromagnetic field Optical tweezers Shields IV, Reyes, Lopez, Lab Chip 5 (2015) 1230 Chemical properties based cell sorting • Specific antibodies magnetically labelled • Magnetic labelling of cells Shields IV, Reyes, Lopez, Lab Chip 5 (2015) 1230 Cell sorting overall • Promising cell sorting using microfluidics – Accessible fabrication – Low reagent consumption – Small footprints – Improved safety over traditional cell sorting (eliminating potentially biohazardous aerosols) • Problems – Many technologies in proof-of-concept stage – Low throughput due to single-channel design – Blocking or clogging of the microchannels – Sample preparation still necessary Biochips and sensing arrays • “Miniaturised laboratory” • Collection of miniaturised sensors arranged in array on solid substrate • Many tests performed simultaneously – Hundreds or thousands of (bio)chemical reactions • Small dimensions • Term overlapping with implanted carriers of information – Replacing drivers licence, ID card, medical records… https://gdvmahesh.wordpress.com http://www.igb.fraunhofer.de Biochip & microarray design • Transduction – Electrochemical – Optical http://clse.epfl.ch/ http://www.gersteltec.ch http://www.scienion.com Microarrays • DNA and protein microarray • Each spot is modified with specific protein marker/DNA/antibody etc. • Localisation related information • Evaluation of photography • Valid for electrochemical transducer – Localisation of microelectrode http://www.igb.fraunhofer.de Microarrays • Necessary digital evaluation of results • Crucial – fabrication of sensor array http://prohardver.hu Deposition of biomolecules at nanoscale • High resolution modification of sensing array • Several methodical approaches – Nanolithography (SPM) – Nanofabrication – Nanodeposition • Activation of the surface (chemical, physical) Nanolithography • AFM, (SECM) • Dip-pen nanolithography • Local electrochemical modification (removal, deposition) http://www.npl.co.uk Danieli, Colleran, Mandler, PhysChemChemPhys 13 (2011) 20345 Carano, Lion, Abid, Girault, Electrochem Commun 6 (2004) 1217 Liquid nanodispersing • Deposition of droplets through nanochannel drilled in AFM tip Fabié, Agostini, Stopel, Blum, Lassagne, Subramaniam, Ondarçuhu, Nanoscale 7 (2015) 4497 Nanofabrication Self assembled monolayers Self-assembly Vericat, Vela, Benitez, Carro, Salvarezza, Chem Soc Rev 39 (2010) 1805 Sabella, Brunetti, Vecchio, Della Torre, Rinaldi, Cingolani, Pompa, Nanoscale Res Lett 4 (2009) 1222 Deposition of biomolecules at nanoscale • Deposition of nanodrops • System for generation of micro- or nanodrops • Highly suitable for “array sensing” • Activation of surface (surface chemistry) Petrelli, Marconi, Salerno, De Pietri Tonelli, Berdondini, Dante, Lab Chip 13 (2013) 4419 Nanodrop • Scienion • http://www.scienion.com/support/movies/ • For high throughput array production • Piezo-dispensing technology