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Lab-on-a-Chip EE C245 Dr. Thara Srinivasan Lecture 20 Picture credit: Anderson et al. Lecture Outline • Reading from reader • Auroux, P.-A., Manz, A. et al. , “Micro Total Analysis Systems,” (2002) pp. 2637-52. • Krishnan, M., et al., “Microfabricated Reaction and Separation Systems,” (2001) pp. 92-98. • Quake, S., R, and A. Scherer, “From Micro- to Nanofabrication Using Soft Materials,” (2001) pp. 1552-69. • Today’s Lecture EE C245 • Lab-on-a-Chip Concept and Examples • Application to Proteomics • Lab-on-a-Chip Subunits • • • • U. Srinivasan © Sample handling Reactors Separation Methods Detection 2 1 Lab-on-a-Chip • Micro total analysis system (µ-TAS) • Vision proposed by Manz, Widmer and Harrison in early ’90’s • Perform sample addition, pretreatment and transport, chemical reactions, separation, and detection on a microscope slide or credit card size chip • Annual conference, MicroTAS, had 700 attendees in ‘02 • Saves reagents and labor • Increases testing throughput • Creates portable systems EE C245 • Applications • • • • • • Genomics and proteomics Environmental assays Medical diagnostics Drug discovery Chemical production Cellular analysis 3 U. Srinivasan © Affymetrix Lab-on-a-Chip EE C245 • Multiple operations performed • Cell lysis • Sample concentration • Enzymatic reactions such as reverse transcription, PCR, DNAse digestion and terminal transferase labeling • Dilution, hybridization, and washing • Dye staining U. Srinivasan © Anderson et al. 4 2 U of M Lab-on-a-Chip EE C245 • Mastrangelo and Burns groups’ integrated device • Nanoliter liquid injector • Sample mixing and positioning system • Temperaturecontrolled PCR reaction chamber • Electrophoretic separation • Fluorescent photodetector 5 U. Srinivasan © EE C245 Microscope-on-a-Chip U. Srinivasan © 6 3 Proteomics • A “proteome” is the set of proteins encoded by a gene • Proteomics • Identifying all the proteins made by a given cell, tissue or organism • Determining how the proteins network among themselves • Finding out precise 3D structures of the proteins • Proteins more complex than genes EE C245 • • • • DNA: 4 bases, proteins: 20 amino acids Even with a protein’s sequence, its function and networks still unknown 3D shape of folded protein difficult to predict All human cells have same genome, but differ in which genes are active and which proteins are made • ~40,000 human genes, each gene can encode several proteins (typical cell makes 100,000’s proteins) 7 U. Srinivasan © Scientific American April 2002 EE C245 U. Srinivasan © 8 4 Necessary Subunits for µ-TAS • Sample handling • • • • Extraction Mixers Valves Pumps EE C245 • Reactors • Separation • Detection 9 U. Srinivasan © Sample Extraction • Means for extracting samples from dilute solutions required EE C245 • At macroscale, centrifugal force is used • For microfluidics, sample extraction is interface to macroscale • Most of the power consumption is spent at this step • Methods include • Filtration • Chromatography U. Srinivasan © 10 5 Extraction Using Filters • Microfabricated filters • Mechanically robust to withstand high pressure drops for filtering µm-sized particles • Very uniform pore sizes determined by EE C245 • Photolithography • Sacrificial layer thickness C.-M. Ho group, UCLA Keller et al., UCB U. Srinivasan © 11 Solid-Phase Extraction • As in chromatography, • Desired components bind reversibly to a coated porous solid and are later flushed out by a change in solvent • Hydrophobic coatings bind nonpolar compounds in aqueous flow EE C245 • Bead chambers • Hydrophobic beads trapped in a flow chamber U. Srinivasan © Harrison group, Univ. of Alberta Stemme group, Sweden 12 6 Extraction Using Porous Polymers • Porous polymers increase available surface area for binding interactions • • EE C245 • Fill channels with polymerization mixture ~ monomers, initiator, and porogenic solvent Irradiate chip with UV light through photomask Surface chemistry may be varied widely Fréchet group, UCB 13 U. Srinivasan © Extraction by Diffusion • Mixing in low Re flows is nearly reversible • Two flows that have been stirred together may be “unstirred”—except for any mixing by diffusion—by reversing the driving force EE C245 • Can we use irreversibility of diffusive mixing in reversibly stirred flows to separate chemical species based on size? U. Srinivasan © 14 7 Extraction by Diffusion EE C245 • As two parallel laminar flows contact, diffusion extracts certain components • Components with higher diffusivity extracted • Micronics H-filter pull elements out of sample into diluent 15 U. Srinivasan © Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection U. Srinivasan © 16 8 Mixing • Mixing of particles, cells and molecules often determines the system efficiency • PCR, DNA hybridization, cell lyses… • Diffusion, the mechanism of mixing at the microscale, still requires relatively long times for thorough mixing. EE C245 • How to assist mixing? • Repeated lamination of flows increases contact area and decreases diffusion length U. Srinivasan © C.-M. Ho Group, UCLA 17 • Chaotic flows can be very efficient mixers EE C245 • Changing surface topography of microchannel floor induces chaotic flows U. Srinivasan © Stroock et al., Whitesides Group, Harvard 18 9 Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection 19 U. Srinivasan © Pumping Mechanisms • Pressure gradients • Electrokinetic forces • Surface tension forces • Electrowetting • Thermocapillary EE C245 • Surface acoustic waves • Magnetohydrodynamic • Dielectrophoresis U. Srinivasan © C. M.Ho 20 10 Centrifugal Forces EE C245 • Gyros, Sweden • When CD spins, centrifugal force causes liquids on their surface to move outwards. • The force can drive liquids through microchannels… • …even breaking through hydrophobic barriers in the channels, releasing different chemicals selectively 21 U. Srinivasan © Electrowetting • Electrical potential can control surface tension on a dielectric solid surface EE C245 • Asymmetric contact angles generate internal pressure imbalance, leading to movement • Fluidic operations can be done on discrete droplets • Low voltages: 25 V DC for v = 30 mm/s; 100V AC for v = 200 mm/s CJ Kim group, UCLA U. Srinivasan © cosθ (V ) = cosθ 0 + εε 0V 2 2γ LV t 22 11 Thermocapillary Pumping • Thermocapillary effect • Local heating reduces surface tension, pulling liquid towards cooler surface • Surface temperature manipulated by embedded heaters EE C245 • Results • v = 600 µm/s for liquid PDMS + Low operating voltage (2-3 V) + Works with polar and non-polar liquids • Thermocapillary mixer ~1000× faster than diffusion Troian group, Princeton U. 23 U. Srinivasan © Thermocapillary Mixer EE C245 • ~1000× faster than diffusion U. Srinivasan © Troian group, Princeton U. 24 12 Surface Acoustic Waves EE C245 • More on ultrasonic fluidic devices at http://wwwbsac.eecs.berkeley.edu/fluidics/ White group, BSAC U. Srinivasan © Sandia Labs 25 Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection U. Srinivasan © 26 13 Elastomer Valves • A good valve needs flexibility and a valve seat that closes completely • Microfabricated poly-Si valves: microactuator forces limited, so stiffness limits minimum size • For elastomers, Young’s Modulus can be tuned over 2 orders of magnitude… • PDMS valves and pumps made by replica molding • • EE C245 • • Crossed channel layout; channels 100 µm wide, 10 µm high When P is applied to upper channel, membrane deflects, closing lower channel Response time 1 ms, applied P = 100kPa Dead volume is zero for on-off valve Unger et al., Quake group U. Srinivasan © 27 Valves and Pumping • Peristaltic pumping with elastomer valves • • • 3 valves on a single channel (closing pattern: 101, 100, 110, 010, 011, 001) 2.35 nL/s at 75 Hz, 1 mN force Avoids drawbacks of EO pumping • Dependence on medium • Electrolytic bubble formation • Difficulty setting voltages when many junctions present EE C245 • Flow stops and gas vents • Hydrophobic patches • Hydrophobic membrane vents • Thermally-generated bubbles U. Srinivasan © Unger et al., Quake group, Caltech 28 14 Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection Jensen group, MIT 29 U. Srinivasan © Immunoassay Reactor • Immunoassays • Important analytical method for clinical diagnostics, environmental analyses, and biochemical studies. • Antigens and antibodies are fixed onto a solid support • ELISA = Enzyme-Linked ImmunoSorbent Assay EE C245 • Point of care testing using microfluidics • • • • Enhanced reaction efficiency Simplified procedures Reduced assay time Lower sample & energy consumption U. Srinivasan © Sato et al., University of Tokyo 30 15 Clinical Diagnosis On-Chip EE C245 • Diagnosis of colon cancer by detection of human carcinoembryonic antigen (CEA) in serum on-chip • Polystyrene beads coated with antibody in microchannel, antigenantibody complex detected optically • Liquid handling significantly simplified • Assay time reduced to ~1% (45 h to 35 min) • Compared to conventional ELISA, detection limit dozens of times lower • High throughput analysis using branching channels for simultaneous analysis Sato et al., University of Tokyo U. Srinivasan © 31 Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection U. Srinivasan © 32 16 Separation by Electrophoresis • Current standard method for protein sizing • Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) • SDS denatures proteins and gives them charge; PAGE separates by size • Protein electrophoresis on chip • Steps: sample loading (protein + SDS), dye labeling (staining), separation, SDS dilution and destaining, and detection EE C245 • Staining and SDS dilution steps occur in 100’s ms, 104 × faster than macroscale • Sequential analysis of 11 samples, sizing accuracy >5%, sensitivity 30 nM Video clip at http://www.chem.agilent.com/scripts/generic.asp?lPage=1566&indcol=N&prodcol=Y 33 U. Srinivasan © Separation by Isoelectric Focusing • Isoelectric focusing (IEF) is electrophoresis in a pH gradient (cathode at higher pH) EE C245 + IEF downscales well since resolution is independent of channel length, in contrast to CE • EP focusing effect counteracted by diffusion, yielding Gaussian band distribution U. Srinivasan © ∆pI min = 3 D Dilute acid lower pH, (+) • Issues Dilute base Higher pH, (-) • A protein’s isoelectric point (pI) is the pH at which it has neutral charge • Charged species stop moving when EP pushes them to their pI • Linear pH gradient built up using ampholytes • IEF concentrates and separates dpH dµ L V dx dpH 34 17 IEF On-Chip • Advantages • Sample mobilization unnecessary • No injection plug so separation does not depend on initial sample shape • Short channel length gives rapid analysis and… • Full field detection by imaging with inexpensive CCD • Challenge EE C245 • High field with shorter separation length leads to increased Joule heating 35 U. Srinivasan © Separation by Entropic Traps • Channels with nanoscale constrictions • Require long DNA to repeatedly change conformation, costing entropic free energy • Longer DNA has higher mobility • Separation EE C245 • No sieving medium needed • 5-kbp sample at 80 V/cm in 30 min • Longer channels for better separations; resolution not as good as CE • Sample concentration • At low E, DNA is trapped into band U. Srinivasan © Craighead group, Cornell 36 18 Separation by Diffusion • Using 2-D “obstacle course” and electric field in –y direction • Asymmetric obstacles rectify Brownian motion (diffusion) of molecules • Faster-diffusing species move more in +x direction EE C245 • Results • Obstacles: 1.5×6 µm² at 45° angle • No sieving medium; low E (1.4 V/cm); may be applied to DNA, proteins, cells, etc. • v = 1-15 µm/s, for a 10 cm sieve • Bandwidth = 200 µm for 15 kbp DNA (RG = 0.31 µm) U. Srinivasan © Chou, Austin groups, Princeton, 37 Craighead group, Cornell Necessary Subunits for µ-TAS • Sample handling • • • • Preparation Mixers Pumps Valves EE C245 • Reactors • Separation • Detection U. Srinivasan © 38 19 Detection: Chemiluminescence • Chemiluminescence (CL) or electrochemiluminescence (ECL) • Ru(bpy)3+2 oxidized chemically or electrochemically to Ru(bpy)3+3 which… • Reacts with amines, amino acids, glucose, PCR products, etc… • …and emits light at 620 nm • Advantages • Laser not required • Instruments much simpler than for LIF • Low to zero background signal; sensitivity high • Scaling benefits ~ microphotodetector for on-chip detection EE C245 • Challenges • Need for robust and/or universal probes • Isolation of ECL electrodes from CE high voltage 39 U. Srinivasan © Electrochemical Detection • Electrochemical detection (EC) • Control potential of working electrode and monitor current as samples pass by • Applied potential is driving force for electrochemical reactions of sample analytes, current reflects concentration of compounds • Benefits and challenges EE C245 • On-chip detection; truly portable • Chemistries need to be developed • Rossier et al. integrated screen-printed carbon ink electrodes into plastic microchannels and demonstrated detection limit of ~1 fmol for ferrocenecarboxylic acid (2001). (EPL, Lausanne) U. Srinivasan © 40 20 Mass Spectrometry • Mass spectrometry (MS) measures mass-to-charge ratios (m/z) of species fragments EE C245 • Dilute solution of analyte (10-4-10-5 M) is sprayed from capillary tip at high potential (3-4 kV) • Liquid forms Taylor cone, fine jet of tiny charged droplets which blow apart due to charge repulsion • “Nanospray” uses smaller glass capillaries for lower flows (20-50 nL/min) 2-30 µm U. Srinivasan © New Objective • Electrospray ionization spectrometry (ESI) is recent, powerful technique 41 Proteomics-on-a-Chip • Integrated chromatography + CE + ESI • • Photolithography and wet-etching of Corning 0211 glass Nanospray emitter placed into a flat-bottomed hole drilled into the exit of separation channel • Bead channel for sample concentration 800 µm wide, 150 µm deep, 22 mm long, etched into the cover plate (2.4 µL volume) Filled with bead suspension slurry Low flow resistance of bead channel allows sample loading without perturbing CE channel. • • • EE C245 • Results • Flowrate ~ 2 µL/min • Throughput ~ 5 min/sample • Sensitivity ~ 25 fmol (5 nM) U. Srinivasan © Harrison Group, U. of Alberta 42 21 ESI On-Chip – 2 • Fabrication • • • • Results • • • EE C245 Polymer chip embossed from silicon master Electrospray tip is flat parylene C triangle (5 µm thick) sandwiched between channel chip and sealing cover Tip is wet by analyte, helping to form and fix position of Taylor cone • Low dead volume connection Stable ion current 30-40 nA measured using 2-2.8 kV potentials Analyte liquid is completely confined on triangular tip Cone volume estimated as 0.06 nL Craighead group, Cornell U U. Srinivasan © 43 More Topics • Cell culturing • Cell handling EE C245 • Dielectrophoresis • Optical tweezers • • • • • • U. Srinivasan © Protein crystallization Interfacing between micro-macroworlds Materials and surfaces Microfluidic/nanofluidic components, modeling Applications Many more… 44 22