Lab-on-a-Chip 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 Lab-on-a-Chip Concept and Examples Application to Proteomics Lab-on-a-Chip Subunits Sample handling Reactors Separation Methods Detection 2
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 Applications Genomics and proteomics Environmental assays Medical diagnostics Drug discovery Chemical production Cellular analysis 3 Affymetrix Lab-on-a-Chip 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 Anderson et al. 4
U of M Lab-on-a-Chip Mastrangelo and Burns groups integrated device Nanoliter liquid injector Sample mixing and positioning system Temperaturecontrolled PCR reaction chamber Electrophoretic separation Fluorescent photodetector 5 Microscope-on-a-Chip 6
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 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 Scientific American April 2002 8
Necessary Subunits for µ-tas Sample handling Extraction Mixers Valves Pumps Reactors Separation Detection 9 Sample Extraction Means for extracting samples from dilute solutions required 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 10
Extraction Using Filters Microfabricated filters Mechanically robust to withstand high pressure drops for filtering µm-sized particles Very uniform pore sizes determined by Photolithography Sacrificial layer thickness C.-M. Ho group, UCLA Keller et al., UCB 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 Bead chambers Hydrophobic beads trapped in a flow chamber Harrison group, Univ. of Alberta Stemme group, Sweden 12
Extraction Using Porous Polymers Porous polymers increase available surface area for binding interactions 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 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 Can we use irreversibility of diffusive mixing in reversibly stirred flows to separate chemical species based on size? 14
Extraction by Diffusion 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 Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection 16
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. How to assist mixing? Repeated lamination of flows increases contact area and decreases diffusion length C.-M. Ho Group, UCLA 17 Chaotic flows can be very efficient mixers Changing surface topography of microchannel floor induces chaotic flows Stroock et al., Whitesides Group, Harvard 18
Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection 19 Pumping Mechanisms Pressure gradients Electrokinetic forces Surface tension forces Electrowetting Thermocapillary Surface acoustic waves Magnetohydrodynamic Dielectrophoresis C. M.Ho 20
Centrifugal Forces 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 Electrowetting Electrical potential can control surface tension on a dielectric solid surface 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 cosθ( V ) = cosθ 0 + 2 εε 0V 2 t γ LV 22
Thermocapillary Pumping Thermocapillary effect Local heating reduces surface tension, pulling liquid towards cooler surface Surface temperature manipulated by embedded heaters 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 Thermocapillary Mixer ~1000 faster than diffusion Troian group, Princeton U. 24
Surface Acoustic Waves More on ultrasonic fluidic devices at http://wwwbsac.eecs.berkeley.edu/fluidics/ White group, BSAC Sandia Labs 25 Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection 26
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 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 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 Flow stops and gas vents Hydrophobic patches Hydrophobic membrane vents Thermally-generated bubbles Unger et al., Quake group, Caltech 28
Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection Jensen group, MIT 29 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 Point of care testing using microfluidics Enhanced reaction efficiency Simplified procedures Reduced assay time Lower sample & energy consumption Sato et al., University of Tokyo 30
Clinical Diagnosis On-Chip 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 31 Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection 32
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 Staining and SDS dilution steps occur in 100 s ms, 10 4 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 Separation by Isoelectric Focusing Isoelectric focusing (IEF) is electrophoresis in a ph gradient (cathode at 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 Issues + IEF downscales well since resolution is independent of channel length, in contrast to CE EP focusing effect counteracted by diffusion, yielding Gaussian band distribution Dilute base Higher ph, (-) pi = 3 min dph D dx L dµ V dph Dilute acid lower ph, (+) 34
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 High field with shorter separation length leads to increased Joule heating 35 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 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 Craighead group, Cornell 36
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 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 (R G = 0.31 µm) Chou, Austin groups, Princeton, Craighead group, Cornell 37 Necessary Subunits for µ-tas Sample handling Preparation Mixers Pumps Valves Reactors Separation Detection 38
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 Challenges Need for robust and/or universal probes Isolation of ECL electrodes from CE high voltage 39 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 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) 40
Mass Spectrometry Mass spectrometry (MS) measures mass-to-charge ratios (m/z) of species fragments Electrospray ionization spectrometry (ESI) is recent, powerful technique 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 New Objective 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. Results Flowrate ~ 2 µl/min Throughput ~ 5 min/sample Sensitivity ~ 25 fmol (5 nm) Harrison Group, U. of Alberta 42
ESI On-Chip 2 Fabrication 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 Results 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 43 More Topics Cell culturing Cell handling Dielectrophoresis Optical tweezers Protein crystallization Interfacing between micro-macroworlds Materials and surfaces Microfluidic/nanofluidic components, modeling Applications Many more 44