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Lab-on-a-Chip

Dr. Thara SrinivasanLecture 20

Picture credit: Anderson et al.

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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

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5Lab-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

• Applications• Genomics and proteomics • Environmental assays • Medical diagnostics • Drug discovery• Chemical production• Cellular analysis

• Saves reagents and labor

• Increases testing throughput

• Creates portable systems

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AffymetrixLab-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.

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U of M Lab-on-a-Chip

• Nanoliter liquid injector

• Sample mixing and positioning system

• Temperature-controlled PCR reaction chamber

• Electrophoretic separation

• Fluorescent photodetector

• Mastrangelo and Burns groups’ integrated device

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Microscope-on-a-Chip

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5Proteomics

• 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)

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Sci

entif

ic A

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ican

Apr

il 20

02

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5Necessary Subunits for µ-TAS

• Sample handling• Extraction• Mixers• Valves• Pumps

• Reactors• Separation • Detection

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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

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5Extraction 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

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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

Stemme group, Sweden

Harrison group, Univ. of Alberta

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5Extraction 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

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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?

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5Extraction by Diffusion

• As two parallel laminar flows contact, diffusion extracts certain components• Components with

higher diffusivity extracted

• Micronics H-filterpull elements out of sample into diluent

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Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

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5Mixing

• 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

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• Chaotic flows can be very efficient mixers• Changing

surface topography of microchannelfloor induces chaotic flows

Stroock et al., Whitesides Group, Harvard

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5Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

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Pumping Mechanisms

• Pressure gradients• Electrokinetic forces• Surface tension forces

• Electrowetting• Thermocapillary

• Surface acoustic waves • Magnetohydrodynamic • Dielectrophoresis

C. M.Ho

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5Centrifugal 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

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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 tVVLVγ

εεθθ2

cos)(cos2

00 +=

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Thermocapillary Pumping

• Thermocapillary effect• Local heating reduces surface tension, pulling liquid

towards cooler surface• Surface temperature manipulated by embedded heaters

Troian group, Princeton U.

• 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

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Thermocapillary Mixer

Troian group, Princeton U.

• ~1000× faster than diffusion

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5Surface Acoustic Waves

• More on ultrasonic fluidic devices at http://www-bsac.eecs.berkeley.edu/fluidics/

White group, BSAC Sandia Labs

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Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

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Elastomer Valves

• 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

• 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…

Unger et al., Quake group

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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

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5Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

Jensen group, MIT

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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

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• Diagnosis of colon cancer by detection of human carcino-embryonic antigen (CEA) in serum on-chip• Polystyrene beads coated with

antibody in microchannel, antigen-antibody 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

Clinical Diagnosis On-Chip

Sato et al., University of Tokyo

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Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

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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, 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

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• 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

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

Dilu

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dpHdVL

dxdpHDpI µ3min =∆

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5IEF 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

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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

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5Separation 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

(RG = 0.31 µm)Chou, Austin groups, Princeton,Craighead group, Cornell

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Necessary Subunits for µ-TAS

• Sample handling• Preparation• Mixers• Pumps • Valves

• Reactors• Separation • Detection

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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

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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 fmolfor ferrocenecarboxylic acid (2001). (EPL, Lausanne)

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5Mass 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

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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

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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

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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…