Content • Fluidics Applications • Scaling in Fluidics • A CD as a Fluidic Platform • Nanofluidics • Challenges in Microfluidic Platforms
DNA RNA protein bacteria, cancer cell, WBC, et al Hybridization Electrophoresis Sequencing Raw sample Cell Separation Cell Lysis, Purification Amplification Sample Preparation Fluidics Applications Memory devices today and tomorrow Diagnostics/Molecular diagnostics today and tomorrow
Fluidics Applications • Lab-on-a-chip: • One system to provide all of the possible required analyses for a given type of problem • All processing steps are performed on the “chip” • No user interaction required except for initialization • High throughput screening (HTS) and diagnostics are two major applications for Lab-on-a-chip • Partitioning of functions between disposable and instrument is very different for HTS and Molecular Diagnostics Instrument Power Propulsion Heater (PCR) Electronics Detection Disposable Cassette Reagents Fluidics PROPULSION Mechanicalpressure Acoustic Centrifugal Electrokinetic
Lab-on-a-chip: Goals Portable Robust Easy to use Flexible Inexpensive Modular? Components: Separation Mixing Reaction(s) Sample injection Sample preparation Detection Pumping Transport (channels) Reservoirs Flow control Intelligence and Memory Power Display Fluidics Applications
Scaling in Fluidics • Most sensing techniques scale poorly in the micro domain (-) • Often large samples are required to get enough target species collected (-) • Short analysis time dictates small devices (+) • Fast heating/cooling (e.g., for PCR) requires small samples (+) • All flow is laminar (little turbulent mixing) (- for mixing) • Surface tension becomes significant (+/-) • No inertia effects (+/-) • Apparent viscosity increases (+/-) • Evaporation is very fast for small samples (-) • Devices are almost always too large for Si to be a solution.
Different Propulsion Options-Pumps • Propulsion Mechanisms-Pumps: • Mechanical (pneumatic/hydraulic)--example shown on the right is the blister pouch (kodak/Johnson&Johnson) • Electrokinetic • Thermal (shape memory alloy, phase changes) • Acoustic • Centrifuge • Electrohydrodynamic • Magnetic • Chemical (hydrogel, osmotic pressure, phase change) • Electrochemical (create bubles through electrolysis)
Different Propulsion Options • Mechanical (blister pouch for example) • Scales as L3 • No fluid contact • Generic • Innovation in the blister pouch • Solves liquid and vapor valving !! • Difficult to further miniaturize • Difficult to multiplex
Different Propulsion Options • Electrokinetic (electro-osmosis): • Requires materials with surface charge • Preferably permanent • Glasses and many polymers have permanent negative surface charge • Positive charges assemble on surface • Applied charges pull assembled charges • Charges at surfaces drag bulk material • Plug flow
Different Propulsion Options • Electrokinetic (DC) • High voltage source is not convenient • Many parameters influence propulsion force • Not generic • Mixing difficult to implement • Fluid contact • Scales as L2 • First products (Caliper) • May solve liquid valving but not for vapors ! • Better for high-throughput screening (HTS) and smaller samples
Different Propulsion Options • Centrifugal • Compatible with a wide range of samples • Mixing easy to implement • Sample preparation easier • Simple and inexpensive CD player for drive • No fluid contact • Established • Generic • Solves liquid valving elegantly • Scales a bit better than l3 • Most functions demonstrated • Cell work easier • Better for diagnostics
Different Propulsion Options • Acoustic (Dick White’s flexural plate wave device for example) • Scales as L2 • No fluidic contact • R & D phase • Generic • Doesn’t solve valving yet • ZnO technology still difficult to reproduce • Easy to further miniaturize
A CD as a Fluidic Platform • Why a CD as a Microfluidic Platform ? • Microscope, smart centrifuge and plastic disposable with fluid storage capability • Comparison with other microfluidic platforms • Example Applications • Most Recent Application: Integrated Molecular Diagnostics (DNA Arrays on a CD) • Lysis • Lysis 1: multiplex • Lysis 2: single circular • Fast hybridization detection • Optical • This is where we areheaded • Conclusions
A CD as a Fluidic Platform • The optical disc drive is a sophisticated laser scanning microscope designed to characterize and identify micrometer sized features at a rate of about a Megahertz (H. Kido and J.Zoval).
A CD as a Fluidic Platform • The voltages from the photodetector are sent to a computer using a fast A/D converter. • The image is then reconstitued using simple graphics software VOLTAGE (H. Kido and J.Zoval). TIME
A CD as a Fluidic Platform • Examples of pictures taken using the CD player. • Vision is another dimension CD fluidics can offer. DNA array Gnat wing White blood cells
Center R1 r R2 A CD as a Fluidic Platform • The optical disc drive is a smart centrifuge.
A CD as a Fluidic Platform • The Compact Disc (CD) is a biocompatible “solid phase” (plastic) • It can substitute for standard consumables such as: slides, micro-wells, centrifuge tubes.
A CD as a Fluidic Platform • List of Lab tasks feasible on a CD • Mixing, • Two-point calibration, • Washing, • Centrifuge, • Sample splitting, • Sample metering, • Molecule separation, • PCR, • Fast Immuno-assays, • Fast DNA- assays, • Cell viability tests
A CD as a Fluidic Platform • Cell lysis on the CD instead of using a vortex ---to make further integration possible • Motivation: To extract DNA from cells in a CD platform • The design below has a single lysis chamber only.
E.coli Lysis A CD as a Fluidic Platform • Type: Chinese Hamster Ovary (CHO-K1) • Size: ~10 µm • Glass Beads: 100 – 220 µm • No. of Rotation Cycles: 300 (5 min.) DNA concentration measured using PicoGreen Quantitation Kit
A CD as a Fluidic Platform • Multiplex design allows the integration of several cell lysis chambers with other analysis tasks on the same platform. • As we saw before the cells can also be visualized before and after lysis using the CD optics.
A CD as a Fluidic Platform • Fast DNA Hybridization Detection • Problem: Time consuming hybridization caused by slow diffusion of DNA molecules in passive DNA array approaches • How to speed up hybridization ? • Electrophoretic • Mixing • Flow Microspots with DNA Capture Probes Target DNA Injection Out Flow-through Hybridization column
A CD as a Fluidic Platform Modeling of DNA transport in flow-through hybridization column Navier Stokes eq.: Species transport equation:
A CD as a Fluidic Platform • Hybridization in a constrained column using CD platform for sample and reagent propulsion • The flow cell consists of a hybridization column 1, hydration buffer chamber 2, sample chamber 3, and two rinse chambers 4&5. • Fast hybridization steps: • Hydration • Sample flow • Two consecutive wash steps
A CD as a Fluidic Platform (i) (ii) (a) (b) • The figure (a) on the left shows the results of hybridization on the CD. • The figure compares a non-specific sequence ssDNA (i) with specific sequence ssDNA (ii) hybridization experiment. • A spinning velocity of 450 RPM was used (corresponding to the flow rate ranging from 0.65 uL/min to 1.3uL/min).
A CD as a Fluidic Platform • Final goal: • Sample to answer nucleic acid analysis test • Multi unit CD combining: • Live/dead viability assay for cell quantization. • Hybrization detection in which Cells are lysed, nucleic acids are purified and mixed with RNAase inhibitor, calibrants, and reporters. Fast hybridization using flow-through column Live/dead viability unit Fast Hybridization detection unit
DNA RNA protein bacteria, cancer cell, WBC, et al Hybridization Electrophoresis Sequencing Raw sample Cell Separation Cell Lysis, Purification Amplification Sample Preparation A CD as a Fluidic Platform • Diagnostics as a powerful new application of a very mature and well established technology: CD, DVD, etc. • Sample to answer for molecular diagnostics in a hand-held is not about if but when --microfluidics will make it possible and the CD approach has the most features that fit the application’s need. • Don’t throw away your reject CD’s (AOL, Barry Manilow, the Bee Gees, etc....).They may have some use after all. Put blood on the tracks !
Nanofluidics • As lithography tools go beyond 1 µm new fluidic possibilities arise. • With fluidic channels of the size of biological polymers we can start interacting with these species. • Figure on the right (H. Craighead) demonstrates DNA separation using nanochannels (artificial hydrogel).
Microfluidic Challenges • Wet reagent storage and dry reagent reconstitution • Tight liquid and vapor valves • Integrated microvalves and micropumps • Packaging • Interconnects (optimize, reduce, eliminate) • Filling / bubbles / dead volume • Leakage • Surface functionalization • Microflow measurement and characterization • Control algorithms, data processing, and communications • Integrated, ultrasensitive detection • Heterogenous material integration • Sensitivity limited by sample volume (front end amplifiers/concentrators?) • Low power • Harness energy from host or ambient • Low power pressure sources