BioMEMS - Applications - Lecture Notes | BE 825, Study notes of Engineering

Download BioMEMS - Applications - Lecture Notes | BE 825 and more Study notes Engineering in PDF only on Docsity! Lecture 23. BioMEMS — Applications Applictions of BioMEMS/NEMS Targets: Cells, Proteins, DNA, Molecules, etc. Mechanisms: Mechanical, Electrichemical, Optical, Thermal, etc. Attributes: Sensitive, Rapid, High throughput, etc. 1. Sorting and concentration using dielectrophoresis (from: Li, H., etc. Sensors and Actuators B 86 (2002) 215-221) Biological cells consist of adjacent structures of materials that have very different electrical properties and exhibit large induced boundary polarizations that are highly dependent on the applied field frequency as well as their physiological states. For example, the cell membrane consists of a very thin lipid bi-layer containing many proteins and is highly insulating with a conductivity of around 10−7 S/m, while the cell interior contains many dissolved charged molecules, leading to a conductivity as high as 1 S/m. Upon death, the cell membrane becomes permeable and its conductivity can increase by a factor of 104 due to the cell contents exchanging freely material with the external medium through the small pores on the membrane. This large change in the dielectric properties on cell death indicates a large change in the dielectric polarizability [5]. Hence, a large difference in DEP responses (positive and negative, respectively) and a selective separation can be achieved between live and dead cells. 1 Fig. A schematic plot of the experimental apparatus in (a) and the top view of the interdigitated microelectrodes in (b). The width of the electrodes and the spacing between two adjacent electrodes are both 15 µm. The silicone rubber wall is 0.3 mm thick. The electrodes are connected to a HP 33120A arbitrary waveform generator. Fig. Negative dielectrophoresis for both live and heat-treated L. innocua cells on interdigitated microelectrodes by applying a 1 V (peak-to-peak) and 10 kHz signal. Live 2 Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'- phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction (2). Micro-PCR 5 Chip layout. (A) Schematic of a chip for flowthrough PCR. Three well-defined zones are kept at 95°, 77°, and 60°C by means of thermostated copper blocks. The sample is hydrostatically pumped through a single channel etched into the glass chip. The channel passing through the three temperature zones defines the thermal cycling process. (B) Layout of the device used in this study. The device has three inlets on the left side of the device and one outlet to the right. Only two inlets are used: one carrying the sample, the other bringing a constant buffer flow. The whole chip incorporates 20 identical cycles, except that the first one includes a threefold increase in DNA melting time. The chip was fabricated in Corning 0211 glass at the Alberta Microelectronic Centre, Canada. All channels are 40 mm deep and 90 mm wide; the etched glass chip and the cover plate are each 0.55 mm thick. Access to the channels is provided by holes (400 mm) drilled into the cover plate. Standard fused-silica capillaries (outside diameter 375 mm, inside diameter 100 mm) are glued with epoxy into the holes of the chip. Virtually no dead volume is introduced by this connection. Two precision syringe pumps (Kloehn 50300, 25 ml) deliver the PCR sample and the buffer solution onto the chip. The pumps are controlled by a program written in Labview running on a PC. Product is collected at the outlet capillary and then analyzed by slab-gel electrophoresis. The copper blocks are heated by 5-W heating cartridges, and the surface temperature is monitored by a Pt100 thin-film resistor mounted on the surface of the block near the chip contact area. Cooling fins passively cool the two blocks at 77° and 60°C. The temperature controllers are built with standard PID (proportional, integral, and derivative) digital temperature controllers (CAL 3200), power supplies, and switching electronics for the heating cartridges. From: Kopp, et al., 1998. Chemical amplification: Continuous-flow PCR on a chip. Science 280, 1046-1048. 3. Micro electrophoresis 6 Injection volume is around 100 pL, Injection to detection distance is 5 cm. 4. BioMEMS on cantilever sensors 7