M6 - BioMEMS

Applications:BioMEMSCSE 495/595: Intro to Micro- and Nano- Embedded SystemsProf. Darrin Hanna

Microfluidics Reynolds number Ratio of kinetic energy to rate of loss of energy due to friction v D / is fluid density v is the average velocity D is the diameter of the channel is the absolute viscosity Below approx. 2,300 flow is laminar slower at edges Higher turbulent

Microfluidics Reynolds number microfluidics usually water-based fluids are used approx. 1 gm/cm3 approx. 0.01 g / (cm s) Example D = 30 m 1 mm/s Reynolds number = 0.03 usually below 1 No mixing Joining streams flow side-by-side with only diffusion special flow structures for mixing increase the area of diffusive mixing

Microfluidics Agilent Cell LabChip. detects cells stained with fluorescent dyes vacuum pulls separate flows of cells and buffer Y-shaped junction

Microfluidics flow of cells pushed to one side of the microchannel by the flow of buffer Individual stained cells are detected as they pass under an excitation beam and fluoresce If channels were same width as a cell the cell would clog

DNA deoxyribonucleic acid built from nucleotides 4 types of nucleotides adenine, thymine, cytosine, guanine A T C G

Genes sequences in the DNA encodes functional product (i.e. protein) Proteins required for structure, function, and regulation of cells, tissues, and organs each protein has unique functionsA TC G

DNA Proteins made up of amino acids 20 amino acids Chromosome self-replicating structure of cells containing the cellular DNA that bears in its nucleotide sequence the linear array of genes

DNA Human genome 23 separate pairs of chromosomes (46 chromosomes) averaging 130 million base pairs in length each = total of about three billion base pairs genes that form the template for proteins are typically 27,000 base pairs long only about 1,000 are used, rest is filler

DNA Each nucleotide molecule has two ends 3 and 5 corresponds to hydroxyl and phosphate groups attached to the 3 and 5 positions of carbon atoms in the backbone two strands joined together by weak interactionsA TC G

Copying DNA amplification Polymerase chain reaction (PCR) 1980 Kary Mullis Awarded Nobel Prize Chemistry 1993 PCR separate the two strands and use each as a template replicate compliments

Copying DNA

Copying DNA raise temperature of the DNA fragment to 95C denature the two strands Incubation occurs next at 60C DNA polymerase such as Taq polymerase ample supply of nucleotides (dNTPs) two complementary primers short chains of nucleotides that match up using complementarity with a very small segment of the DNA fragment defines the starting point for the replication process

Copying DNA raise temperature of the DNA fragment to 95C denature the two strands

Copying DNA DNA polymerase enzyme (Taq) catalyzes construction begins from the position of the primer proceeds 5 3 direction Replication of a portion of the single strand is rapid ~ 50 bases per second cycle ends with two identical copies of only the sections between (and including) the primers in addition to the starting DNA template

Copying DNA each cycle increases the number of identical copies with a factor of 2n, n is the number of cycles after 20 cycles, about one million copies have been created efficiency drops after about 20 cycles 30 to 40 cycles are typically sufficient

Copying DNA

Copying DNASHOW PCR CLIP

Copying DNA Mini-PCR small chambers greater ratio of surface area to volume surface area affects the rate of heat conduction volume determines the amount of heat necessary for a thermal cycle enables faster thermal cycling in PCR less sample and volume of expensive reagents integrated system detection scheme electrophoretic separation tagging process is simplified, making it faster, less expensive, and more repeatable

Copying DNA PCR on a silicon chip ~ 1994 Lawrence Livermore National Laboratory (LLNL) thermally cycle a solution between the denaturing and incubation temperatures approximately 95C and 60C one chamber, with a volume of 25 to 100 l, is made of two silicon chips with etched grooves bonded together SiN window bare silicon inhibits PCR amplification disposable polypropylene liner added to chamber slows the heat/cool rate from an all-silicon version to about 8C/s

Copying DNA

Copying DNA polysilicon heater on a silicon nitride membrane for heating the fluid inside the chamber with external sensor platinum heater can do both heating and sensing operations temperature variations as high as 10C across the chamber temperature uniformity move heater away from the membrane so that heat flows through the Si walls fan can be added for more rapid cooling modifications yield tighter closed-loop temperature control enables faster cycling, from around 35s per cycle to as little as 17s per cycle Large-scale devices ~ 4 min per cycle!

Detection - dyes add TaqMan dyes (probes) link to certain sections of a DNA strand like the primers do results in fluorescence of green light from each replicated DNA strand when excited by blue or ultraviolet light intensity of the fluorescence is proportional to the number of replicated DNA matching strands typically no detectable fluorescence signal for the first 2025 cycles after cycling on the order of 515 minutes, the signal appeared and rapidly grows if there is a match simultaneous DNA amplification for multiple

DNA Sequencing amplify DNA strand and chemically label the amplified DNA fragments with specific fluorescent or radioactive tags detect labeled DNA products electrophoresis separates DNA (charged), in suspension under the effect of an electric field

DNA Sequencing In solution, a hydrogen ion dissociates from DNA backbone net negative charge on DNA strand charge-to-mass ratio is approximately the same for strands of different lengths when driven with an electric field through a molecular sieve, larger molecules move more slowly groups of small molecules move farther than larger ones over time

DNA Sequencing gel electrophoresis DNA products put in at edge of porous gelatinous sheet 20 to 100 cm long electric field is limited to only 540 V/cm heating problem capillary electrophoresis products are fed into thin capillary tube 10 to 300 m in diameter and ~ 50 cm long applied electric field of up to 1,200 V/cm higher fields can be used with smaller cross sections due to the ability to remove heat more rapidly tag DNA with tag to light up strands across gel radioactive or florescence

DNA Sequencing Sanger method of sequencing for fragments up to about 1,000 bases long many identical copies of single, denatured sections of DNA replication is started from the 5 end, just as in PCR a small concentration of bases in the solution of one type is altered so that the replication of that DNA strand stops when the replication-halting base is used results in copies of the original strands of varying length that always end in a particular base

DNA Sequencing Sanger method of sequencing (contd) same is done in separate solutions with small concentrations of replication-halting bases of the other types four groups of variable-length copies undergo electrophoresis in four parallel channels sequences of each length are separated for reading results from the four channels are compared to infer the entire sequence of the strand

DNA Sequencing

DNA Sequencing Miniaturization capillary electrophoresis length of the sample emitted can be kept short on the order of 100 m reduces distance for the fragments of different lengths to travel to separate reduces length of the channel decreases the applied voltage to maintain a high electric field from few kilovolts down to hundreds of volts faster separation with shorter distances overall volume of DNA and reagents decreases significantly to one microliter or less

DNA Sequencing capillary electrophoresis on a chip ~ 1992 University of California, Berkeley first in 1994 to demonstrate DNA sequencing by capillary electrophoresis on a glass chip two orthogonal channels etched with HF acid into a first glass substrate a short channel for injecting fluid and a long channel for separating the DNA fragments. A second glass substrate covers the channels

DNA Sequencing secure second glass substrate to first substrate adhesive or thermal bonding holes etched or drilled with a diamond-core drill in the top glass substrate fluid access ports both channels are typically 50 m wide and 8 m deep can be as wide as 100 m and as deep as 16 m separation channel is 3.5 cm long surfaces of the channels have a coating to eliminate charging prevent electroosmosis injection and separation channels are filled with sieving matrix of hydroxyethylcellulose by applying a vacuum to one end

DNA Sequencing

DNA Sequencing fluid containing DNA fragments put into the injection channel fragments are electrophoretically pumped by means of an electric field of 170 V/cm 3060s injection-channel loading time is critical too short, more short DNA fragments are injected in the next step too long, the sample is biased toward longer fragments applied voltage is switched to separation channel applied electric field directs fragments from the intersection of the two channels into the separation channel

DNA Sequencing electric field of 180 V/cm ~ 2 min to complete the separation of the DNA fragment compare to 8 to 10 hours to complete an equivalent separation using conventional gel electrophoresis compare to 1 to 2 hours with conventional capillary electrophoresis optical imaging of a fluorescent tag on each DNA fragment is used to detect separated products inside the channel up to 1,000 nucleotides long

DNA Sequencing Agilent DNA 1000 LabChip DNA concentration range of 0.550 ng/l size range of 251,000 base pairs size accuracy of 15% resolution better than 10% over most of the range sample volume is 1 l and takes 30 min to analyze

DNA Hybridization Arrays preassembled nucleotides attached to a substrate DNA sections to be identified are tagged with a fluorescent dye at one end lengths range of a hundred to thousands of bases buffer solution on the substrate sections of some of the unknowns hybridize to the complementary sequences on the substrate

DNA Hybridization Arrays substrate is then rinsed and illuminated locations of fluorescence indicate hybridization and thus which sequences are present detection of specific gene mutations search for known pathogens

DNA Hybridization Arrays Using a standard photolithographic mask UV light is shone through 20-m square openings remove the protection groups activating selected sites on the substrate

DNA Hybridization Arrays A solution containing one type of nucleotide with a removable protection group is flushed across the surface nucleotides bond to activated sites in each square that was exposed but not in the other areas

DNA Hybridization Arrays process is repeated to start chains of other three-nucleotides repeated exposure with different masks to remove the protection groups and flushing with the four nucleotide solutions grow DNA strands typically 25 nucleotides long

DNA Microelectrodes

DNA Microelectrodes

DNA Microelectrodes