Automated Detection of Single Nucleotide Polymorphism
Imec and Panasonic are jointly developing a fully integrated sample-to-answer device to perform molecular diagnostic tests. Recently, they’ve created prototype of the device. The chip is about half the size of a credit card that performs fast, simple and sensitive detection of genetic markers, specifically single nucleotide polymorphisms (SNPs).
SNPs are variations in a single DNA base among individuals. Detecting SNPs makes it possible to check for hereditary diseases or predispositions as well as stratify individuals according to their likely pharmacogenomic responses to treatments. Drugs whose efficacy depends on the presence/absence of SNPs in specified genes are, for example, warfarin (an anticoagulant), tamoxifen and cetuximab (breast and colon-rectal cancer treatment), albuterol (an anti-asthmatic). SNPs genotyping allows the choice of the most adequate therapy and an accurate dosage determination.
Currently SNP genotyping has to be done in a specialized lab, so it actually takes three to four days. The prototype fully automates all stages of SNPs genotyping, from sample preparation through to detection. The aim is to reduce time of the analysis, determining a patient’s SNPs in a time frame of about an hour after receiving the blood.
The disposable cartridge (Figure 1) is made by heterogeneous integration of a Si chip, a polymer layer and a printed circuit board. This unique combination of materials and technologies harness on one side the power of semiconductor manufacturing processes for fabricating fluidic structures with precise feature control and on the other side the characteristics of polymers for providing fluid motion. The final result will be a high performance, low cost, mass manufacturable device. The silicon chip houses microfluidic conduits, filters, mixers and thermal reactors. The polymer layer houses reservoirs, pumps, valves and detectors. The printed circuit board routes the contacts of electrical components in the cartridge to those of a benchtop instrument which provides bias and signal processing.
The Si fabrication platform is based on deep reactive ion etching and anodic bonding. Fluidic structures are first sculpted in Si and then sealed by a Pyrex wafer. A backside etch allows both access to fluidics and implementation of thermal insulation trenches around microreactors (see details in Figure 1). The platform is based on two Si-etch steps, one for ‘coarse structures’, down to 3-5 µm critical dimensions and one for ‘fine structure’, down to 0.5 µm. This wide range of critical features facilitates microfluidic devices design and makes the custom microfluidics platform highly flexible and ready for use in a large variety of applications. Figure 2 shows some of the fabricated components.
Pumps and valves are based on the stack of several layers of conductive polymer actuators, actuation voltage is low (1-2 V) thus enabling mobile, battery powered applications. The pumps generate a high pressure (10-50 MPa); a sizeable flow can hence be sustained even in structures with large fluidic impedance.
The actual diagnostic test is initiated by injecting an individual’s blood sample into the chip, and subsequently placing the chip into the benchtop instrument. Once inside the chip, the blood is mixed with pre-stored reagents and passed into a microreactor. Here, the cells are lysed by applying a temperature of 95°C for two minutes, their genetic content is released and the SNP containing regions in the DNA are cut out and amplified through a targeted PCR reaction. We have achieved high-speed PCR, where 30 temperature cycles are completed in only nine minutes. This result is due to the careful design of the heating and cooling system (based on a commercial, microfabricated thermoelectric cooler, Micropelt) and to the small microreactor thermal mass obtained thanks to the trenches which separate it from the rest of the chip. The amplified DNA is then purified by retaining cell debris in an integrated sieve. The part of the chip so far described is a simple and effective ‘sample preparation module’ of very generic use.
Purified DNA is sent to the detection section of the chip where it is mixed with specific reagents and a second PCR reaction is performed. Primers are designed in such a way that amplification occurs only if the SNP is present. The pyrophosphoric acid produced during this second PCR is later detected electrochemically. The two-PCR process needs a combined optimization to avoid the primers and phyrophosforic acid related to the first PCR influence, the second PCR reaction, and the detector reading.
The electrochemical detector is a small cavity (0,5, 1 µL volume), containing three electrodes implemented in the polymer part of the chip. Reagents are dry-stored in the detector cavity. Figure 3a shows the difference in the electrochemical signal for two common SNPs.
The same scheme described above is used for multiple SNP detection. During the first PCR, multiple segments of DNA are amplified together. Each segment, referring to a different SNP, has a different length. Segments are separated according to length by using a micropillar filter with pillar spacing and diameter in the µm range and then detected. The filter acts as the separation column in a liquid chromatography experiment. Figure 3b shows the separated peaks corresponding to different SNPs.
Panasonic is currently working towards the commercialization of a single SNP detector exploiting excellent performances of the prototype.