This paper focuses on the design of an EIS (electrolyte on insulator on Silicon) structure as a detection method for pathogenic DNA. Current rapid detection methods rely on fluorescent labeling to determine binding affinity. Fluorescent quenching is seen by a change in activity as opposed to non-quenched states. Sensitive optical equipment is required to detect and distinguish these colour changes because they cannot be seen by the naked eye. The disadvantages of this is (1) a portable, independent device cannot be made since samples have to be brought back to the benchtop and (2) the obvious cost of acquiring and maintaining these optical detection systems. A low cost, portable electrical detection method has been investigated. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a small field effect DNA detection sensor. The sensor responds to fluctuating capacitances caused by a depletion layer thickness change at the surface of the silicon substrate as a result of DNA adsorption onto the dielectric oxide/APTES (Aminopropylthioxysilane) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes. The negative charge exhibited by the DNA forces negative charge carriers in the silicon substrate to move away from the surface. This causes a depletion layer in n-type substrate to thicken and for a p-type to thin and can be observed as a change in capacitance. A low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The EIS sensor is designed to be later integrated into a complete lab on chip solution. A full lab on chip can incorporate the sensor to perform DNA quantity based measurements. Nucleic acids can be amplified by the on chip PCR system and then fed into the sensor to work out the DNA concentration. The sensor surface contains capture probes that will bind to the pathogen. They are held onto the sensor surface by the positively charged layer. The sensor will have onboard electronics to process the signals and determine the result of the measurements. The sensitivity of the sensor is on par with similar capacitance sensing technologies and is expected to be improved with later enhancements.
This research focuses on the use of EIS (electrolyte on insulator on Silicon) structure as a detection platform for DNA binding.
The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitances caused by a depletion layer thickness change at the surface of the silicon substrate and also through DNA adsorption onto the dielectric oxide/amino surface.
In this paper we present the fundamentals of the Capacitance-Voltage plot technique and how it can be used a method for detecting DNA binding and surface charge transits. The CV plot is a widely used technique in the microelectronics industry for characterizing and profiling capacitor devices. It is mainly used to test the quality of these devices and give an indication of failing processing conditions. Its high sensitivity and ability to provide a wealth of information makes it a suitable choice for our research [11].
We also looked at using 2 types of amino layers and compared their effectiveness as DNA adhesives based on surface charge. The two types we chose to investigate were Poly-L-Lysine and 3-Aminopropylthioxysilane. Their compounds are quite similar in nature in that they contain a NH2 terminated group which is easily protonated in physiological buffers. PolyLysine and APTES are both commonly used in labs to coat slides for adhering cells and also used as monolayer linkers for tethering further compounds. PolyLysine tends to be more expensive than APTES, but safer to use since it isn't as corrosive as APTES. Our results show that APTES was a suitable choice for our experiments.
The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make
detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon
can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel,
label-free and simple to fabricate way to make a field effect DNA detection sensor.
The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon
substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse
to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative
charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This
causes an n-type depletion layer substrate to thicken and a p-type to thin.
The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted
using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain
Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not
affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of
pathogens we’ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface
by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode.
Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with
“floating” biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging
effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors that share a
common pseudo-reference electrode is used to cancel out this effect.
This paper will look at a differential system for multi-arrayed biosensors fabricated on silicon.
Dye-labeled dideoxy terminators are the preferred reporter groups for DNA sequencing. This labeling technique can result in uneven peak heights in the electropherograms due to sequence context effects on the biochemical distribution of extension vs termination events. Large variations in peak heights can result in difficulty in identifying very small peaks adjacent to big peaks. Using substrate structure to minimize the sequence context effects on dye-terminator incorporation, we have studied the effects of combinations of base, dye, and dye linkage on terminator incorporation during sequencing. We have identified a propargyl ethoxyamino linker that proved useful in improving peak height evenness and enhancing the terminator activity. New dye-linker-base combinations were optimized for overall peak evenness and relative electrophoretic mobility shifts. The more even peak patterns improve base calling and heterozygote analysis using dye terminator chemistry.
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