Detecting DNA the Electrochemical Way

In recent years, intensive efforts have been focused on developing ultrasensitive deoxyribonucleic acid (DNA) biosensors/ arrays that are capable of quantitative gene expression analysis.

These biosensors have potential applications ranging from genotyping to molecular diagnostics. Abnormalities in the expression of specific genes, for example, have been linked to a large and increasing number of diseases.

Quantifi cation of gene expression is a promising basis for early diagnosis. Among the various gene expression profiling tools developed, polymerase chain reaction (PCR)- based fluorescent microarrays are the most widely studied systems, as they offer the highest degree of sensitivity and multiplexing capability, and the widest dynamic range.

Arrays containing numerous unique probe sequences have been constructed to enable simultaneous assessment of tens of thousands genes down to a few copies. They have been used to identify gene expression patterns associated with specific biological functions on a global scale. However, there are several technical limitations that prevent researchers from using PCR-based microarrays as a clinical tool as it is difficult to reliably profile low abundance genes. Consequently, much effort has been focused on realizing accurate, sensitive, selective, robust and portable biosensing devices for laboratory and point-ofcare applications.

Electrical Techniques

In principle, the use of electrical techniques, rather than fl uorescence allows for simple, rapid, and portable DNA detection platforms. Recent advances in nanotechnology have opened new opportunities for electrical detection system for biomolecules.

These nano-sensors that use electrical readouts exhibit the potential for high sensitivity and selectivity in DNA detection down to the sub-picomolar region.

The following section will describe enzyme- catalyzed polymerization of conductive polymer, polyaniline, and template-guided formation of the resulting conductive polymer nano-wires for the electrical detection of nucleic acids.

Polymerization is performed on a nano-gap structure with interdigitated gold electrodes with a gap of 0.5 μm fabricated on silicon dioxide/silicone (SiO2/Si) substrate.

Electrical signals from the probes are sent to a complementary metal-oxide semiconductor (CMOS) interface circuit, where multi-phase measurement is carried out to measure the variation of the electrical parameters.

The system is illustrated in Figure 1. Here, it is demonstrated that the resistance and capacitance changes are detected as a result of nucleic acids concentration.

According to Science in February this year, the electrical readout method has attracted much attention lately in nucleic acid detection. The electrical readout system has the potential to achieve miniaturization, higher sensitivity and selectivity. It is cost effective as it avoids the expensive, complex setup of an optical readout method.

Micro fabrication technology in electrical readout method leads to micro scale electrical detection-based biosensors. According to the Nano Letter in June 2005, detection of conductivity changes using such micro scale biosensors is demonstrated by Mirkin and co-workers, where detection of nucleic acids was performed with a pair of microelectrodes separated by a micro-gap using gold (Au) nano-particle-conjugated oligonucleotide detection probes.

Besides a Au nano-particle, nanostructured conducting materials have also aroused intense attention in bridging the gap between probes. While pursuing controllable synthetic procedures under mild conditions, the authors of the article exploited nucleic acids as templates for fabricating nanoscale conducting materials due to its physicochemical stability, linear molecular structure and mechanical rigidity.

The Experiment

According to research published in the Journal of Physical Chemistry in 1999 and Nature in 1998, nucleic acids can indeed serve as the templates for compounds cadmium sulphide (CdS) nano-particles, nano-particle arrays, and metal nano-wires synthesis.

By using horseradish peroxidase (HRP) as a catalyst, polyaniline is synthesized along a double-stranded DNA molecule under mild conditions.

In the experiment described in Cooperation Treaty in July 2007 and the Journal of the American Chemical Society in June 2007, a biosensor with interdigitated gold electrodes with gap of 0.5 μm fabricated on SiO2/Si substrate as platform is used for performing DNA hybridization. The electrical signals are then registered by a CMOS interface circuit, where multi-phases measurement is adopted to measure its electrical parameters (as described in Electronics Letters in March 2008).

Initially, single-stranded peptide nucleic acid (PNA) capture probes were immobilized in the nano-gap through silane coupling agent. Following hybridization with target nucleic acids, a cocktail of aniline, H2O2 and HRP in pH-4.0 0.10-M acetate buffer was applied to the biosensor.

The HRP catalyses the polymerization of aniline that was adsorbed on the hybridized anionic nucleic acid. The resulting conductive polymer polyaniline was deposited as nanowires along the hybridized target nucleic acid, bridging the nano-gap.

After a brief doping treatment, the resistance and capacitance between the electrodes is measured, which directly correlates to the amount of the target nucleic acid in solution as illustrated in Figure 3.

Observations

Interestingly, by using the conductive polymer method, there is a formation of sensor capacitance (in the range of picofarads to nanofarads) in parallel with the sensor resistance (in the range of gigaohms) across the nano-gap sensor, as illustrated in Figure 1, with detailed illustrations shown in Figure 2.

Different nucleic acid concentration therefore causes change in resistance and capacitance. A resistor-capacitor (RC)- to-time constant interface circuit was thus designed and implemented in 0.18-μm CMOS to characterize the conductive polymerlabeled DNA hybridization activity on the microarray sensor.

The nano-biosensor tends to have large variation in electrical parameters with different DNA concentration. Large resistance and capacitance associates with the DNA hybridization activity prevent the use of conventional readout circuit.

A time-constant based readout circuit, which is configured in two measurement phases, is employed to achieve both requirements. In the first phase of measurement, the biosensor is configured in shunt configuration to extract the capacitance, as the second phase of the measurement extracts the resistance with a series configuration.

Due to the DNA hybridization activity, this readout circuit enables the measurement of a wide range of capacitance and resistance. The circuit uses four switches and a comparator, which consumes less than 40 μA at 1.8-V single supply. This ultra lowpower characteristic is important as an array measurement requires array of readout circuit to average out the measurement error and device variation.

It was found that the average reading between the nano-gap electrodes after peptide-nucleic acids (PNA) immobilization was 0.01 nF and 22.91 G . After performing a hybridization step with the target DNA at concentration of 10-11 M, 10-12 M and 10-13 M, polyaniline is allowed to deposit followed by a doping step with HCl vapor. The average readings were measured again and an increase of capacitance to 2.8 nF, 0.82 nF and 0.36 nF were observed with a decrease of resistance to 1.02 G , 3.35 G , and 7.66 G , respectively. The average reading on the control sample was found to be at 0.12 nF and 22.91 G

Conclusion

In this article, an electrical biosensor with its readout circuit is presented, as the ultralow power and large dynamic range RC-totime constant interface circuit is used to measure the conductive-polymer-based sensor capacitance and resistance. Since the interface circuit requires only simple circuitries, it is suitable for sensor array implementation.

From the experimental results, this circuit is able to differentiate various DNA concentrations down to sub-picomolar. This highlights the electrochemical way of DNA detection as a potential low cost but high sensitivity detection scheme, opening the door to routine gene expression profiling and molecular diagnostics.