In-Depth Electrochemical Investigation of Surface Attachment Chemistry via Carbodiimide Coupling.

Aminoferrocene is used as an electroactive indicator to investigate carbodiimide coupling reactions on a carboxylic acid-functionalized self-assembled monolayer. The commonly used attachment chemistry with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used for surface activation. A number of conditions are investigated, including EDC and NHS concentration, buffer solutions, incubation timing, and aminoferrocene concentration. Ferrocene is a well-documented electroactive species, and the number of surface-bound ferrocene species can be calculated using electrochemical methods. This capability allows determination of optimal conditions, as well as providing a method for comparing and investigating novel carboxylated surfaces. An EDC-mediated procedure with ∼5 mM EDC and NHS (1:1) made in water, with a full acid monolayer, with 250 µM aminoferrocene for 40 min was found to give the highest ferrocene attachment. An application of this is demonstrated for preparing a probe-DNA-coated surface for DNA sensing. By backfilling with aminoferrocene, a differential quantification of the amount of probe DNA available for sensing can be obtained. This provides an elegant method to monitor an important aspect, namely, probe surface characterization, which will be highly useful for biosensing purposes.

Detection of target-probe oligonucleotide hybridization using synthetic nanopore resistive pulse sensing.

Despite the plethora of DNA sensor platforms available, a portable, sensitive, selective and economic sensor able to rival current fluorescence-based techniques would find use in many applications. In this research, probe oligonucleotide-grafted particles are used to detect target DNA in solution through a resistive pulse nanopore detection technique. Using carbodiimide chemistry, functionalized probe DNA strands are attached to carboxylated dextran-based magnetic particles. Subsequent incubation with complementary target DNA yields a change in surface properties as the two DNA strands hybridize. Particle-by-particle analysis with resistive pulse sensing is performed to detect these changes. A variable pressure method allows identification of changes in the surface charge of particles. As proof-of-principle, we demonstrate that target hybridization is selectively detected at micromolar concentrations (nanomoles of target) using resistive pulse sensing, confirmed by fluorescence and phase analysis light scattering as complementary techniques. The advantages, feasibility and limitations of using resistive pulse sensing for sample analysis are discussed.

Development of an electrochemical polypyrrole-based DNA sensor and subsequent studies on the effects of probe and target length on performance.

DNA sensors have a wide scope of applications in the present and emerging medical and scientific fields, such as medical diagnostics and forensic investigations. However, much research-to-date on DNA sensor development has focused on short target DNA strands as model genes. In this communication we study the effect of the length of oligonucleotide probe and target strands as a significant step towards real world applications for DNA detection. The sensor technology described uses the conducting polymer polypyrrole as both a sensing element and transducer of sensing events - namely the hybridization of complementary target oligonucleotide to probe oligonucleotide. Detection is performed using electrical impedance spectroscopy. Initially sensor development is performed, wherein we demonstrate an improvement in stability and sensitivity as well as show a reduction in non-specific DNA binding for fabricated sensors, through use of a specific dopant and post-growth treatment. Subsequently, we show that longer target DNA strands display increased response, as do sensors containing longer probe DNA strands. It is suggested that these results are a feature of the increase in negative charges associated with the longer DNA strands. The results of this comparative study are aimed to guide future design of analogous sensors.