Changqing Zhan

Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: Optimization and data analysis

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.

Real-Time Biosensor Platform: Fully Integrated Device for Impedimetric Assays

An impedimetric biosensor platform for bioaffinity assays has been developed that is based on real-time, label-free electrochemical detection performed via a direct interface to electronic digital data processing. The sensor array consists of 15 gold microelectrode pairs (Fig. 1) that are enclosed in three reaction chambers and biofunctionalized with specific probes. The impedance change caused by specific capture of target analyte molecules on the functionalized electrode surface is recorded in real time. The measuring instrument is capable of continuous and simultaneous stimulation and recording of all electrodes on the array. A corresponding mathematical algorithm and a software package for data analysis have been developed. The software performs (i) filtering of the instrument noise, and (ii) extraction of the exponential component of the impedance signal. Thus, the algorithm can quantify both rate of target to probe binding, and target to probe affinity. The described fully integrated platform can be used as a basic research tool for development of various bio-affinity impedimetric assays. To facilitate such applications, we have developed a streamlined manufacturing technology, and a set of assay protocols for detection of microbes based on nucleic acid hybridization. The assay was shown to detect and distinguish between two closely related but different Escherichia coli strains. The assay sensitivity was sufficient for reliable measurements of specific PCR products amplified from microbial genomic DNA. The sensor array platform is adaptable for detection of a wide range of analytes of practical significance, and it has potential for further integration with amplification (i.e. PCR) and sample preparation modules.

Orientation- and position-controlled alignment of asymmetric silicon microrod on a substrate with asymmetric electrodes

In this paper, we demonstrate the orientation-controlled alignment of asymmetric Si microrods on a glass substrate with an asymmetric pair of electrodes. The Si microrods have the shape of a paddle with a blade and a shaft part, and the pair of electrodes consists of a narrow electrode and a wide electrode. By applying AC bias to the electrodes, the Si microrods suspended in a fluid align in such a way to settle across the electrode pair, and over 80% of the aligned Si microrods have an orientation with the blade and the shaft of the paddle on the wide and the narrow electrodes, respectively. When Si microrods have a shell of dielectric film and its thickness on the top face is thicker than that on the bottom face, 97.8% of the Si microrods are aligned with the top face facing upwards. This technique is useful for orientation-controlled alignment of nano- and microsized devices that have polarity or a distinction between the top and bottom faces.

Asymmetric AC electrophoresis with insulated electrodes: Toward positional control of microand nanoscale devices

This paper demonstrates electrophoresis of silicon micro-rods by applying asymmetric AC bias to two electrodes capped with a thin dielectric film. The silicon micro-rods migrate bi-directionally when asymmetric AC bias is applied to the electrodes. The insulated electrodes significantly contribute to elimination of bubbling and contamination originating from electrochemical reactions, which makes adoption of the technique to mass production processes realistic. This technique is widely applicable to positional control of small objects including micro- and nanoscale devices.

Orientation-controlled dielectrophoretic alignment of silicon microrod on a substrate with high positional accuracy

We demonstrate the orientation-controlled dielectrophoretic alignment of asymmetric Si microrods on a glass substrate with an asymmetric pair of electrodes. By applying AC bias to the electrodes, over 80% of the Si microrods align on the electrode pair so that a particular end of the microrod relates to a certain part of the electrode; the thick and thin ends overlap the thick and thin electrodes, respectively. Furthermore, the orientation of the top and bottom face of the Si microrod is also controllable when the thicknesses of the dielectric film on the top and bottom faces are different.

Bidirectional migration of Au colloids and silicon microrods in liquid using asymmetrical alternating current electric field with insulated electrodes

In this study, we demonstrate the migration of Au colloids and silicon microrods in deionized (DI) water and isopropyl alcohol (IPA) by applying asymmetrical AC bias to two electrodes capped with a thin dielectric film. Both Au colloids and silicon microrods successfully migrate from one electrode to the other when asymmetrical AC bias is applied to the electrodes. Furthermore, the direction of the migration can be easily reversed by inverting the wave form. The insulated electrodes have the potential to prevent contamination and bubbling originating from electrochemical reactions, which makes the adoption of the technique for mass production processes easy and realistic. The bidirectional migration acts similarly to electrophoresis and is effective even in DI water and IPA in which conventional DC electrophoresis with insulated electrodes is ineffective. This technique is widely applicable to the positional control of small objects including nano- and micro-sized devices.