Kum Cheong Tang

An electrical biosensor for the detection of circulating tumor cells

In this report, we demonstrate a semi-integrated electrical biosensor for the detection of rare circulating tumor cells (CTCs) in blood. The sample was first enriched through a combination of immunomagnetic isolation and size filtration. The integration of both methods provided a high enrichment performance with a recovery rate above 70%, even for very low numbers of cancer cells present in the original sample (10 spiked MCF7 cells in 0.5 mL of blood). In the same system, the sample was then transferred to a microchip for further magnetic concentration, followed by immunochemical trapping and electronic detection by impedance spectroscopy. Three levels of spiked CTC number (30±2, 124±29, 273±23) in 10 μL of filtered blood sample were distinguished by monitoring the impedance change of the microelectrode array (MEA). The integration of different functions in a single system provided a methodology to process milliliter-sized blood samples at the macroscale and interface with the microdimensions of a highly sensitive electronic detector. The results showed that the whole system was able to detect different levels of spiked cancer cells without the use of time- and cost-intensive fluorescence labeling and image analysis. This has the potential to provide clinicians with a standalone system to monitor changes in CTC numbers throughout therapy conveniently and frequently for efficient cancer treatments.

Lateral patch-clamping in a standard 1536-well microplate format

Lateral patch-clamping has emerged as a chip-based platform for automation of the conventional patch-clamp technique, the gold standard for studying cellular ion channels. The conventional technique, as it relies on skilled-maneuver of glass micropipettes to patch cells, is extremely delicate, low in throughput, and thus cannot be used for primary screening of compounds against ion channels. Direct integration of glass capillaries on silicon provides lateral junctions for automated trapping and patching of cells. We demonstrate here a method of scaling up the lateral junctions to a standard 1536-well microtiter plate format. A single unit of 1536-well plate has been formed here on a 9 mm by 9 mm microstructured silicon with the inclusive of 16 wells molded in a capping layer made of polydimethylsiloxane (PDMS). The silicon substrate provides integrated glass capillaries (total 12) and their associated microfluidic network. Each glass capillary has an independent access through a dedicated well in PDMS and leads to a centralized channel in which cell suspension can be delivered through one of the remaining 4 wells. The unit has been tested on RBL-1 cells by recording whole-cell activity from inwardly rectifying endogenous potassium channels. A revised test protocol has been prescribed to avoid inaccurate readings due to altered ionic composition of the recording buffer when a typical suction is applied to capture cells.

Label-free impedance detection of low levels of circulating endothelial progenitor cells for point-of-care diagnosis.

This paper presents a novel microfluidic system for rapid label-free detection of endothelial progenitor cells (EPCs) from small volumes of white blood cells samples, to obtain a bedside cardiovascular diagnostic solution. The system was built on a single 1 cm(2) microelectrode array silicon chip, integrated with negative dielectrophoresis for cell trapping, surface immunochemistry for selective cell capture, and fluidics for cell washing and impedance detection. The level of circulating EPC level in blood is a biomarker of clinical interest, linked to the assessment of risk factors in cardiovascular diseases which are a major global concern. Rare EPCs are usually detected through in vitro culture or flow cytometry, which are too time-consuming to bring timely reports in acute diseases. Although microfluidics approaches have enabled reduced processing time and enhanced portability, their sensitivity and processing volumes are still inadequate for rare cell detection at a bedside setting. Using small highly sensitive microelectrodes, our novel integrated system achieved the detection of 720 EPCs in a small 12 microl sample of 72,000 peripheral blood mononuclear cells (PBMC), i.e. equivalent to a concentration of EPCs of 0.1% of 100 microl blood. This demonstrated that clinically significant level of EPCs (<0.5% of PBMC) could be detected for the first time on a detection system at bedside set-up, showing great potential in applications for point-of-care diagnosis.

Aptamer-based array electrodes for quantitative interferon-γ detection

Present work describes the methylene blue tagged thiolated aptamer-modified gold micro-array based biosensor for specific detection of IFN-γ. The microchips with the microelectrode array were fabricated using standard silicon microfabrication technologies, and modified with methylene blue tagged aptamer using standard gold thiol chemistry. Electrodes were characterized and tested using Cyclic Voltammetric (CV) and Square Wave Voltammetry (SQW) measurements in a standard three-electrode format at room temperature. On an aptamer modified electrode, aptamer density was estimated to be about 4.4 × 10(12)molecules/cm(2). In IFN-γ studies, oxidation peak currents were found to decrease and more than 50% signal suppression was achieved at 500 ng/ml. Further, the magnitude of signal suppression was found to be logarithmically proportional to the IFN-γ in the concentration range of 1-500 ng/ml, with a detection limit of 1.3 ng/ml (i.e. 0.8 fmol in used sample volume of 10 µl). Biosensor showed negligible signal changes (5%) in a very high non-specific protein background, while still able to differentiate target protein IFN-γ at 5 ng/ml. The results indicated that our sensor binds selectively to target molecules, and the non-specific binding where adsorption of BSA protein molecules may be effectively omitted from consideration.

Microelectrothermofluidic packaging for single chip integrated viral RNA extraction and RT-PCR microdevices

This paper presents a microelectrothermofluidic packaging of integrated microdevice for viral ribonucleic acid (RNA) extraction from blood samples and its amplification through a reverse transcription (RT)-polymerase chain reaction (PCR) method for application to the point-of-care (POC) infectious disease diagnostics. Integrated microdevice consists of solid phase extraction microchannel structure, microfluidic mixer, and meander-shaped microchamber for RT-PCR. Metallic microheater and resistive temperature detection (RTD) microsenosr is monolithically integrated with fluidic components. In order to secure both electrical and fluidic interconnections, spring-loaded electrical pin and drilled microchannel structure has been design in polycarbonate micropackaging structure. In the biotesting, we have demonstrated to detect dengue serotype III virus from the 80µl of blood sample with 80pfu virus concentration.

Design of a fully-enclosed disposable bio-micro fluidic cartridge with self-contained reagents for infectious diseases diagnostic applications

To meet the requirements of infectious diseases identification, a sealed and fully enclosed cartridge with self-contained reagents was developed. The inlet and outlet ports of the cartridge are self-sealing. The waste produced during the diagnostic process was collected in a waste bag, which is enclosed in the cartridge. Two different types of waste bags were designed to reduce the overall thickness of the cartridge. A silicon chip with filter, binder and mixer components was integrated into the microfluidic cartridge for the extraction of the nucleic acid sample from the infectious virus sample. By using a switch valve the nucleic acid sample and waste were split and collected in a PCR tube and a waste bag respectively. The PCR tube, modified with self-sealing elastomer cap, was used for collecting the nucleic acid. The whole extraction process was carried out automatically in a small table-top actuator system. With the dengue virus as the input sample, RNA (ribonucleic acid) is extracted and subjected to RT-PCR (reverse transcription polymerase chain reaction) which detected the diagnostic size of the target amplicon.