Biomedical subjects

I-Ming Hsing

Publications and source records attributed to I-Ming Hsing.

10 recordsLinked to original sources

PubMed2006

Electrochemical real-time polymerase chain reaction.

In this work, we report the first electrochemistry-based real-time polymerase chain reaction technique for sequence-specific nucleic acid detection. This new technique builds upon the advantages of the well-established fluorescence-based counterpart, such as short assay time (simultaneous target DNA amplification and detection). In addition, this electrochemical approach could employ simple and miniaturizable instrumentation compared to the bulky and expensive optics required in the fluorescence-based schemes. We have demonstrated a proof-of-concept experiment showing that the utilization of solid-phase extension of the electrode surface-immobilized capture probe with Fc-dUTP during PCR resulted in the accumulation of the redox marker on the transducer surface. This new technique can be applied to a microfabricated PCR electrochemical device for point-of-care diagnostics as well as on-site environmental monitoring and biowarfare agent detection.

DNA↗

PubMed2006

A DNA biochip for on-the-spot multiplexed pathogen identification.

Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole-probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

Bacillus subtilis↗

PubMed2006

Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications.

A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.

Biosensing Techniques↗

PubMed2005

A microsystem compatible strategy for viable Escherichia coli detection.

This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.

Biosensing Techniques↗

PubMed2005

Flow-based and sieving matrix-free DNA differentiation by a miniaturized field flow fractionation device.

DNA separation is typically done by gel electrophoresis based on its charge property. In our previous work, we reported that dielectrophoresis could be used to manipulate polystyrene nanoparticles' motion by using a miniaturized electrical field flow fractionation device (micro-EFFF) with a segmented electrode operated under a pulsed voltage (PV). In this work, we report the manipulation and separation of DNA molecules using the micro-EFFF. DNA motion was in situ visualized inside the device. Results revealed that dielectrophoresis governed DNA motion, which was strongly correlated with the pulse frequency but not the duty cycle of a PV. A longer retention time of DNA molecules was measured under a PV. The retention time increased with the length of DNA molecules. As the micro-EFFF is flow-based and sieving-matrix-free, it has a potential to be applied to sample preparation in a micrototal analysis system or when fractionated molecules are needed for downstream analysis.

DNA, Bacterial↗

PubMed2005

Effects of gold nanoparticle and electrode surface properties on electrocatalytic silver deposition for electrochemical DNA hybridization detection.

In this paper we report the catalytic effects of various gold nanoparticles for silver electrodeposition on indium tin oxide (ITO)-based electrodes, and successfully apply this methodology for signal amplification of the hybridization assay. The most widely used gold nanoparticle-based hybridization indicators all promote silver electrodeposition on the bare ITO electrodes, with decreasing catalytic capability in order of 10 nm gold, DNA probe-10 nm gold conjugate, streptavidin-5 nm gold, and streptavidin-10 nm gold. Of greater importance, these electrocatalytic characteristics are affected by any surface modifications of the electrode surfaces. This is illustrated by coating the ITO with an electroconducting polymer, poly(2-aminobenzoic acid)(PABA), as well as avidin molecules, which are promising immobilization platforms for DNA biosensors. The catalytic silver electrodeposition of the gold nanoparticles on the PABA-coated ITO surfaces resembles that on the bare surfaces. With avidin covalently bound to the PABA, it is interesting to note that the changes in electrocatalytic performance vary for different types of gold nanoparticles. For the streptavidin-5 nm gold, the silver electrodeposition profile is unaffected by the presence of the avidin layer, whereas for both the 10 nm Au and DNA probe-10 nm gold conjugate, the deposition profiles are suppressed. The streptavidin-5 nm gold is employed as the hybridization indicator, with avidin-modified (via PABA) ITO electrode as the immobilization platform, to enable signal amplification by the silver electrodeposition process. Under the conditions, this detection strategy offers a signal-to-noise ratio of 20. We believe that this protocol has great potential for simple, reproducible, highly selective and sensitive DNA detection on fully integrated microdevices in clinical diagnostics and environmental monitoring applications.

DNA↗

PubMed2004

Mechanistic investigation of nanoparticle motion in pulsed voltage miniaturized electrical field flow fractionation device by in situ fluorescence imaging.

In our previous study, we reported a miniaturized electrical field flow fractionation device (micro-EFFF) that used a pulsed voltage (PV) to increase the effective electric field and, hence, improved the separation performance. In this work, we developed two micro-EFFFs with planar or segmented electrode design and investigated the particle movement in the flow channels under a PV. Numerical simulation was used to understand the electric field distribution in the micro-EFFFs. When the calculations for the micro-EFFF with a segmented electrode (segmented micro-EFFF) and the micro-EFFF with planar electrodes (planar micro-EFFF) are compared, a stronger electric field at the top electrode segments is found in the segmented micro-EFFF, with the strongest field at the edges of the electrode segments. Nanoparticle motion in both devices was in situ visualized by using a fluorescence microscope equipped with a CCD-camera. Results reveal that electrophoresis governs the nanoparticle movement in the planar micro-EFFF and dielectrophoresis dominates the movement in the segmented micro-EFFF. Two models are postulated to explain the experimental observations of the nanoparticle movement. The mechanistic understanding of controlling nanoparticle motion in a miniaturized environment will help the design and application of micro-EFFF for the separation of charged biomolecules (proteins and DNAs).

Journal Article↗

PubMed2003

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection.

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

Electrochemistry↗

PubMed2002

Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.

In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.

DNA Probes↗

PubMed2002

Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray.

A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the micro-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the micro-DAAD with different samples of 3-microL volume. Temperature sensors and heating elements of the micro-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.

Genotype↗