Dayu Liu

Determination of SARS-coronavirus by a microfluidic chip system.

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS‐CoV). The system includes a laser‐induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription‐polymerase chain reaction (RT‐PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS‐CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT‐PCR on SARS‐CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home‐made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS‐CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT‐PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.

Analysis of multiplex PCR fragments with PMMA microchip.

Abstract Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5μL, and the limit-of-detection (LOD) of DNA template was 0.7ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.

On-chip coupling of free-solution transient ITP and CGE for highly efficient separation of dsDNA with variable sample loading amounts

We developed an on‐chip DNA analysis method, in which free‐solution transient isotachophoresis (FstITP) were coupled with CGE. Using chloride ions in the sample matrix and HEPES in the background electrolyte, respectively, as the leading and terminating ions, dsDNAs were isotachophoretically preconcentrated in free‐solution and then separated in sieving polymer. The coupling of FstITP and CGE enabled higher separation efficiency due to higher preconcentration rate in free‐solution. The FstITP‐CGE analysis offered adjustable signal intensities by varying sample injection time. With the maximum sample loading volume, the LOD of the FstITP‐CGE analysis was determined to be 0.24 ng/mL by confocal laser‐induced fluorescence detection. The FstITP‐CGE method is simple, robust and flexible, thus well suited to the analysis of highly saline DNA samples at different concentration level.

A simple and sensitive transient ITP method for on‐chip analysis of PCR samples

We present a sensitive, simple and robust on‐chip transient ITP (TITP) method for the analysis of polymerase chain reaction (PCR) samples. This TITP analysis was performed on a poly (methyl methacrylate) microchip with cross structure. The PCR sample was injected into a 4 mm free‐solution buffer channel to increase sample loading. When applied separation voltage, the sample ions were stacked by TITP using chloride ions in the sample matrix as the leading ions and HEPES in the background electrolyte as the terminating ions. The stacked sample plug was then separated by zone electrophoresis (ZE) in hydroxypropylmethyl cellulose polymer. The whole operation was carried out in single background electrolyte. Sample injection, preconcentration / separation were performed continuously with the sequential switching of voltage. By injection of the sample into the free‐solution buffer channel, sampling bias was avoided and the injection time was reduced. With this TITP method, the detection sensitivity was improved without loss of resolution, a 20‐fold signal enhancement was achieved and the average limit of detection was estimated to be 0.24 pg/μL (S/N = 3). The TITP method is simple, fast and sensitive, thus is well suited to direct analysis of the highly saline PCR samples.