Chunsun Zhang

Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics.

The ability to perform DNA amplification on a microfluidic device is very appealing. In this study, a compact continuous-flow polymerase chain reaction (PCR) microfluidics was developed for rapid analysis of genetically modified organisms (GMOs) in genetically modified soybeans. The device consists of three pieces of copper and a transparent polytetrafluoroethylene capillary tube embedded in the spiral channel fabricated on the copper. On this device, the P35S and Tnos sequences were successfully amplified within 9min, and the limit of detection of the DNA sample was estimated to be 0.005 ng microl(-1). Furthermore, a duplex continuous-flow PCR was also reported for the detection of the P35S and Tnos sequences in GMOs simultaneously. This method was coupled with the intercalating dye SYBR Green I and the melting curve analysis of the amplified products. Using this method, temperature differences were identified by the specific melting temperature values of two sequences, and the limit of detection of the DNA sample was assessed to be 0.01 ng microl(-1). Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way.

Integrated microfluidic reverse transcription-polymerase chain reaction for rapid detection of food- or waterborne pathogenic rotavirus.

The development of microfluidic tools for nucleic acid analysis has become a burgeoning area of research during the postgenome era. Here we have developed a microfluidic device that integrates reverse transcription (RT) and polymerase chain reaction (PCR) with online fluorescence detection to realize a rapid detection system for performing both genetic amplification and product analysis. The microfluidic device mainly comprises a grooved copper heating block for RT and a heated cylinder for amplification. To expedite the analysis process, we combined the continuous-flow PCR with an online fluorescence detection system that allows analysis of amplification products within 1 min. Rotaviruses are worldwide enteric pathogens in humans and animals responsible for a significant burden of disease through person-to-person transmission and exposure to contaminated foods and water. In this study, rotavirus from stool specimens was successfully amplified and detected using the RT-PCR microfluidic system within 1 h, and the limit of detection of the RNA concentration was estimated to be 3.6×10(4) copies μl(-1). Compared with a large-scale apparatus, the integrated microfluidic system presented here can perform rapid nucleic acid amplification and analysis, possibly making it a crucial platform for future diagnosis application.

Chemiluminescence detection for microfluidic cloth-based analytical devices (μCADs).

In this work, we report the first demonstration of chemiluminescence (CL) detection for microfluidic cloth-based analytical devices (μCADs). Wax screen-printing is used to make cloth channels or chambers, and enzyme-catalyzed CL reactions are imaged using an inexpensive charge coupled device (CCD). We first evaluate the relationship between the wicking rate and the length/width of cloth channel. For our device, the channel length and width between the loading and detection chambers are optimized to be 10mm and 3mm. Thus, the detection procedure can be accomplished in about 15s on a cloth-based device (15 × 30 mm(2)) by using 25-μL sample spotted on it. Next, several parameters affecting cloth-based CL intensity are studied, including exposure time, pH, and concentrations of luminol and enzyme. Under optimal conditions, a linear relationship is obtained between CL intensity and hydrogen peroxide (H2O2) concentrations in the range of 0.5-5mM with a detection limit of 0.46 mM. Finally, the utility of cloth-based CL is demonstrated for determination of H2O2 residues in meat samples. On our device, the chicken meat soaked for 6h with 3% H2O2 can be detected. Moreover, the supernatant of grinded meat sample can be directly applied, without need for other treatments. We believe that μCADs with CL detection could provide a new platform of rapid and low-cost assays for use in areas such as food detection and environmental monitoring.

Chemiluminescence cloth-based glucose test sensors (CCGTSs): A new class of chemiluminescence glucose sensors

Chemiluminescence (CL) has been widely applied in many fields, but it is rarely used in a very simple, economical but effective way. In this work, for the first time, the CL cloth-based glucose test sensors (CCGTSs) are developed as a new class of CL glucose sensors, with no need for complicated, expensive device fabrication and peripheral equipment. When integrated with desirable hydrophobic barrier in the flow channel and gravity/capillary flow induced by a difference in height between the loading zone and the detection zone, a single cloth-based device can perform the whole CL process involving two steps of enzyme reactions. The wax screen-printing approach is used to fabricate ultra-cheap CCGTSs, the glucose detection involves the enzymatic oxidation of glucose to gluconic acid and H2O2 followed by the horseradish peroxidase (HRP)-catalyzed oxidation of luminol by H2O2, and the emitted CL signals are heightened with p-iodophenol (PIP) and imaged using an inexpensive, portable CCD camera. Under optimized conditions, glucose can be determined over the range of 0.1-100mM, with a detection limit of 0.0948mM and an analysis time of less than 5.5min. Finally, the applicability and validity of the CCGTSs are demonstrated for the measurement of glucose in clinical urine and serum samples. Thus, the proposed sensors could provide great promise in applications in many areas, and may facilitate the achievement of point-of-care testing.

A sample-to-answer, real-time convective polymerase chain reaction system for point-of-care diagnostics

Timely and accurate molecular diagnostics at the point-of-care (POC) level is critical to global health. To this end, we propose a handheld convective-flow real-time polymerase chain reaction (PCR) system capable of direct sample-to-answer genetic analysis for the first time. Such a system mainly consists of a magnetic bead-assisted photothermolysis sample preparation, a closed-loop convective PCR reactor, and a wireless video camera-based real-time fluorescence detection. The sample preparation exploits the dual functionality of vancomycin-modified magnetic beads (VMBs) for bacteria enrichment and photothermal conversion, enabling cell pre-concentration and lysis to be finished in less than 3min. On the presented system, convective thermocycling is driven by a single-heater thermal gradient, and its amplification is monitored in real-time, with an analysis speed of less than 25min, a dynamic linear range from 106 to 101 copies/µL and a detection sensitivity of as little as 1 copies/µL. Additionally, the proposed PCR system is self-contained with a control electronics, pocket-size and battery-powered, providing a low-cost genetic analysis in a portable format. Therefore, we believe that this integrated system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics.

Bipolar electrochemiluminescence on thread: A new class of electroanalytical sensors

This paper introduces a new and simple concept for fabricating low-cost, easy-to-use capillary microchannel (CMC) assisted thread-based microfluidic analytical devices (CMCA-μTADs) for bipolar electrochemiluminescence (BP-ECL) application. The thread with patterns of carbon screen-printed electrodes and bare thread zones (BTZs) is embedded into a CMC. Such CMCA-μTADs can produce a strong and stable BP-ECL signal, and have an extremely low cost (

Facile and sensitive chemiluminescence detection of H2O2 and glucose by a gravity/capillary flow and cloth-based low-cost platform

0.01 per device). Interestingly, the CMCA-μTADs are ultraflexible, and can be bent with a 135° bending angle at the BTZ or with a 150° bending angle at the middle of bipolar electrode (BPE), with no loss of analytical performance. Additionally, the two commonly-used ECL systems of Ru(bpy)32+/TPA and luminol/H2O2 are applied to demonstrate the quantitative ability of the BP-ECL CMCA-μTADs. It has been shown that the proposed devices have successfully fulfilled the detection of TPA and H2O2, with detection limits of 0.00432mM and 0.00603mM, respectively. Based on the luminol/H2O2 ECL system, the CMCA-μTADs are further applied for the glucose measurement, with the detection limit of 0.0205mM. Finally, the applicability and validity of the CMCA-μTADs are demonstrated for the measurements of H2O2 in milk, and glucose in human urine and serum. The results indicate that the proposed devices have the potential to become an important new tool for a wide range of applications.

Micropumps, microvalves, and micromixers within PCR microfluidic chips : Advances and trends

This paper describes the development of a cloth-based CL strategy that can quantify H2O2 or glucose in a low-cost, easy-to-use, rapid and sensitive manner. Wax screen-printing is applied to fabricate the ultralow-cost cloth-based devices, and thus the as-prepared sensors have a very low cost (about

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

0.01 per assay). Moreover, an inexpensive CCD camera is used to image the light from the gravity/capillary flow-triggered enzyme-catalysed oxidation of luminol by H2O2. Under optimized conditions, the developed CL platform can fulfill the determination of H2O2, with a linear range of 0.01–10 mM and a detection limit of 9.07 μM. Similarly, glucose can be detected in the range of 0.01–10 mM, with a detection limit of 9.74 μM. Additionally, the RSDs of these two assays are 3.36%, 3.66% and 2.64% for 0.01, 0.1 and 5 mM H2O2, and 3.01%, 2.80% and 3.68% for 0.1, 1 and 5 mM glucose, respectively. Moreover, the devices can maintain ∼95% of their initial response for three weeks, and tolerate a series of interfering species at reasonable concentrations. Therefore, the proposed platform has an acceptable sensitivity, linear range, reproducibility and stability. Finally, this platform is successfully applied to the detection of H2O2 in milk and glucose in human serum.