Search

In this work, a cloth-based chemiluminescence (CL) biosensor has been firstly presented for highly sensitive determination of long PCR amplicons. Under the action of a hybridization chain reaction, a good deal of hemin/G-quadruplex DNAzyme molecules are produced, which can effectively enhance the CL signal. Moreover, effective cloth-based DNA biosensors can be fabricated by sequential wax screen-printing and surface-modification processes. Especially, the integration of a desirable hydrophobic barrier and gravity/capillary flow onto the flow channel of the cloth-based device makes the biosensor easy to be fabricated and to be associated with a flow CL. For the luminol/H2O2-based CL system, the signals are triggered by the hemin/G-quadruplex DNAzyme and are recorded by a low-cost CCD. Under optimized conditions, the determination range of target DNA is 0.002–20,000 pM and its limit of detection is calculated to be 1.1 fM. The results show that the proposed CL biosensor has a good analytical performance, such as high detectability and specificity, wide linear range, and receivable reproducibility and stability. Finally, the proposed biosensor is proven by the fact that this method can successfully detect the target DNA prepared from the Listeria monocytogenes–spiked milk samples. Therefore, it is believed to have the potential application prospects in food safety and environmental monitoring. [ABSTRACT FROM AUTHOR]

Abstract: 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 1min. 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 1h, and the limit of detection of the RNA concentration was estimated to be 3.6×104 copiesμl−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. [Copyright &y& Elsevier]

Abstract: We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45°C along the gradient direction on the copper flake (45×40×4mm). When the temperature of the hot zone is 90–97°C and the temperature of the cold zone is 60–70°C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70°C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems. [Copyright &y& Elsevier]

Abstract: 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 0.005ng μl−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 0.01ng μl−1. Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way. [Copyright &y& Elsevier]

Traditional detection methods for food-borne pathogens are usually expensive and laborious, so there is an urgent need for an economical, facile and sensitive method. In this work, a novel cloth-based supersandwich electrochemical aptasensor (CSEA) is firstly developed for direct detection of pathogens. Carbon ink- and wax-based screen-printing is used to make cloth-based electrodes and hydrophilic/hydrophobic regions respectively to fabricate the sensing devices. Two well-designed, specific single-stranded DNA sequences arise a cascade hybridization reaction to form the DNA supersandwich (DSS) whose grooves can be inserted by methylene blue (MB), which effectively amplifies the current signal to greatly improve the detection sensitivity. Taking the detection of Salmonella typhimurium (S. typhimurium) as an example, the aptamers bind to S. typhimurium to form the target-aptamers complex, which can simultaneously bind to the capture probe and DSS, resulting in detection of S. typhimurium. Moreover, the addition of tail sequences of aptamer makes the proposed CSEA versatile. Under optimized conditions, the electrochemical signal increases linearly with the logarithm of S. typhimurium concentration over the range from 102 to 108 CFU mL−1, with a limit of detection of 16 CFU mL−1. Additionally, the CSEA efficiently determined the levels of S. typhimurium in milk samples. Experimental results illustrate that the fabricated CSEA is sensitive, specific, reproducible and stable. Moreover, when Ru(bpy) 3 2+ replaces MB, the electrochemiluminescence (ECL) can be performed. Thus, for the proposed sensing strategy, the dual-mode detection of electrochemistry and ECL is easily realized. [Display omitted] • The aptamer is firstly applied onto μCADs for bacterial detection. • The additional tail sequences of the aptamer make the CSEA be versatile. • The DNA supersandwich is used to amplify the current signal. • The sensing interface can be expanded to achieve the dual-modal detection. • The sample preparation is simplified, without DNA extraction and amplification. [ABSTRACT FROM AUTHOR]