Yuepu Pu

A disposable amperometric immunosensor for chlorpyrifos-methyl based on immunogen/platinum doped silica sol-gel film modified screen-printed carbon electrode.

A disposable amperometric immunosensor for sensitive detection of chlorpyrifos-methyl (CM) has been developed by combining dual signal amplification of platinum colloid with an enzymatic catalytic reaction. The immunosensor was fabricated by modification of the screen-printed carbon electrodes (SPCE) with nanocomposites made by skillful doping of bovine serum albumin conjugated chlorpyrifos-methyl antigen (BSA-Ag) and platinum colloid into silica sol-gel. The scanning electron microscope (SEM) images and electrochemical measurements showed that platinum colloid domains in the nanocomposite material could enhance electron transfer and change the brittleness of the silica sol-gel. The immobilisation of BSA-Ag on the nanocomposite retained its immunoactivities, which allowed the immobilised BSA-Ag to effectively capture unbound Ab-HRP in the detection solution. A linear response to CM concentration was exhibited, ranging from 0.4 to 20ng/mL. Detection of CM with the presented method in soil or grape samples treated with CM matched the reference values well, which indicated that the proposed disposable immunosensor hold promising applications in environmental and food monitoring.

A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII

DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate.

Fabrication of CdTe@SiO2 nanoprobes for sensitive electrogenerated chemiluminescence detection of DNA damage.

Detection of DNA damage is significant for the evaluation of genotoxicity of new chemicals in the early stages of its development. An electrogenerated chemiluminescence (ECL) biosensor was fabricated to detect specific sequences of DNA by using CdTe@SiO2 as nanoprobes for signal amplification. This DNA biosensor was constructed by self-assembly of an aminated capture DNA on the glass carbon electrode. DNA detection was realized by outputting a remarkable ECL signal of the CdTe@SiO2 labeled probe DNA. When the target DNA was introduced into the system, it was complementary to the probe DNA at the one-half-segment and complementary to the capture DNA at the other half-segment, resulting in the formation of a stable duplex complex. As a result, the CdTe@SiO2 labeled probe was proximate to the electrode surface and the ECL was observed. This DNA biosensor was proved to have a low detection limit (0.03 nM) and a wide dynamic range (from 0.1 nM to 2 μM). Most importantly, the sensing system could differentiate the single base mismatched DNA from the complementary DNA. It was successfully applied to study the damage to DNA caused by several genotoxicity chemicals, which was rapid, simple, reliable and sensitive compared to the classical biological methods.

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag(+)-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

Improving the fluorometric determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine by using a 3D DNA nanomachine

AbstractThe authors describe a fluorometric method for improving the determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine (8-OHdG). A nicking endonuclease (NEase)-powered 3-D DNA nanomachine was constructed by assembling hundreds of carboxyfluorescein-labeled single strand oligonucleotides (acting as signal reporter) and tens of swing arms (acting as single-foot DNA walkers) on a gold nanoparticle (AuNP). The activity of this DNA nanomachine was controlled by introducing the protecting oligonucleotides. In the presence of aptamer against 8-OHdG, the protecting oligonucleotides are removed from the swing arms by toehold-mediated strand displacement reaction. In the next step, detached DNA walker hybridizes to the labelled DNA so that the DNA nanomachine becomes activated. Special sequences of signal reporter in the formed duplex can be recognized and cleaved by NEase. As a result, the DNA walker autonomously and progressively moves along the surface of the AuNP, thereby releasing hundreds of signal reporters and causing a rapid increase in green fluorescence. This 3-D nanomachine is highly efficient because one aptamer can release hundreds of signal reporters. These unique properties allowed for the construction of a DNA nanomachine-based method for sensitively detecting 8-OHdG in concentrations as low as 4 pM. This is three orders of magnitude lower compared to previously reported methods. Graphical abstractSchematic of a fluorometric method for determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine. A nicking endonuclease powered 3D-DNA nanomachine was used to improve the sensitivity. Limit of detection is three orders of magnitude lower than reported methods.