Debin Zhu

A new kind of aptamer-based immunomagnetic electrochemiluminescence assay for quantitative detection of protein

A new kind of aptamer-based immunomagnetic electrochemiluminescence (IM-ECL) assay for quantitative detection of protein is developed. The assay consists of a double aptamer sandwich format in which a biotin-labeled aptamer is used for rapid and specific capture of target protein, and a [Ru(bpy)(3)](2+) (TBR)-labeled aptamer is used for ECL detection. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 80 pmol/L of PDGF-BB. A calibration curve with a linearity range from 0.1 to 1000 nmol/L is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this new method holds great promise in protein detection.

Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform

MicroRNAs (miRNAs) are a kind of small molecules that involve in many important life activities. They have higher expression levels in many kinds of cancers. In this study, we developed an isothermal enzyme-free amplification (EFA) and label-free graphene oxide (GO)-based SYBR Green I fluorescence platform for detection of miRNA. MiRNA-21 was used as an example to demonstrate the feasibility of the method. Results show that the sensitivity of miRNA-21 is 1pM, and the linearity range is from 1pM to 1nM. The method can specifically discriminate miRNA-21 from miRNA-210 and miRNA-214. Three tumor cell lines of A549, HepG2 and MCF7 were detected by the method. The sensitivities of them were 10(2) cells, 10(3) cells and 10(3) cells respectively. Clinical tumor samples were also tested by this method, and 29 of 40 samples gave out positive signals. The method holds great promise in miRNA detection due to its convenience, rapidness, inexpensive and specificity.

Low-power laser irradiation inhibiting Abeta25-35-induced PC12 cell apoptosis via PKC activation.

Apoptosis is a contributing pathophysiological mechanism of Alzheimers disease (AD). Recently, low-power laser irradiation (LPLI) has been applied to moderate AD, but the underlying mechanism remains unknown. In this study, the techniques of fluorescence resonance energy transfer (FRET) and real-time quantitative RT-PCR were used to investigate the anti-apoptotic mechanism of LPLI. Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Aβ25-35) for induction of apoptosis before LPLI treatment. The cell viability assays and morphological examinations show that low fluence of LPLI (0.156 J/cm2-0.624 J/cm2) could inhibit the cells apoptosis. An increase of PKC activation was dynamically monitored in the cells treated with PMA (specific activator of PKC), LPLI only or Aβ25-35 followed by 5 min LPLI treatment, respectively. However, the effect of LPLI activating PKC could be inhibited by Gö 6983 (specific inhibitor of PKC). Similar results were obtained by using Western blot analysis. Furthermore, LPLI involved an increase in mRNA of the cell survival member bcl-xl and a decrease in the up-regulation of cell death member bax mRNA caused by Aβ25-35. Further data show that low fluence of LPLI could reverse the increased level of bax/bcl-xl mRNA ratio caused by Aβ25-35 treatment. In addition, Gö 6983 could inhibit the decreased level of bax/bcl-xl mRNA ratio. Taken together, these data clearly indicate that LPLI inhibited Aβ25-35-induced PC12 cell apoptosis via PKC-mediated regulation of bax/bcl-xl mRNA ratio.

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)(3)](2+) (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer-protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin-streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1 pM of PDGF-BB. A calibration curve with a linearity range from 1 pM to 10 nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.

A novel mutant allele specific amplification and electrochemiluminescence method for the detection of point mutation in clinical samples

A novel mutant allele specific amplification (MASA) and electrochemiluminescence (ECL) method for point mutation detection is proposed. Briefly, the target gene was amplified by a biotinylated mutant specific sense primer and a Ru(bpy)(3)(2+) (TBR)-labeled universal antisense primer. Only the mutant allele can be selectively amplified by the mutant specific primer pair. Then, the MASA product was captured onto the streptavidinylated magnetic beads through biotin-streptavidin linkage and detected by measuring the ECL emission of TBR. The method was applied to detect a possible point mutation at codon 12 of K-ras oncogene in 30 colorectal cancer (CAC) clinical samples. The experimental results show that the method can detect K-ras mutant in a 5000-fold excess of wild-type allele. Furthermore, different kinds of mutations can be clearly discriminated. The point mutation was found in 15 (50%) out of 30 CAC samples. This novel MASA-ECL method could potentially become a sensitive, specific, simple, rapid and safe approach for point mutation detection.

Point-of-care testing for streptomycin based on aptamer recognizing and digital image colorimetry by smartphone

The rapid detection of antibiotic residual in everyday life is very important for food safety. In order to realize the on-site and visual detection of antibiotic, a POCT method was established by using digital image colorimetry based on smartphone. Streptomycin was taken as the analyte model of antibiotics, streptomycin aptamer preferentially recognized analyte, and the excess aptamer hybridized with the complementary DNA to form the dsDNA. SYBR Green I combined with the dsDNA and then emitted obvious green fluorescence, thus the fluorescence intensity decreased with the increasing of streptomycin concentration. Then a smartphone-based device was constructed as the fluorescence readout. The smartphone camera acquired the images of the fluorescence derived from the samples, and the Touch Color APP installed in smartphone read out the RGB values of the images. There was a linear relationship between the G values and the streptomycin concentrations in the range of 0.1-100µM. The detection limit was 94nM, which was lower than the maximum residue limit defined by World Health Organization. The POCT method was applied for determining streptomycin in chicken and milk samples with recoveries in 94.1-110%. This method had the advantages of good selectivity, simple operation and on-site visualization.

Ultrasensitive electrochemiluminescence detection of Staphylococcus aureus via enzyme-free branched DNA signal amplification probe

As a main cause of foodborne diseases, pathogenic bacteria have threatened the health and well-being of human communities. There is a need of fastness, accuracy and sensitivity in the method of detecting pathogenic bacteria. Classical signal amplification assays usually employ enzymes as biocatalysts to generate amplified signals, but the strict experimental conditions and complicated instruments restrict their application. In this work, we demonstrated an enzyme-free branched DNA (bDNA)-based signal amplification electrochemiluminescence (ECL) assay for ultrasensitive detection of pathogenic bacteria. Firstly, the capture probes and the amplification probes group were carefully designed by our research group. The detecting ECL signal of Staphylococcus aureus (S. aureus) was amplified by bDNA technique through the layer-by-layer signal amplification. The sensitivity was greatly improved by the use of multiple Ru(bpy)32+ (TBR)-labeled ECL probes. Secondly, the whole process of the detection was carried out in the absence of enzyme, without the need to control the reaction conditions strictly. Thirdly, the designed amplification probes group could be used for the analysis of other pathogenic bacteria, virus, tumor markers, biomarkers, etc. For the detection of S. aureus, the limit of detection (LOD) of the method was 2u202fpM for standard DNA, with the linear range from 20u202fpM to 100u202fnM. Last but not least, the LOD of the S. aureus asymmetric PCR products was 5u202fpM, with the linear range from 10u202fpM to 50u202fnM. The sensitivity was 1-2 orders in magnitude higher than that of the common detection assays.

Rapid and quantitative measuring of telomerase activity using an electrochemiluminescent sensor

Telomerase, a ribonucleoprotein enzyme that adds telomeric repeats to the 3end of chromosomal DNA for maintaining chromosomal integrity and stability. This strong association of telomerase activity with tumors establishing it is the most widespread cancer marker. A number of assays based on the polymerase chain reaction (PCR) have been developed for the evaluation of telomerase activity. However, those methods require gel electrophoresis and some staining procedures. We developed an electrochemiluminescent (ECL) sensor for the measuring of telomerase activity to overcome these problems such as troublesome post-PCR procedures and semi-quantitative assessment in the conventional method. In this assay 5-biotinylated telomerase synthesis (TS) primer serve as the substrate for the extension of telomeric repeats under telomerase. The extension products were amplified with this TS primer and a tris-(22-bipyridyl) ruthenium (TBR)-labeled reversed primer. The amplified products was separated and enriched in the surface of electrode by streptavidin-coated magnetic beads, and detected by measuring the ECL signals of the TBR labeled. Measuring telomerase activity use the sensor is easy, sensitive, rapid, and applicable to quantitative analysis, should be clinically useful for the detection and monitoring of telomerase activity.

Electrochemiluminescence-PCR detection of genetically modified organisms

The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can’t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

Evaluation of UVA-induced oxidative stress using a highly sensitive chemiluminescence method

Oxidative stress is mainly mediated by reactive oxygen species (ROS). Evaluation of oxidative stress is helpful for choosing an appropriate method to protect the organism from the oxidative damage. In this study, a highly sensitive and simple chemiluminescence method is presented for the evaluation of radiation-induced oxidative stress in human peripheral lymphocytes. The lymphocytes were irradiated by ultraviolet radiation (320-400nm, UVA) with different doses. The ROS generated by the lymphocytes was detected by chemiluminescence method, using a highly sensitive chemiluminescence probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one (MCLA). The cell viability was detected with Cell Counting Kit-8 (CCK-8). The malondialdehyde (MDA), a marker of lipid peroxidation and oxidative stress, and the total antioxidant capacity (TAC), a parameter that is taken as evidence of oxidative stress, were measured too. The results show that both chemiluminescence intensity, cell mortality and MDA concentration of lymphocytes grow with the increase of UVA dose range from 0.5 to 8 J/cm2, while the TAC decreases. There exists a positive relationship between cell oxidative damage degree and the chemiluminescence intensity of lymphocytes. This highly sensitive chemiluminescence method would potentially provide an easy way to evaluate the level of UVA-induced oxidative stress readily, sensitively and rapidly