Zhouping Wang

Multiplexed Fluorescence Resonance Energy Transfer Aptasensor between Upconversion Nanoparticles and Graphene Oxide for the Simultaneous Determination of Mycotoxins

We presented a new aptasensor for mycotoxins, which was based on multiplexed fluorescence resonance energy transfer (FRET) between multicolor upconversion fluorescent nanoparticles (UCNPs) as donors and graphene oxide (GO) as the entire and effective acceptor. BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.7), Tm(0.02) upconversion nanoparticles were synthesized and functionalized, respectively, with immobilized ochratoxin A (OTA)-aptamers and fumonisin B(1) (FB(1))-aptamers. On the basis of the strong π-π stacking effect between the nucleobases of the aptamers and the sp(2) atoms of GO, the aptamer modified-UCNPs can be brought in close proximity to the GO surface. The strong upconversion fluorescence both of BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.2), Tm(0.02) can be completely quenched by the GO, because of a good overlap between the fluorescence emission of multicolor UCNPs and the absorption spectrum of GO. In contrast, in the presence of OTA and FB(1), the aptamers preferred to bind to their corresponding mycotoxins, which led to changes in the formation of aptamers, and therefore, aptamer modified-UCNPs were far away from the GO surface. Our study results showed that the fluorescence intensity of BaYF(5):Yb Er and BaYF(5):Yb Tm were related to the concentration of OTA and FB(1). We therefore developed a sensitive and simple platform for the simultaneous detection of OTA and FB(1) with multicolor UCNPs and GO as the FRET pair. The aptasensor provided a linear range from 0.05 to 100 ng·mL(-1) for OTA and 0.1 to 500 ng·mL(-1) for FB(1); the detection limit of OTA was 0.02 ng·mL(-1) and FB(1) was 0.1 ng·mL(-1). As a practical application, the aptasensor was used to monitor OTA and FB(1) level in naturally contaminated maize samples with the results consistent with that of a classic ELISA method. More importantly, the novel multiplexed FRET was established for the first time based on multiplexed energy donors to the entire energy acceptor; this work was expected to open up a new field of FRET system applications for various targets.

Simultaneous Aptasensor for Multiplex Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles Labels

A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for Staphylococcus aureus, Vibrio parahemolyticus, and Salmonella typhimurium. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50-10(6) cfu mL(-1). Improved by the magnetic separation and concentration effect of Fe3O4 magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL(-1) for S. aureus, V. parahemolyticus, and S. typhimurium, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors.

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus.

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY(0.78)F(4):Yb(0.2),Tm(0.02) UCNPs modified aptamer 1 and NaY(0.28)F(4):Yb(0.70),Er(0.02) UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10(1)-10(5) cfu mL(-1) (R(2)=0.9964), and the signal was in the range of 10(1)-10(5) cfu mL(-1) (R(2)=0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL(-1) for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF(4) UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.

Selection and Characterization of Aptamers against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX)

In this paper, a high-affinity ssDNA aptamer binding to Salmonella typhimurium was obtained by a whole-bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. After nine rounds of selection with S. typhimurium as the target, a highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homology and secondary structure similarity. Eleven sequences from different families were selected for further characterization via flow cytometry analysis. The results showed that the sequence ST2P demonstrates affinity for S. typhimurium much more strongly and specifically than other sequences tested. The estimated Kd value of this particularly promising aptamer was 6.33 ± 0.58 nM. To demonstrate the potential use of the aptamers in the quantitative determination of S. typhimurium, a fluorescent bioassay with the aptamer ST2P was prepared. Under optimal conditions, the correlation between the concentration of S. typhimurium and fluorescent signal was found to be linear within the range of 50-10(6) cfu/mL (R(2) = 0.9957). The limit of detection (LOD) of the developed method was found to be 25 cfu/mL. This work demonstrates that this aptamer could potentially be used to improve the detection of S. typhimurium.

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels.

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B(1) (AFB(1)) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY(0.78)F(4):Yb(0.2), Tm(0.02) and NaY(0.28)F(4):Yb(0.7),Er(0.02) UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB(1) antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB(1) and OTA under optimal conditions was as low as 0.01 ng mL(-1), and the effective detection range was from 0.01 to 10 ng mL(-1). The proposed method was successfully applied to measure AFB(1) and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF(4) UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.

An aptamer-based electrochemical biosensor for the detection of Salmonella.

Salmonella is one of the most common causes of food-associated disease. An electrochemical biosensor was developed for Salmonella detection using a Salmonella-specific recognition aptamer. The biosensor was based on a glassy carbon electrode modified with graphene oxide and gold nanoparticles. Then, the aptamer ssDNA sequence could be linked to the electrode. Each assembly step was accompanied by changes to the electrochemical parameters. After incubation of the modified electrode with Salmonella, the electrochemical properties between the electrode and the electrolyte changed accordingly. The electrochemical impedance spectrum was measured to quantify the Salmonella. The results revealed that, when more Salmonella were added to the reaction system, the current between the electrode and electrolyte decreased; in other words, the impendence gradually increased. A detection limit as low as 3 cfu/mL was obtained. This novel method is specific and fast, and it has the potential for real sample detection.

Selection and Identification of a DNA Aptamer Targeted to Vibrio parahemolyticus

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Ionic liquids based simultaneous ultrasonic and microwave assisted extraction of phenolic compounds from burdock leaves

The ionic liquids based simultaneous ultrasonic and microwave assisted extraction (IL-UMAE) technique was first proposed and applied to isolate compounds. The ionic liquids comprising a range of four anions, five 1-alkyl-3-methylimidazolium derivatives were designed and prepared. The results suggested that varying the anion and cation both had apparent effects on the extraction of phenolics. The results also showed that irradiation power, time and solid-liquid ratio significantly affected the yields. The yields of caffeic acid and quercetin obtained by IL-UMAE were higher than those by regular UMAE. Compared with conventional heat-reflux extraction (HRE), the proposed approach exhibited higher efficiency (8-17% enhanced) and shorter extraction time (from 5h to 30s). The results indicated ILUMAE to be a fast and efficient extraction technique. Moreover, the proposed method was validated by the reproducibility and recovery experiments. The ILUMAE method provided good recoveries (from 96.1% to 105.3%) with RSD lower than 5.2%, which indicated that the proposed method was credible. Based on the designable nature of ionic liquids, and the rapid and highly efficient performance of the proposed approach, ILUMAE provided a new alternative for preparation of various useful substances from solid samples.

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels.

A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.

A sensitive gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus

In this study, a gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus (S. aureus) using tyramine signal amplification (TSA) technology has been developed. First, the biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of the microtiter plate via biotin-avidin binding. Then, the target bacteria (S. aureus), biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and avidin-catalase were successively introduced into the wells of the microtiter plate. After that, the existing catalase consumed the hydrogen peroxide. Finally, the freshly prepared gold (III) chloride trihydrate was added, the color of the reaction production would be changed and the absorbance at 550 nm could be measured with a plate reader. Under optimized conditions, there was a linear relationship between the absorbance at 550 nm and the concentration of S. aureus over the range from 10 to 10(6) cfu mL(-1) (with an R² of 0.9947). The limit of the developed method was determined to be 9 cfu mL(-1).