Yonggang Hu

Sensitive chemiluminescence immunoassay for E. coli O157:H7 detection with signal dual-amplification using glucose oxidase and laccase.

A novel, sensitive chemiluminescence (CL) immunoassay for Escherichia coli O157:H7 detection with signal dual-amplification using glucose oxidase (GOx) and laccase was investigated. The method was based on the characterization of a luminol-H2O2-laccase reaction. Compared with the horseradish peroxidase-based biosensor, laccase exhibited high catalytic activity in strong alkaline medium, which was compatible with the luminol system. The capture antibody was immobilized onto the magnetic bead (MB) surfaces. The detection antibody was linked with GOx through biotin-avidin recognition. Accordingly, the bioconjugation of MB-caputure antibody- E. coli O157:H7-detection antibody-GOx catalyzed the substrate glucose, thereby generating H2O2. E. coli O157:H7 was then detected by measuring the CL intensity after H2O2 formation. Under optimal conditions, the calibration plot obtained for E. coli O157:H7 was approximately linear from 4.3 × 10(3) colony-forming unit (CFU) mL(-1) to 4.3 × 10(5) CFU mL(-1), and the total assay time was <2.0 h without any enrichment. The limit of detection for the assay was 1.2 × 10(3) CFU mL(-1) (3σ), which was considerably lower than that of enzyme-linked immunosorbent assay method (1.0 × 10(5) CFU mL(-1)) (3σ). A series of repeatability measurements of using 1.7 × 10(4) CFU mL(-1) E. coli O157:H7 exhibited reproducible results with a relative standard deviation (RSD) of 3.5% (n = 11). Moreover, the proposed method was successfully used to detect E. coli O157:H7 in synthetic samples (spring water, apple juice, and skim milk), which indicated its potential practical application. This protocol can be applied in various fields of study.

Sensitive and selective detection of Ag+ in aqueous solutions using Fe3O4@Au nanoparticles as smart electrochemical nanosensors.

Owing to the selective deposition reaction on the surface of magnetic nanoparticles, we reported a simple and selective magnetic electrochemical method for the detection of Ag(+) ions in aqueous solutions. The analyte deposited on the nanoparticles was brought to the surface of a homemade magnetic electrode and detected electrochemically in 0.1 mol/L KCl solution based on the reaction of Ag0 transferred to AgCl. Under the optimal conditions, the linear response range of Ag(+) ions was 0.117-17.7 μmol/L (R(2)=0.9909) with a detection limit of 59 nmol/L (S/N=3). A series of repeatability measurements 1.0 μmol/L Ag(+) gave reproducible results with a relative standard deviation (RSD) of 4.5% (n=11). The interference from other metal cations can be eliminated by adding EDTA as a co-additive to mask the metal cations. The recoveries ranging from 98.6% to 103.99% after standard additions demonstrate that this sensor has great potential in practical applications. The advantages of this developed method include remarkable simplicity, low cost, and no requirement for probe preparation, among others.

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: Application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.

Whole-cell method for phenol detection based on the color reaction of phenol with 4-aminoantipyrine catalyzed by CotA laccase on endospore surfaces.

A green method for phenol spectrophotometric determination was developed based on the color reaction of phenol with 4-aminoantipyrine catalyzed by addition of Bacillus amyloliquefaciens endospores in the presence of O2. The catalytic activity of the endospores may be attributed to the presence of coat protein A on the cell surfaces. This deduction was confirmed by cotA gene knock-out from B. amyloliquefaciens using the homologous double-exchange method. Under optimal conditions, linear responses were obtained over phenol concentrations ranging from 5.0×10(-5)gL(-1) to 1.0×10(-2)gL(-1) (r=0.9984) with a detection limit of 2.1×10(-5)gL(-1) (3σ). Repeatability measurements of 1.0mgL(-1) phenol provided reproducible results with a relative standard deviation of 5.3% (n=11). Standard addition tests indicated recoveries ranging from 92.78% to 107.60%. The proposed whole-cell method was successfully used to detect total phenol in synthetic samples. Results confirmed the potential use of the developed method in practical applications.

Quantum dot incorporated Bacillus spore as nanosensor for viral infection.

In this paper, we report a high-throughput biological method to prepare spore-based monodisperse microparticles (SMMs) and then form the nanocomposites of CdTe quantum dot (QD)-loaded SMMs by utilizing the endogenous functional groups from Bacillus spores. The SMMs and QD-incorporated spore microspheres (QDSMs) were characterized by using transmission electron microscopy, high-resolution transmission electron microscopy, fluorescence microscopy, fluorescence and UV-visible absorption spectroscopy, zeta potential analysis, Fourier-transform infrared spectroscopy, potentiometric titrations, X-ray photo-electron spectroscopy. The thermodynamics of QD/SMM interaction and antigen/QDSM interaction was also investigated by isothermal titration microcalorimetry (ITC). Fluorescent QDSMs coded either with a single luminescence color or with multiple colors of controlled emission intensity ratios were obtained. Green QDSMs were used as a model system to detect porcine parvovirus antibody in swine sera via flow cytometry, and the results demonstrated a great potential of QDSMs in high-throughput immunoassays. Due to the advantages such as simplicity, low cost, high throughput and eco-friendliness, our developed platform may find wide applications in disease detection, food safety evaluation and environmental assessment.

A quantum dot-spore nanocomposite pH sensor.

A new quantum dot (QD)-based pH sensor design is investigated. The sensor is synthesized based on the self-assembly of green QDs onto treated spores to form QD@spore nanocomposites. The nanocomposites are characterized using laser scanning confocal microscopy, transmission electron microscope, and fluorescence spectroscopy, among others. Fluorescence measurements showed that these nanocomposites are sensitive to pH in a broad pH range of 5.0-10.0. The developed pH sensors have been satisfactorily applied for pH estimation of real samples and are comparable with those of the commercial assay method, indicating the potential practical application of the pH sensors.

Laccase-Assisted Rapid Synthesis of Colloidal Gold Nanoparticles for the Catalytic Reduction of 4-Nitrophenol

A green method for the rapid preparation of uniform-sized colloidal gold nanoparticles under ambient conditions was presented and validated using laccase as a reduction agent in alkaline medium. UV-Vis spectrophotometry, field-emission high-resolution transmission electron microscopy, X-ray diffraction, selected area electron diffraction, energy dispersive X-ray analysis, zetasizer, and Fourier transform infrared spectroscopy were used to characterize the gold nanoparticles. The gold nanoparticles were spherical, crystalline, uniform, and monodisperse with an average size of 12.24 nm. These nanoparticles were successfully used to reduce 4-nitrophenol in the presence of NaBH4 and exhibited an excellent catalytic activity.

A competitive chemiluminescence enzyme immunoassay method for β‐defensin‐2 detection in transgenic mice

A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2.

A novel fluorimetric method for laccase activities measurement using Amplex Red as substrate.

In this paper, a novel fluorescence-based method for laccase assay was presented. The method was based on the transformation of Amplex Red into a highly fluorescent and colored resorufin catalyzed by laccase in the presence of O2. The catalysis and transformation mechanism were investigated in detail. The kinetic parameters of the Amplex Red catalysis by laccase were determined using the Lineweaver-Burk equation. Vmax and Km were estimated to be 15.63μmolmin-1 and 76.88μmolL-1, respectively. Under optimal conditions, a good linear correlation was found between fluorescence intensity and laccase activities within 5.62-702UL-1 (r=0.9992), with a detection limit of 1.76UL-1 (S/N=3). A series of repeatability measurements (351UL-1 laccase) gave reproducible results with a relative standard deviation (RSD) of 1.9% (n=11). The recoveries ranged from 93.7% to 100.0% after standard additions. Common existing species such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, Fe2+, l-lysine, glycine, glucose, phenol, humic acid, lignin peroxidase, manganese peroxidase alkaline phosphatase, cellulose, glucose oxidase, urease, catalase, invertase, and horseradish peroxidase did not significantly exhibit interference. The test solution (i.e., Amplex Red stock solution) could stabilize at least three months via storage in dark at 4±0.1°C. These results confirmed that the laccase-Amplex Red system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that this method can has great potential in practical applications for the assay of laccase activity. The proposed method was further successfully used to detect laccase activities in 38 soil samples. We noticed that the laccase activity significantly correlated with total nitrogen content (r=0.559; p<0.01) of soil, indicating laccase activity assay holds great promise as an index of soil analysis. These findings indicate that this presented method has great perspective in ecological investigation and fundamental research of soil environment.

Self-assembled Ti4+@Biospore Microspheres for Sensitive DNA Analysis

Ti4+ can be chemically adsorbed and assembled on the surface of the modified spore to form highly monodispersed Ti4+@spore microspheres. Moreover, we for the first time found that these biomicrospheres exhibit differential affinities toward ssDNA and dsDNA. As a principle-of-proof, we exploited the self-assembled Ti4+@spore microspheres for a hybridization analysis. Interestingly, in the hybridization analysis, residual ssDNA probes are selectively adsorbed on Ti4+@spore microspheres at pH 5.0 and then removed via centrifugation. By taking advantage of this property, the signal-to-noise ratio for DNA analysis was considerably increased by reducing the noise caused by the residual ssDNA probes. The proposed method features easy operation, high specificity, and sensitivity and thus exhibits potential for further applications on DNA biosensing.