Donglei Jiang

Mast cell-based electrochemical biosensor for quantification of the major shrimp allergen Pen a 1 (tropomyosin)

A novel cell-based electrochemical biosensor was developed to quantify major shrimp allergen Pen a 1 (tropomyosin) and to assess its immunoglobulin E (IgE)-mediated hypersensitivity. Rat basophilic leukemia (RBL-2H3) mast cells, encapsulated in type I collagen, were immobilized on a self-assembled l-cysteine/gold nanoparticle (AuNPsCys)-modified gold electrode to monitor IgE-mediated mast cell sensitization and activation. The exposure of dinitrophenol-bovine serum albumin (DNP-BSA), as a model antigen that stimulates mast cells, induced a robust and long-lasting electrochemical impedance signal in a dose-dependent manner which efficiently measured degranulation of anti-DNP IgE-stimulated mast cells. Then this mast cell-based biosensor was applied into quantification for the shrimp allergen with anti-shrimp tropomyosin IgE-sensitization. The electrochemical impedance spectroscopy (EIS) results showed that the impedance value (Ret) increased with the concentration of purified shrimp allergen Pen a 1 (tropomyosin) in range of 0.5-0.25 μg mL(-1) with the detection limit as 0.15 μg mL(-1), and the electrochemical result was confirmed by β-hexosaminidase assay and scanning electron microscopic morphological (SEM) analysis. Thus, a simple, label-free, and sensitive method for the determination of shrimp allergens was proposed and demonstrated here, implying a highly versatile biosensor for food allergen detection and prediction.

Magnetic molecularly imprinted polymer nanoparticles based electrochemical sensor for the measurement of Gram-negative bacterial quorum signaling molecules (N-acyl-homoserine-lactones).

We have developed a novel and economical electrochemical sensor to measure Gram-negative bacterial quorum signaling molecules (AHLs) using magnetic nanoparticles and molecularly imprinted polymer (MIP) technology. Magnetic molecularly imprinted polymers (MMIPs) capable of selectively absorbing AHLs were successfully synthesized by surface polymerization. The particles were deposited onto a magnetic carbon paste electrode (MGCE) surface, and characterized by electrochemical measurements. Differential Pulse Voltammetry (DPV) was utilized to record the oxidative current signal that is characteristic of AHL. The detection limit of this assay was determined to be 8×10(-10)molL(-1) with a linear detection range of 2.5×10(-9)molL(-1) to 1.0×10(-7)molL(-1). This Fe3O4@SiO2-MIP-based electrochemical sensor is a valuable new tool that allows quantitative measurement of Gram-negative bacterial quorum signaling molecules. It has potential applications in the fields of clinical diagnosis or food analysis with real-time detection capability, high specificity, excellent reproducibility, and good stability.

Development of a novel electrochemical sensor using pheochromocytoma cells and its assessment of acrylamide cytotoxicity

We report on a sensitive, simple, label-free cell-based electrochemical sensor to monitor the toxic effect of acrylamide on the Pheochromocytoma cells. The surface of the electrode was modified with gold nanoparticles and electrochemically reduced graphene oxide. Cyclic voltammetry, impedance spectroscopy and differential pulse voltammetry were applied to characterize the modified electrode. Reduced graphene oxide was proved to increase electron-transfer rate between the cell and the surface of electrode, while gold nanoparticle retain cell bioactivity. The sensor exhibited good correlation to the logarithmic value of cell numbers ranging from 1.6×10(4) to 1.6×10(7) cells mL(-1), with R.S.D value of 1.68%. The value of differential pulse voltammetry (cell adsorption concentration of 1.6×10(7) cells mL(-1)) decreased with the concentration of acrylamide in range of 0.1-5 mM with the detection limit as 0.04 mM. Scanning electron microscope-based morphological and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis confirmed the results of the electrochemical study. This sensor was proved to be a useful tool for probing the toxicity of cells, and assisted in the development of a labeling-free, simple, rapid and immediate detection method.

A novel and simple cell-based electrochemical impedance biosensor for evaluating the combined toxicity of DON and ZEN.

In this study, a novel and simple cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON) and zearalenone (ZEN) on BEL-7402 cells. The sensor was fabricated by modification with AuNPs, p-aminothiophenol, and folic acid in succession. The BEL-7402 cells which had a good activity were adhered on the electrode through the high affinity between the folate receptor and folic acid selectivity. We used the collagen to maintain the cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate the individual and combined toxicity of DON and ZEN. Our results indicate that DON and ZEN caused a marked decrease in the cell viability in a dose-dependent manner. The value of electrochemical impedance spectroscopy decreased with the concentration of DON and ZEN in range of 0.1-20, 0.1-50 μg/ml with the detection limit as 0.03, 0.05 μg/ml, respectively, the IC50 for DON and ZEN as obtained by the proposed electrochemical method were 7.1 μg/ml and 24.6 μg/ml, respectively, and the combination of two mycotoxins appears to generate an additive response. The electrochemical cytotoxicity evaluation result was confirmed by biological assays. Compared to conventional methods, this electrochemical test is inexpensive, highly sensitive, and fast to respond, with long-term monitoring and real-time measurements. The proposed method provides a new avenue for evaluating the toxicity of mycotoxins.

Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs.

In this study, a novel electrochemical rat basophilic leukemia cell (RBL-2H3) cell sensor, based on fluorescent magnetic beads, has been developed for the detection and evaluation of different allergens in foodstuffs. Fluorescein isothiocyanate (FITC) was successfully fused inside the SiO2 layer of SiO2 shell-coated Fe3O4 nanoparticles, which was superior to the traditional Fe3O4@SiO2@FITC modification process. The as-synthesized fluorescent magnetic beads were then encapsulated with lipidosome to form cationic magnetic fluorescent nanoparticles (CMFNPs) for mast cell magnetofection. The CMFNPs were then characterized by SEM, TEM, VSM, FTIR, and XRD analyses, and transfected into RBL-2H3 cells through a highly efficient, lipid-mediated magnetofection procedure. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was then employed to adsorb the CMFNP-transfected RBL-2H3 cells activated by an allergen antigen for electrochemical assay. Results show that the exposure of model antigen-dinitrophenol-bovine serum albumin (DNP-BSA) to anti-DNP IgE-sensitized mast cells induced a robust and long-lasting electrochemical impedance signal in a dose-dependent manner. The detection limit was identified at 3.3×10(-4) ng/mL. To demonstrate the utility of this mast cell-based biosensor for detection of real allergens in foodstuffs, Anti-Pen a1 IgE and Anti-PV IgE-activated cells were employed to quantify both shrimp allergen tropomyosin (Pen a 1) and fish allergen parvalbumin (PV). Results show high detection accuracy for these targets, with a limit of 0.03 μg/mL (shrimp Pen a 1) and 0.16 ng/mL (fish PV), respectively. To this effect, we conclude the proposed method is a facile, highly sensitive, innovative electrochemical method for the evaluation of food allergens.

Preparation and application of acrylamide molecularly imprinted composite solid-phase extraction materials

A novel acrylamide (AA) molecularly imprinted material based on silica microparticles was synthesized successfully by a surface polymerization method using propionamide (PAM) as a template molecule, methacrylic acid (MAA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. The molecularly imprinted polymers (MIP) on the surfaces of silica microparticles were characterized using infrared spectroscopy and scanning electron microscopy, and the results suggested that the MIP was successfully grafted onto the silica surface. The amount of AA adsorbed on the MIP was measured using high-performance liquid chromatography (HPLC) and equilibrium binding experiments. The results of static adsorption equilibrium experiments and Scatchard analysis showed that compared with non-imprinted polymers, the imprinted polymer had a significantly higher adsorption capacity for AA. Scatchard analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 0.182 and 0.099 μmol mL−1, and the maximum apparent binding capacities were 16.18 and 54.18 μmol g−1. The new adsorbent was successfully used in solid-phase extraction (SPE) to selectively enrich and determine AA in fried food samples. The experimental results indicated that the MIP-SPE column gave recoveries higher than 93% with a relative standard deviation (RSD) of less than 2.24%, much better than those obtained using a commercial C18-SPE column, which gave a recovery of less than 24% with RSD < 2.07%. The developed MIP-SPE/HPLC protocol improved the selectivity and eliminated the effects of template leakage on quantitative analysis, and could be used for the determination of AA in complex food samples.

Mast-Cell-Based Fluorescence Biosensor for Rapid Detection of Major Fish Allergen Parvalbumin

In this study, we developed a rat basophilic leukemia cell (RBL-2H3) fluorescence sensor to detect and identify the major fish allergen parvalbumin (PV). We constructed and transfected a CD63-enhanced green fluorescent protein (EGFP) plasmid into RBL cells through a highly efficient, lipid-mediated, DNA-transfection procedure. Stable transfectant RBL cells were then obtained for a cell fluorescence assay with confocal laser scanning microscopy. Results show that the cell surface expression of CD63 reflects degranulation, indicating that a fluorescence assay with these cells could efficiently measure the activation of antigen-stimulated transfectant cells and detect antigens with a nanogram level. Therefore, this cell-based fluorescence biosensor technique for detecting fish PV exhibits promise for quantifying fish PV after anti-PV immunoglobulin E (IgE) stimulation. Results show that fluorescence intensities increased with purified PV concentrations from 1 to 100 ng/mL, with a detection limit of 0.35 ng/mL [relative standard deviation (RSD) of 4.5%], confirmed by β-hexosaminidase assays. These rat basophilic leukemia (RBL) mast cells transfected with the CD63-EGFP gene and responded to PV only when they were sensitized with the specific IgE antibody. This demonstrates the utility of this highly sensitive biosensor for food allergen detection and prediction.

Longitudinal surface plasmon resonance assay enhanced by magnetosomes for simultaneous detection of Pefloxacin and Microcystin-LR in seafoods.

A simple longitudinal surface plasmon resonance (LSPR) assay for the simultaneous detection of Pefloxacin and Microcystin-LR in seafoods has been developed for the first time using antibody-functionalized gold nanorods as signal probes and antigen-ovalbumin modified biological magnetosomes as signal amplification probes. The gold nanorods exhibit two different LSPR peaks, at around 695nm and 863nm, the positions of which were sensitive to changes in the local environment but can be subjected to simultaneous UV-vis detection. The biological magnetosomes produced by the magnetotactic bacteria not only act as a substrate for the immobilization of artificial antigen, but also enable signal enhancement and rapid separation, because of good dispersivity, biocompatibility and superparamagnetic properties. Under optimal conditions, magnetosome-enhanced LSPR assays showed a good linear response over the range 1-20ngmL(-1) (R(2)=0.9978 and R(2)=0.9992) with little adsorption to Enrofloxacin, Sarafloxacin, Ciprofloxacin, Norfloxacin, Microcystin-RR, Microcystin-LW, and Microcystin-LF, and compared with magnetosome-free LSPR assays, the response signal was amplified 2.5-5.0 fold. Furthermore, LSPR assays were successful in the analysis of Pefloxacin and Microcystin-LR in naturally contaminated seafood samples and high recoveries were achieved. Indications are that this LSPR assay promises reliable simultaneous detection of Pefloxacin and Microcystin-LR in seafoods, and holds the potential of novel applications in exploiting this multiple simultaneous UV-vis detection.

An electrochemical sensor based on molecularly imprinted membranes on a P-ATP–AuNP modified electrode for the determination of acrylamide

A novel electrochemical sensor for acrylamide (AM) detection based on molecularly imprinted polymer (MIP) membranes was constructed. p-Aminothiophenol (P-ATP) and AM were assembled on the surface of a gold nanoparticle (AuNP) modified glass carbon electrode (GCE) by the formation of Au–S bonds and hydrogen-bonding interactions, and polymer membranes were formed by electropolymerization in a polymer solution containing P-ATP, HAuCl4, tetrabutylammonium perchlorate (TBAP) and a dummy template molecule propanamide (PMA). A novel molecularly imprinted sensor (MIS) was obtained after the removal of PMA. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were used to monitor the electropolymerization process and its optimization, which was further characterized by scanning electron microscopy (SEM). The linear response range of the MIS was between 1 × 10−12 and 1 × 10−7 mol L−1, with a detection limit of 0.5 × 10−12 mol L−1. This research provides a fast, sensitive and real-time method for the detection of AM in a real sample without complex pretreatment and with average recoveries higher than 95% and a relative standard deviation (RSD) lower than 3.73%. All the obtained results indicate that the MIS is an effective electrochemical technique to determine AM in real-time and in a complicated matrix.

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.