Lichao Fang

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.

An electrochemical immunosensor for sensitive detection of Escherichia coli O157:H7 using C60 based biocompatible platform and enzyme functionalized Pt nanochains tracing tag.

A sensitive and efficient electrochemical immunosensor was designed for amperometric detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). The immunosensing platform was first composed of fullerene (C60), ferrocene (Fc) and thiolated chitosan (CHI-SH) composite nano-layer which could offer rich -SH functional groups and maintain good biocompatibility. Then the Au nanoparticles coated SiO2 nanocomposites (Au-SiO2) were assembled on the CHI-SH/Fc/C60 composite. Next, the large amount of avidin (SA) was coated on the Au-SiO2 surface, which was used to immobilize biotinylated capture antibodies of E. coli O157:H7 (bio-Ab1) through the covalent reaction between biotin and avidin. With surface area enhancement by C60 and Au-SiO2, and directional immobilization by avidin-biotin system, the amount of immobilized bio-Ab1 can be enhanced obviously. For signal amplification, the glucose oxidase (GOD) loaded Pt nanochains (PtNCs) were used as tracing tag to label signal antibodies (Ab2). With a sandwich-type immunoreaction, the concentration volume of heat-killed E. coli O157:H7 ranged from 3.2 × 10(1) to 3.2 × 10(6)CFU/mL with a limit of detection down to 15 CFU/mL (S/N=3), which could be well accepted for early clinical detection. The studied system provides new opportunities, and might speed up disease diagnosis, treatment and prevention with pathogen.

A novel electrochemical DNA biosensor based on HRP-mimicking hemin/G-quadruplex wrapped GOx nanocomposites as tag for detection of Escherichia coli O157:H7

A novel sensitive electrochemical DNA biosensor was developed for amperometric detection of Escherichia coli O157:H7 (E. coli O157:H7). The graphene oxide (GOx) was utilized as nanocarrier to immobilize thionine (Thi) and the Au nanoparticles coated SiO2 nanocomposites (Au-SiO2) by electrostatic adsorption and the adsorption among nanomaterials. Then a large amounts of signal DNA (S2) and G-quadruplex were immobilized on the GOx-Thi-Au@SiO2 nanocomposites. Finally, hemin was intercalated into the G-quadruplex to obtain the hemin/G-quadruplex structure as HRP-mimicking DNAzyme. On the basis of the signal amplification strategy of GOx-Thi-Au@SiO2 nanocomposites and DNAzyme, the developed DNA biosensor could respond to 0.01 nM (S/N=3) with a linear calibration range from 0.02 to 50.0 nM E. coli O157:H7, which could be well accepted for early clinical detection. The studied system provides new opportunities, and might speed up disease diagnosis, treatment and prevention with pathogen.

An Electrochemical Strategy using Multifunctional Nanoconjugates for Efficient Simultaneous Detection of Escherichia coli O157: H7 and Vibrio cholerae O1

The rapid and accurate quantification of the pathogenic bacteria is extremely critical to decrease the bacterial infections in all areas related to health and safety. We have developed an electrochemical strategy for simultaneous ultrasensitive detection of E. coli O157:H7 and Vibrio cholerae O1. This approach was based on the specific immune recognition of different pathogenic bacteria by multifunctional nanoconjugates and subsequent signal amplification. By employing the proposed biosensor, the concentrations of these pathogenic bacteria could be established on a single interface in a single run with improved sensitivity and accuracy. The successful approach of the simultaneous detection and quantification of two bacteria by an electrochemical biosensor demonstrated here could be readily expanded for the estimation of a variety of other pathogenic bacteria, proteins, and nucleotides. Because of their high sensitivity, electrochemical biosensors may represent a new avenue for early diagnosis of diseases.

Preparation and Application of Amino- and Dextran-Modified Superparamagnetic Iron Oxide Nanoparticles

Amino-dextran-functionalized superparamagnetic iron oxide nanoparticles (SPION) were synthesized by two-steps: Dextran-modified SPION was obtained by “one-step” co-precipitation method. Then, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS) was added to the resultant dextran-SPION to prepare amino-dextran-functionalized SPION (AEAPS/Dex-SPION). The particles were characterized by vibrating sample magnetometer (VSM), transmission electron micrographs (TEM), atomic force micrographs (AFM), gas chromatography and atomic absorption spectrophotometry. The size of the modified particles varied in a range of 30 to 40 nm and did not change significantly after modification. The binding rate of AEAPS was 91.15%. A monoclonal antibody against S100 (anti-S-100), a gold standard for the diagnosis of melanocytic lesions, was conjugated to the AEAPS/Dex-SPION to prepare immuno-SPION. From the result in melanoma B16 cell, the immuno-SPION was proven to have a bio-targeting activity. Such AEAPS/Dex-SPION might be very useful for bio-magnetically targeted detection in early melanocytic lesions.

An electrochemical strategy with tetrahedron rolling circle amplification for ultrasensitive detection of DNA methylation

Sensitive and specific detection of DNA methylation in genomic DNA is imperative for rapid epigenetic evaluations. Here, a novel sensitive electrochemical strategy was developed for ultrasensitive detection of DNA methylation in genomic DNA via padlock probe primer generating rolling circle amplification (RCA). Typically, after bisulfite treatment of methylated DNA, the methylation-specific linear padlock is only circularized in the presence of methylated DNA and subsequently serves as a template containing a DNA tetrahedron for RCA. The DNA tetrahedron is utilized as a nanocarrier that can be immobilized on a gold electrode to generate RCA product to load hemin, an iron-containing porphyrin with chlorine, forming the G-quadruplex as a horseradish peroxidase like DNAzyme, which reduces methylene blue (MB) in the presence of H2O2 to yield a distinct current signal. Using the developed DNAzyme with the RCA signal amplification strategy, the DNA biosensor can achieve a detection limit as low as 0.1 fM for the ultrasensitive electrochemical detection of methylated DNA sequence with a detection range from 10-15 M to 10-9 M. At the same time, the satisfactory specificity, reproducibility, stability and recovery performances indicated its satisfied potentials for clinical diagnosis. Most importantly, this method can be further applied to analyse other genomic DNA also.