Taihua Li

Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk

We demonstrated the new antibody/gold nanoparticle/magnetic nanoparticle nanocomposites (antibody/AuNP/MNPs) and their application in the detection of the foodborne pathogen, Staphylococcus aureus (S. aureus), in milk. The nanocomposites were synthesized by coating the MNPs with bovine serum albumin (BSA) then adsorbing the AuNPs and anti-S. aureus antibodies on their surface. Using the completed immunomagnetic nanostructures, S. aureus inoculated in the milk sample was captured and isolated from the medium using the permanent magnet. The nanoparticle-bound cells as well as the unbound cells in the supernatant were enumerated via surface plating to evaluate the target binding capacity of the nanocomposites. The capture efficiencies of the antibody/AuNP/MNPs were 96% and 78% for S. aureus in PBS and the milk sample respectively, which were significantly higher than those of the antibody-coupled MNPs without any AuNP. The captured cells were also applied to the selective filtration system to produce color signals that were used for the detection of the target pathogen. During the filtration, the cells bound to the antibody/AuNP/MNPs remained on the surface of the membrane filter while unbound nanoparticles passed through the uniform pores of the membrane. After the gold enhancement, the cells-particles complex resting on the membrane surface rendered a visible color, and the signal intensity became higher as the target cell concentration increased. The detection limits of this colorimetric sensor were 1.5×10(3) and 1.5×10(5)CFU for S. aureus in PBS and the milk sample respectively. This sensing mechanism also had the high specificity for S. aureus over the other pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The assay required only 40min to obtain the results. With the use of the appropriate antibodies, our immunomagnetic nanocomposites-based detection strategy can provide an easy, convenient, and rapid sensing method for a wide range of pathogens.

A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A.

Binding of an analyte on the surface of a nanoparticle typically promotes a change in the local refractive index, which gives rise to a shift in the wavelength of the localized surface plasmon resonance (LSPR) absorption band. The magnitude of the LSPR wavelength change is dependent on both the location of the analyte relative to the surface of the nanoparticle and the degree of alteration of the refractive index. We have employed this phenomenon as the basis for designing a new, label-free approach for the detection of the toxic mold mycotoxin, ochratoxin A (OTA) that employs a gold nanorod (GNR) and an aptamer target binding mechanism. In this system, binding of OTA causes an accumulation of OTA and G-quadruplex structure of the aptamer. This process results in a longitudinal wavelength shift of the LSPR peak associated with a change in the local refractive index near the GNR surface. By using this method, OTA can be quantitatively detected at concentrations lower than 1 nM. In addition, the results of this effort show that aptamer functionalized GNR substrate is robust in that it can be regenerated for reuse over seven times by heating in methanol at 70 °C to remove OTA. Moreover, the proposed biosensor system exhibits high selectivity for OTA over other mycotoxins. Finally, the sensor can be employed to detect OTA in ground corn samples with excellent recovery levels.

Label-free homogeneous FRET immunoassay for the detection of mycotoxins that utilizes quenching of the intrinsic fluorescence ofantibodies

The phenomenon of fluorescence quenching of an antibody by a specific ligand was applied in developing a technique for detection of mycotoxins, such as aflatoxin B₁ (AFB₁), ochratoxin A, and zearalenone. Studies showed that the intrinsic fluorescence of tryptophan (Trp) residues in antibodies, promoted by excitation at 280 nm, is quenched upon binding of specific mycotoxin ligands. Fluorescence quenching in FRET system takes place in these systems as a consequence of the overlap of the emission spectra of antibody donors with the absorption spectra of the mycotoxins. Further studies focusing on the detection of AFB₁ revealed that the Fab fragment, the variable region of the antibody where specific binding of AFB₁ occurs, can be utilized to increase the sensitivity of the detection system. The results demonstrate that fluorescence of the Fab fragment is almost completely quenched by AFB₁ whereas emission from intact anti-AFB₁ is only partially quenched by this mycotoxin. The limits of detection (LODs) were found to be 0.85 and 0.09 ng mL⁻¹ for assays using the intact antibody and the Fab fragment, respectively, corresponding to a 10-fold enhancement. A practical application of the Fab fragment based assay system was demonstrated by its use in the detection of AFB₁ in spiked barley grain samples. The observations made in this effort show that the new, label-free, non-competitive, and homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of organic substances such as mycotoxin.

The use of an engineered single chain variable fragment in a localized surface plasmon resonance method for analysis of the C-reactive protein

A gold nanorod (GNR) based LSPR sensor has been developed for label-free detection of C-reactive protein (CRP). The sensor utilizes a single chain variable fragment (scFv) as a receptor to bind CRP. The results of this effort show that CRP in human serum can be quantitatively detected at lower than 1 ng mL(-1).

Electrical Immunosensor Based on a Submicron-gap Interdigitated Electrode and Gold Enhancement

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations).

The effects of pH and surfactants on the absorption and fluorescence properties of ochratoxin A and zearalenone

The pH and surfactant dependencies of the absorption and fluorescence properties of ochratoxin A (OTA) and zearalenone (ZEN), the main mycotoxins found as contaminants in foods and feeds, were evaluated. Three surfactants with different ionic properties were investigated, namely sodium dodecyl sulfate (SDS, anionic), Tween 20 (nonionic) and hexadecyltrimethylammonium bromide (CTAB, cationic). The results show that the effects of pH on the absorption wavelength maxima and fluorescence efficiencies of the mycotoxins, which are a consequence of the presence of acidic phenol and/or carboxyl containing fluorophores, are dependent on the ionic nature of the added surfactants. Specifically, the fluorescence responses to pH changes of OTA and ZEN are similar in the presence or absence of Tween 20 and SDS. By contrast, the pH-dependent fluorescence properties of these mycotoxins are altered when CTAB is present in the solutions. Moreover, unlike OTA, ZEN in aqueous solution displays almost no fluorescence. However, fluorescence enhancement takes place when surfactants are present in aqueous solutions of this mycotoxin. The results of this study demonstrate that the different microenvironments, present in the organized micellar systems created by the individual surfactants, can be potentially employed to modulate the sensitivities and selectivities of the fluorescence detection of OTA or ZEN.

Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for the Determination of Zearalenone

This study demonstrates the use of antigen-antibody binding for the detection of zearalenone. Based on the principle of the fluorescence resonance energy transfer (FRET) phenomenon between antibody and antigen, an immunoassay, in which zearalenone coupled with the anti-zearalenone antibody, was developed, optimized, and applied. Owing to intrinsic fluorescence properties in basic pH conditions with the optimal cationic surfactant, anti-zearalenone and zearalenone played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of analyte increased, the antigen/antibody emission intensity ratio (I 430 nm/I 350 nm) was enhanced due to larger amounts of zearalenone/anti-zearalenone complexes. This assay, based on the ratio of intensities (I 430 nm/I 350 nm), displayed high specificity and sensitivity with a detection limit of 0.8 ng mL−1 for zearalenone. The results obtained from analysis of spiked wheat grain samples were found to be in good agreement with those obtained by employing a direct competitive enzyme-linked immunosorbent assay. The label-free, noncompetitive, and homogeneous FRET immunoassay strategy served as a powerful tool for the simple, rapid, and sensitive quantitative determination of zearalenone in food and feed matrices.

A rapid detection of neopterin based on a label-free and homogeneous FRET immunoassay system

Herein, we have developed a label-free and homogeneous fluorescence resonance energy transfer (FRET) immunoassay for the detection of neopterin (NPT), which is an early and valuable biochemical marker of cellular immunity. Owing to intrinsic fluorescence properties of antibody and NPT, anti-NPT antibody (anti-NPT) and analyte played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of NPT increases, the fluorescence intensity at ~350 nm decreases owing to the formation of increasing amounts of the anti-NPT/NPT complex in which FRET takes place. The assay system was found to display a high specificity and a low detection limit (0.14 ng mL-1) for NPT. A practical application of the FRET immunoassay system was demonstrated by its use in the detection of NPT in spiked human serum samples. The observations made in these efforts show that the homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of NPT.