Signal amplification by enzymatic reaction in an immunosensor based on localized surface plasmon resonance (LSPR).

An enzymatic reaction was employed as a means to enhance the sensitivity of an immunosensor based on localized surface plasmon resonance (LSPR). The reaction occurs after intermolecular binding between an antigen and an antibody on gold nano-island (NI) surfaces. For LSPR sensing, the gold NI surface was fabricated on glass substrates using vacuum evaporation and heat treatment. The interferon-γ (IFN-γ) capture antibody was immobilized on the gold NIs, followed by binding of IFN-γ to the antibody. Subsequently, a biotinylated antibody and a horseradish peroxidase (HRP) conjugated with avidin were simultaneously introduced. A solution of 4-chloro-1-naphthol (4-CN) was then used for precipitation; precipitation was the result of the enzymatic reaction catalyzed the HRP on gold NIs. The LSPR spectra were obtained after each binding process. Using this method, the enzyme-catalyzed precipitation reaction on the gold NI surface was found to effectively amplify the change in the signal of the LSPR immunosensor after intermolecular binding.

Ultrasensitive Detection of Escherichia coli O157:H7 by Immunomagnetic Separation and Selective Filtration with Nitroblue Tetrazolium/5-Bromo-4-chloro-3-indolyl Phosphate Signal Amplification.

Here, we report an enhanced colorimetric method using enzymatic amplification with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation for the ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective filtration. Biotinylated anti- E. coli O157:H7 antibody and streptavidin-alkaline phosphatase were conjugated to the surface of magnetic nanoparticles, and E. coli O157:H7-conjugates complexes remained on the membrane filter surface. The resultant light brown spots on the membrane filter were amplified with NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with an increasing E. coli O157:H7 concentration. E. coli O157:H7 was detected in pure samples with limits of detection of 10 and 6.998 colony-forming units (CFU)/mL through visual observation and measurement of optical density, respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli O157:H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This enhanced colorimetric method has potential for on-site detection of food contaminants and environmental pollutants.

Triple sensitivity amplification for ultrasensitive electrochemical detection of prostate specific antigen.

In general, current difference (ΔI) due to immunoreactions is significant in determining biosensor sensitivity. In this work, a new strategy of triple sensitivity amplification for ultrasensitive electrochemical detection of prostate specific antigen (PSA) was developed. Au-poly(methylene blue) (Au-PMB) was implemented as a redox species with strong current signal at -0.144V and used to fabricate the substrate of the biosensor. Conductive reduced graphene oxide-Au nanocomposites (Au-rGO) were coated on the Au-PMB modified glassy carbon electrode (GCE) to amplify current signal. After peptides (CEHSSKLQLAK-NH2) were fixed on the Au-rGO/Au-PMB/GCE, the fixed peptides reacted with glutaraldehyde to immobilize polydopamine-Au-horse radish peroxidase nanocomposites (PDA-Au-HRP). The electrochemical sensing interface for PSA was realized. Due to smaller resistance compared to antibodies, the peptides which can be cleaved specifically by PSA were employed. After the incubation of PSA, a large ΔI was obtained and behaved as the decrease of current signal. Then the remaining PDA-Au-HRP accelerated an enzyme-catalyzed precipitation reaction between 4-chloro-1-naphthol and H2O2. A further decrease in current signal (namely the increase in ΔI) resulted from the poorly conductive precipitation adhering onto the biosensor. The designed biosensor presented a wide linear range from 1.0fgmL-1 to 100ngmL-1 with an ultralow detection limit of 0.11fgmL-1.

Impedimetric aptasensor for nuclear factor kappa B with peroxidase-like mimic coupled DNA nanoladders as enhancer.

In this work, we developed a sensitive and universal aptasensor for nuclear factor kappa B (NF-κB) detection based on peroxidase-like mimic coupled DNA nanoladders for signal amplification. The dsDNA formed by capture DNA S1 and NF-κB binding aptamer (NBA) was firstly assembled on electrode surface. The presence of target NF-κB then led to the leave of NBA from electrode surface and thus provided the binding sites for immobilizing initiator to trigger in situ formation of DNA nanoladders on electrode surface. Since the peroxidase-like mimic manganese (III) meso-tetrakis (4-Nmethylpyridyl)-porphyrin (MnTMPyP) interacts with DNA nanoladders via groove binding, the insoluble benzo-4-chlorohexadienone (4-CD) precipitation derived from the oxidation of 4-chloro-1-naphthol (4-CN) could be formed on electrode surface in the presence of H2O2, resulting in a significantly amplified EIS signal output for quantitative target analysis. As a result, the developed aptasensor showed a low detection limit of 7pM and a wide linear range of 0.01-20nM. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective and stable detection of different types of target proteins.

Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).

Signal enhancement strategy for a micro-arrayed polydiacetylene (PDA) immunosensor using enzyme-catalyzed precipitation.

This paper describes a signal enhancement strategy to improve the sensitivity of an antibody-based immunosensor that uses polydiacetylene (PDA) liposomes to detect a target protein (human immunoglobulin E [hIgE]). To achieve ultrasensitive detection, multiple stimuli applied to PDA immunosensor chips offer a signal enhancement method that combines the primary immune reaction between antigen and antibody with the sandwich method of polyclonal antibody (pAb)-conjugated horseradish peroxidase (HRP). In the second step, fluorescence is enhanced by the mechanical pressure from the precipitate formed by enzyme catalysis. In order to detect hIgE, the surface of immobilized PDA liposomes was conjugated with monoclonal antibodies against hIgE, and fluorescence signals were detected after the antigen-antibody reaction. In this step, hIgE concentrations as low as 10 ng/mL were detected. Fluorescence signals slightly increased when anti-hIgE pAb-HRP was used as an amplifying agent after primary immunoresponse. After secondary immunoresponse, HRP-catalyzed oxidation of 3,3'-diaminobenzidine produced an insoluble precipitate that strongly stimulated PDA liposomes by their weight and pressure, thereby dramatically increasing the fluorescence signal. Thus, PDA liposome immunosensor could detect hIgE concentrations as low as 0.01 ng/mL, representing a 1000-fold increase in sensitivity over the signal generated by the primary immunoresponse. This study indicates that increasing the external mechanical force applied to PDA liposomes by enzyme-catalyzed precipitate formation enhanced the sensitivity of the PDA liposome immunosensor chip. This strategy can be applied to the detection of other biomolecules in experimental or clinical settings where ultrasensitive and highly specific biosensing is required.