Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: A femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG(3)-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and Fr-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG(6)-COOH and HS-OEG(3)-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52 ± 3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 g/100 g of shellfish meat.

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×106–1×102 oocysts ml-1. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Abnormal anti-Stokes Raman scattering of carbon nanotubes

Abnormal anti-Stokes Raman scattering (AASR) was unambiguously observed in carbon nanotubes (CNT's). In contrast to traditional Raman scattering theory, the absolute value of the Raman frequency of the anti-Stokes peak is not the same as that of the corresponding Stokes peak. It was demonstrated that AASR scattering originates from the unique nanoscale cylindrical structure of CNT's that can be considered naturally as a graphite structure with an intrinsic defect from its rolling. The double-resonance Raman scattering theory was applied to interpret the scattering mechanism of the AASR phenomenon successfully and quantitatively.