Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO_2 nanoparticles

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO2 particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H2O2 forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO2 particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10-6-7.3×10-4 g/L and with a detection limit of 1.5×10-6 g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results.

Nanobody-based immunosensing methods for safeguarding public health

Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical potential.Compared with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing strategy.This minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.

Cyanidin-horseradish peroxidase-hydroperoxide reaction system and its application in enzyme-linked immunosensing assays

A cyanidin-based horseradish peroxidase(HRP)-catalyzed reaction system was established in this work.In B-R buffer solutions(pH 6.8),a UV-visible absorbance peak of cyanidin(CAG) at 540 nm(Ap1) appeared.After the oxidation reaction of CAG catalyzed by HRP in the presence of H2O2,a significant absorbance peak at 482 nm(Ap2) occurred.The ratio R(AP2/AP1)was proportional to the HRP concentration.The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen(FMDVAg) as a model analyte.In sandwich immunoreaction,the analyte FMDVAg and food and mouth disease virus antibody(FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina(Con A) bound with HRP-FMDVAb.After de-absorbing and separating,the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements.The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H2O2,and the substrate CAG is converted to products.Based on this principle,a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe3O4 magnetic nanoparticles.The linear range of the FMDVAg determination is 1.5×10-8-2.7×10-6 g/mL with the relatively standard deviation of 3.7%(n = 11).The detection limit is 3.1×10-9 g/mL.Additional advantages of the typical substrate such as OPD,OAP and TMB are good water-solubility and stability.

An ultrasensitive time-resolved fluorescent immunoassay method for determination aflatoxins B1 in edible oil

Edible oil is one major nutritional ingredient to human and widely consumed directly. The contamination of aflatoxin B1 （AFB1） in edible oils has been attracted exten-sive efforts due to its hazard to human health and life. To avoid the digestion of edible oils contaminated by AFB1 the development of rapid and sensitive sensing method for AFB1 is required. Herein, a quantitative, sensitive and rapid method for AFB1 detection in edible oils was proposed by using ultrasensitive time-resolved fluorescent immunosensing （TRFIS） method. This method poses unique advantages from both time-resolved fluorescent sens-ing method and immunochromatographic assay format. The nanospheres were modified with fluorescent europium and then captured the home-made monoclonal antibody against AFB1 （3G1）. After optimization, by using a competitive immunosensing manner, this TRFIS method has a detectable linear range of 0.54-20.0 μg/kg with minimum detectable concen-tration of 0.18μg/kg. It can be completed merely within 10 min with recovery from 87.0% to 121.9%. The agreement was observed between the results by TRFIS and high perfor-mance liquid chromatography （HPLC） methods. This research provides a promising sens-ing method for sensitive and rapid determining AFB1 in edible oils.

Efficiently Writing Bragg Grating in High-Birefringence Elliptical Microfiber for Label-Free Immunosensing with Temperature Compensation

Immunosensor is a powerful tool in healthcare and clinic,food and drug industry,and environmental protection.Label-free fiber-optic immunosensors have shown a myriad of advantages,such as high sensitivity,anti-electromagnetic interference,and afield measurement via the fiber network.However,the fiber-optic based sensor may bear the temperature cross-talk,especially under the warming condition for bio-activating the immune molecules.In this study,we proposed a highly birefringent microfiber Bragg grating for immunosensing with the temperature-compensation.The birefringent microfiber was drawn from the elliptical cladding multimode fiber that was ablated by the CO2 laser.The considerably large energy overlap region offered by the original multimode fiber favored the efficient inscription of FBG with high reflectivity.The dual reso-nances derived by the orthogonal polarization states presented similar temperature responsivities but significantly different ambient refractive index sensitivities,allowing the temperature-compensational RI sensing.The human immunoglobulin G(IgG)molecules were anchored on the surface of the microfiber grating probe by the covalent functionalization technique to enable the specific detection of the anti-IgG molecule.The proposed method promises a high-efficiency and low-cost design for the microfiber Bragg grating-based biosensor without being subjected to the temperature cross-sensitivity.

Nanoparticles-based nanochannels assembled on a plastic flexible substrate for label-free immunosensing

A novel, cheap, disposable and single-use nanoparticles-based nanochannel platform assembled on a flexible substrate for label-free immunosensing is pre- sented. This sensing platform is formed by the dip-coating of a homogeneous and assembled monolayer of carboxylated polystyrene nanospheres （PS, 200 and 500 nm-sized） onto the working area of flexible screen-printed indium tin oxide/polyethylene terephthalate （ITO/PET） electrodes. The spaces between the self-assembled nanospheres generate well-ordered nanochannels, with inter-PS particles distances of around 65 and 24 nm respectively. The formed nanochannels are used for the effective immobilization of antibodies and subsequent protein detection based on the monitoring of [Fe（CN）6]^4- flow through diffusion and the decrease in the differential pulse voltammetric signal upon immunocomplex formation. The obtained sensing system is nanochannel-size dependent and allows human immunoglobulin G （IgG） （chosen as a model analyte） to be detected at levels of 580 ng/mL. The system also exhibits an excellent specificity against other proteins present in real samples and shows good performance with a human urine sample. The developed device represents an integrated and simple biodetection system which overcomes many of the limitations of previously reported nanochannels-based approaches and can be extended in the future to several other immuno and DNA detection systems.

Signal Amplification for Highly Sensitive Immunosensing

To dissolve the bottleneck problem of life and biomedical science in detection of biomolecules with low abundance and acquisition of ultraweak biological signals,highly sensitive analytical methods coupling with the specificity of biological recognition events have been quickly developed by the exploring of signal amplification strategies.These strategies have extensively been introduced into the development of highly sensitive immunosensing methods by combining with highly specific immunological recognition.They can be divided into two groups.One group of strategies attempts to transfer the immunological recognition event into large number of reporter probes or signal probes for signal readout by employing nano/micro-materials as vehicles for multi-labeling and/or molecular biological amplification for increasing the abundance of the signal molecules.The other uses nanomaterials or enzyme mimics as catalytic tools/tags to obtain enhanced detection signal.This review focuses on the significant advances in design of signal amplification strategies for highly sensitive immunosensing.