| The immunochromatographic assay, with many advantages over traditional immunoassays such as rapid operation, simplicity of the procedure, stability of the labeling material, low cost, no requirements for skilled technicians or expensive equipment, has been accepted in the fields of disease diagnosis, food safety and environment monitoring for qualitative and quantitative detection. In gold nanoparticle-based immunochromatographic assay the gold nanoparticle (AuNPs) labeled antigen or antibody is used as the tracer, and developed based on the specific reaction between an antigen and its antibody. Most of these techniques were developed using nitrocellulose membrane (NCM) as the carrier material. The high protein binding capacity of NCM may facilitate the immobilization of antibodies. However, because of the complicated production process the binding between protein and NCM is greatly affected by the pore diameter of NCM, sample solution, the condition of blocking, environmental conditions including temperature and relative humidity. Meanwhile, the NCM is subject to breakage, compression, and scoring during processing. Towing phenomena, diffusion phenomena, and significant deviation (inter and intra) always exist in chromatography because of the complicated and heterogeneous structure of NCM. For these reasons, NCM-based immunochromatographic assays have relatively poor sensitivity, reproducibility, and repeatability, which limits their use in real applications. Therefore, it is urgent and necessary to develop a more simple, rapid, sensitive and low-cost screening technology techniques.In this study, a new strategy is proposed for the design and fabrication of an immunochromatographic assay based on a glass capillary. This is mainly motivated by the unique advantages of the glass capillary in comparison to other materials:low cost, less sample usage, easily modified, and favorable optical properties. More importantly, the uniform smooth surface and the high rigidity and ionic strength tolerance of the glass matrix make it more robust than flexible materials regarding possible interference from the environment and the sample matrix, which can be very helpful for improving the accuracy and precision of the analytical operations. Herein, a nano-gold capillary immunochromatographic assay (CICA) emulating the lateral-flow format was designed and fabricated, the main variables affecting the CICA response were investigated, and the performing conditions were optimized. The main results are listed as follows:Three different diameters of gold nanoparticle were prepared by sodium citrate reduction method, and the AuNPs-labeled anti-PV conjugate was prepared under the optimal conditions. The specific bonding strength between antigen and its AuNP-labeled anti-PV conjugate was estimated by ELISA. The anti-PV antibody labeled with AuNPs of 13 nm with the highest stability and antigen binding activity was chosen for further experiments. A novel non-instrumental bioanalysis based on colloidal-gold immunochromatography in a modified glass capillary was developed and named capillary immunochromatographic assay (CICA). Goat anti-rabbit IgG and parvalbumin (PV) were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively.The CICA was constructed, and main variables for the performance were optimized. Using an important allergen of fish products (parvalbumin, PV) as the target, the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 70 ng mL-1 and 40 ng mL-1, respectively. The coefficient of variation (CV) for the intra-assay and inter-assay, the accuracy and stability of CICA were confirmed. The developed technique was implemented and validated with different foodstuffs, including Scophthalmus maximus (Linnaeus), surimi products, and livestock, confirming sufficient accuracy and precision of results and verifying the method to be efficacious. These results enabled us to propose CICA as a new and promising technique for simple, rapid, and on-site screening of PV in biological samples.Furazolidone (FZD), possible carcinogen and mutagen to humans, has been banned from using in food animals, but its continued misuse is widespread in aquacultures. A novel rapid, regenerable colloidal-gold capillary immunochromatographic assay (CICA) based on competitive format was developed for the furazolidone, a low-molecular weight compound, to verify its universal applicability, high sensitivity and tolerance for complex matrix samples. Goat anti-mouse IgG and coating antigen were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively. The main variables for the performance were optimized and the metabolite of furazolidone (3-amino-2-oxazolidinone, AOZ) was chosen as the target. Under the optimized conditions, the analytical efficiency of the developed technique was investigated and the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 0.65 ng mL-1 and 0.26 ng mL-1, respectively. The entire operation, including sample preparation, was consistently performed in 2 h. Meanwhile, the proposed CICA exhibited good precision, selectivity, acceptable stability, reproducibility and regenerability. The CICA has also been successfully implemented and validated to the AOZ analysis with different foodstuffs, which enabled us to propose it as a new and promising technique for simple, rapid, and on-site screening of furazolidone in biological samples.The modified glass capillary with many excellent characteristics was used as supporting material of the CICA, which makes it possible to reuse. The regeneration of immunoassays has greatly attracted attention of many scholars, which can decrease deviation and save materials, especially for expensive biological materials. The reusability of the CICA was investigated by different dissociation solutions, and 0.1M Gly-HCl (pH=2.8) containing 1% DMSO and methanol was chosen as regeneration solution in currently practiced. The main variables for the regeneration process were optimized, and the varying time was in 60mn after incubation, the optimal dissociation temperature was 20 ℃, the optimal dissociation time was 5min. It was found that this method could remove the AuNP-labeled anti-PV conjugate from the wall of CICA completly, and regenerate the CICA for 3 times with the most acceptable percentage variation. So the sensitivity and stability of the post-regeneration CICA was essentially unchanged from the initial result.The glass capillary with many advantages including low cost, easily modified, the uniform smooth surface and favorable optical properties, was proposed as a support in immunochromatographic assay. Surface modification of capillary is one of the key factors affecting quality of CICA. Capillary was modified with amino group, epoxy group and aldehyde group. Immunochromatographic assays were established based on different modified glass capillary, and was used for FZD detection. To summarize, the modification with space arms and dendrimer was beneficial to the stability of CICA, but little influence on sensitivity and regeneration.A novel non-instrumental capillary immunochromatographic assay based on a modified glass capillary was developed and the main variables for the performance were optimized detailedly. The proposed capillary immunochromatographic assay was successfully realized the rapid detection for macromolecular and small molecule with high sensitivity requirements in aquatic products. Appropriate modification method of capillary and regeneration were also discussed in detail. Thus the proposed CICA offers a high sensitivity, fast response and good stability, which broadened the insight of immunochromatographic assay, and established a theoretical basis and operational basis for establishing rapid detection method with high sensitivity and stability. |
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