New Chemiluminescence Immunoassay Methods Based On Atomic-scale Catalysts

In view of many merits such as low background interference,high sensitivity,and ideal specificity,chemiluminescence immunoassay（CLIA）has substantially been utilized in various fields such as drug analysis,clinical diagnosis,food safety and so forth.It’s inefficient of traditional reactions to convert chemical energy into light energy.And thus,catalysts are required to enhance the CL signal for improving the sensitivity of detection.Although natural enzymes have massively served as typical catalysts with high catalytic activity,they exhibit some drawbacks such as complex preparation process,high cost and poor stability.The catalytic effect of these reported nanomaterials with CL catalytic performance is still not ideal,and there are shortcomings such as uneven active sites,unsatisfactory catalytic activity and unstable properties.Therefore,it is of great significance to develop catalysts with excellent catalytic activity,uniform active sites and stable properties.Single-atom catalysts（SACs）are a class of catalysts in which active metal atoms exist as atomic sites on the supports.They show the highest atomic utilization and excellent catalytic activity.The catalytic activity of SACs is significantly improved compared with traditional nanocatalysts due to its atomically dispersed size and uniform coordination structure.Therefore,SACs are currently used in the fields of organic catalysis,energy conversion,biotherapy and biochemical analysis.In this study,SACs with prominent catalytic activity and cluster-assisted SACs were obtained based on two synthesis strategies,and the catalytic mechanism for CL reaction was explored.Subsequently,they were used as probes to develop CLIA methods for ultra-sensitive detection of chlorothalonil（CTL）and imidacloprid（ICP）in Chinese herbal medicines.The related work has been published in Analytical Chemistry.The specific research contents are as follows:1.A chemiluminescence immunoassay for the detection of chlorothalonil based on ultra-thin layered double hydroxides loaded Co SACsIn this experiment,ultra-thin monolayer Co Al layered double hydroxides（Co Al-LDHs）were synthesized by using formamide as a layer growth inhibitor to control interlayer growth.The aberration-corrected scanning transmission electron microscopy and synchrotron X-ray absorption spectroscopy were used to prove that Co atoms were dispersed on the LDHs in the form of single atoms,and the active site was Co-N₄.The results of radical scavenger experiments and electron paramagnetic resonance spectroscopy showed that Co SACs accelerated the decomposition of H₂O₂ to produce a large number of reactive oxygen species（ROS）,and then significantly catalyze the luminol CL reaction（1642.6 times）.Based on its excellent catalytic effect,SACs were labeled on CTL antibody by the electrostatic interaction method as a CL signal probe,and a CLIA method for sensitive detection of CTL was developed.The experimental results showed that the detection range of CTL was 0.05-50 ng m L^-1,and the detection limit was 4.9 pg m L^-1（3σ）.Other common pesticides showed low interference,indicating a satisfactory selectivity of the Co SACs-based CLIA protocol.Finally,Chuanxiong Rhizoma,Chinese angelica and Radix Astragali were used as mock samples for spiked recovery experiments.The recoveries were 90.60-109.92,95.74-106.68 and 86.37-91.80%,respectively,indicating that the ideal practicability of the sensitive CLIA for quantitating pesticide residues.2.A chemiluminescence immunoassay for the detection of imidacloprid based on synergistic dual-site atom catalystIn this experiment,Co₃N@Co SACs with Prussian blue nanoparticles as the carrier were synthesized by a facile doping method.The catalytic active sites were proved to be Co₃N clusters and Co-O₄ single atom sites by synchrotron X-ray absorption spectroscopy.In addition,Co₃N@Co SACs significantly increased the luminol CL signal by 2155.0 times,which is significantly better than the enhancement effect of pure Co SACs（98.4 times）.The results of radical scavenger experiments and electron paramagnetic resonance spectroscopy show that the catalytic mechanism of Co₃N@Co SACs is that the double-site atom catalyst can accelerate the decomposition of H₂O₂into ¹O₂ and O₂^·-,thus showing excellent catalytic performance.Subsequently,Co₃N@Co SACs were labeled on ICP antibody by the electrostatic interaction method and used as CL signal probe to establish CL immunochromatographic test strips platform for rapid and sensitive quantitative detection of ICP.The quantitative range of ICP is 0.05-10 ng m L^-1 with a detection limit of 1.7 pg m L^-1（3σ）.The interference levels for other common pesticides are negligible,indicating a satisfactory selectivity of the Co₃N@Co SACs-based protocol.Finally,the method has been successfully applied on the spiked recovery of chrysanthemum,honeysuckle and Lycium chinensis samples.The recoveries were 95.19-103.05,83.42-106.66 and 91.36-92.17%,respectively,indicating that the method shows good practicability.In summary,two synthetic schemes were adopted to obtain two atomic catalysts in the study.These two atomic-level catalysts exhibited significant catalytic effect on the luminol CL system.The successful anchoring of single atoms and the active sites of the catalyst were proved by aberration-corrected scanning transmission electron microscopy and synchrotron X-ray absorption spectroscopy.At the same time,the catalytic mechanism of the catalysts was further explored.The CLIA methods based on atomic scale catalysts show the advantages of high sensitivity,ideal specificity and wide linear range.This study expands the application of SACs in the field of CLIA and provides new ideas for the synthesis of SACs suitable for analysis.