A novel enhanced chemiluminescent immunoassay (CLIA) for ultrasensitive and excellent precisive determination of cardiac troponin I (cTnI) was reported. The method made full use of poly[(N-isopropyl acrylamide)-co-(methacrylic acid)] (P(NIPAM-co-MAA)) microspheres as new potential signal enhancers and magnetic fluorescent nanoparticles as internal standards for better precision. This protocol involved a sandwich format, in which the antigen in the sample was captured by the immobilized antibodies on the surface of magnetic fluorescent beads and recognized by the other antibodies labeled with acridinium ester (AE)-loaded P(NIPAM-co-MAA) microspheres. The combination of the remarkable sensitivity of the enhanced CLIA method and the use of P(NIPAM-co-MAA) microspheres as anti-cTnI carriers for acridinium ester signal amplification provided an extremely sensitive limit of blank (LoB) at 0.097 pg mL⁻¹, a limit of detection (LoD) at 0.116 pg mL⁻¹, and a limit of quantitation (LoQ) at 0.606 pg mL⁻¹, much greater than those achieved by the classical chemiluminescence immunoassay (CLIA, Getein). Moreover, the intra-day variable coefficient can be improved to 1.21–2.12%, and inter-day variability was 2.01–3.49% under the application of magnetic fluorescent beads as an internal standard. The sensitivity and precision have reached a high level, comparable with the current commercial detection kits. The results showed a good correlation with a commercial chemiluminescence assay (CLIA, Abbott), with a correlation coefficient of 0.9883. This proposed method has been successfully applied to the clinical determination of cTnI in the human serum.

The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.

Amperometric sensors evaluate current changes that occur as a result of redox reactions under constant applied potential. These changes in current intensity are stoichiometrically related to the concentration of analytes. Owing to their unique features, such as fast reaction velocity, high specificity, abundant existence in nature, and feasibility to be immobilized, enzymes are widely used by researchers to improve the performance of amperometric sensors. Unfortunately, natural enzymes have intrinsic disadvantages due to their protein structures. To overcome these proteinic drawbacks, scientists have developed nanozymes, which are nanomaterials with enzymatic properties. As the result of significant advances in materiology and analytical science, great progress has been achieved in the development of nanozyme-based amperometric sensors with outstanding performance. To highlight achievements made in recent years, we first summarize the development directions of nanozyme-based amperometric sensors. Then, H₂O₂ sensors, glucose sensors, sensors combining natural enzymes with nanozymes, and sensors targeting untraditional specific targets will be introduced in detail. Finally, the current challenges regarding the nanozymes utilized in amperometric sensors are discussed and future research directions in this area are suggested.