Chemiluminescent Nanogels as Intensive and Stable Signal Probes for Fast Immunoassay of SARS-CoV-2 Nucleocapsid Protein.

It is highly desired to exploit good nanomaterials as nanocarriers for immobilizing chemiluminescence (CL) reagents, catalysts and antibodies to develop signal probes with intensive and stable CL properties for immunoassays. In this work, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and Co2+ bifunctionalized polymethylacrylic acid nanogels (PMAANGs-ABEI/Co2+) were synthesized via a facile strategy by utilizing carboxyl group-rich PMAANGs as nanocarriers to immobilize ABEI and Co2+. The obtained PMAANGs-ABEI/Co2+ showed extraordinary CL performance. The CL intensity is 2 orders of magnitude higher than that of previously reported ABEI and Cu2+-cysteine complex bifunctionalized gold nanoparticles with high CL efficiency. This was attributed to the excellent catalytic ability of Co2+ and polymethylacrylic acid nanogels, as well as the improved CL catalytic efficiency from a decreased spatial distance between ABEI and the catalyst. The as-prepared nanogels also possess abundant surface reaction sites and good CL stability. On this basis, a sandwich immunoassay for the nucleocapsid protein of SARS-CoV-2 (N protein) was developed by using magnetic bead connected primary antibody as a capture probe and PMAANGs-ABEI/Co2+ connected secondary antibody as a signal probe. The linear range of the proposed method for N protein detection was 3.16-316 ng/mL, and its detection limit was 2.19 ng/mL (S/N = 3). Moreover, the developed immunoassay was performed with a short incubation time of 5 min, which greatly reduced the detection time for N protein. By using corresponding antibodies, the developed strategy might be applied to detect other biomarkers.

Charge-Dependent Signal Changes for Label-Free Electrochemiluminescence Immunoassays.

Label-free electrochemiluminescence (ECL) immunoassays (lf-ECLIA), based on biomarker-induced ECL signal changes, have attracted increasing attention due to the simple, rapid, and low-cost detection of biomarkers without secondary antibodies and complicated labeling procedures. However, the interaction rule and mechanism between analytical interfaces and biomarkers have rarely been explored. Herein, the interactions between biomarkers and analytical interfaces constructed by assembly of a nanoluminophore and antibody-functionalized gold nanoparticles on an indium tin oxide electrode were studied. The nanoluminophore was synthesized by mixing Cu2+/l-cysteine chelate and N-(4-Aminobutyl)-N-ethylisoluminol-bifunctionalized gold nanoparticles with chitosan. It was found that positively charged biomarkers increased the ECL intensity, whereas negatively charged biomarkers decreased the ECL intensity. The assembly pH influenced the biomarker charges, which determined the ECL enhancement or inhibition. The detection pH only affected the ECL intensity but not the ECL changing trends. Based on the ECL signal changes, a charge-dependent lf-ECLIA was established, which exhibited inhibition responses to negatively charged human immunoglobulin G and copeptin and enhancement responses to positively charged cardiac troponin I, heart-type fatty acid binding protein, brain natriuretic peptide, and SARS-CoV-2 N protein. The linear range was 0.1-1000 pg/mL, and the detection limits were distributed in 0.024-0.091 pg/mL. Besides, a mechanism of the charge-dependent ECL enhancement and inhibition effects is proposed, which is very important for the development of new lf-ECLIA methodologies.

The mechanism and effects of remdesivir-induced developmental toxicity in zebrafish: Blood flow dysfunction and behavioral alterations.

The antiviral drug remdesivir has been used to treat the growing number of coronavirus disease 2019 (COVID-19) patients. However, the drug is mainly excreted through urine and feces and introduced into the environment to affect non-target organisms, including fish, which has raised concerns about potential ecotoxicological effects on aquatic organisms. Moreover, studies on the ecological impacts of remdesivir on aquatic environments have not been reported. Here, we aimed to explore the toxicological impacts of microinjection of remdesivir on zebrafish early embryonic development and larvae and the associated mechanism. We found that 100 µM remdesivir delayed epiboly and impaired convergent movement of embryos during gastrulation, and dose-dependent increases in mortality and malformation were observed in remdesivir-treated embryos. Moreover, 10-100 µM remdesivir decreased blood flow and swimming velocity and altered the behavior of larvae. In terms of molecular mechanisms, 80 differentially expressed genes (DEGs) were identified by transcriptome analysis in the remdesivir-treated group. Some of these DEGs, such as manf, kif3a, hnf1ba, rgn, prkcz, egr1, fosab, nr4a1, and ptgs2b, were mainly involved in early embryonic development, neuronal developmental disorders, vascular disease and the blood flow pathway. These data reveal that remdesivir can impair early embryonic development, blood flow and behavior of zebrafish embryos/larvae, probably due to alterations at the transcriptome level. This study suggests that it is important to avoid the discharge of remdesivir to aquatic ecosystems and provides a theoretical foundation to hinder remdesivir-induced ecotoxicity to aquatic environments.

Label-Free Immunoassay for Sensitive and Rapid Detection of the SARS-CoV-2 Antigen Based on Functionalized Magnetic Nanobeads with Chemiluminescence and Immunoactivity.

Direct detection of SARS-CoV-2 in biological specimens is often challenging due to the low abundance of viral components and lack of enough sensitivity. Herein, we developed a new type of chemiluminescent functionalized magnetic nanomaterial for sensitive detection of the SARS-CoV-2 antigen. First, HAuCl4 was reduced by N-(aminobutyl)-N-(ethylisoluminol) (ABEI) in the presence of amino magnetic beads (MB-NH2) to generate ABEI-AuNPs, which were directly assembled on the surface of MB-NH2. Then, Co2+ was modified onto the surface to form MB@ABEI-Au/Co2+ (MAA/Co2+). MAA/Co2+ exhibited good chemiluminescence (CL) and magnetic properties. It was also found that it was easy for the antibody to be connected with MAA/Co2+. Accordingly, MAA/Co2+ was used as a sensing interface to construct a label-free immunoassay for rapid detection of the N protein in SARS-CoV-2. The immunoassay showed a linear range from 0.1 pg/mL to 10 ng/mL and a low detection limit of 69 fg/mL, which was superior to previously reported methods for N protein detection. It also demonstrated good selectivity by virtue of magnetic separation, which effectively removed a sample matrix after immunoreactions. It was successfully applied for the detection of the N protein in spiked human serum and saliva samples. Furthermore, the immunoassay was integrated with an automatic CL analyzer with magnetic separation to detect the N protein in patient serums and rehabilitation patient serums with satisfactory results. Thus, the CL immunoassay without a complicated labeling procedure is sensitive, selective, fast, simple, and cost-effective, which may be used to combat the COVID-19 pandemic. Finally, the CL quenching mechanism of the N protein in the immunoassay was also explored.

Automatic label-free immunoassay with high sensitivity for rapid detection of SARS-CoV-2 nucleocapsid protein based on chemiluminescent magnetic beads.

Accurate and efficient early diagnosis is crucial for the control of COVID-19 pandemic. However, methods that can balance sensitivity, high throughput, detection speed and automation simultaneously are still scarce. Here, we report an automatic label-free chemiluminescence immunoassay (CLIA) for rapid SARS-CoV-2 nucleocapsid protein (NP) detection with high sensitivity and throughput. N-(4-aminobutyl)-N-ethylisoluminol and Co2+ dual-functionalized chemiluminescent magnetic beads (dfCL-MB) were first applied to the detection of protein by a novel and simple strategy. Sulphydryl polyethylene glycol was coated on the surface of dfCL-MB so as to assemble dfCL-MB and antibody conjugated gold nanoparticles through Au-S bond. Considering the high-risk application scenarios, the immunosensor was integrated with an automatic chemiluminescence analyzer so that the whole testing procedure could be carried out automatically without manual operation. A linear correlation between CL intensities and the logarithm of NP concentration was obtained in the range of 0.1-10,000 pg/mL with a detection limit of 21 fg/mL. The whole process cost 25 min and the sample compartment can bear 24 samples simultaneously. The spiked human serum samples and serum samples from COVID-19 patients were determined with satisfactory recoveries of 91.1-109.4%, suggesting that the proposed label-free CLIA is of great potential for SARS-CoV-2 NP detection in practice.

COVID-19 Outbreak and Management Approach for Families with Children on Long-Term Kidney Replacement Therapy.

BACKGROUND AND OBJECTIVES: During the coronavirus disease 2019 outbreak, the treatment of families with children on long-term KRT is challenging. This study was conducted to identify the current difficulties, worries regarding the next 2 months, and mental distress experienced by families with children on long-term KRT during the coronavirus disease 2019 outbreak and to deliver possible management approaches to ensure uninterrupted treatment for children on long-term KRT. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A multicenter online survey was conducted between February 10 and 15, 2020, among the families with children on long-term KRT from five major pediatric dialysis centers in mainland China. The primary caregivers of children currently on long-term KRT were eligible and included. Demographic information, severe acute respiratory syndrome coronavirus 2 infection status, current difficulties, and worries regarding the next 2 months were surveyed using a self-developed questionnaire. The Patient Health Questionnaire-9 and the General Anxiety Disorder Scale-7 were used to screen for depressive symptoms and anxiety, respectively. RESULTS: Among the children in the 220 families included in data analysis, 113 (51%) children were on dialysis, and the other 107 (49%) had kidney transplants. No families reported confirmed or suspected cases of coronavirus disease 2019. Overall, 135 (61%) and 173 (79%) caregivers reported having difficulties now and having worries regarding the next 2 months, respectively. Dialysis supply shortage (dialysis group) and hard to have blood tests (kidney transplantation group) were most commonly reported. A total of 29 (13%) caregivers had depressive symptoms, and 24 (11%) had anxiety. After the survey, we offered online and offline interventions to address their problems. At the time of the submission of this paper, no treatment interruption had been reported. CONCLUSIONS: The coronavirus disease 2019 outbreak has had physical, mental, logistical, and financial effects on families with children on long-term KRT.