Exploring the entropy-driven amplification reaction and trans-cleavage activity of CRISPR-Cas12a for the development of an electrochemiluminescence biosensor for the detection of the SARS-CoV-2 RdRp gene in real samples and environmental surveillance

In this paper, we present the first idea of using a DNA triple helix structure to inhibit CRISPR-Cas12a activity and apply it to the design of an electrochemiluminescent biosensor for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) gene in real samples and environmental surveillance. We employed a segment from the RdRp gene of SARS-CoV-2 by an entropy-driven reaction, which was paired with double-stranded DNA that can activate CRISPR-Cas12a activity by Hoogsteen pairing to form triple-stranded DNA, thereby inhibiting the binding interaction of the double-stranded DNA with CRISPR-Cas12a, which in turn inhibits the trans cleavage activity of CRISPR-Cas12a. The inhibited CRISPR-Cas12a is unable to cut the nucleic acid modified on the electrode surface, resulting in the inability of the ferrocene (Fc) modified on the other end of the nucleic acid to move away from the electrode surface, and thus failing to cause electrochemiluminescence changes in GOAu-Ru modified on the electrode surface. The extent of the electrogenic chemiluminescence change can reflect the concentration of the gene to be tested. Using this system, we achieved the detection of the SARS-CoV-2 RdRp gene with a detection limit of 32.80 aM.

SARS-CoV-2 monitoring by automated target-driven molecular machine-based engineering.

Biosensors based on nucleic acid-structured electrochemiluminescence are rapidly developing for medical diagnostics. Here, we build an automated DNA molecular machine on Ti3C2/polyethyleneimine-Ru(dcbpy)3 2+@Au composite, which alters the situation that a DNA molecular machine requires laying down motion tracks. We use this DNA molecular machine to transduce the target concentration information to enhance the electrochemiluminescence signal based on DNA hybridization calculations. Complex bioanalytical processes are centralized in a single nucleic acid probe unit, thus eliminating the tedious steps of laying down motion tracks required by the traditional molecular machine. We found a detection limit of 0.68 pM and a range of 1 pM to 1 nM for the analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific DNA target. Recoveries range between 96.4 and 104.8% for the analysis of SARS-CoV-2 in human saliva. Supplementary Information: The online version contains supplementary material available at 10.1007/s10311-022-01434-9.

COVID-19 countermeasures of Chinese national athletes: Prevention, treatment, and return to play.

Under the condition of normalized epidemic, how athletes train and compete well has been in the spotlight. This article reported the symptom, hospitalization and training situation of seven confirmed cases of coronavirus-disease-2019 (COVID-19) among Chinese national teams. Moreover, the paper summarized the experience of Chinese national teams in terms of epidemic prevention and control, treatment of infection, and safe return to play. Through a scientific combination of medication and non-medical treatment, seven athletes were all discharged from the hospital. These discharged athletes underwent strict isolation and scientific training before returning to sports teams. Before returning to play, continuous monitoring of physical and mental condition was required. All seven athletes returned to play safely and performed excellently. As for hosting large-scale sporting events, the entire enclosed-loop management from immigration to competition was proposed in this paper. This study could serve as a standard of epidemic prevention and control, treatment for infection and safe return to play during competition and training around the world.

Hybridization chain reaction circuit-based electrochemiluminescent biosensor for SARS-cov-2 RdRp gene assay.

In this work, we designed an ECL ratiometric biosensor with a three-stranded Y-type DNA (Y-DNA) probe and induced a hybridization chain reaction (HCR) for the highly sensitive detection of SARS-CoV-2 nucleic acid. The important component of this system is the self-assembled Y-Shaped probe based on three nucleic acids. Y1, Y2, and Y3 can be linked by complementary base pairing to Hairpin1 (H1), Hairpin2 (H2), and Ru modified DNA (Ru1), respectively. H1 and H2 can trigger the HCR reaction when activated by the SARS-CoV-2 RdRp gene and the 5' end of Ru1. The 5' end of Ru1 is modified with the Ru complex, which can produce a strong electrochemiluminescence luminescence signal at 620 nm under an applied voltage. Through the amplification of Y-DNA-induced HCR reaction, Ru1 on the electrode surface gradually increased, the ECL signal at 460 nm was gradually quenched, and the signal at 620 nm was steadily generated. The SARS-CoV-2 RdRp gene can be quantified according to the degree of decrease of ECL signal at 460 nm and the increase of ECL signal at 620 nm. Combining the two signal amplification strategies, this ratiometric ECL biosensor can accurately and efficiently detect the target gene with a detection limit of 59 aM.

A pH-engineering regenerative DNA tetrahedron ECL biosensor for the assay of SARS-CoV-2 RdRp gene based on CRISPR/Cas12a trans-activity.

In this work, we constructed an exonuclease III cleavage reaction-based isothermal amplification of nucleic acids with CRISPR/Cas12a-mediated pH-induced regenerative Electrochemiluminescence (ECL) biosensor for ultrasensitive and specific detection of SARS-CoV-2 nucleic acids for SARS-CoV-2 diagnosis. The triple-stranded nucleic acid in this biosensor has an extreme dependence on pH, which makes our constructed biosensor reproducible. This is essential for effective large-scale screening of SARS-CoV-2 in areas where resources are currently relatively scarce. Using this pH-induced regenerative biosensor, we detected the SARS-CoV-2 RdRp gene with a detection limit of 43.70 aM. In addition, the detection system has good stability and reproducibility, and we expect that this method may provide a potential platform for the diagnosis of COVID-19.

A strategy combining 3D-DNA Walker and CRISPR-Cas12a trans-cleavage activity applied to MXene based electrochemiluminescent sensor for SARS-CoV-2 RdRp gene detection.

Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.

Entropy-driven amplified electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with self-assembled DNA tetrahedron scaffolds.

Dependable, specific and rapid diagnostic methods for severe acute respiratory syndrome ß-coronavirus (SARS-CoV-2) detection are needed to promote public health interventions for coronavirus disease 2019 (COVID-19). Herein, we have established an entropy-driven amplified electrochemiluminescence (ECL) strategy to detect the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 known as RdRp-COVID which as the target for SARS-CoV-2 plays an essential role in the diagnosis of COVID-19. For the construction of the sensors, DNA tetrahedron (DT) is modified on the surface of the electrode to furnish robust and programmable scaffolds materials, upon which target DNA-participated entropy-driven amplified reaction is efficiently conducted to link the Ru (bpy)32+ modified S3 to the linear ssDNA at the vertex of the tetrahedron and eventually present an "ECL on" state. The rigid tetrahedral structure of the DT probe enhances the ECL intensity and avoids the cross-reactivity between single-stranded DNA, thus increasing the sensitivity of the assays. The enzyme-free entropy-driven reaction prevents the use of expensive enzyme reagents and facilitates the realization of large-scale screening of SARS-CoV-2 patients. Our DT-based ECL sensor has demonstrated significant specificity and high sensitivity for SARS-CoV-2 with a limit of detection (LOD) down to 2.67 fM. Additionally, our operational method has achieved the detection of RdRp-COVID in human serum samples, which supplies a reliable and feasible sensing platform for the clinical bioanalysis.

Entropy-driven electrochemiluminescence ultra-sensitive detection strategy of NF-κB p50 as the regulator of cytokine storm.

2019 novel coronavirus (2019-nCoV) with strong contagion in the crowd, has ravaged worldwide and severely impacts the human health and epidemic prevention system, by producing a series of significant stress reactions in the body to induce further cytokine storm. Transcription factors (TFs) served as essential DNA binding proteins play an integral role in regulating cytokine storm, and the detection of it in the human coronavirus environment provides especially valuable approaches to diagnosis and treatment of 2019-nCoV and development of antiviral drugs. In this work, an entropy-driven electrochemiluminescence (ECL) biosensor was constructed for ultra-sensitive bioassay of NF-κB p50. The strategy primarily capitalizing the splendid double-stranded DNA (dsDNA) binding properties of transcription factors, employing GOAu-Ru composite material as ECL emitter, utilizing entropy-driven reactions for signal amplification method, offered a repeatable proposal for TFs detection. In the absence of TFs, the released DNA1 further went in the entropy-driven reaction, contributing to an "ECL off" state. However, in the presence of TFs, the dsDNA avoided being digested, which blocked DNA1 for participating in the entropy-driven reaction, and the system exhibited an "ECL on" state. Most importantly, the ECL bioanalytical method denoted broad application prospects for NF-κB p50 detection with a lower detection limit (9.1 pM).