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.

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.

Rational engineering the DNA tetrahedrons of dual wavelength ratiometric electrochemiluminescence biosensor for high efficient detection of SARS-CoV-2 RdRp gene by using entropy-driven and bipedal DNA walker amplification strategy.

Fast and effective detection of epidemics is the key to preventing the spread of diseases. In this work, we constructed a dual-wavelength ratiometric electrochemiluminescence (ECL) biosensor based on entropy-driven and bipedal DNA walker cycle amplification strategies for detection of the RNA-dependent RNA polymerase (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The entropy-driven cyclic amplification reaction was started by the SARS-CoV-2 RdRp gene to generate a bandage. The bandage could combine with two other single-stranded S1 and S2 to form a bipedal DNA walker to create the following cycle reaction. After the bipedal DNA walker completed the walking process, the hairpin structures at the top of the DNA tetrahedrons (TDNAs) were removed. Subsequently, the PEI-Ru@Ti3C2@AuNPs-S7 probes were used to combine with the excised hairpin part of TDNAs on the surface of Au-g-C3N4, and the signal change was realized employing electrochemiluminescence resonance energy transfer (ECL-RET). By combining entropy-driven and DNA walker cycle amplification strategy, the ratiometric ECL biosensor exhibited a limit of detection (LOD) as low as 7.8 aM for the SARS-CoV-2 RdRp gene. As a result, detecting the SARS-CoV-2 RdRp gene in human serum still possessed high recovery so that the dual-wavelength ratiometer biosensor could be used in early clinical diagnosis.

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.

"Covalent biosensing" enables a one-step, reagent-less, low-cost and highly robust assay of SARS-CoV-2.

We have established a new protocol for detecting severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) using a peptidomimetic to covalently detect a viral marker protease.

Thiol-sensitive probe enables dynamic electrochemical assembly of serum protein for detecting SARS-Cov-2 marker protease in clinical samples.

The poor situational awareness about the spreading of the virus especially in the underdeveloped regions calls for novel virus assays of low cost and simple operation. Currently, such assays are exclusively restricted to nucleic acid detection. In this investigation, a virus protein serum assay has been proposed in a one-step and reagent-less route. Specifically, in this assay, the main protease of the virus is targeted by a short probe mimicking its substrate. While the probe-protein interaction brings them together, a fluorescent thiol targeting molecule reacts with the free thiol groups on the target protein near the probe, generating a fluorescence signal proportional to the concentration of the target. This induces an electroactive 2D peptide nano-network on the sensing surface only in the presence of the target protein. The sensitivity of the method is enhanced through potential electrochemical scanning during incubation with serum samples. The successful detection of the virus marker protein in the serum of the infected patients encourages further development of incorporation of this method into clinical practice.

Electrochemiluminescence platform for transcription factor diagnosis by using CRISPR-Cas12a trans-cleavage activity.

Herein, we exploited the double-stranded DNA (dsDNA) binding property of transcription factor (TF), combined with the trans cleavage characteristic of CRISPR-Cas12a, for the detection of NF-κB p50.

Nanoparticle-based fluorescence probe for detection of NF-κB transcription factor in single cell via steric hindrance.

A novel nanoparticle-based fluorescence probe was developed for NF-κB transcription factor detection and in situ imaging via steric hindrance. The probe contains gold nanoparticles (AuNPs) to quench fluorescence, and nucleic acids immobilized on the surface of AuNPs to output fluorescence. In the basal state, Cy5 labeled DNA1 folds its long chain into a hairpin structure and quenches fluorescence by forcing the Cy5 fluorophore close to the surface of AuNPs. After the probe enters the cell, the NF-κB transcription factor can bind to the κB site in the DNA duplex of the nucleic acids. The steric hindrance caused by NF-κB leads to the extension of the long chain of DNA1 and the removal of the Cy5 fluorophore from the surface of AuNPs, thereby restoring the fluorescence of the probe. By measuring NF-κB in cell lysis in vitro, the probe obtains a detection limit of 0.38 nM and the linear range from 0.5 to 16 nM. Repeated measurements showed the recovery in the cell nuclear extract was between 93.38 and 109.32%, with relative standard deviation less than 5%. By monitoring the sub-localization of the Cy5 fluorophore in single cell, the probe system can effectively distinguish active NF-κB (nucleus) and inactive NF-κB (cytoplasm) through in situ imaging. The well-designed probe will make up for the shortcomings of the existing technology, and reveal the regulatory role of transcription factors in many disease processes.

Functional Carbazole-Fullerene Complexes: A New Perspective of Carbazoles Acting as Nano-Octopus to Capture Globular Fullerenes.

Fullerene host-guest constructs have attracted increasing attention owing to their molecular-level hybrid arrangements. However, the usage of simple carbazolic derivatives to bind with fullerenes is rare. In this research, three novel carbazolic derivatives, containing a tunable bridging linker and carbazole units for the capturing of fullerenes, are rationally designed. Unlike the general concave-convex interactions, fullerenes could interact with the planar carbazole subunits to form 2-dimensional hexagonal/quadrilateral cocrystals with alternating stacking patterns of 1 : 1 or 1 : 2 stoichiometry, as well as the controllable fullerene packing modes. At the meanwhile, good electron-transporting performances and significant photovoltaic effects were realized when a continuous C60⋅⋅⋅ C60 interaction channel existed. The results indicate that the introduction of such carbazolic system into fullerene receptor would provide new insights into novel fullerene host-guest architectures for versatile applications.

Rational Engineering of the DNA Walker Amplification Strategy by Using a Au@Ti3C2@PEI-Ru(dcbpy)32+ Nanocomposite Biosensor for Detection of the SARS-CoV-2 RdRp Gene.

The detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for preventing and controlling infectious diseases and disease treatment. In this work, a Au@Ti3C2@PEI-Ru(dcbpy)32+ nanocomposite-based electrochemiluminescence (ECL) biosensor was rationally designed, which realized sensitive detection of the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. In addition, a DNA walker was also used to excise the hairpin DNAs under the action of Nb.BbvCI endonuclease. Furthermore, model DNA-Ag nanoclusters (model DNA-AgNCs) were used to quench the initial ECL signal. As a result, the ECL biosensor was used to sensitively detect the SARS-CoV-2 RdRp gene with a detection range of 1 fM to 100 pM and a limit of detection of 0.21 fM. It was indicated that the ECL biosensor had a great application potential for clinical medical detection. Furthermore, the DNA walker amplification also played a reliable candidate strategy for other detection methods.

Electrochemiluminescence aptasensor for Siglec-5 detection based on MoS2@Au nanocomposites emitter and exonuclease III-powered DNA walker.

Lectins are highly specific binding proteins for glycoproteins which widely exist in living organisms, playing a vital role in exploring the biological evolution process, such as cellular proliferation, differentiation, carcinogenesis and apoptosis. Therefore, the content monitoring of lectin becomes particularly significant and urgent in the bioanalytical application. In this work, we fabricated an aptasensor, majorly capitalizing the eminent affinity between sialic acid-binding immunoglobulin (Ig)-like lectin 5 (Siglec-5) and nucleic acids aptamer (K19), with nontoxic MoS2@Au nanocomposites as electrochemiluminescence (ECL) emitters based on exonuclease III (Exo III)-powered DNA walker for the bioassays of Siglec-5. The DNA track was constructed on the emitters' surface, providing a reliable platform for the DNA walker's autonomous move. In the assay, the primer DNA in the DNA duplex was replaced by Siglec-5 due to the aptamer interactions and repeatedly released to participate in the movement of the DNA walker, further triggering cascade signal amplification. Finally, our aptasensor indicates significant potential for assays of Siglec-5 with a detection limit of 8.9 pM.

γ-Tocotrienol reverses multidrug resistance of breast cancer cells through the regulation of the γ-Tocotrienol-NF-κB-P-gp axis.

The problem of multidrug resistance (MDR) presents a major obstacle in the chemotherapy of cancer. The MDR phenotype is often linked to the overexpression of ATP-binding cassette (ABC) transporters, that pumps out and decreased intracellular drug accumulation. γ-Tocotrienol, an unsaturated tocopherol belonging to the vitamin E family, has been shown to reverse the MDR of MCF-7/Adr cell. To reveal the role of γ-tocotrienol-NF-κB-P-gp axis in the reversal process, the expression level of mdr1/P-gp was determined by real-time PCR and western blot, while NF-κB activity was detected by immunofluorescence and NF-κB transcriptional activity reporter assay. Besides, mdr1 promoter activity and P-gp transport capacity were measured with the effect of γ-tocotrienol and NF-κB agonist/antagonist. Results showed that γ-tocotrienol effectively inhibited the expression levels of mdr1 mRNA and P-gp protein. It is demonstrated that γ-tocotrienol also suppressed mdr1 promoter activity and the efflux activity of P-gp. In addition, the activation of NF-κB signaling pathway and the transcriptional activity of NF-κB were both reduced by γ-tocotrienol. Evidences also showed that the NF-κB pathway is really involved in the regulation of the expression and function of mdr1/P-gp. Taken together, we confirmed that γ-tocotrienol reversed the MDR of MCF-7/Adr through the signaling pathway of NF-κB and P-gp.

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).

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.

Exploring the trans-cleavage activity of CRISPR-Cas12a for the development of a Mxene based electrochemiluminescence biosensor for the detection of Siglec-5.

Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a's side-cutting feature to build a 2D ultra-thin Ti3C2Tx (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.

Dual-Wavelength Electrochemiluminescence Ratiometric Biosensor for NF-κB p50 Detection with Dimethylthiodiaminoterephthalate Fluorophore and Self-Assembled DNA Tetrahedron Nanostructures Probe.

In this work, we fabricated a dual-wavelength electrochemiluminescence ratiometric biosensor based on electrochemiluminescent resonance energy transfer (ECL-RET). In this biosensor, Au nanoparticle-loaded graphitic phase carbon nitride (Au-g-C3N4) as a donor and Au-modified dimethylthiodiaminoterephthalate (TAT) analogue (Au@TAT) as an acceptor were investigated for the first time. Besides, tetrahedron DNA probe was immobilized onto Au-g-C3N4 to improve the binding efficiency of the transcription factor and ECL ratiometric changes on the basis of the ratio of ECL intensities at 595 and 460 nm, which were obtained through the formation of a sandwich structure of DNA probe-antigen-antibody. Our biosensor achieved the assay of NF-κB p50 with a detection limit of 5.8 pM as well as high stability and specificity.

Determination of the concentration of transcription factor by using exonuclease III-aided amplification and gold nanoparticle mediated fluorescence intensity: A new method for gene transcription related enzyme detection.

Herein, we report a new probe for the determination of the concentration of NF-κB p50, one kind of DNA-binding transcription factors (TFs), by using Exonuclease III (Exo III)-aided amplification and gold nanoparticle mediated fluorescence intensity. Since TFs play critical roles in various biological processes, the detection of TFs can provide a lot of useful biological information for studding gene expression regulation related disease. In our system, in the presence of transcription factor, Exo III based amplification reaction was trigged. This enzymatic digestion results in the release of intermediate DNA and ultimately liberating the fluorophore (which, separated from the quencher of AuNP and BHQ2, now fluoresces). The released intermediate DNA then hybridizes with another strand3, whence the cycle starts anew. So, the fluorescence intensity reflects the NF-κB p50 concentration with a detection limit of 1.32 pM. Importantly, this method might be further extended to selectively detect various dsDNA-binding proteins by simply changing the binding-site sequences of strand1/strand2 duplex probes.

Endovascular stent repair of celiac arterial aneurysm.

The aim of this study was to evaluate the safety and clinical effectiveness of endovascular stent repair of celiac arterial aneurysm (CAA).From January 2015 to December 2018, 11 patients (7 males, 4 females with a mean age of 52.2 ±â€Š7.9 years) underwent endovascular stent repair of CAA in our center. A covered stent was used to occlude the CAA neck. Follow-up was performed 2 weeks and 1, 3, 6, and 12 months following surgery and yearly thereafter. Rates of technical success, treatment-related complications, and long-term outcome were analyzed.Each patient was placed with 1 stent for repair of CAA. Stent was placed in the celiac and common hepatic arteries for 10 patients and was placed in the celiac artery for 1 patient. The duration of the procedure ranged from 50 to 75 minutes (mean 63.2 ±â€Š7.2 minutes). The rate of technical success of the endovascular stent repair was 100%. No patient experienced CAA rupture or instant endoleak during or after stent insertion. Abdominal pain was relieved progressively after stent insertion. All patients were followed-up for 6 to 48 months (mean 22.4 ±â€Š10.8 months). All patients were alive during the follow-up. No endoleaks were experienced during follow-up with 100% stent patency rate. No patient suffered splenic, hepatic, or bowel infarction during follow-up.Endovascular stent repair is a safe, simple, and effective treatment for patients with CAA.