Reporter Probe Research Articles

Entropy-driven assisted T7 RNA polymerase amplification-activated CRISPR/Cas13a activity for SARS-CoV-2 detection in human pharyngeal swabs and environment by an electrochemiluminescence biosensor

In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.

Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.

Versatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high “on” signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.

Quadruple cascade strand displacement amplification reaction mediated polyfluorene derivative-based multiple FRET for synchronous determination of multiple biomarkers

Currently reported detections for biomarkers of Alzheimer’s disease (AD) were usually single-target detections, which were prone to generate misdiagnosis. This work developed quadruple cascade strand displacement amplification (QC-SDA)-mediated polyfluorene derivative-based multiple fluorescence resonance energy transfer (FRET) for simultaneous determination of AD biomarkers miRNA-16 and amyloid-beta oligomer species (AβOs). Carboxyl-functionalized poly [9,9-bis(3′-(N,N-dimethylamino)propyl)− 2,7-fluorene]-alt-2,7-(9,9-dioctylfluorene)] polymer nanoparticles (c-PFN PNPs) served as fluorescent donor and two fluorescent dyes served as fluorescent acceptors. When the targets combined with templates to continuously trigger QC-SDA, multiple infinite cycles occurred to generate numerous triggers. The resulting triggers initiated rolling circle amplification (RCA) to generate repeated sequences, which further hybridized with helper probes and reporter probes to achieve substantial immobilization of fluorescent acceptors on c-PFN PNPs, thereby realizing multiple FRET from c-PFN PNPs to acceptors with only single excitation. As FAM and Texas Red corresponded to miRNA-16 and AβOs, the high-throughput detections of biomarkers were achieved with limit of detection of 110 aM and 1.3 fM, respectively. The c-PFN PNPs coupling FAM and Texas Red provided an ideal multiplex FRET platform for synchronous detection of multi-target. What's more, the creative incorporation of amplification strategies QC-SDA and RCA endowed multiplex FRET with high sensitivity and ease of operation for bioanalysis.

Ultrasensitive detection of pathogenic bacteria by primer exchange reaction coupled with PGM

This study proposes an innovative approach for highly sensitive and selective detection of pathogenic bacteria based on primer exchange reaction (PER) coupled with a personal glucose meter (PGM). The modified nucleic acid capture probe (CP, 3ʹ-end modified biotin) was immobilized onto the surface of magnetic beads (MBs) and hybridized with the specific aptamer of Escherichia coli O157:H7 (AP) to form a double-stranded structure on the surface of MBs. The AP preferentially bound to E. coli O157:H7, thereby exposing some CP. Further, CP hybridized with a 5ʹ-end sequence of long single-stranded DNA, which was autonomously synthesized by PER, and captured a nucleic acid reporter probe (5ʹ-end modified biotin) via nucleic acid hybridization. Subsequently, numerous SA-labeled invertase conjugates were added. Next, glucose was produced on adding the substrate (sucrose) of the invertase, which was detected using PGM. Under optimal conditions, the experimental results showed the corresponding detection of E. coli O157:H7 with limits as low as 10 CFU/mL and linear ranges 10–107 CFU/mL. The proposed biosensor was used to detect pathogenic bacteria in milk samples successfully.

Nanoreporter for Real-Time Monitoring of Inflammasome Activity and Targeted Therapy.

Inflammasome activation is associated with a myriad of inflammatory diseases. However, existing methods provides a limited understanding of spatiotemporal kinetics of inflammasome activation, with restricted scope for early detection of associated treatment efficacy. This limitation offers an opportunity for the development of biocompatible in-vivo inflammasome monitoring tools with translational prospects. To achieve this, they report developing a pair of lipid-based nanoparticle systems, a reporter nanoparticle consisting of a caspase-1 activatable probe alone, and a theranostic nanoparticle combining the probe with an inflammasome-inhibiting drug. This biocompatible platform enhances the probe's residence time in circulation by preventing its opsonization and allowing its sustained release over time. Their results demonstrate the specificity of reporter nanoparticles towards caspase-1 activity and provides early-on monitoring of inflammasome activation both in-vitro as well as in-vivo. Additionally, the delivery of disulfiram, an inflammasome-inhibiting drug, along with reporter probe using theranostic nanoparticles enables real-time tracking of treatment efficacy in the gouty-arthritis inflammatory model. In summary, they report an unparalleled pair of the inflammasome-associated reporter and theranostic platforms suited not only for diagnostic applications but can also detect inflammasome-targeted treatment efficiency in real-time. These findings establish two novel, sensitive nanotools for non-invasive evaluation of inflammasome-targeted immunotherapy.

Simultaneous Imaging and Visualizing the Association of Survivin mRNA and Telomerase in Living Cells by Using a Dual-Color Encoded DNA Nanomachine.

Simultaneous imaging and especially visualizing the association of survivin mRNA and telomerase in living cells are of great value for the diagnosis and prognosis of cancer because their co-expression facilitates the development of cancer and identifies patients at high risk of tumor-related death. The challenge is to develop methods that enable visualizing the association of multiplex targets and avoid the distorted signals due to the different delivery efficiency of probes. Herein, we engineered a DNA triangular prism nanomachine (DTPN) for simultaneous multicolor imaging of survivin mRNA and telomerase and visualizing their association in living cells. Two recognizing probes targeted survivin mRNA and telomerase, and the reporter probe was assembled on the DTP in equal amounts, ensuring the same delivery efficiency of the probes to the living cells. The results showed that this DTPN could quantify intracellular survivin mRNA expression and telomerase activity. Moreover, it also enabled us to visualize the effect of the down-regulation of one target on the expression of another target under different drug stimulations. The results implied that our DTPN provided a promising platform for cancer diagnosis, prognosis, drug screening, and related biological research.

A label-free fluorescence strategy for analysis of aflatoxin M1 by self-protected DNAzyme and aptamer recognition triggered DNA walker cascade amplification

Monitoring the information of aflatoxin M1 (AFM1) is of great significance for food safety since its widespread presence in milk and other dairy products. In view of this, we developed a label-free fluorescence DNA walker based on self-protected DNAzyme for determination of AFM1. By inserting the AFM1 aptamer into DNAzyme, the complete DNAzyme was split into two separated parts and designed to be an arch-like shield to avoid its degradation in complex matrix. This excellent structure of DNAzyme and the report probe were pre-assembled on the surface of gold nanoparticles (AuNPs). Through the high efficiency affinity between AFM1 and its aptamer, the DNAzyme could be activated and show its high catalytic cleavage towards its substrate of report probe. The releasing G-quardruplex (G4) of this report probe from AuNPs could combine with berberine and exhibit obvious fluorescence values. Under optimized experimental conditions, the AFM1 detection limit was achieved at 6.40 ng/L. This DNA walker exhibited good selectivity, reproductivity and stability. Furthermore, the feasibility for analysis in real sample of our method was performed towards the target spiked in milk, indicating its potential application for real sample determination.

Portable high-throughput multimodal immunoassay platform for rapid on-site COVID-19 diagnostics

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a causal agent of Coronavirus Disease 2019 (COVID-19) has led to the global pandemic. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. To suit rapid on-site COVID-19 diagnostics under various application scenarios with an all-in-one device and simple detection reagents, we propose a high-throughput multimodal immunoassay platform with fluorescent, colorimetric, and chemiluminescent immunoassays on the same portable device and a multimodal reporter probe using quantum dot (QD) microspheres modified with horseradish peroxidase (HRP) coupled with goat anti-human IgG. The recombinant nucleocapsid protein fixed on a 96-well plate works as the capture probe. In the condition with the target under detection, both reporter and capture probes can be bound by such target. When illuminated by excitation light, fluorescence signals from QD microspheres can be collected for target quantification often at a fast speed. Additionally, when pursuing simple detection without using any sensing devices, HRP-catalyzed TMB colorimetric immunoassay is employed; and when pursuing highly sensitive detection, HRP-catalyzed luminol chemiluminescent immunoassay is established. Verified by the anti-SARS-CoV-2 N humanized antibody, the sensitivities of colorimetric, fluorescent, and chemiluminescent immunoassays are respectively 20, 80, and 640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip. Additionally, such a platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site within 50 min relying on field-operable processing and field-portable devices. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.

In Vivo Visualization of RNAi Efficiency Using a Pumilio/FBF Protein-Based Reporter

As a strategy that induces gene silencing by the delivery of small interfering RNA (siRNA) targeting a specific gene locus into cells or tissues, RNA interference (RNAi) technology holds the potential to be a powerful tool in a range of intractable disorder therapeutics. However, reliable noninvasive probes for visualizing the siRNA delivery and silencing efficiency have become a major obstacle in siRNA-based treatment. Here, we describe the development of an RNA-binding protein Pumilio/FBF (PUF)-based reporter probe for the monitoring of siRNA delivery efficiency and functional screening of effective siRNA target sites in vivo. This reporter consisted of a Firefly luciferase (Fluc) gene whose expression is regulated by the unique interaction architecture of the PUF protein with its Nanos response element (NRE) target RNA. We showed that a robust and rapid increase in the luminescence signal was detected by the successful delivery of siRNA against the enhanced green fluorescent protein (EGFP) or p53 genes into mammalian cells or the livers of mice. The delivery efficiencies of various commercial transfection vehicles were quantitatively evaluated with this reporter. In addition, we also employed in vivo bioluminescence imaging to screen and identify the most potent siRNA targeting p53. Our study indicates that the positive-readout reporter represents a promising indicator for siRNA optimization and visualization, advancing the development of siRNA therapeutic products.

Development of Nucleic‐Acid‐Based Electrochemical Biosensors for Clinical Applications

AbstractNucleic acids are remarkable molecules. In addition to Watson–Crick base pairing, the different structural motifs of these molecules can bind non‐nucleic acid targets or catalyze chemical reactions. Additionally, nucleic acids are easily modified with different molecules or functional groups. These properties make nucleic acids, particularly DNA, ideally suited for use in electrochemical biosensors, both as biorecognition elements and redox reporter probes. In this Minireview, we will review the historical evolution of nucleic acids as probes in electrochemical biosensors. We will then review the specific examples of nucleic‐acid‐based biosensors that have been evaluated for clinical use in the areas of infectious disease, cancer, or cardiovascular health.

Development of Nucleic-Acid-Based Electrochemical Biosensors for Clinical Applications.

Nucleic acids are remarkable molecules. In addition to Watson-Crick base pairing, the different structural motifs of these molecules can bind non-nucleic acid targets or catalyze chemical reactions. Additionally, nucleic acids are easily modified with different molecules or functional groups. These properties make nucleic acids, particularly DNA, ideally suited for use in electrochemical biosensors, both as biorecognition elements and redox reporter probes. In this Minireview, we will review the historical evolution of nucleic acids as probes in electrochemical biosensors. We will then review the specific examples of nucleic-acid-based biosensors that have been evaluated for clinical use in the areas of infectious disease, cancer, or cardiovascular health.

Allosteric probe-modified liposome loading bufalin-fluorouracil complex for targeted colorectal cancer therapy*

Abstract Objective Bufalin, the main active anti-tumor monomer of toad venom, is crucial in cancer treatment. However, intrinsic issues, such as poor solubility and systematic toxicity, have considerably mitigated its anticancer functions and caused unwanted side effects. It is essential to develop innovative targeting systems to precisely and efficiently deliver anticancer drugs to achieve satisfying therapeutic efficiency. Methods This work established a novel and more efficient system for simultaneously detecting and killing colorectal cancer cells. The proposed method designed two allosteric probes, a report probe and a recognize probe. The method exhibited high sensitivity towards cell detection via the recognizing probe identifying target cancer cells and the report probe’s signal report. Combining bufalin and fluorouracil endowed better tumor cell inhibition. Results We observed significantly enhanced fluorescence dots surrounding the HCT-116 cell membranes. No fluorescence increments in the other three cells were identified, indicating that the established liposome complex could specifically bind with target cells. In addition, the best ratio of bufalin to fluorouracil was 0.15 and 0.5, respectively. This improved the anti-tumor effects and achieved more than 60% tumor cell inhibition. Conclusion This method will provide new opportunities for intracellular biomolecule detection and targeted cancer cell therapy.

Sequence-specific amperometric detection based on a double-probe mode and enzyme-mediated multiple signal electrocatalysis for the double-stranded DNA of PML/RARα-related fusion gene

A new electrochemical DNA biosensor based on double-probe mode and enzyme-mediated multiple signal electrocatalysis is constructed for the highly sensitive determination of double-stranded (ds-) PML/RARα fusion gene. Through the ingenious design of two groups of detection probes, including two thiolated capture probes anchored on dual standalone detection units integrated into one customized gold electrode and four biotinylated reporter probes, hybridizing with different segments of the same target single-stranded DNA (ssDNA) simultaneously, the hybridization efficiency between the probes and target is improved by preventing the reannealing of the two separate target ssDNA. Compared with a single reporter probe, this method can dramatically increase the amount of biotin and introduce numerous streptavidin-labelled horseradish peroxidase (HRP), thereby significantly amplifying electrochemical signals with low background signals. The combination of the dual-probe mode, multiple signal amplification strategy, and the inherent electrocatalytic activity of the HRP results in the prominent electrochemical sensing performance in detecting large-fragment target dsDNA with a detection limit as low as 71 fM. Furthermore, taking advantage of the new detection strategy, polymerase chain reaction (PCR) products and enzyme-digested PCR products from NB4 cells can be effectively analysed, showing great promise for the development of a new class of point-of-care platforms for disease-/drug-related genes.

22AG G-quadruplex RNA/QnMorpholine–mediated fluorimetric detection of miR-21

Herein we report a simple ligation/transcription-mediated system, using a 22AG G-quadruplex RNA secondary structure and a fluorescence-inducing QnMorpholine probe, for the detection of miR-21. In the presence of the target miR-21, two oligonucleotide probes (promoter and reporter) were ligated, thereby transcribing the 22AG RNA sequence, a complement of the reporter probe. In contrast, in the absence of this target-induced ligation, the reporter complement could not be transcribed to produce the 22AG RNA sequence. Subsequent addition of the QnMorpholine probe resulted in binding with the 22AG G-quadruplex RNA, thereby generating high fluorescence; no fluorescence occurred in the absence of this secondary structure. Hence, the presence of miR-21 was evidenced by a target-induced high-intensity signal. This simple one-pot fluorimetric system, which could detect miR-21 of up to 3.08 femtomolar in less than 30 min, holds promise as a diagnostic tool for selective and sensitive miRNA detection.

The synergy of Fe3O4@Au and molybdate as HRP-mimetic catalysts for gold nanorods etching: Development of an ultrasensitive genosensor for detection of Helicobacter pylori

Etching changes the optical properties of plasmonic nanoparticles by oxidizing their atoms. In iodine-mediated gold nanorods (AuNRs) etching, H2O2 oxidizes I– to I3–, which turns Au0 to Au+. In this work, the ability of Fe3O4@Au nanoparticles for iodide oxidation was proved. Also, the synergy between peroxidase-mimic Fe3O4@Au nanoparticles and molybdate ions on iodide oxidation was utilized to enhance the etching rate and design a rapid, cost-effective, and ultrasensitive genosensor for the detection of Helicobacter pylori (H. pylori). In the presence of glmM fragment as a conserved H. pylori biomarker, DNA sandwiches consisting of capture probe-conjugated Fe3O4@Au nanoparticles, target sequence, and glucose oxidase (GOx)-labeled reporter probe were formed. After separating the GOx-labeled DNA sandwiches carried on Fe3O4@Au nanoparticles using an external magnet, the required substances for etching were added. Eventually, the GOx-generated H2O2 initiates the Fe3O4@Au- and molybdate-catalyzed iodine-mediated AuNRs etching. The blue shift of AuNRs’ longitudinal plasmonic peak was detected by a spectrophotometer. Under the optimal condition, an excellent sensitivity toward H. pylori was obtained with a linear range and detection limit of 40–2040 aM and 31.8 aM, respectively. Also, the fabricated genosensor showed satisfactory feasibility against real biopsy samples, validated by polymerase chain reaction (PCR) and bacterial culture.

Cascaded molecular logic gates using antibiotics as inputs based on exonuclease III and DNAzyme

We successfully constructed several cascaded molecular logic gates (2INHIBIT-2AND, 2AND-2OR, and 2OR-2INHIBIT) using three different antibiotics as the inputs. In the presence of kanamycin (KAN), chloramphenicol (CHL), or oxytetracycline (OXY), the aptamer-antibiotic recognition will release the trigger DNA to active the hairpin DNA hybridization. Exonuclease III (Exo III)-mediated catalysis reaction was introduced in the logic system to generate Mg2+-dependent DNAzyme, which was used to cleave the fluorescence signal reporter probe. For input, the presence and absence of the antibiotic was defined as 1 and 0, respectively. For output, the fluorescence intensity higher or lower than the threshold value was defined as 1 and 0, respectively. In the 2INHIBIT-2AND logic circuit, the 101 input combination generates an output of 1 and other input combinations generate an output of 0. In the 2AND-2OR logic circuit, the input combinations of 001, 011, 110, 101, and 111 generate an output of 1 and other input combinations generate an output of 0. In the 2OR-2INHIBIT logic circuit, the input combinations of 010, 100, and 110 generate an output of 1 and other input combinations generate an output of 0. Our constructed logic system exhibits high selectivity and can work even in complex water samples. With the advantages of multiple biocomputation capabilities, high flexibility, and easy scalability, this logic gate system provides a new analytical method for the intelligent detection of different antibiotics.

An ultrasensitive lateral flow immunoassay platform for foodborne biotoxins and pathogenic bacteria based on carbon-dots embedded mesoporous silicon nanoparticles fluorescent reporter probes

Lateral flow immunoassays (LFIAs) are routine methods for rapid foodborne pollutants screening, with detection limits that are closely associated with the label probes used. The exploitation of high performance and robust probe is highly desirable, and remains a great challenge. Herein, we reported an emerging fluorescent nanobeads i.e. carbon-dots (CD) covalently incorporated mesoporous silicon nanoparticles (CD-MSNs) for LFIAs. CD-MSNs revealed brighter fluorescence, larger particle size and more modification sites in comparison with those of single CD. After bio-functionalisation, CD-MSNs probes were introduced to construct LFIA test strips, and designed for ultrasensitive detection of aflatoxin B1 (AFB1) and Staphylococcus aureus (S. aureus), two representative foodborne pollutants, based on the competitive and sandwich models, respectively. Very competitive quantitative detection limits i.e. 0.05 ng/mL and 102 cfu/mL were correspondingly obtained. Additionally, the test strips were successfully applied to rapidly and accurately screen AFB1 and S. aureus in food samples, highlighting their practicality.

An Emerging Fluorescent Carbon Nanobead Label Probe for Lateral Flow Assays and Highly Sensitive Screening of Foodborne Toxins and Pathogenic Bacteria.

By virtue of the fascinating merits of low cost, rapid screening, and on-site detection, fluorescence lateral flow assays (FLFAs) have attracted considerable attention. Their detection limits are closely associated with the label probes used. The development of high-performance and robust phosphors remains a great challenge. Herein, we presented a new label probe, i.e., fluorescent carbon nanobeads (FCNBs), for FLFAs. Monodispersive, water-soluble, and highly emissive FCNBs were facilely prepared via a hydrothermal carbonization manner. Their abundant amino groups were beneficial for versatile surface functionalization. After being modified by biomolecules, the fabricated FCNB reporter probes were adopted for the construction of lateral flow test strips toward representative foodborne toxins, i.e., aflatoxin B1 (AFB1), and pathogenic bacteria, i.e., Staphylococcus aureus (S. aureus), respectively. The detection limits (0.01 ng/mL for AFB1 and 102 cfu/mL for S. aureus) were about 1 or 2 orders of magnitude lower than most reported methods. Furthermore, the proposed test strips were successfully applied for the quantitative, accurate, and rapid screening of AFB1 and S. aureus in food samples. This work provided a promising label probe for FLFAs and would open the opportunity to exploit a sensing platform by modifying different ligands onto the FCNBs.

An electrochemical biosensor for the highly sensitive detection of Staphylococcus aureus based on SRCA-CRISPR/Cas12a

As one of the major foodborne pathogens, Staphylococcus aureus (S. aureus) can cause infectious diseases. In the current study, a novel electrochemical biosensor based on saltatory rolling circle amplification (SRCA) combined with CRISPR/Cas12a system was developed for the accurate detection of S. aureus. The thio-modified reporter probes (SH-ssDNA-MB) was immobilized on the surface of gold nanoparticle-modified electrode through the Au–S bond. In the presence of S. aureus, the target DNA double strands obtained by SRCA can be specifically recognized with Cas12a/crRNA complex. The trans-cleavage activity of Cas12a induces SH-ssDNA-MB to be cleaved from the electrode surface, resulting in a decrease in the current signal. Subsequently, the ratio of the current can be calculated as the detection result. Under optimal conditions, the detection limits were 2.51 fg/μL for genomic DNA and 3 CFU/mL for S. aureus in pure cultures, respectively. Moreover, the method demonstrated satisfactory specificity, acceptable stability and reproducibility. In comparison with ISO methods, the sensitivity, specificity and accuracy of the developed method were 100%, 97.8% and 98%, respectively. In conclusion, the developed novel electrochemical biosensor provides a potential powerful platform for the accurate detection of S. aureus.