Nucleic Acid Signal Research Articles

A review of urinary HPV testing for cervical cancer management and HPV vaccine surveillance: rationale, strategies, and limitations.

Human papillomavirus (HPV) infections are the leading cause of cervical cancer, the fourth most common cancer among women worldwide. Despite concerted efforts to combat this preventable disease through HPV vaccination and cancer screening have helped reduce morbidity and mortality levels, the burden persists in both developing and developed countries due to insufficient vaccination and screening coverage. Urinary HPV testing has emerged as a noninvasive detection method, offering significant advantages in cervical cancer management and vaccine surveillance. Notably, it boasts high acceptance rates, ease of self-collection, user-friendly implementation, and relatively low cost. Various urinary HPV detection methods have been explored, predominantly relying on nucleic acid amplification and signal amplification, targeting a variety of biomarkers in urine, such as HPV DNA, RNA, and oncoproteins. Existing literature underscores urine as a promising specimen for HPV testing, demonstrating comparable detection performance to cervical and vaginal samples in several studies. However, the lack of standardized and authoritative protocols in sample collection, storage, preparation, DNA extraction, and amplification necessitates further evaluation for the comprehensive utilization of urinary HPV testing in clinical and epidemiological settings. This study aims to review pertinent publications and offer insights into the rationale, common strategies, and limitations of urinary HPV testing, with the ultimate goal of maximizing its utility in practice.

Filter Membrane-Based Colorimetric Approach for Point-of-Care Detection of Biomarkers Using CRISPR-Cas12a.

CRISPR-Cas-based point-of-care testing (POCT) strategies have been widely explored for the detection of diverse biomarkers. However, these methods often require complicated operations, such as careful solution transfer steps, to achieve high sensitivity and accuracy. In this study, we combine a filter membrane-based POCT method with CRISPR-Cas12a for colorimetric detection of biomarkers. For the nucleic acid target, the trans-cleavage activity of CRISPR-Cas12a is directly triggered, cutting the single-stranded DNA linkers on glucose oxidase (GOx)-modified polymer nanoparticles. Due to the size difference between GOx and the polymer nanoparticles, GOx can be separated using a filter membrane. The filtrate containing GOx reacts with the substrate to generate a colorimetric signal. For the non-nucleic acid target, the non-nucleic acid signal is converted into a nucleic acid signal that activates CRISPR-Cas12a, resulting in a colorimetric signal. The entire operation is easy to perform, and the signal can be directly observed via the naked eye, which circumvents the use of costly instruments. The developed strategy holds great promise for accurate and accessible POCT detection of disease biomarkers in resource-limited settings.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

DNA Polymerase-Endonuclease Efficiently Synthesizes DNA to Prepare DNA Materials and Develop Novel Signal Amplification System.

DNA biosynthesis, a focus of fundamental and applied research, typically involves DNA polymerases by using templates, primers, and dNTPs. Some polymerases can polymerize dNTPs for DNA de novo synthesis, although this is generally to occur randomly. This novel synthesis method has garnered our attention and practical use. Herein, we observed that the addition of endonuclease significantly enhances the efficiency of the de novo synthesis reaction catalyzed by the DNA polymerase. We further investigated the reaction conditions that influence this efficiency. Building on the optimal reaction conditions, we developed a rapid and efficient strategy for preparing DNA hydrogel. Further, coupled with the CRISPR-Cas system, we developed a nucleic acid signal amplification system characterized by versatility, sensitivity, specificity, and no risk of aerosol contamination. We successfully detected viral nucleic acids in clinical samples. In summary, our study demonstrates the significant potential of DNA polymerase- and endonuclease-catalyzed DNA de novo synthesis in diverse applications.

Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

Point-of-care testing of methamphetamine and cocaine utilizing wearable sensors

The imperative for the point-of-care testing of methamphetamine and cocaine in drug abuse prevention necessitates innovative solutions. To address this need, we have introduced a multi-channel wearable sensor harnessing CRISPR/Cas12a system. A CRISPR/Cas12a based system, integrated with aptamers specific to methamphetamine and cocaine, has been engineered. These aptamers function as signal-mediated intermediaries, converting methamphetamine and cocaine into nucleic acid signals, subsequently generating single-stranded DNA to activate the Cas12 protein. Additionally, we have integrated a microfluidic system and magnetic separation technology into the CRISPR system, enabling rapid and precise detection of cocaine and methamphetamine. The proposed sensing platform demonstrated exceptional sensitivity, achieving a detection limit as low as 0.1 ng/mL. This sensor is expected to be used for on-site drug detection in the future.

Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus using an Argonaute-Centered Portable and Visual Biosensor.

The development of novel method for drug-resistant bacteria detection is imperative. A simultaneous dual-gene Test of methicillin-resistant Staphylococcus aureus (MRSA) is developed using an Argonaute-centered portable biosensor (STAR). This is the first report concerning Argonaute-based pathogenic bacteria detection. Simply, the species-specific mecA and nuc gene are isothermally amplified using loop-mediated isothermal amplification (LAMP) technique, followed by Argonaute-based detection enabled by its programmable, guided, sequence-specific recognition and cleavage. With the strategy, the targeted nucleic acid signals gene are dexterously converted into fluorescent signals. STAR is capable of detecting the nuc gene and mecA gene simultaneously in a single reaction. The limit of detection is 10 CFU/mL with a dynamic range from 10 to 107 CFU/mL. The sample-to-result time is <65min. This method is successfully adapted to detect clinical samples, contaminated foods, and MRSA-infected animals. This work broadens the reach of Argonaute-based biosensing and presents a novel bacterial point-of-need (PON) detection platform.

Recent advances in nucleic acid signal amplification-based aptasensors for sensing mycotoxins.

Mycotoxin contamination in food products may cause serious health hazards and economic losses. The effective control and accurate detection of mycotoxins have become a global concern. Even though a variety of methods have been developed for mycotoxin detection, most conventional methods suffer from complicated operation procedures, low sensitivity, high cost, and long assay time. Therefore, the development of simple and sensitive methods for mycotoxin assay is highly needed. The introduction of nucleic acid signal amplification technology (NASAT) into aptasensors significantly improves the sensitivity and facilitates the detection of mycotoxins. Herein, we give a comprehensive review of the recent advances in NASAT-based aptasensors for assaying mycotoxins and summarize the principles, features, and applications of NASAT-based aptasensors. Moreover, we highlight the challenges and prospects in the field, including the simultaneous detection of multiple mycotoxins and the development of portable devices for field detection.

Argonaute-DNAzyme tandem biosensing for highly sensitive and simultaneous dual-gene detection of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a common zoonotic multidrug-resistant bacterium, puts a great threat to public health and food safety. Rapid and reliable detection of MRSA is crucial to guide effective patient treatment at early stages of infection and control the spread of MRSA infections. Herein, we developed a Simultaneous dual-gene and ulTra-sensitive detection for methicillin-resistant Staphylococcus aureus using Argonaute-DNAzyme tandem Detection (STAND). Simply, loop-mediated isothermal amplification (LAMP) was used for the amplification of the species-specific mecA and nuc gene, followed by STAND enabled by the site-specific cleavage of programable Argonaute. The Argonaute-DNAzyme tandem reaction rendered a conceptually novel signal amplification and transduction module that was more sensitive (1 or 2 order of magnitude higher) than the original Argonaute-based biosensing. With the strategy, the target nucleic acid signals gene were dexterously converted into fluorescent signals. STAND could detect the nuc gene and mecA gene simultaneously in a single reaction with 1 CFU/mL MRSA and a dynamic range from 1 to 108 CFU/mL. This method was confirmed by clinical samples and challenged by identifying contaminated foods and MRSA-infected animals. This work enriches the arsenal of Argonaute-mediated biosensing and presents a novel biosensing strategy to detect pathogenic bacteria with ultra-sensitivity, specificity and on-site capability.

Constructing DNAzyme-driven three-dimensional DNA nanomachine-mediated paper-based photoelectrochemical device for ultrasensitive detection of miR-486-5p

As a unique class of dynamic nanostructures, biomimetic DNA walking machines that exhibit geometrical complexity and nanometre precision have gained great success in photoelectrochemical (PEC) bioanalysis. Despite certain achievements, the slow reaction kinetics and low processivity severely restrict the amplification efficiency of the DNA walker-mediated biosensors. Herein, by taking advantage of efficient DNA rolling machines, a three-dimensional (3D) DNA nanomachine-mediated paper-based PEC device for speedy ultrasensitive detection of miR-486–5p was successfully constructed. To achieve it, a novel In2S3/SnS2 sensitized heterojunction was firstly in-situ grown on the Au-modified paper fibers and implemented as the photoanode with effective separation of photogenerated carriers to achieve an enhanced initial photocurrent. Subsequently, the copper hexacyanoferrate(II)-modified CuO nanosphere was introduced as a multifunctional signal regulator via the competitive capture of electron donors and photon energy with the photoelectric layer for efficiently quenching the PEC signal. With the introduction of targets, the DNAzyme-driven DNA nanomachine with editable motion modes was gradually activated and it could continuously cleave the tracks DNA labeled quenching probes, finally achieving the recovery of PEC signal. As a proof of concept, the elaborated paper-based PEC device presented a wide linear range from 0.1 fM to 100 pM and a detection limit of 35 aM for miR-486–5p bioassay. This work provides an innovative insight to the exploitation of DNA nanobiotechnology and nucleic acid signal amplification strategy.

Analysis of differentially expressed genes discovers Latroeggtoxin VI-induced changes and SYNJ1 as a main target in PC12 cells

BackgroundPrevious preliminary work found that Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin from the eggs of spider Latrodectus tredecimguttatus, could promote the synthesis and release of dopamine in PC12 cells. However, the underlying mechanisms have not been fully clear. Here, the effects of LETX-VI on the gene expression profile and dopamine in PC12 cells were analyzed with the differential transcriptome-based strategies.ResultsAfter treatment of PC12 cells with LETX-VI for 24 h, a total of 356 differentially expressed transcripts were identified. Of them 165 were up-regulated and 191 down-regulated. Relevant GO analysis indicated that LETX-VI modulated the expression of certain genes and thereby affected multiple biological processes in PC12 cells, including protein metabolism, nucleic acid metabolism, substance transport, signaling, neurotransmitter metabolism and release. When western blot analysis was employed to confirm the abundance levels of synaptojanin 1 and synuclein alpha interacting protein, the representatives of highly up- and down-regulated transcript-encoded proteins that are closely related with dopamine respectively, it was found that the level of synaptojanin 1 in the PC12 cells treated with LETX-VI was increased, whereas that of synuclein alpha interacting protein was not obviously altered, suggesting that synaptojanin 1 may be much more involved in the effects of LETX-VI on dopamine. After synaptojanin 1 level was knocked down using siRNA, the levels of both total and released dopamine were significantly decreased, indicating that synaptojanin 1 is a protein positively modulating the synthesis and secretion of dopamine. When the PC12 cells with knocked down synaptojanin 1 were treated by LETX-VI, the adverse effects of synaptojanin 1 knockdown on dopamine were attenuated, confirming that LETX-VI promotes the synthesis and secretion of dopamine at least partially by enhancing the expression of the gene SYNJ1 encoding synaptojanin 1.ConclusionsThis work demonstrates that LETX-VI exerts multiple regulatory effects on the cellular processes in PC12 cells by altering the gene expression profile. LETX-VI modulates the expression of the genes closely related to the synthesis, transport and release of neurotransmitters especially dopamine in PC12 cells, with the gene SYNJ1 encoding synaptojanin 1 as a main target.

The Protective Effect of Virus Capsids on RNA and DNA Virus Genomes in Wastewater.

Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the T90 for MS2 (+)ssRNA increased by 424× and the T90 for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.

Facile Strategy to Output Fluorescein from Nucleic Acid Interactions.

Biomolecular operations, which involve the conversion of molecular signals or interactions into specific functional outputs, are fundamental to the field of biology and serve as the important foundation for the design of diagnostic and therapeutic systems. To maximize their functionalities and broaden their applicability, it is crucial to develop novel outputs and facile chemical transformation methods. With this aim, in this study, we present a straightforward method for converting nucleic acid signals into fluorescein outputs that exhibit a wide range of functionalities. This operation is designed through a DNA-templated reaction based on riboflavin-photocatalyzed oxidation of dihydrofluorescein, which is readily prepared by simple NaBH4 reduction of the fluorescein with no complicated chemical caging steps. The templated photooxidation exhibits high efficiency (kapp = 2.7 × 10-3/s), generating a clear fluorescein output signal distinguishable from a low background, originating from the high stability of the synthesized dihydrofluorescein. This facile and efficient operation allows the nucleic acid-initiated activation of various fluorescein functions, such as fluorescence and artificial oxidase activity, which are applied in the design of novel bioanalytical systems, including fluorescent and colorimetric DNA sensors. The operation presented herein would expand the scope of biomolecular circuit systems for diagnostic and therapeutic applications.

Epithelioid neoplasm of the spinal cord in a child with spinal muscular atrophy treated with onasemnogene abeparvovec

Spinal muscular atrophy is an autosomal recessive disease resulting in motor neuron degeneration and progressive life-limiting motor deficits when untreated. Onasemnogene abeparvovec is an adeno-associated virus serotype 9-based gene therapy that improves survival, motor function, and motor milestone achievement in symptomatic and presymptomatic patients. Although the adeno-associated virus genome is maintained as an episome, theoretical risk of tumorigenicity persists should genomic insertion occur. We present the case of a 16-month-old male with spinal muscular atrophy who was diagnosed with an epithelioid neoplasm of the spinal cord approximately 14months after receiving onasemnogene abeparvovec. In situ hybridization analysis detected an onasemnogene abeparvovec nucleic acid signal broadly distributed in many but not all tumor cells. Integration site analysis on patient formalin-fixed, paraffin-embedded tumor samples failed to detect high-confidence integration sites of onasemnogene abeparvovec. The finding was considered inconclusive because of limited remaining tissue/DNA input. The improved life expectancy resulting from innovative spinal muscular atrophy therapies, including onasemnogene abeparvovec, has created an opportunity to analyze the long-term adverse events and durability of these therapies as well as identify potential disease associations that were previously unrecognized because of the premature death of these patients.

Dual-Signal Cascaded Nucleic Acid Amplification Circuit-Loaded Metal-Organic Frameworks for Accurate and Robust Imaging of Intracellular MicroRNA.

Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.

Smartphone-assisted point-of-care testing of nucleic acids based on hybridization chain reaction, magnetic beads, and gold nanorods etching

Colorimetric biosensors are simple but effective tools that are gaining popularity due to their ability to provide low-cost, rapid, and accurate detection for viruses like the Novel coronavirus, Influenza A, and Dengue virus, especially in point-of-care testing (POCT) and visual detection. In this study, a smartphone-assisted nucleic acid POCT was built using hybridization chain reaction (HCR), magnetic beads (MBs), and oxidized 3,3′,5,5′-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (GNRs). The application of HCR without enzyme isothermal characteristics and MBs with easy separation, can quickly amplify nucleic acid signal and remove other reaction components. The blue shift of longitudinal localized surface plasmon resonance (LSPR) based on GNRs showed significant differences in etching color for different concentrations of target nucleic acid, which convert the signal into a visually semi-quantitative colorimetric result, achieving quantitative analysis with the color recognition software built into smartphones. This strategy, which only takes 40 min to detect and is two-thirds less time than the PCR, was successfully applied for the detection of the Dengue target sequence with a detection limit of 1.25 nM and exhibited excellent specificity for distinguishing single-base mutations, indicating broad application prospects in clinical laboratory diagnosis and enriching the research of nucleic acid POCT.

Biochemical characterization and discrimination of Leishmania major parasites and infected macrophages with Raman spectroscopy and chemometrics

Leishmaniasis is classified as one of the neglected tropical disease (NTD), which are caused by a group of parasitic protozoans called Leishmania. The high case load and severity of the disease make Leishmaniasis second only to malaria in terms of both severity and infectivity. However, due to the low economic interest in research and development, it may become a major world-wide health threat. Current diagnostics including serological assessment of infected tissue by either light microscopy, or antibody tests or by the culturing of potential infection via in vitro or in vivo animal inoculation, parasitological tests using samples aspirated from the spleen and bone marrow, Immunological tests such as the Montenegro test, Fluorescence assays, and polymerase chain reaction (PCR) techniques are suffer from several limitations including time and expense. Herein, we first apply Raman microscopy to distinguish the two L. Major parasitic forms namely promastigotes and amastigotes and secondly, distinguish infected from non-infected macrophages using multivariate data analysis including Principal Component Analysis (PCA) and unsupervised hierarchical cluster imaging analysis (UHCA). The maximum variance between infected and uninfected macrophage groups are visible in the lipid region (92.20 %) as compared to the fingerprint region (46.13 %) along PC1. The contributions from nucleic acids can be found at 805 cm−1 (phosphodiester - Z-marker), 767 cm−1 (pyrimidine ring breathing mode), 742 cm−1 (ring breathing mode of DNA/RNA bases), and 568 cm−1 (cytosine/guanine). These amplified nucleic acid signals in infected macrophages indicate the presence of infection compared to the uninfected macrophage group. Similarly, the maximum variance between amastigotes and promastigotes groups are observed in the lipid region (88.45%) as compared to the fingerprint region (28.34 %). Moreover, the UHCA of infected macrophages revealed the accumulation of lipid bodies or droplets inside or close proximity parasitophorous vacuole, which is consistent with the reported literature. Once established macrophages were infected with Leishmania in vitro and the differences between infected and non-infected established with high reproducibility. The reported spectral differences between infected and non-infected macrophages lays the ground work for developing a diagnostic tool for detection of leishmaniasis in a buffy coat preparation and also offers the potential of monitoring the effects of new therapeutics.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Enhancing biosensing with fourfold amplification and self-powering capabilities: MoS2@C hollow nanorods-mediated DNA hexahedral framework architecture for amol-level liver cancer tumor marker detection

Two-dimensional carbon-coated molybdenum disulfide (MoS2@C) hollow nanorods are combined with nucleic acid signal amplification strategies and DNA hexahedral nanoframework to construct a novel self-powered biosensing platform for ultra-sensitive dual-mode detection of tumor suppressor microRNA-199a. The nanomaterial is applied on carbon cloth and then modified with glucose oxidase or using as bioanode. A large number of double helix DNA chains are produced on bicathode by nucleic acid technologies including 3D DNA walker, hybrid chain reaction and DNA hexahedral nanoframework to adsorb methylene blue, producing high EOCV signal. Methylene blue also is reduced and an increased RGB Blue value is observed. For microRNA-199a detection, the assay shows a extensive linear range of 0.0001–100 pM with a low detection limit of 4.94 amol/L (S/N = 3). The method has been applied to the detection of actual serum samples, providing a novel method for the accurate and sensitive detection of tumor markers.

A triple amplification strategy using GR-5 DNAzyme as a signal medium for ultrasensitive detection of trace Pb2+ based on CRISPR/Cas12a empowered electrochemical biosensor

Lead ions (Pb2+) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb2+ is essential. With their trans-cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb2+. In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb2+ into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb2+ detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.