ssDNA Probe Research Articles

Tuberculosis detection from raw sputum samples using Au-electroplated screen-printed electrodes as E-DNA sensor.

Tuberculosis (TB) remains a leading cause of death globally, especially in underdeveloped nations. The main impediment to TB eradication is a lack of efficient diagnostic tools for disease diagnosis. In this work, label free and ultrasensitive electrochemical DNA biosensor for detecting Mycobacterium tuberculosis has been developed based on the electrodeposition of gold nanoparticles on the surface of carbon screen-printed carbon electrode (Zensors) for signal amplification. Particularly, screen-printed electrodes were modified by electrochemical deposition of Au to enhance the conductivity and facilitate the immobilization of ssDNA probes via Au-S bonds. The electrochemically modified SPEs were characterized using Scanning electron microscopy/Energy Dispersive X-Ray Analysis (SEM/EDX) and X-Ray Diffraction (XRD). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used to investigate the DNA hybridization between single-stranded (ssDNA) probe and target DNA (tDNA). Under the ideal conditions, DPV exhibited a correlation coefficient R2 = 0.97, when analyzed with different tDNA concentrations. The proposed DNA biosensor exhibits a good detection range from 2 to 10nm with a low detection limit of 1.91nm, as well as high selectivity that, under ideal conditions, distinguishes non-complementary DNA from perfectly matched tDNA. By eliminating the need for DNA purification, this work paves the path for creating disposable biosensors capable of detecting DNA from raw sputum samples.

Exploiting the size exclusion effect of protein adsorption layers for electrochemical detection of microRNA: A new mechanism for design of E-DNA sensor

The assay performance of electrochemical DNA (E-DNA) sensors is deeply influenced by the state of DNA probes immobilized on electrode. Moreover, the immobilization procedures for DNA probes are tedious and vary according to the probes and analytes. In this work, we find that the adsorption layers of bovine serum albumin (BSA) on gold electrode (AuE) possess a size exclusion effect to distinguish between single-stranded (-ss) DNA probes and the DNA fragments generated from enzymatic digestion of ssDNA probes. In detail, the BSA layers act as a gatekeeper that hinders the adsorption of a ssDNA probe on AuE but permits the DNA fragments with much smaller sizes to pass through the adsorption layers and adsorb on AuE. This finding is developed into a novel E-DNA sensor for microRNA (miRNA) detection by coupling with duplex-specific nuclease (DSN)-assisted target recycling strategy. The ssDNA probe in solution phase is enzymatically digested during the DSN-assisted target recycling process initiated by target miRNA-21, generating plenty of DNA fragments. The adsorption of these DNA fragment on BSA/AuE is permitted, which arouses electrochemical signals after binding with [Ru(NH3)6]3+ to indicate the recognition of miRNA-21. The developed E-DNA sensor possesses a wide calibration range from 0.001 to 100 pM and a low detection limit of 0.48 fM. Significantly, accurate evaluation of miRNA-21 expression levels in cancer cell lines and non-small-cell lung carcinomas (NSCLC) serum samples are successfully achieved using the developed method. This work provides a new mechanism for constructing sensitive E-DNA sensor without tedious probe immobilization procedures.

Preparation of electrochemical sensor based on glassy carbon electrode and its specificity and sensitivity for directional detection of antibiotic resistance genes spreading in the water environment.

Antibiotic-resistant bacteria/resistance genes (ARB/ARGs) have been paid much attention due to the environmental risks they might bring. They were demonstrated to be widespread in surface water and wastewater. Determining the concentrations of ARGs is the first step to evaluate the degree of pollution. In this study, electrochemical detection technology was studied due to its advantages of low cost, fast response, and satisfactory selectivity. Additionally, the electrochemical sensor technology was used to determine the concentration of a ubiquitous ARG (ampicillin gene blaTEM) in the water environment. A kind of electrochemical sensor was prepared on a glassy carbon electrode (GCE). The results of X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV) curves indicated that the single-stranded DNA (ssDNA) probe can be successfully immobilized on the surface of the GCE. In addition, the performance of hybridization between the ssDNA probe and the target DNA at diverse temperatures was compared, of which 35°C was the optimum. Moreover, the change of charge transfer resistance (ΔRct) for the GCE sensor hybridizing with complementary DNA was much higher than that of DNA with the mismatched base, which indicated that the electrochemical sensor prepared in this study was specific. The sensitivity of the sensor was also proved by the strong correlation between the concentrations of ARGs and ΔRct (with the correlation coefficient (R2) of 0.9905). All in all, this study is meaningful for the comprehend on the detection of ARGs through the electrochemical method.

High-Performance Circulating Tumor DNA Liquid Biopsy Based on Graphitic Carbon Nitride Nanosheet for Monitoring Gastric Cancer-Related Gene Mutations

Early diagnosis and timely monitoring of cancer progression are the most effective ways to improve the cure rate of cancer patients. And it is essential to create convenient, sensitive, accurate, as well as noninvasive or minimally invasive tests for better respecting patients’ wishes and optimizing diagnosis. The fluorescent biosensor discovered in our study on the basis of graphitic carbon nitride nanosheet (CNNS) could be used to detect the gastric cancer-associated circulating tumor DNA (ctDNA) in human blood by highly specific binding to fluorescein-labeled single-stranded DNA detection probes. The ssDNA detection probe was adsorbed on the surface of CNNS through weak Π–Π stacking, thereby obtaining efficient fluorescence quenching. With the presence of the target DNA, the ssDNA probe showed weak affinity for CNNS and restored fluorescence by base complementary pairing with target ssDNA through strong hydrogen bonds. The results show that the nanometer detection is a convenient, low-cost and high-efficiency technology, which is promising in biological detection and analysis.

Quantification of mRNA in Single Cells Based on Dimerization-Induced Photoluminescence Nonblinking of Quantum Dots.

Photoluminescence (PL) intermittency (or "blinking") is a unique characteristic of single quantum dot (QD) emission. Here, we report a novel single-molecule detection strategy for the intracellular mRNA of interest using the mRNA-induced nonblinking QD dimers as probes. The working principle of the method is that the DNA hybrid of the target DNA (or mRNA) with a biotin-modified ssDNA probe can induce two blinking streptavidin-modified QDs (SAV-QDs) conjugated. The formed QD dimer as a bright spot showed a nonblinking emission property, observed with total inner reflection fluorescence microscopy (TIRFM). In theory, one nonblinking spot indicated a target DNA (or mRNA). The experimental results from single-spot fluorescence trajectory analysis and single-particle brightness analysis based on TIRFM and fluorescence correlation spectroscopy (FCS) techniques verified this dimerization process of QDs or its induced nonblinking emission. Employing a target DNA with the same base sequences to Survivin mRNA as a model, the detection strategy was used to detect the target DNA concentration based on the linear relationship between the percentage of the nonblinking spots and the target DNA concentration. This single-molecule detection strategy was also successfully used for determining Survivin mRNA in a single HeLa cell. The method can simplify the hybridization steps, eliminate self-quenching and photobleaching of fluorophores, and reduce the influence of unspecific binding on the detection.

Rational Design of Porous Poly(ethylene glycol) Films as a Matrix for ssDNA Immobilization and Hybridization.

Poly(ethylene glycol) (PEG) films, fabricated by thermally induced crosslinking of amine- and epoxy-terminated four-arm STAR-PEG precursors, were used as porous and bioinert matrix for single-stranded DNA (ssDNA) immobilization and hybridization. The immobilization relied on the reaction between the amine groups in the films and N-hydroxy succinimide (NHS) ester groups of the NHS-ester-decorated ssDNA. Whereas the amount of reactive amine groups in the films with the standard 1:1 composition of the precursors turned out to be too low for efficient immobilization, it could be increased noticeably using an excess (2:1) concentration of the amine-terminated precursor. The respective films retained the bioinertness of the 1:1 prototype and could be successfully decorated with probe ssDNA, resulting in porous, 3D PEG-ssDNA sensing assemblies. These assemblies exhibited high selectivity with respect to the target ssDNA strands, with a hybridization efficiency of 78–89% for the matching sequences and full inertness for non-complementary strands. The respective strategy can be applied to the fabrication of DNA microarrays and DNA sensors. As a suitable transduction technique, requiring no ssDNA labeling and showing high sensitivity in the PEG-ssDNA case, electrochemical impedance spectroscopy is suggested.

An ultrasensitive electrochemical self-signal circulating tumor DNA recognition strategy employing black phosphorous nanosheets assembled with flavin adenine dinucleotide

A self-signal electrochemical sensing platform was constructed for direct recognition of circulating tumor DNA (ctDNA) employing black phosphorous nanosheets (BPNS) assembled with flavin adenine dinucleotide (FAD) prepared by ultrasonication approach. FAD provided a highly efficient and stable dispersing medium for acquiring highly dispersed BPNS in aqueous phase. The obtained FAD/BPNS nanocomposite exhibited favorable electrochemical redox activity and was utilized as the interface for the immobilization and hybridization of DNA. The amine-terminated probe ssDNA was covalently assembled onto the FAD/BPNS nanocomposite with plentiful phosphonate groups accompanied by the decrease of the self-signal. The self-redox signal of the nanointerface regenerated after the probe hybridized with the complementary sequence as a result of DNA transformation. Electrochemical response enhanced with complementary DNA concentration from 1.0 × 10-18 to 1.0 × 10-8 mol/L with a detection limit of 2.6 × 10-19 mol/L. The DNA determination platform revealed outstanding sensitivity, specificity and stableness, and was successfully employed in the detection of ctDNA associated with colorectal cancer. The developed biosensing strategy is simple to accomplish and has the potency for the application for diverse morbific gene without sophisticated label procedure.

Enhanced Sensing Performance of a Microchannel-Based Electrochemiluminescence Biosensor for Adenosine Triphosphate via a dsDNA Superstructure Amplification Strategy.

The sensing performance of a microchannel-based electrochemiluminescence (ECL) biosensor is related to the change ratio of charge density on the surface of microchannels caused by a target recognition reaction. In this study, adenosine triphosphate (ATP) served as a model target. The dsDNA superstructures containing a capture probe (CP, containing an ATP aptamer sequence) and alternating units of ssDNA probes of P1 and P2, CP/(P1/P2)n, were grafted onto the inner wall of microchannels first. The CP in dsDNA superstructures captured ATP molecules, causing the release of dsDNA fragments containing alternating units of P1 and P2, (P1/P2)n. The target recognition reaction significantly changed the charge density of microchannels, which altered the ECL intensity of the (1,10-phenanthroline)ruthenium(II)/tripropylamine system in the reporting interface. The ECL intensity of the constructed system had a linear relationship with the logarithm of ATP concentration ranging from 1 fM to 100 pM with a detection limit of 0.32 fM (S/N = 3). The biosensor was successfully applied to detect ATP in rat brains.

Electrochemical DNA Biosensor Based on Platinum-gold Bimetal Decorated Graphene Modified Electrode for the Detection of Vibrio parahaemolyticus Specific tlh Gene Sequence

Background: By using bimetal nanocomposite modified electrode, the electrochemical DNA biosensor showed the advantages of high sensitivity, low cost, rapid response and convenient operation, which was applied for disease diagnosis, food safety, and biological monitoring. Objective: A nanocomposite consisting of platinum (Pt)-gold (Au) bimetal and two-dimensional graphene (GR) was synthesized by hydrothermal method, which was modified on the surface of carbon ionic liquid electrode and further used for the immobilization of probe ssDNA related to Vibrio parahaemolyticus tlh gene to construct an electrochemical DNA sensor. Method: Potassium ferricyanide was selected as electrochemical indicator, cyclic voltammetry was used to study the electrochemical behaviours of different modified electrodes and differential pulse voltammetry was employed to test the analytical performance of this biosensor for the detection of target gene sequence. Results: This electrochemical DNA biosensor could detect the Vibrio parahaemolyticus tlh gene sequence as the linear concentration in the range from 1.0×10-13 mol L-1 to 1.0×10-6 mol L-1 with the detection limit as 2.91×10-14 mol L-1 (3σ). Conclusion: This proposed electrochemical DNA biosensor could be used to identify the special gene sequence with good selectivity, low detection limit and wide detection range.

Synthesis of Ag@carbonized Polymer Dots and their Electrochemical Sensing of miRNA

AbstractPolyethyleneimine (PEI) is a cationic polymer with a lot of amino groups in its molecular structure that is secure, sterile, and biocompatible. However, it is difficult for PEI molecules to be directly applied in electroanalysis due to their poor electroactivity. Herein, the PEI‐based carbonized polymer nanodots (CPDs) with core‐shell structure were prepared by hydrothermal method using PEI as the precursors. The results showed that the as‐prepared PEI‐based CPDs can not only exhibit the unique properties of carbon nanodots but also inherit the properties of PEI. Subsequently, based on the strong coordination interaction between the abundant amino groups on the surface of PEI and silver ions, the electroactive Ag@carbonized polymer dots (Ag@CPDs) were prepared. Furthermore, to investigate the electrochemical behaviors and application of Ag@CPDs, the DNA molecule (double‐stranded and single‐stranded DNA) was taken as an example. It was found that the interaction of DNA with Ag@CPDs had a significant influence on the current intensity of Ag@CPDs. Based on this phenomenon, a sensitive electrochemical technique for miRNA detection was established and effectively employed to detect miRNA in human serum samples based on the discriminating effect of Ag@CPDs towards ssDNA probes and ssDNA probe/miRNA complex (dsDNA).

Exonuclease III-assisted CRISPR/Cas12a electrochemiluminescence biosensor for sub-femtomolar mercury ions determination

A novel ECL biosensor was developed for ultrasensitive mercury ions (Hg2+) determination based on gold nanoparticles-coated nitrogen-doped carbon dots (Au@CNDs) as luminophores and exonuclease III (Exo III) as well as CRISPR/Cas12a as signal amplifiers. The biosensor was fabricated by pre-modifying Au@CNDs hybrids and ferrocene-labeled single-stranded DNA (Fc-ssDNA) layer in sequence on the interface of a gold working electrode. The initial Au@CNDs ECL of the biosensor is quenched by Fc. Then, a thymine-rich ssDNA probe was designed for the recognition of Hg2+. In the presence of Hg2+, this thymine-rich ssDNA probe was folded to a DNA duplex based on T-Hg2+-T mismatches. Then, Exo III catalyzes the digestion of duplex of hairpin DNA, liberating short single-stranded DNA (ssDNA) product and releasing Hg2+. The produced ssDNA can be recognized by CRISPR/Cas12a and further trigger the collateral cleavage ability of CRISPR system, which can indiscriminately cut short Fc-ssDNA on the electrode, producing an ECL signal of Au@CNDs. Under the optimized conditions, a detection limit of 0.45 fM was realized for Hg2+ determination. The efficiency and robustness of this biosensor were further verified by using environmental water and human serum samples, which demonstrated its potential application in the environment and biological science.

CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene.

Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.

An aptamer-assisted biological nanopore biosensor for ultra-sensitive detection of ochratoxin A with a portable single-molecule measuring instrument

Biological nanopore-based single-molecule detection technology has shown ultrahigh sensitivity to various target analyte. But the detection scope of interesting targets is limited due to the lack of effective signal conversion strategies. In addition, conventional nanopore detection instruments are cumbersome, resulting nanopore detection can only be performed in laboratory. Herein, a customizable nanopore current amplifier is constructed to lower the cost and increase the portability of the nanopore instrument, and then an immobilized aptamer-based signal conversion strategy is proposed for α-hemolysin (α-HL) nanopore to detect small molecules (ochratoxin A, OTA). The presence of OTA in sample would trigger the release of probe single-strand DNA (ssDNA) from magnetic beads, which could subsequently cause current blockage in nanopore. The results show that the signal frequency of probe ssDNA has a linear relationship with the OTA concentration in the range of 2 × 101～2 × 103 pmol/L. Compared to other methods, our sensing system has achieved an ultra-sensitive detection of OTA with the detection limit as low as 1.697 pmol/L. This strategy could broaden the scope of nanopore detection and have the potential for rapid and in-situ detection of other food contaminants in the future.

Development of a Novel Electrochemical Sensor Based on Functionalized Carbon Black for the Detection of Guanine Released from DNA Hydrolysis

AbstractIn this work, we reported a new strategy based on DNA strands hydrolysis in combination with the employment of functionalized carbon black modified screen‐printed electrode (f‐CB/SPE) for guanine detection. Using glass microbeads as a solid substrate of ssDNA probe fixation, and under appropriate conditions of temperature, time, and hydrochloric acid concentration, DNA hydrolysis has been successfully used to release the guanine, allowing its oxidation at f‐CB/SPE using differential pulse voltammetry. The selectivity was successfully tested with a non‐complementary sequence, and a detection limit of 0.7 nM of the target DNA has been reached.

Fabrication and quantitative performance analysis of a low-cost, flexible CNT network-based DNA sensors using ink-jet printing

This paper reports the design, fabrication and quantitative performance analysis of a low-cost, flexible carbon nanotube (CNT) network-based deoxyribonucleic acid (DNA) sensor. These sensors comprise an array of ink-jet printed silver (Ag) electrodes on a transparent polyethylene terephthalate (PET) flexible substrate, where a CNT network acts as a sensing layer. DNA hybridization is studied by immobilizing single-stranded DNA (ssDNA) probes on the CNT surface; these probes recognize their complementary DNA target. The analytic hierarchy process (AHP) was used to analyze the quantitative performance of flexible CNT biosensors. Various influencing factors (performance indicators) have been identified and quantified in the performance of flexible CNT biosensors (before bending, during bending and after bending). Additionally, the noise and other external factors contributing to the measured real signal have been quantified. The interpretation of the overall outcome will enable the improvement of the performance of flexible biosensors fabricated through large-scale manufacturing for possible commercialization.

Interfacial DNA Framework-Enhanced Background-to-Signal Transition for Ultrasensitive and Specific Micro-RNA Detection.

Interfacial DNA self-assembly is fundamental to solid nucleic acid biosensors, whereas how to improve the signal-to-noise ratio has always been a challenge, especially in the charge-based electrochemical DNA sensors because of the large noise from the negatively charged DNA capture probes. Here, we report a DNA framework-reversed signal-gain strategy through background-to-signal transition for ultrasensitive and highly specific electrical detection of microRNAs (miRNAs) in blood. By using a model of enzyme-catalyzed deposition of conductive molecules (polyaniline) targeting to DNA, we observed the highest signal contribution per unit area by the highly charged three-dimensional (3D) tetrahedral DNA framework probe, relative to the modest of two-dimensional (2D) polyA probe and the lowest of one-dimensional (1D) single-stranded (ss)DNA probe, suggesting the positive correlation of background DNA charge with signal enhancement. Using such an effective signal-transition design, the DNA framework-based electrochemical sensor achieves ultrasensitive miRNAs detection with sensitivity up to 0.29 fM (at least 10-fold higher than that with 1D ssDNA or 2D polyA probes) and high specificity with single-base resolution. More importantly, this high-performance sensor allows for a generalized sandwich detection of tumor-associated miRNAs in the complex matrices (multiple cell lysates and blood serum) and further distinguishes the tumor patients (e.g., breast, lung, and liver cancer) from the normal individuals. These advantages signify the promise of this miRNA sensor as a versatile tool in precision diagnosis.

Development of paper-based DNA sensor for detection of O. tsutsugamushi using sustainable GQDs@AuNPs nanocomposite

The graphene quantum dots (GQDs) was synthesized using potato starch and water by hydrothermal method and further used for reduction of tetracholoroauric acid to form graphene quantum dots-gold (GQDs@AuNPs) nanocomposite. The GQDs/GQDs@AuNPs were analyzed using FTIR, UV–Vis, Flourometry and HR-TEM. The synthesized GQDs@AuNPs were further used for fabrication of cost-effective screen-printed paper electrode (SPPE) based DNA sensor for the detection of O. tsutsugamushi using htrA gene specific 5′NH2 linked DNA probe. Modification of SPPE using GQDs@AuNPs nanocomposite and ssDNA probe was monitored using EIS, FTIR, FE-SEM and AFM. The sensor detection limit (LOD) was assessed as 0.002 ng/μl from the standard calibration curve with the correlation coefficient, R2 = 0.993. The sensitivity of the DNA sensor was calculated as 7700 μA/cm2/ng for ssGDNA of O. tsutsugamushi using cyclic voltammetry. The sensor validation was done using scrub typhus patient's blood DNA samples. The sensor showed good storage stability at 4 °C for six months with just a loss of 12% of the initial current values. The SPPE/DNA sensor developed is very specific, sensitive, stable and detects O. tsutsugamushi in less time.

DIRECT2: A novel platform for a CRISPR–Cas12-based assay comprising universal DNA–IgG probe and a direct lateral flow test

CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT2). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR–Cas12 activity detection and its bioanalytical applications.

Test Strip Platform Spin-Off for Telomerase Activity Detection: Development of an Electrochemical Biosensor.

Telomerase overexpression has been associated directly with cancer, and the enzyme itself is recognized within the scientific community as a cancer biomarker. BIDEA’s biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using electrochemical impedance spectroscopy (EIS). This BBS is an interdigital gold (GID) electrode array similar in size and handling to a portable glucose sensor. For the detection of the biomarker, BBS was modified by the immobilization of a telomere-like single strand DNA (ssDNA) on its surface. The sensor was exposed to telomerase-positive extract from commercially available cancer cells, and the EIS spectra were measured. Telomerase recognizes the sequence of this immobilized ssDNA probe on the BBS, and the reverse transcription process that occurs in cancer cells is replicated, resulting in the ssDNA probe elongation. This surface process caused by the presence of TA generates changes in the capacitive process on the electrode array microchip surface, which is followed by EIS as the sensing tool and correlated with the presence of cancer cells. The telomerases’ total cell extraction protocol results demonstrate significant changes in the charge-transfer resistance (Rct) change rate after exposure to telomerase-positive extract with a detection limit of 2.94 × 104 cells/mL. Finally, a preliminary study with a small set of “blind” uterine biopsy samples suggests the feasibility of using the changes in the Rct magnitude change rate (Δ(ΔRct/Rcti)/Δt) to distinguish positive from negative endometrial adenocarcinoma samples by the presence or absence of TA.

Programming the trans-cleavage Activity of CRISPR-Cas13a by Single-Strand DNA Blocker and Its Biosensing Application.

The precise and controllable programming of the trans-cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg2+ concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the trans-cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked trans-cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the trans-cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.