Nucleic Acid Biosensor Research Articles

Recent Advances in the CRISPR/Cas-Based Nucleic Acid Biosensor for Food Analysis: A Review.

Food safety is a major public health issue of global concern. In recent years, the CRISPR/Cas system has shown promise in the field of molecular detection. The system has been coupled with various nucleic acid amplification methods and combined with different signal output systems to develop a new generation of CRISPR/Cas-based nucleic acid biosensor technology. This review describes the design concept of the CRISPR/Cas-based nucleic acid biosensor and its application in food analysis. A detailed overview of different CRISPR/Cas systems, signal amplification methods, and signal output strategies is provided. CRISPR/Cas-based nucleic acid biosensors have the advantages of high sensitivity, strong specificity, and timeliness, achieving fast analysis of a variety of targets, including bacteria, toxins, metal ions, pesticides, veterinary drugs, and adulteration, promoting the development of rapid food safety detection technology. At the end, we also provide our outlook for the future development of CRISPR/Cas-based nucleic acid biosensors.

Rapid Label-free and Amplification-free Detection of SARS-CoV-2 Using Solution-Gated Graphene Transistor-Based Nucleic Acid Biosensors in a Portable Biosensing Microsystem

Abstract The COVID-19 pandemic has highlighted the significance of swift and efficient large-scale screening to prevent epidemic outbreaks. Solution-gated graphene transistors (SGGTs) have emerged as a valuable asset in creating point-of-care (POC) sensing platforms for detecting SARS-CoV-2, owing to their notable stability and sensitivity in solution environments. However, the poor interface engineering of sensing electrodes is of great concern for reliable functionalization, leading to poor sensitivity. In this study, we present an interface engineering strategy for realizing the functionalization of SGGT sensing electrodes with high stability and sensitivity to construct a SARS-CoV-2 nucleic acid biosensor. The developed biosensor exhibits the capability to detect ORF1ab gene fragments at a concentration as low as 10−16 M without requiring additional nucleic acid amplification. Furthermore, the entire detection process can be accomplished using a portable biosensing microsystem within 30 minutes. This research holds promise for advancing the study of FET-based biosensors and facilitating their practical application in clinical settings.

CRISPR: The frontier technology of next-generation RNA detection

Rapid and accurate molecular diagnostics are crucial for disease diagnosis and precision medicine. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins have emerged as highly effective tools for molecular diagnostics. Numerous nucleic acid detection instruments and biosensors utilizing the CRISPR/Cas system have been developed. The profiling activity of CRISPR/Cas effectors has facilitated the creation of instrument-free, sensitive, precise, and rapid nucleic acid diagnostics. This review summarizes recent advancements in CRISPR technology for RNA detection, focusing on the application of Cas12 and Cas13 systems in two scenarios: in combination with isothermal amplification technology and without amplification. It also explores the significant potential of CRISPR as a next-generation technology for RNA detection and anticipates future developments. The ongoing advancements in CRISPR are expected to enhance precision and convenience in RNA testing, impacting both biomedical research and public health practices.

Visual detection of microRNAs using gold nanorod-based lateral flow nucleic acid biosensor and exonuclease III-assisted signal amplification.

An ultrasensitive method for the visual detection of microRNAs (miRNAs) in cell lysates using a gold nanorod-based lateral flow nucleic acid biosensor (GN-LFNAB) and exonuclease III (Exo III)-assisted signal amplification. The Exo III-catalyzed target recycling strategy is employed to generate a large number of single-strand DNA products, which can be detected by GN-LFNAB visually. With the implementation of a unique recycling strategy, we have demonstrated that the miRNA in the concentration as low as 0.5pM can be detected without the need for instrumentation, providing a detection limit that surpasses previous reports. The new biosensor is ultrasensitive and can be applied to the reliable monitoring of miRNAs in cell lysates with high accuracy. The approach offers a simple and rapid tool for cancer diagnosis and clinical biomedicine, thanks to its flexibility, simplicity, cost-effectiveness, and convenience. This new method has the potential to significantly improve the detection and monitoring of cancer biomarkers, ultimately contributing to more effective cancer diagnosis and treatment.

Digitized Kinetic Analysis Enhances Genotyping Capacity of CRISPR-Based Biosensing.

CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of crRNA. However, existing CRISPR-based genotyping methods are limited by the mismatch tolerance of Cas effectors, necessitating a comprehensive screening of crRNAs to effectively distinguish between wild-type and point-mutated sequences. To circumvent the limitation of conventional CRISPR-based genotyping, here, we introduce Single-Molecule kinetic Analysis via a Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages the differential kinetics of the signal increase in CRISPR/Cas systems, which is modulated by the complementarity between crRNA and the target sequence. It employs single-molecule digital measurements to discern mutations based on kinetic profiles that could otherwise be obscured by variations in the target concentrations. We applied SMART-dCRISPR to genotype notable mutations in SARS-CoV-2, point mutation (K417N) and deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, and Omicron BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, we introduced a portable digital real-time sensing device to streamline SMART-dCRISPR and enhance its practicality for point-of-care settings. The combination of a rapid and sensitive isothermal CRISPR-based assay with single-molecule kinetic analysis in a portable format significantly enhances the versatility of CRISPR-based nucleic acid biosensing and genotyping.

Antisense oligonucleotide conjugated gold nanoconstructs-based electrochemical biosensor for detection of SARS-CoV-2

Nanoconstructs of gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 specific antisense oligonucleotides (ASO) have been utilized to develop sensitive electrochemical nucleic acid biosensor for the detection of SARS-CoV-2 RNA. AuNPs were prepared through a one-pot synthesis method by utilizing Poly-L-Lysine (PLL) biopolymer and as synthesised AuNP were characterized by various analytical techniques such as UV–Vis spectroscopy, X-ray Diffraction (XRD) analysis, Fourier Transform Infra-Red spectroscopy (FT-IR), zeta potential, and Transmission Electron Microscopy (TEM). Poly-L-Lysine functionalized AuNPs (PLL-AuNPs) nanoconstructs platform was employed for immobilization of SARS-CoV-2 specific antisense oligonucleotides (ASO-conjugated PLL-AuNPs) via electrostatic interactions. The PLL-AuNPs were drop casted on glassy carbon electrode (GCE) following immobilization of ASO for fabrication of electrochemical biosensor. The ASO-conjugated PLL-AuNPs nanoconstructs were characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques. The responsiveness of ASO-conjugated PLL-AuNPs nanoconstructs in presence SARS-CoV-2 RNA was monitored using the DPV, SWV and EIS technique, where methylene blue was employed as an electrochemical indicator for DNA-RNA hybridization detection. The biosensor exhibits a detection range for SARS-CoV-2 RNA infection ranging from 0 to 100 nM, with a limit of detection at 30.2 nM. The electrode, modified with ASO-conjugated PLL-AuNPs, was employed for the detection of SARS-CoV-2 RNA from clinical samples collected from COVID-19-positive individuals.

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy.

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 μL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation.

The escalating threat of emerging diseases, often stemming from contaminants and lethal pathogens, has precipitated a heightened demand for sophisticated diagnostic tools. Within this landscape, the functional nucleic acid (FNA) biosensor, harnessing the power of single-stranded DNA (ssDNA), has emerged as a preeminent choice for target analyte detection. However, the dependence on ssDNA has raised difficulties in realizing it in biological samples. Therefore, the production of high-quality ssDNA from biological samples is critical. This review aims to discuss strategies for generating ssDNA from biological samples for integration into biosensors. Several innovative strategies for ssDNA generation have been deployed, encompassing techniques, such as asymmetric PCR, Exonuclease-PCR, isothermal amplification, biotin-streptavidin PCR, transcription-reverse transcription, ssDNA overhang generation, and urea denaturation PAGE. These approaches have been seamlessly integrated with biosensors for biological sample analysis, ushering in a new era of disease detection and monitoring. This amalgamation of ssDNA generation techniques with biosensing applications holds significant promise, not only in improving the speed and accuracy of diagnostic processes but also in fortifying the global response to deadly diseases, thereby underlining the pivotal role of cutting-edge biotechnology in public health and disease prevention.

Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.

Portable, and ultrasensitive HR-HPV tests based on nucleic acid biosensors.

Cervical cancer is the third most common cancer threatening women's health globally, and high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer worldwide. Given the recurrent nature of HR-HPV infection, accurate screening is essential for its control. Since the commonly used polymerase chain reaction (PCR) technique is limited by professional equipment and personnel, convenient and ultrasensitive detection methods for HR-HPV are still highly needed. As new molecular detection methods, nucleic acid amplification-based biosensors have the advantages of high sensitivity, rapid operation, and portability, which are helpful for point-of-care testing in rural and remote areas. This review summarized nucleic acid biosensors for HR-HPV screening based on a variety of nucleic acid amplification strategies involved in improved PCR, loop-mediated isothermal amplification, recombinase polymerase amplification, hybridization chain reaction, catalyzed hairpin assembly, and CRISPR/Cas systems. In combination with microfluidic technology, lateral flow assays, electrochemical analysis and other sensing technologies, HR-HPV nucleic acid biosensors have the advantages of high throughput, short response time, high sensitivity and easy operation in the field. Although there are still shortcomings, such as high cost and poor reproducibility, this approach will be suitable for on-site screening of HR-HPV infection or cervical cancer and for auxiliary clinical diagnosis in complex environments and poor areas in the future.

Electrochemical Nucleic Acid Biosensor on a Microfluidic Chip to Understand the Coupling between Electrochemistry and Microfluidics

Electrochemical Nucleic Acid Biosensor on a Microfluidic Chip to Understand the Coupling between Electrochemistry and Microfluidics

A Label‐Free Deoxyribozyme Sensor for m6A Demethylase Activity Detection†

Comprehensive SummaryA label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening.

DNA nanotechnology for nucleic acid analysis: sensing of nucleic acids with DNA junction-probes.

DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.

A label-free and immobilization-free approach for constructing photoelectrochemical nucleic acid sensors utilizing DNA-silver nanoparticle affinity interactions.

Efficient and affordable nucleic acid detection methods play a pivotal role in various applications. Herein, we developed an immobilization-free and label-free strategy to construct a photoelectrochemical nucleic acid biosensing platform based on interactions between silver nanoparticles and DNA. First, CRISPR-Cas12a exhibited a trans-cleavage effect on adenine nucleotide sequences upon recognizing the target DNA. The resulting adenine nucleotide sequences of varying lengths then engaged in interactions with silver nanoparticles, leading to a solution characterized by distinct light transmittance. Subsequently, the solution was positioned between the light source and the photoelectrode, strategically impacting the photon absorption step within the photoelectrochemical process. Consequently, the detection of nucleic acid was accomplished through the analysis of the resultant photocurrent signal. The developed platform exhibits a detection limit of 0.06 nM (S/N = 3) with commendable selectivity. The innovative use of adenine nucleotide sequences as cost-effective probes interacting with silver nanoparticles eliminates the need for complex interfacial immobilization processes, significantly simplifying the fabrication of DNA sensors. The outcomes of our research present a promising pathway for advancing the development of economically feasible miniature DNA sensors.

Pseudouridine-modified RNA probe for label-free electrochemical detection of nucleic acids on 2D MoS2 nanosheets.

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS2 surface and causes a change in interfacial electrochemical charge transfer in the MoS2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS2. Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS2 surface. Ψ-RNA maintained robust interactions with the MoS2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification.

A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.

Development of a nucleic acid-based screen printed electrochemical biosensor using Ti3C2Tx-MXene for the detection of SARS-CoV-2

The SARS-CoV-2 virus poses a major risk to human health which has put a tremendous strain on already stretched healthcare resources. Despite continuous improvements in Coronavirus Disease-19 (COVID-19) detection, treatment technology and ongoing global immunization efforts, the lethal virus has undergone several mutations and is still claiming millions of lives worldwide. In this respect, the development of a hybridization-based nucleic acid biosensor for SARS-CoV-2 virus is the major focus of the current effort in response to the critical demand for developing quick and accurate diagnostic methods. Herein, we employ Screen Printed Carbon Electrode (SPCE) coated with Ti3C2Tx-MXene nanosheets followed by amino-functionalized probe DNA (NH2-pDNA) as a robust surface for the sensing of SARS-CoV-2. Electrochemical impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry were employed for assessing the electrochemical behavior of NH2-pDNA/Ti3C2Tx/SPCE bioelectrode. Meanwhile, the analytical sensor response for SARS-CoV-2 was studied using impedance within target DNA concentration of 0.1 pM − 1 µM. The developed biosensor displayed detection limit of 0.004 pM, showing a high degree of selectivity in the presence of non-complementary interferents with a shelf life of 40 days. Additionally, a good linearity for the detection of target sequence in spiked serum samples was established; as a result, exhibiting huge potential for use in the contemporary healthcare sector for fast, accurate and reliable diagnosis of COVID-19 in a point-of-care scenario.

Aromatic Ring–Mediated Nonspecific Signaling Mechanism and Nafion‐Dominated Solution in Graphene Field‐Effect Transistor–Based Nucleic Acid Biosensors

AbstractGraphene field‐effect transistors (G‐FETs) have attracted widespread attention in disease diagnosis, benefiting from these advantages of high sensitivity, label‐free, easy integration, and direct detection of nucleic acids (NAs) in liquid environments. However, the problem of nonspecific signals in G‐FETs is not fundamentally solved due to a lack of systematic theoretical research to support the development of effective solutions. Thus, researchers have to rely on speculative mechanisms to minimize nonspecific signals in experiments as much as possible. Herein, the nonspecific signal mechanism caused by eight types of π–π interaction paths mediated by aromatic rings is theoretically determined. Based on theoretical simulation results, the feasibility of blocking nonspecific signal paths through Nafion functionalization methods is experimentally verified. Experiments confirm that Nafion‐modified G‐FETs (NMG‐FETs) have excellent performance in avoiding nonspecific signals compared to traditional G‐FETs. Furthermore, the NMG‐FET achieves ultra‐sensitive detection of Down syndrome–related DNA down to 1 aM and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA down to 5 aM, and shows good specificity in base recognition. This study is expected to promote the theoretical advancement of the nonspecific signal mechanism in G‐FET NA detection and offer a practical strategy for improving signal purity and accuracy.

Instrument-free detection of African swine fever virus in raw blood samples via CRISPR/Cas12a

Nucleic acid amplification coupled with clustered regularly interspaced short palindromic repeats (CRISPR) detection has been widely applied in nucleic acid biosensing for its high sensitivity and specificity. However, the separated two-step increases the complexity of hands-on operations and the risks of amplicon-formed aerosol transmission, which hinders the popularization of nucleic acid detection for a wider range of scenarios. In this study, we propose a microchip-assisted detection platform for African swine fever virus (ASFV) detection. It involves ultrafast nucleic acid extraction, on-chip loop-mediated isothermal amplification (LAMP), CRISPR detection, and lateral flow assay-based visualized result readout. The liquid transfer between different reaction chambers of the microchip is driven by the gravity of liquid solutions. Therefore, complex liquid transfer and instruments are not required. The platform, which is named as All-In-Free (all nucleic acid detection steps included, instrument-free), can detect 10 copies/µL ASFV in approximately 45 min. For 32 real blood samples, the detection results of the All-In-Free platform are 96.3% positive and 100% negative consistent with polymerase chain reaction (PCR) testing. The All-In-Free detection platform has the advantages of low cost, high efficiency, and easy-to-operation, which is promising for popularization in low-resourced areas.

Comparison of DNA-Gold Nanoparticle Conjugation Methods: Application in Lateral Flow Nucleic Acid Biosensors.

Lateral flow nucleic acid biosensors (LFNABs) have attracted extensive attention due to their rapid turnaround time, low cost, and results that are visible to the naked eye. One of the key steps to develop LFNABs is to prepare DNA-gold nanoparticle (DNA-AuNP) conjugates, which affect the sensitivity of LFNABs significantly. To date, various conjugation methods-including the salt-aging method, microwave-assisted dry heating method, freeze-thaw method, low-pH method, and butanol dehydration method-have been reported to prepare DNA-AuNP conjugates. In this study, we conducted a comparative analysis of the analytical performances of LFNABs prepared with the above five conjugation methods, and we found that the butanol dehydration method gave the lowest detection limit. After systematic optimization, the LFNAB prepared with the butanol dehydration method had a detection limit of 5 pM for single-strand DNA, which is 100 times lower than that of the salt-aging method. The as-prepared LFNAB was applied to detect miRNA-21 in human serum, with satisfactory results. The butanol dehydration method thus offers a rapid conjugation approach to prepare DNA-AuNP conjugates for LFNABs, and it can also be extended to other types of DNA biosensors and biomedical applications.