Detection Of Nucleic Acid Molecules Research Articles

A Universal CRISPR/Cas12a‐Assisted Methodology Based on Duplex Switch Structure to Detect Multiple Types of Targets

AbstractRecent years, molecular detection technology has been playing an unprecedentedly important role in disease prevention and public health. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems such as CRISPR/Cas12a and CRISPR/Cas13a, have been increasingly used in the detection of nucleic acid molecules because of its collateral cleavage ability in recent years. Herein, we develop a universal CRISPR/Cas12a‐assisted methodology based on a nucleic acid duplex switch structure that can distinguish different categories of targets, such as DNA, RNA and small molecules. It is worth noting that for nucleic acid detection, this method can significantly identify single base substitutions with high specificity, compared with other Cas12a‐assisted biosensing systems. The experimental results suggest that this method has great specificity for different targets, promising to be applied to rapid molecular diagnosis.

Amplification-Free Detection of SARS-CoV-2 Down to Single Virus Level by Portable Carbon Nanotube Biosensors.

The rapid and sensitive detection of trace-level viruses in a simple and reliable way is of great importance for epidemic prevention and control. Here,a multi-functionalized floating gate carbon nanotube field effect transistor (FG-CNT FET) based biosensor is reported for the single virus level detection of SARS-CoV-2 virus antigen and RNA rapidly with a portable sensing platform. The aptamers functionalized sensors can detect SARS-CoV-2 antigens from unprocessed nasopharyngeal swab samples within 1 min. Meanwhile, enhanced by a multi-probe strategy, the FG-CNT FET-based biosensor can detect the long chain RNA directly without amplification down to single virus level within 1 min.Thedevice, constructed with packaged sensor chips and a portable sensing terminal, can distinguish 10 COVID-19 patients from 10 healthy individuals in clinical tests both by the RNAs and antigens by a combination detection strategy with an combined overall percent agreement (OPA) close to 100%.Theresults provide a general and simple method to enhance the sensitivity of FET-based biochemical sensors for the detection of nucleic acid molecules and demonstrate that the CNT FG FET biosensor is a versatile and reliable integrated platform for ultrasensitive multibiomarker detection without amplification and has great potential for point-of-care (POC) clinical tests.

Ultra-low concentration detection of DNA without its amplification using a biological nanopore.

Nanopore sensing is a powerful tool for single-molecule detection. A single molecule is recognized and detected from the information obtained when the target molecule passes through the nanoscale pore by electrophoresis. However, practically it is challenging to measure the target molecules at ultra-low concentrations because it takes a long time for the target molecule to enter the pore. Several elaborate techniques with a biological nanopore, α-hemolysin (αHL), have been proposed to detect the subpicomolar or femtomolar nucleic acids. Wang et al. used a salt gradient between solutions that sandwiched the αHL pore to enhance the electric field and increased the capture frequency of the targets, resulting in the detection of nucleic acid molecules at 0.1 pM. Zhang et al. showed the detection of target nucleic acids at 1 fM using the αHL pore with isothermal amplification of the targets. In contrast, we have recently reported the detection of 0.5 fM nucleic acids without their amplification reaction in the presence of a 500 nM probe complementary binding to the target. The excess amount of the probe relative to the target molecules was assumed to increase the capture frequency of the target/probe duplexes. Here, we attempt to reveal the mechanism of detecting DNA molecules at ultra-low concentrations with the αHL nanopore using probes with complementary sequences to the target molecules. We found that the complementary strand of the target in the probe sequence enhances the capture frequency of the target/probe duplexes by interacting with the target/probe molecules. In addition, higher concentrations of the probe relative to the target molecule (>107-fold) resulted in more frequent capture. This approach potentially advances nanopore technology toward its application in the direct detection of nucleic acid biomarkers at ultra-low levels in body fluids.

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets

Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) and insitu 3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

(Invited) Ultrasensitive Detection of Nucleic Acid Molecules with Interferometric Plasmonic Microscopy

The detection of nucleic acid molecules plays important role in clinical diagnosis of diseases and fundamental biomedical research. The widely used polymerized chain reaction technique requires hours of amplification process, and is prone to aerosol pollution. Here, we presented the amplification-free digital sensing of nucleic acid molecules with the interferometric plasmonic microscopy (iPM). A sandwich assay was performed with the primary DNA probes coated on the plasmonic sensing surface and the secondary DNA probes tethered Au nanoparticles. Hybridization between DNA probes and single analyte molecules was monitored by the imaging of single Au nanoparticle with iPM system. By digitally counting the events of hybridization, the detection limit at the fM level could be achieved. Without amplification, this assay could be done within one hour. Furthermore, by analyzing the real-time binding behaviro during single hybridization event, non-specific bindings could be excluded from the specific ones, resulting in the improved specificity. This technique could be further improving by a careful design of the kinetic binding properties between DNA probes and analyte molecules.

Recent Advances in the Fabrication and Application of Graphene Microfluidic Sensors.

This review reports the progress of the recent development of graphene-based microfluidic sensors. The introduction of microfluidics technology provides an important possibility for the advance of graphene biosensor devices for a broad series of applications including clinical diagnosis, biological detection, health, and environment monitoring. Compared with traditional (optical, electrochemical, and biological) sensing systems, the combination of graphene and microfluidics produces many advantages, such as achieving miniaturization, decreasing the response time and consumption of chemicals, improving the reproducibility and sensitivity of devices. This article reviews the latest research progress of graphene microfluidic sensors in the fields of electrochemistry, optics, and biology. Here, the latest development trends of graphene-based microfluidic sensors as a new generation of detection tools in material preparation, device assembly, and chip materials are summarized. Special emphasis is placed on the working principles and applications of graphene-based microfluidic biosensors, especially in the detection of nucleic acid molecules, protein molecules, and bacterial cells. This article also discusses the challenges and prospects of graphene microfluidic biosensors.

Label-Free Detection of miRNA Using Surface-Enhanced Raman Spectroscopy.

miRNA plays a vital role in many biological processes by regulating the expression of target genes and is considered to be a promising disease biomarker. Although surface-enhanced Raman spectroscopy (SERS) has been widely used for the detection of nucleic acid molecules, obtaining a characteristic SERS signal of unlabeled RNA in a nondestructive state still remains a challenge. Herein, titanium ions were used as aggregating agents to induce the aggregation of silver nanoparticles, forming hot spots, and the fingerprint information on miRNAs was successfully obtained. This method was used to determine the RNA sequence of homopolymeric bases and the peak position of each base in the Raman spectrum. The obtained RNA spectrum was normalized with the signal intensity of ribose at 959 cm-1, and the base contents of a series of mature miRNA sequences were quantified. Subsequently, the characteristic SERS signal of the RNA hybridization event was obtained by studying the process of RNA hairpin structure formation, and the role of the precursor mir-21 in regulating the target streptomycin sulfate was successfully analyzed. The changes in the SERS signal intensity at the interaction site were accurately identified. This method has potential applications in biological functions of miRNAs, molecular diagnosis, disease treatment, and targeted drug design.

Integrated microfluidic systems with sample preparation and nucleic acid amplification.

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment.

Robust label-free microRNA detection using one million ISFET array.

Detection of nucleic acid molecules is one of the most pervasive assays in biology, medicine, and agriculture applications. Currently, most comely used DNA/RNA detection platforms use fluorescence labeling and require lab-scale setting for performing the assay. There is a need for developing less expensive, label-free, and rapid detection of biomolecules with minimal utilization of resources. Use of electrical approaches for detection of biomolecules by utilizing their inherent charge is a promising direction for biosensing assays. Here, we report a 1024 × 1024 array of Ion Sensitive Field Effect Transistors (ISFET) as label free sensors for detection of nucleic acid molecules. Using PNA probe functionalized on these ISFET array, we robustly detected miRNA Let-7b by measuring changes in drain current after hybridization of target molecules with concentration as low as 1nM. We demonstrate that mismatched or non-complementary target molecules resulted in statistically smaller changes. Most importantly, the high-density sensor array shows unprecedented reliability and robustness with P values <0.0001 for all experiments. Practical implementation of this platform could have a wide range of applications in high-throughput nucleic acid genotyping, detection of amplified pathogenic nucleic acid, detection of cell-free DNA, and electrical readouts for current hybridization-based DNA biomolecular assays.

On-nylon membrane detection of nucleic acid molecules by rolling circle amplification

Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3′ end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.

Porous silicon ring resonator for compact, high sensitivity biosensing applications.

A ring resonator is patterned on a porous silicon slab waveguide to produce a compact, high quality factor biosensor with a large internal surface area available for enhanced recognition of biological and chemical molecules. The porous nature of the ring resonator allows molecules to directly interact with the guided mode. Quality factors near 10,000 were measured for porous silicon ring resonators with a radius of 25 μm. A bulk detection sensitivity of 380 nm/RIU was measured upon exposure to salt water solutions. Specific detection of nucleic acid molecules was demonstrated with a surface detection sensitivity of 4 pm/nM.

Electronic Detection of Nucleic Acid Molecules with a Field-Effect Transistor

ABSTRACTCurrently, systems for the detection of nucleic acid sequences, known as DNA-chips, are getting lots of attention. Such systems usually involve either an enzymatic or chemical labelling reaction as part of the detection process. The next generation of DNA-chips aims at a labelfree, fully electronic readout system. Several new approaches to signal generation that avoid a labelling step have been developed in recent years. Besides other surface sensitive measurements the possibility of electrochemical impedance and field-effect measurements for the detection of biomolecules have been discussed. The fully electronic detection of charged biomolecules based on the field-effect principle offers a labelfree method, which combines the unique sensitivity and selectivity of biomolecular recognition reactions with an electronic chip-based readout. In this approach one type of molecules is fixed at a surface and the biomolecular reaction with complementary molecules is detected by change in the drain-source current of the transistor. This change can occur by a change of the interface capacitance of the transistor gate or by change of the surface potential during adsorption of the molecules. At the moment a complete theoretical description of the detection principle is still under discussion. However, the fully electronic readout of biomolecular reactions offers a unique principle for the construction of many different sensors for bioassays. We are working on an approach to detect the hybridization of DNA sequences using electrolyte-oxide-semiconductor field-effect transistor (EOSFET) arrays. This method allows direct and in situ detection of specific DNA sequences without any labelling.