Sensitive DNA Detection Research Articles

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

Label-free terahertz microfluidic biosensor for sensitive DNA detection using graphene-metasurface hybrid structures

Metasurface assisted terahertz (THz) real-time and label-free biosensors have attracted intense attention. However, it is still challenging for specific detection of highly absorptive liquid samples with high sensitivity in the THz range. Here, we incorporated graphene with THz metasurface into a microfluidic cell for sensitive biosensing. The proposed THz graphene-metasurface microfluidic platform can effectively reduce the volume of the sample solution and boost the interaction between biomolecules and THz waves, thus enhancing the sensitivity. As a proof of concept, comparative experiments using other three kinds of microfluidic cells (pure microfluidic cell, metasurface-based microfluidic cell and graphene-based microfluidic cell) were conducted to explore and verify the sensing mechanism, which evidences the high sensitivity of delicate sensing based on the hybrid graphene-metasurface THz microfluidic device. Furthermore, to perform biosensing applications on that basis, specific aptamers were modified on the graphene-metasurface, enabling DNA sequences of foodborne pathogen Escherichia coli O157:H7 to be recognized. Based on the THz microfluidic biosensor, 100 nM DNA short sequences can be successfully detected. The sensing results of antibiotics and DNA based on the graphene-metasurface microfluidic biosensor confirm the superiority of the proposed design and considerable promise in THz biosensing. The novel sensing platform provides the merits of enabling highly sensitive, label-free, low-cost, easy to use, reusable, and real-time biosensing, which opens an exciting prospect for nanomaterial-metasurface hybrid structure assisted THz label-free biosensing in liquid environment.

Development of a new and sensitive method for the detection of pork adulteration in gelatin and other highly processed food products

ABSTRACT Porcine adulteration has become a major concern amongst communities including Muslims and Jews. Reliable detection of the presence of porcine DNA in gelatin and highly processed food samples is essential for Halal and kosher food control. In this study, a forensic approach involving DNA isolation and real-time polymerase chain reaction (PCR) was performed to detect the presence of porcine DNA in commercial gelatin and processed foods containing porcine by-products. The method was compared with commercially sensitive porcine DNA detection kits (Biotecon and R-Biopharm) which are commonly used in food control laboratories for Halal and kosher authentication. The results indicated that a newly developed method called TübiGel was at least 10 times more sensitive for porcine DNA detection. The TübiGel method was found to have a detection limit of 0.01% porcine gelatin, whilst the Biotecon method had 0.1% and R-Biopharm method detected >5% porcine gelatin. The forensic DNA isolation approach of the TübiGel method was found to be a critical step. In addition, real-time PCR of TübiGel method was also found to detect porcine DNA better than real-time PCR of commercial kits.

High-fidelity detection of DNA combining the CRISPR/Cas9 system and hairpin probe

Methods that enable specific and sensitive detection of DNA are greatly required for high-fidelity sequence measurement and single-nucleotide variations (SNVs) genotyping. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems have provided revolutionary tools for detecting nucleic acids. However, most of the current CRISPR/Cas-based DNA biosensing platforms suffer from inherent off-target effects of Cas proteins and require pre-amplification processes, which compromise the analytical fidelity. In this work, a CRISPR/Cas9-triggered hairpin probe-mediated biosensing method (namely CHP) was used to directly read the original DNA sequences, while effectively neutralizing the off-target effect and achieving high sensitivity. This technique can quantify DNA targets with a limit of detection (LOD) at the attomole level and identify SNVs with allelic fractions as low as 0.01%~0.1%. Moreover, we show that the CHP system is applicable in detecting mutations in serum samples without DNA isolation steps. Collectively, the CHP system is a sensitive and high-fidelity platform, which promises a great potential for providing robust tool for DNA sequence analysis and SNVs genotyping.

Plasmonic Ag nanocube enhanced SERS biosensor for sensitive detection of oral cancer DNA based on nicking endonuclease signal amplification and heated electrode

Surface-enhanced Raman scattering (SERS) biosensing has attracted great attention due to its multiplexed capability, high sensitivity, and rapid readout. In this study, plasmonic Ag nanocube (AgNC) enhanced SERS biosensing platform was proposed by coupling nicking endonuclease-assisted target recycling signal amplification with heated electrode for sensitive detection of DNA species related to oral cancer. SERS tags were manufactured by signal DNA (sDNA) conjugating onto the surfaces of AgNCs, and 4-mercaptobenzoic acid (4-MBA) assembled on the surfaces of AgNC/sDNA as a Raman reporter. The sharp feature of edges and corners of AgNCs with high electromagnetic field provided SER hot spots with high quality, and stronger SERS signal could be achieved. In addition, the activity of nicking endonuclease Nt.BstNBI could be greatly enhanced by elevating the temperature of the surface of the heated Au electrode (HAuE) during Nt.BstNBI-assisted target recycling amplification process, resulting in dramatic amplification of target DNA detection. All of these contributed to the high sensitivity of the developed SERS biosensor. A limit of detection (LOD) of 3.1 fM (S/N = 3) could be obtained with an electrode temperature of 45 °C. This proposed SERS biosensor was anticipated to act as a promising biosensing platform for early clinical diagnosis of oral cancer.

A label-free electrochemical biosensor based on novel DNA nanotweezer coupled with G-quadruplex for sensitive DNA detection

Abstract In this work, a label-free electrochemical biosensor for DNA assay was constructed based on a novel structural DNA nanotweezer (DNT) coupled with split G-quadruplex. The novel DNT consists of a long single strand with two hairpin structures in the middle as a recognition element and two short single strands with split G-rich sequences at outsides as a signal reporter. The target triggered two hairpin structures hybridized with each other, it caused the DNT transformation from “open” to “close”. The “closed” DNT facilitated the split G-rich sequences combined together and provided electrochemical signals. Here, the hairpin structures were not only used for specific target identification but also avoided the false positive signals and improved the sensitivity. Additionally, the split G-rich sequences were put at outsides instead of insides, which significantly improved the signal-background ratio. Moreover, the construction of this biosensor was convenient and easy to control, which was beneficial to reduce the error generated by the operation process and improve stability and reproducibility. Under the optimal conditions, the proposed assay achieved a low detection limit (22 aM) and a wide linear range (1 fM to 1 nM) for circulating tumor DNA determination. This efficient and convenient DNT can be used as a template in electrochemical biosensors fabrication.

Cas12aFDet: A CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c

The CRISPR/Cas12a system has displayed remarkable potential in the development of new methods for nucleic acid detection owing to the trans-cleavage activity of Cas12a. Despite the tremendous development in recent years, existing CRISPR/Cas12a-based methods have several limitations such as the time-consuming process, which takes up to 2 h, and the risk of aerosol contamination during DNA amplicon transfer. Herein, we propose a CRISPR/Cas12a-based fluorescence detection platform named “Cas12aFDet” for rapid nucleic acid detection that overcomes these limitations. By integrating PCR or recombinase-aided amplification (RAA) methods with Cas12a-mediated cleavage in a sealed reaction tube, Cas12aFDet-based detection of amplified products could be accomplished within 15 min, while avoiding amplicon contamination. The detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet were determined to be 3.37 × 101 cfu/mL and 1.35 × 102 cfu/mL of Listeria monocytogenes serotype 4c in pure culture, respectively. Most importantly, RAA-based Cas12aFDet exhibited 0.64 aM sensitivity for DNA detection, and showed high specificity for detection of other serotypes of Listeria and non-Listeria strains. Furthermore, the feasibility of the RAA-based Cas12aFDet method was evaluated in spiked and natural samples, enabling the quantitative detection of 1.35 × 108–1.35 × 103 cfu/g fresh grass carp of the target L. monocytogenes serotype 4c, and the results obtained for 22 natural aquatic samples were highly consistent with those of the culture-based serotyping method. The established Cas12aFDet platform is expected to provide a new paradigm for the sensitive and specific detection of pathogens in food safety and clinical diagnosis.

Development of a Novel Multiple Cross-Linking Spiral Amplification for Rapid and Sensitive Detection of HPV16 DNA.

PCRThere has been increasing interest in the head and neck squamous cell carcinoma (HNSCC) that is caused by high-risk human papillomavirus (HR-HPV) and has posed a significant challenge to Otolaryngologists. A rapid, sensitive, and reliable method is required for the detection of HR-HPV in clinical specimens to prevent and treat HPV-induced diseases. In this study, a multiple cross-linking spiral amplification (MCLSA) assay was developed for the visual detection of HPV-16. In the MCLSA assay, samples were incubated under optimized conditions at 62°C for 45 min, and after mixing with the SYBR Green I (SGI) dye, the positive amplicons showed bright green fluorescence while the negative amplicons exhibited no obvious change. The specificity test revealed that the developed MCLSA technique had high specificity and could effectively distinguish all five HPV-16 strains from other pathogenic microorganisms. In terms of analytical sensitivity, the limit of detection (LoD) of MCLSA assay was approximately 5.4 × 101 copies/tube, which was 10-fold more sensitive than loop-mediated isothermal amplification (LAMP) and RT-PCR. The detection results of laryngeal cancer specimens collected from 46 patients with suspected HPV infection in the Liaoning region demonstrated that the positive detection rates of MCLSA and hybridized capture 2 kit were 32.61% (15/46). The true positive rate of the MCLSA assay was higher than that of RT-PCR (100% vs. 93.33%) and LAMP (100% vs. 86.67%). Therefore, the MCLSA assay developed in the present study could be a potentially useful tool for the point-of-care (PoC) diagnosis of HR-HPV, especially in resource-limited countries.

A colorimetric IsoPCR for the rapid and sensitive visual detection of high-risk HPV16 in clinical samples with hydroxynaphthol blue

HPV16 infection is found in more than 50 % of cervical cancer cases worldwide, triggering the development of numerous molecular techniques for viral diagnosis. The present study focuses on the development of a colorimetric IsoPCR for HPV16 DNA detection. The methodology combines the advantages of PCR and LAMP, while the most significant aspect of the new established methodology is the visual detection of amplification products through hydroxynapthol blue dye, thus minimizing the time and labor needed. An experimental cut-off value was tested through reconstitution experiments, while the specificity was evaluated by assessing clinical samples. The analytical sensitivity of the new colorimetric IsoPCR was found to be 0.1 viral DNA copy per reaction, while the specificity was 100 % for the detection of HPV16 DNA. The assay enabled the amplification of viral DNA in cases with viral load lower than 1 copy. In conclusion, the new established colorimetric IsoPCR can be regarded as an attractive molecular tool that facilitates the specific, rapid and highly sensitive visual detection of HPV16 DNA even at the very early stages of viral infection.

A sensitive platform for DNA detection based on organic electrochemical transistor and nucleic acid self-assembly signal amplification.

Highly sensitive detection of DNA is of great importance for the detection of genetic damage and errors for the diagnosis of many diseases. Traditional highly sensitive organic electrochemical transistor (OECT)-based methods mainly rely on good conductivity materials, which may be limited by complex synthesis and modification steps. In this work, DNA biosensor based on OECT and hybridization chain reaction (HCR) signal amplification was demonstrated for the first time. Au nanoparticles were electrochemically deposited on the Au gate electrode to increase the surface area. Then, the HCR products, long negatively charged double-stranded DNA, were connected to the target by hybridization, which can increase the effective gate voltage offset of OECT. This sensor exhibited high sensitivity and even 0.1 pM target DNA could be directly detected with a significant voltage shift. In addition, it could discriminate target DNA from the mismatched DNA with good selectivity. This proposed method based on HCR in DNA detection exhibited an efficient amplification performance on OECT, which provided new opportunities for highly sensitive and selective detection of DNA.

Sensitive detection of p53 DNA based on spatially confined fluorescence resonance energy transfer and multivalent assembly of branched DNA.

A key challenge for the discrete distribution-based Förster resonance energy transfer system (D-FRET) is the reduced intensity and stability of signal probes in complex biological matrices. Here, we present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output. It consists of multivalent FRET pairs labeled with FAM or TAMRA. In this assay, p53 DNA was chosen as a model hairpin probe (HP), and two kinds of branched DNA probes (ssDNA-FAM, ssDNA-TAMRA) were involved. Under the action of p53 DNA, the unfolded HP acts as a primer to initiate polymerization extension of KFP polymerase and cleavage of Nb.BbvCI endonuclease, which produces plenty of ssDNA (primer-DNA). The branched DNA is designed to have the same binding core and different sticky ends, the core part of which can self-assemble to form X-shaped branched DNA (X-FAM or X-TAMRA), and the sticky ends of which are complementary to the primer-DNA. Therefore, the primer-DNAs released during the polymerization cleavage process will combine a large number of X-FAM and X-TAMRA in a limited space through complementary base pairing. Fluorescence was transferred from FAM to TAMRA, and a strong FRET response was generated by the locational effects. The proposed SC-FRET system based on the multivalent assembly of branched DNA exhibited a strong FRET response with an LOD of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research.

Selenium-atom-modified thymidine enhances the specificity and sensitivity of DNA polymerization and detection.

Nucleobase mismatches can jeopardize DNA polymerization specificity, causing mutations and errors in DNA replication and detection. Herein we report the first synthesis of novel 2-Se-thymidine triphosphate (SeTTP), describe the single-selenium atom-specific modification strategy (SAM) against T/G mismatches, and demonstrate SAM-assisted polymerization and detection with much higher specificity and sensitivity. SAM can effectively suppress the formation of non-specific products in DNA polymerization and detection. Thus, SAM enhances the specificity of DNA synthesis by approximately 10 000 fold, and in turn, it allows the detection of clinical COVID-19 viral RNA in low copy numbers (single-digit copies), while the conventional RT-qPCR does not.

Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.

DNA-targeted formation and catalytic reactions of DNAzymes for label-free ratiometric electrochemiluminescence biosensing

A label-free ratiometric electrochemiluminescence (ECL) sensing strategy for the sensitive detection of target DNA (T-DNA) was proposed on the basis of G-quadruplex/hemin-regulated ECL emissions of CdS quantum dots (QDs) and luminol with their common coreactant of H2O2. The ECL biosensor was constructed through stepwise assemblies of CdS QDs and hairpin DNA (H-DNA) on a glassy carbon electrode, and subsequent introduction of T-DNA resulted in the development of G-quadruplex/hemin DNAzymes via the specific recognition of T-DNA and H-DNA in the presence of hemin and K+ ions. The formed DNAzymes not only prompted the catalytic oxidation of hydroquinone followed by deposition of insoluble oxidation oligomers on the electrode surface to attenuate the cathodic ECL emission of CdS QDs but also triggered the catalytic oxidation of luminol to enhance the anodic ECL emission. The label-free ratiometric ECL biosensor for the detection of T-DNA showed a wide response range from 1 to 10,000 fM (10−15 M) with a low detection limit of 0.2 fM and exhibited excellent selectivity against mismatched base sequences. This work provides a reliable and sensitive sensing platform for the detection of targets in analytical community by means of rational design of DNA sequences.

Detection and Diagnostic Utilization of Cellular and Cell-Free Tumor DNA.

Because cancer is caused by an accumulation of genetic mutations, mutant DNA released by tumors can be used as a highly specific biomarker for cancer. Although this principle was described decades ago, the advent and falling costs of next-generation sequencing have made the use of tumor DNA as a biomarker increasingly practical. This review surveys the use of cellular and cell-free DNA for the detection of cancer, with a focus on recent technological developments and applications to solid tumors. It covers (a) key principles and technology enabling the highly sensitive detection of tumor DNA; (b) assessment of tumor DNA in plasma, including for genotyping, minimal residual disease detection, and early detection of localized cancer; (c) detection of tumor DNA in body cavity fluids, such as urine or cerebrospinal fluid; and (d) challenges posed to the use of tumor DNA as a biomarker by the phenomenon of benign clonal expansions.

Novel Homogeneous Anion Exchange Membranes for Reproducible and Sensitive Nucleic Acid Detection via Current-Voltage Characteristic Measurement.

One-pot synthesis of novel hydrogel-based anion exchange membranes (AEMs), with only a single-phase monomer mixture, was used to eliminate surface heterogeneity and generate reproducible electroconvective microvortices in the over-limiting region of the current-voltage characteristic (CVC) curves. Diallyldimethylammonium chloride (DDA) was used as the main component to provide the cation charge groups, and 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethyl acrylate (EGDMA) were used as the auxiliary structure monomers. The uniform membrane structure allowed reproducible and sensitive DNA detection and quantification, as probe-target surface complexes can gate the ion flux and produce large voltage shifts in the over-limiting region. Suppressed membrane curvature due to controlled swelling is a crucial part to avoid the reduction of depletion region for maintaining the influence of target gene hybridization. Fourier-transform infrared (FTIR) spectroscopy verified the synthesized membrane structure, with a residual vinyl group that allows easy carboxylation via additional photografting reaction. Consequently, a significantly higher DNA probe functionalization efficiency is obtained on the homogeneous AEMs, evidenced by the increasing nitrogen element content and bonding via X-ray photoelectron spectroscopy (XPS). The DDA content was optimized to provide a sufficient coulomb force between AEM and nucleic acid backbone to promote the specific binding efficiency but without high dimensional swelling which might change the surface geometry and restrict the voltage shifting for sensing in the over-limiting region, and the optimal DDA/HEMA ratio was found to be 4/10. The synthesized AEM sensor for recombinant 35S promoter sequence identification exhibited a reproducible calibration standard curve with dynamic range between 30 fM and 1 μM and high selectivity with only 0.01 V shift for 1 μM nontarget oligo.

Highly sensitive lung cancer DNA detection via GO enhancing eATRP signal amplification

Atom transfer radical polymerization (ATRP) is an effective polymerization technology, and low ppm ATRP attracts unceasing interest in biological field. In this work, a fast and effective electrochemical biosensor based on graphene oxide (GO) via electrochemically mediated ATRP (eATRP) realized the ultrasensitive detection of lung cancer DNA. Peptide nucleic acid (PNA) was used as capture probes to specifically recognize target DNA, GO was connected to PNA/DNA heteroduplexes via phosphate-Zr4+-carboxylate chemistry to introduce eATRP initiators. Under a potentiostatic condition, eATRP was initiated with the help of reducibility of electrons, and numerous monomers labeled with ferrocene moieties were grafted onto GO. GO was synthesized by modified Hummer’s method and was characterized by Fourier transform infra-red spectra (FT-IR) and X-ray diffraction (XRD). The analytical performance of this biosensor was evaluated by square wave voltammetry (SWV) under optimal conditions, its limit of detection (LOD) was 0.213 aM (R2 = 0.994) with a linear range from 1.0 aM to 100 fM. High selectivity of this biosensor indicated a good application potential of genotyping of single nucleotide polymorphisms (SNPs), and simple operation and highly sensitive analytical performance endowed this biosensor a superior application prospect. More importantly, a novel thinking for material application in biological detection was provided in this work.

DNA recovery from Droplet Digital™ PCR emulsions using liquid nitrogen.

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing.

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

Ultrasensitive Photoelectrochemical Assay for DNA Detection Based on a Novel SnS2/Co3O4 Sensitized Structure.

In this work, an ultrasensitive photoelectrochemical (PEC) assay was established for sensitive DNA detection based on a novel SnS2/Co3O4 sensitized structure as a photoactive matrix and benzo-4-chlorohexadienone (4-CD) precipitate as a signal quencher. Noticeably, the photoelectric conversion efficiency of the SnS2/Co3O4 sensitized structure was dramatically enhanced due to the effective sensitization of Co3O4 toward SnS2, thus attaining an intense photocurrent response, which was sixfold higher than that of pristine SnS2. Additionally, with the assistance of Nt.BstNBI enzyme-assisted target cycling process, a limited amount of target DNA (a fragment of p53 gene) could be converted into extensive output DNA, which could hybridize with capture DNA to yield abundant DNA duplex for loading mimetic enzyme manganese porphyrin (MnPP). Subsequently, 4-chloro-1-naphthol (4-CN) could be catalyzed to form 4-CD precipitate by MnPP on the modified electrode surface with the existence of H2O2. Then, the 4-CD precipitate severely hampered electron transfer, causing a prominently diminished photocurrent response for DNA determination. The elaborated PEC assay not only extended the application of SnS2 in the PEC biosensing field but also manifested a wide linear range of 100 aM to 1 nM with a low detection limit of 30 aM, exhibiting enormous potential for the detection of various biomarkers or other targets in bioanalysis and disease diagnosis fields.