Circular Strand-displacement Research Articles

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

One-step self-assembly of quantum dot-based spherical nucleic acid nanostructure for accurate monitoring of long noncoding RNA MALAT1 in living cells and tissues

Simple and sensitive measurement of long non-coding RNA (lncRNA) is critical for early detection of malignancies. Herein, we demonstrate one-step self-assembly of quantum dot (QD)-based spherical nucleic acid (SNA) nanostructure for accurate monitoring of lncRNAs in living cells and tissues. When target lncRNA is present, it binds with a dumbbell probe to expose the complementary domain of Cy5-labeled primer, which subsequently induces cascade primer exchange reaction to produce abundant Cy5-labeled initiators. The Cy5-labeled initiators subsequently hybridize with hairpin probes on the QD surface to activate isothermal circular strand-displacement polymerization reaction, generating the QD-DNA-Cy5 nanostructures and inducing efficient Förster resonance energy transfer (FRET) between donor QD and acceptor Cy5. The obtained FRET signals are accurately quantified by single-molecule imaging. Notably, the single QD-based SNA nanostructure functions not only as a signal transmitter but also as a protector against non-specific amplification. Moreover, this assay utilizes only one DNA polymerase to achieve two-stage amplification, avoiding careful modulation of multiple enzymes. The self-assembly of QD nanosensor can be accomplished in single-step and single-tube manners at room temperature, eliminating precise temperature control and labor-intensive reaction protocols. This QD nanosensor achieves high sensitivity with a limit of detection (LOD) of 65.25 aM, and it is capable of quantifying lncRNA expression at single-cell level, differentiate tumor cells from normal cells, and distinguish breast cancer patients from healthy individuals, providing a versatile paradigm for biomedical research and early clinic diagnostics.

Self-Assembly of Multivalent Aptamer-Tethered DNA Monolayers Dedicated to a Fluorescence Polarization-Responsive Circular Isothermal Strand Displacement Amplification for Salmonella Assay.

Pathogenic bacteria are pathogens widely spread that are capable of causing mild to life-threatening diseases in human beings or other organisms. Rationally organizing the simple helical motif of double-stranded DNA (dsDNA) tiles into designed ensemble structures with architecturally defined collective properties could lead to promising biosensing applications for pathogen detection. In this work, we facilely engineered multivalent hairpin aptamer probe-tethered DNA monolayers (MHAP-DNA monolayers) and applied them to build a fluorescence polarization-responsive circular isothermal strand displacement amplification (FP-CSDA) for Salmonella assay. In this system, the MHAP-DNA monolayers were constructed based on a dsDNA tile-directed self-assembly. A FAM-labeled reporting probe (RPFAM) with an inherent low FP signal serves as the signaling unit. The presence of target Salmonella leads to the trapping of F RPFAM into the super DNA monolayers via a target-triggered CSDA to peel off the tethered hairpin-structured aptamer probes (HAPs) responsible for the binding of RPFAM. As a result, the FP signal of the FAM fluorophore can be remarkably amplified due to the recycling of target Salmonella and the capacity of structural DNA materials to strongly restrict the free rotation of the FAM fluorophore but without a fluorescence quenching effect. Experimental results demonstrate that the FP assay is able to detect Salmonella with a low limit of detection (LOD) of 7.2 × 100 CFU/mL and high specificity. As a proof-of-concept study, we envision our study using DNA nanoarchitecture as the foundation to modulate CSDA-based FP assays, promising to open up a new avenue for disease diagnosis, food safety detection, and biochemical studies.

Ultrasensitive electrochemical platform for the p53 gene via molecular beacon-mediated circular strand displacement and terminal deoxynucleotidyl transferase-mediated signal amplification strategy.

As an important tumor suppressor gene, the p53 gene is considered to be a typical biomarker for the early diagnosis and prognosis evaluation of severe cancer. Herein, an electrochemical biosensor was proposed for the ultrasensitive detection of the p53 gene based on molecular beacon-mediated circular strand displacement polymerization combined with terminal deoxynucleotide transferase-mediated template-free DNA extension. Firstly, the p53 gene opened the hairpin structure of the molecular beacon, thereby exposing the binding sequence region of the primer DNA. The circular strand displacement polymerization occurred in the presence of the primer DNA and phi29 DNA polymerase, subsequently resulting in the circulation of the p53 gene. With the catalysis of the terminal deoxynucleotide transferase, the 3'-OH terminal sequence of the molecular beacon elongated to produce long single-stranded DNA under the template-free DNA extension. Methylene blue bound with such DNA strands generated a strong differential pulse voltammetry (DPV) signal with a peak potential of -0.28 V. Under the optimal detection conditions, the DPV signal of methylene blue showed good linear relationships with the logarithm value of the p53 gene in two concentration ranges of 0.05 fM to 3 pM and 5 fM to 100 fM, and the detection limit of the p53 gene was as low as 0.018 fM. This electrochemical biosensor possessed high recognition ability for the p53 gene in its analogues and was successfully applied for p53 gene analysis in human serum samples.

Isothermal circular strand displacement–based assay for microRNA detection in liquid biopsy

Extracellular miRNAs are promising targets for developing new assays for the early diagnosis and prognosis of diseases based on liquid biopsy. The detection of miRNAs in liquid biopsies is challenged by their short sequence length, low concentration, and interferences with bodily fluid components. Isothermal circular strand displacement polymerization has emerged as a convenient method for nucleic acid amplification and detection. Herein, we describe an innovative strategy for microRNA detection directly from biological fluids based on hairpin probe–assisted isothermal amplification reaction. We designed and optimized the assay to detect target analytes in 1 µL of the complex media’s biological matrix using a microfluidic device for the straightforward analysis of multiple samples. We validated the assay to detect circulating miR-127-5p in synovial fluid, recently indicated as a predictive biomarker for osteoarthritis disease. The combined use of a mutant polymerase operating with high yield and a primer incorporating locked nucleic acid nucleosides allowed detection of miR-127-5p with 34 fmol L−1 LOD. We quantified circulating miR-127-5p directly in synovial fluid, thus demonstrating that the assay may be employed for the convenient detection of 4.3 ± 0.5 pmol L−1 concentrated miRNAs in liquid biopsy samples.Graphical abstract

Isothermal chemiluminescent assay based on circular stand-displacement polymerization reaction amplification for cel-miRNA-39-3p determination in cell extracts

A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10−13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR.

HIGHLY SENSITIVE METHODS FOR MICRORNA DETECTION USING ISOTHERMAL METHODS FOR NUCLEIC ACIDS AMPLIFICATION

This work will present the results of the development of two new highly sensitive heterogeneous methods for the determination of miRNA-141 and miRNA-39 using the isothermal circular strand-displacement amplification and the catalytic hairpin assembly methods for analyte amplification.

Ultrasensitive electrochemical biosensor for attomolar level detection of let 7a based on toehold mediated strand displacement reaction circuits and molecular beacon mediated circular strand displacement polymerization

In this study, an ultrasensitive electrochemical miRNA biosensor based on toehold mediated strand displacement reaction circuits (SDRCs) and molecular beacon mediated isothermal circular strand displacement polymerization reaction (ICSDPR) has been proposed. During the SDRCs module, the cascade strand displacement reaction induces the recycling of the target let 7a and generation of a large amount of strand A (SA). The SA recognition opens the hairpin capture probe immobilized on the gold electrode, thus, varying the distance between the redox molecules and electrode surface. The primer mediated ICSDPR is observed to further generate a large amount of SA, thus, leading to a reduction in the signal. Considering these merits, the proposed method is observed to exhibit a log-linear linearity from 10 aM to 100 pM and ultrahigh sensitivity towards let 7a down to 6.2 aM, with a capability of distinguishing the let 7a family members, thereby, providing a new electrochemical route for early cancer screening.

An integrated target recognition and polymerase primer probe for microRNA detection

Extremely sensitive and visual measurements of microRNA (miRNA) in situ for early detection and monitoring of diseases remains a major challenge. To address this issue, this work reports a rapid, highly sensitive and selective microRNA (miRNA) biosensing strategy based on isothermal circular strand-displacement polymerization (ICSDP), and miRNA imaging was performed inside cells. In this work, a double hairpin DNA probe (HP1/HP2 complex) embedded with a sensing region and polymerase primer region was designed. Briefly, after the specific binding of target miRNA with the HP1/HP2 probe, HP1/HP2 itself can function as a primer to initiate the ICSDP with the help of Klenow Fragment (KF), yielding target miRNA for new rounds of ICSDP. In this process, one target can produce multiple signal outputs (1: n), achieving low abundance of miRNA detection. Under optimized conditions, the proposed strategy showed high sensitivity with a detection limit of 5 pM within 15 min and can also easily distinguish the control miRNA from the target miRNA. This method can be further applied to image the intracellular miRNA of interest in situ inside the cancer cells.

Chemiluminescent microplate-based assay of DNA based on isothermal circular strand-displacement polymerization reaction (ICSDPR)

Sensitive microplate-based chemiluminescent assay coupled with isothermal circular strand-displacement polymerization reaction (ICSDPR) for DNA detection was developed. The assay sensitivity was improved using a triple amplification strategy based on employment of ICSDPR, streptavidin-polyperoxidase conjugate and an enhanced chemiluminescent reaction. To increase the accuracy all stages of the assay (one-pot format) were carried out in the same microplate well. The proposed assay detected the target DNA in fM-pM ranges and distinguish target DNA from related mismatched DNAs, demonstrating high sensitivity and high selectivity. This format is highly suitable for the development of sensitive and high-throughput kits of nucleic acids which can be easily automated.

A highly sensitive assay of DNA based on inductively coupled plasma mass spectrometry detection with gold nanoparticle amplification and isothermal circular strand-displacement polymerization reaction

In this work, a simple, sensitive and specific assay for DNA was proposed by combining inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticle (AuNPs) amplification and isothermal circular strand-displacement polymerization reaction (ICSDPR). First, AuNPs were decorated with hairpin-structured DNA (HP-DNA) through Au–S bond to form the AuNPs probe. The ICSDPR was conducted on AuNPs probe in a homogeneous phase to realize the dual amplification and simplify the analytical process at the same time. By using a biotin modified primer, AuNPs were connected with biotins after the ICSDPR, then captured by the streptavidin modified magnetic beads (SA-MBs), and finally detected by ICP-MS. Many key factors including probe volume, hybridization time, polymerase amount, primer concentration, enzyme reaction time, SA-MBs capture time and desorption time were optimized. Under the optimized condition, the proposed method could detect target DNA as low as 45 zmol (8.9 fM in 5 μL) in a relative short time (about 4.5 h) with good specificity, and the linear range of this method is 0.1–10000 pM, the relative standard deviations are in the range of 3.6–6.4%. The proposed method was applied for determination of target DNA in human serum samples, the recovery for the spiked human serum samples is in the range of 84–120%. It demonstrates a good application potential of the developed method for biological studies and clinical diagnosis of human pathogenic diseases.

Cancer-Specific MicroRNA Analysis with a Nonenzymatic Nucleic Acid Circuit.

Sensitive detection of gastric cancer-related biomarkers in human serum provides a promising means for early cancer diagnosis. Herein, we report the design of a nucleic acid circuit for gastric cancer-related microRNA-27a (miRNA-27a) detection based on dual toehold-mediated circular strand displacement amplification (CSDA). In the presence of miRNA-27a, the hybridization between miRNA-27a and probe DNA on magnetic beads through toehold 1 leads to the release of fluorescent DNA and the exposure of a new toehold 2 on linker DNA. After hybridization with catalytic DNA, CSDA is initiated and target miRNA-27a is released to participate in the next cyclic reaction; therefore, a greatly enhanced fluorescence signal is produced. The efficient magnetic separation makes the sensitive detection of miRNA-27a be accomplished within 45 min. With the efficient CSDA, the detection limit of the system (0.8 pM) is ∼100 folds lower than that of the system based on strand displacement without CSDA (79.3 pM). Furthermore, the system also showed good stability and sensitivity to discriminate single-base mismatch, which allows the detection of miRNA-27a in human serum samples. This study provides a novel platform and approach for the rapid quantitative determination of miRNA, which has great potential in clinical diagnosis and disease treatment.

Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization.

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.

Autonomous table-tennis-motion-type DNA machine for the efficient amplification detection of cancer related gene

Herein, an autonomous table-tennis-motion-type DNA machine (ATT DNA machine) was proposed to integrate target recycling amplification (TRA) and triple strand-displacement amplification (T-SDA) into one signaling system for the efficient amplification detection of p53 gene. Firstly, X-shaped DNA duplex probe (XSP) was formed via the simple annealing of Probe A and Probe B. Upon addition of p53, the hybridization of p53 with Probe A results in the peeling of Probe B from XSP. Then, the primer binds to the Probe B to trigger a circular strand-displacement amplification, during which new primers are generated. Moreover, the newly-produced primers can in turn activate the target recycling amplification and triple strand-displacement amplification via bouncing back and forth between different Probe As and Probe Bs in a manner analogous to the table tennis motion. This newly-developed DNA machine can be used to detect p53 down to 0.5 pM with an excellent specificity, offering a 100-fold enhanced sensitivity compared to a non-amplification one-to-one hybridization based system. Moreover, this DNA machine is able to be applied for target analysis in complex environments. Additionally, the potential for practical applications is also verified, demonstrating a promising alternative tool for early diagnosis of cancers.

Branched DNA Junction-Enhanced Isothermal Circular Strand Displacement Polymerization for Intracellular Imaging of MicroRNAs.

The microRNA profiles within living cells are informative for diagnosis and prognosis of human cancers. In the present work, we developed a new sensing strategy based on branched DNA junction-enhanced isothermal circular strand displacement polymerization (B-ICSDP) for the detection and intracellular imaging of microRNAs in living cells of interest. A circular DNA template consisting of three repetitive fragments serves as the scaffold for the self-assembly of sophisticated signaling probes, resulting a shrunk branched DNA junction. Target microRNA triggers the opening of molecular beacon, not only restoring the quenched fluorescence but also activating a circular polymerization-based strand displacement reaction. Thus, patulous branched DNA junction is abundantly formed, generating the amplified signal. It is noteworthy that great heaps of branched product assemblies can be also achieved in living cells, and the intracellular enzymatic assembly based strategy is able to be used to recognize specific microRNA-expressed cancer cells. Moreover, different microRNAs coexisting in the same living cells can be simultaneously screened without any interference from each other by confocal laser scanning microscopy. The measured data from confocal fluorescence imaging of different cancer cells demonstrates that the B-ICSDP-based system is a promising alternative for in vivo analysis of microRNAs in complicated biological samples.

Ultrasensitive assay based on a combined cascade amplification by nicking-mediated rolling circle amplification and symmetric strand-displacement amplification

MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics.

A multiple signal amplified colorimetric aptasensor for antibiotics measurement using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic tags and CSDP target-triggered cycles

An ultrasensitive colorimetric aptasensor was developed for antibiotics detection, with chloramphenicol (CAP) as model target, using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic signal tags and target-triggered circular strand-displacement polymerization (CSDP) for signal amplification. The system consists of two components which can partially hybridize with each other: one is capture probe which was formed through immobilizing hairpin DNA containing aptamer sequence on magnetic beads (MB-cDNA), another is signal tag which was constructed through labeling single strand DNAzyme (G-quadruplex/Hemin) which can partially hybrid with cDNA on platinum nanoparticles functionalized Fe-MIL-88 (MIL-88-Pt-DNAzyme). All components of MIL-88, Pt and DNAzyme in the tag can act as peroxidase mimic to triply catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 into a blue-colored oxidized TMB (oxTMB) for the colorimetric readout. Thus distinctive signal can be observed by naked eye even in presence of 0.02 nM tags. In the presence of target and primer, cDNA loop can open to form cDNA/CAP intermediates, enabling primer to hybridize with the exposed sequences of the cDNA, which initiated target assisted CSDP recycles. Then numerous signal tags were released into supernatant to catalyze TMB for color development. There was a liner relationship between the absorbance and the concentration of CAP in the range of 0.1 pM (0.0323 pg/mL) to 1000 pM (323 pg/mL) with the detection limit of 0.03 pM (0.0097 pg/mL). The ultra-high sensitivity was ascribed to the multiplex catalytic activities from the tags and CSDP based signal amplification. Furthermore, this method can produce signals being observed by naked eye to facilitate in-situ detection and be further extended to detect other antibiotics in food just by simply replacing cDNA on the sensing system.

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognizethe target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

A Lateral Flow Biosensor for the Detection of Single Nucleotide Polymorphisms.

A lateral flow biosensor (LFB) is introduced for the detection of single nucleotide polymorphisms (SNPs). The assay is composed of two steps: circular strand displacement reaction and lateral flow biosensor detection. In step 1, the nucleotide at SNP site is recognized by T4 DNA ligase and the signal is amplified by strand displacement DNA polymerase, which can be accomplished at a constant temperature. In step 2, the reaction product of step 1 is detected by a lateral flow biosensor, which is a rapid and cost effective tool for nuclei acid detection. Comparing with conventional methods, it requires no complicated machines. It is suitable for the use of point of care diagnostics. Therefore, this simple, cost effective, robust, and promising LFB detection method of SNP has great potential for the detection of genetic diseases, personalized medicine, cancer related mutations, and drug-resistant mutations of infectious agents.