Strand Displacement Amplification Research Articles

Aggregation-Induced Enhancement Electrochemiluminescence of Ag Nanoribbons as a Signal Probe for Ultrasensitive Detection of the Halogenase Gene Segment from Aspergillus Ochratoxin.

Here, an electrochemiluminescence (ECL) biosensor was developed based on aggregation-induced enhancement ECL of Ag nanoribbons (Ag NRs) as a signal probe and the mismatched bases-fueled DNA walker as the amplification strategy for ultrasensitive detection of the halogenase gene segment (target) from aspergillus ochratoxin to evaluate fungi capable of producing ochratoxin in dairy products. Compared with the existing agminated NCs, the Ag NRs displayed dramatically enhanced ECL emission due to effective electron transfer and less energy dissipation of excited-state Ag NRs based on the metallophilic interaction of Ag(I)-Ag(I). Impressively, the target was conversed mismatched bases-DNA walker to trigger a significant ECL response through toehold-mediated strand displacement amplification. Thus, the strategy realized trace analysis of the halogenase gene segment with a detection limit of 45 aM, which ushered a new detection method for toxin determination in the previous stage of toxin biosynthesis to protect people from toxic damage.

Calixarene-based porous coordination compound as microreactors: Enrichment-enhanced electrochemiluminescence of iridium(III) complex for bioanalysis

Improving the electrochemiluminescence (ECL) efficiency of iridium complexes such as Ir(ppy)3 is still valuable for expanding their applications in the ECL field. Herein, a calixarene-based porous coordination compound (referred to SWU-1) with ordered pore structure and large porosity was innovatively developed as microreactors, which not only acted as a carrier to efficiently load Ir(ppy)3 on its surface and further achieve aggregation-induced ECL (AIECL) of Ir(ppy)3, but also enriched coreactant tripropylamine (TPrA) in its hydrophobic cavity. As a result, the prepared Ir@SWU-1 NCYLs exhibited a significantly improved ECL efficiency and aqueous dispersion as compared to Ir(ppy)3. The complex of Ir@SWU-1 NCYLs and polyethyleneimine (PEI) was used to modify the electrode for assembling Pt nanoparticles and further capturing hairpin H2·H2O2 was introduced into test solution to quench the ECL emission from Ir@SWU-1 NCYLs/TPrA system, leading to a signal-off state. When the target amyloid-β oligomers (AβO) existed, amounts of single-strand S1 were released from the target cycle, triggering the strand displacement amplification (SDA) on the magnetic beads. The single-strand ST output from the SDA reaction would open hairpin H2 on the electrode surface and initiate the rolling circle amplification (RCA) reaction so that scores of long chains rich in G bases were generated and further captured hemin to form hemin/G-quadruplex. With the consumption of H2O2 by hemin/G-quadruplex, the ECL signal was recovered, thus achieving the sensitive detection of AβO. This work developed novel ECL microreactors to load Ir(ppy)3 and enrich coreactant TPrA. The Ir@SWU-1 NCYLs with high ECL efficiency and excellent dispersity provided a fascinating ECL sensing platform for bioanalysis, expanding the application of iridium(III) complexes in ECL field.

Assembly of DNA triangular pyramid frustum for ultrasensitive quantification of exosomal miRNA

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

A universal ratiometric method for Micro-RNA detection based on the ratio of electrochemical/electrochemiluminescence signal, and toehold-mediated strand displacement amplification

An ultra-selective and reproductive ratiometric platform was introduced based on the ratio of Ru(phen)32+ electrochemiluminescence (ECL) signal and methylene blue (MB) electrochemistry (EC) signal, which was amplified using a specific and efficient toehold-mediated strand displacement (TMSD). The stable DNA nanoclews (NCs) were efficiently loaded with MB (MB-NCs) as EC signal tags after being synthesized utilizing a simple rolling circle amplification reaction. Besides, Ti3C2-based nanocomposite could apply as a superb carrier for both Ru(phen)32+ and gold nanoparticles (Ti3C2–Au–Ru), resulting in a nearly constant ECL internal reference to eliminate the possible interferences. The Ti3C2–Au–Ru was attached to the surface of the electrode using Nafion, which exhibited excellent conductivity, and hairpin DNAs (hDNAs) were fixed on AuNPs via an Au–S bond. The designed biosensor was finally applied for miRNA-18a detection as a target model.The TMSD method made it possible to concurrently convert and amplify a single miRNA-18ainput into a large amount of output DNAs with high selectivity. These output DNAs were designed to unfold the stem-locked area of hDNAs. The opened hDNAs then hybridized with the MB-NCs to produce an EC signal. In the proposed biosensing system, by raising the target concentration of miRNA, the EC signal gradually rose, the ECL signal remained nearly constant, and the ratiometric detection method markedly promoted biosensor accuracy. Linear correlations of the ratio value of the EC/ECL with miRNA-18a concentrations between 20 aM and 50 pMwere observed, with the limit of detection of 9 aM. The biosensor was applied to detect miRNA-18a in real serum samples with satisfactory results.

A multivalent dual lock-and-key system and its toehold-mediated orthogonal activation for highly specific strand displacement amplification

DNA strand displacement reaction plays an essential role in dynamic DNA nanotechnology and has proven powerful in DNA nano-computers and biosensors. However, the poor specificity and/or kinetics hamper its practical applications. Here we report a multivalent dual lock-and-key (Multi-DLK) system that can be accurately activated to perform a toehold-mediated orthogonal DNA circuitry (ODC) for highly specific and rapid nucleotide polymorphism assay. The “dual lock” can be opened only when target as the unique “key” is introduced and perform matching operation twice in an exact order separately by using the two fragments of target as inputs. The two operations are designed based on toehold-mediated strand displacement reaction, separately named TSDR1 and TSDR2, offering an orthogonal activation strategy for accurate identification. After that, the palindromic end of linker strand is released and taken as self-primer by DNA polymerase to perform a highly target-specific strand displacement amplification (TSSDA). We demonstrate that Multi-DLK system comes with the benefits of orthogonal activation strategy and TSSDA-based amplifier, which make the system ideally suitable for gene typing with a high specificity and efficiency. Comparative analysis and the final application of Multi-DLK system for real samples have shown the great potential for accurate and timely diagnosis.

Integrating CRISPR/Cas12a with strand displacement amplification for the ultrasensitive aptasensing of cadmium(II)

Cadmium ion (Cd(II)) is a pernicious environmental pollutant that has been shown to contaminate agricultural lands, accumulate through the food chain, and seriously threaten human health. At present, Cd(II) monitoring is dependent on centralized instruments, necessitating the development of rapid and on-site detection platforms. Against this backdrop, the present study reports on the development of a fluorometric aptasensor designed to target Cd(II), which is achieved through the integration of strand displacement amplification (SDA) and CRISPR/Cas12a. In the absence of Cd(II), the aptamer initiates SDA, resulting in the generation of a profusion of ssDNA that activates Cas12a, leading to a substantial increase in fluorescence output. Conversely, the presence of Cd(II) curtails the SDA efficiency, culminating in a significant reduction in fluorescence output. The proposed approach has been demonstrated to enable the selective detection of Cd(II) at concentrations of 60pM, with the performance of the aptasensor validated in real water and rice samples. The proposed platform based on aptamer-target interaction holds immense promise as a signal-amplified and precise method for the detection of Cd(II) and has the potential to transform current hazard detection practices in food samples.

Multiplex signal amplification strategy-based early-stage diagnosis of Parkinson's disease on a SERS-enabled LoC system.

In this paper, a multiplex signal amplification strategy was developed for the determination of miR-214 and miR-221 on a surface-enhanced Raman scattering (SERS)-enabled lab-on-a-chip (LoC) system to realize the early-stage diagnosis of Parkinson's disease (PD). The gold nanobipyramids (GNBPs) with great monodispersity were functionalized with Raman reporter molecules and hairpin DNA 1, serving as the SERS nanotags. The presence of targets can initial the strand displacement amplification (SDA) reaction and the numerous short-stranded trigger DNA (tDNA) can be released under the action of polymerase and nicking enzyme. Then, the tDNA can trigger the catalytic hairpin assembly (CHA) event between the SERS nanotags and the capture nanoprobes (Magnetic beads (MBs) modified with hairpin DNA 2), resulting in the aggregation of GNBPs on the MBs surface. The multiplex signal amplification contributed by the SDA-CHA strategy and the magnet-induced aggregation effect can ultimately lead to the significant improvement of the detection sensitivity and the limit of detection (LOD) was low to aM level with reproducibility and specificity meanwhile. Furthermore, a MPTP-induced PD mice model was established to verify the practicability and the expression level of miR-214 and miR-221 at different stages analyzed with the LoC system was confirmed by qRT-PCR.

Aggregation-Induced Electrochemiluminescence of Copper Nanoclusters by Regulating Valence State Ratio of Cu(I)/Cu(0) for Ultrasensitive Detection of MicroRNA.

In this work, Cu nanoclusters (Cu NCs) with strong aggregation-induced electrochemiluminescence (AIECL) as emitters were used to construct an ECL biosensor for ultrasensitive detection of microRNA-141 (miR-141). Impressively, the ECL signals enhanced with the increased content of Cu(I) in the aggregative Cu NCs. When the ratio of Cu(I)/Cu(0) in aggregative Cu NCs was 3.2, Cu NCs aggregates showed the highest ECL intensity, in which Cu(I) could enhance the cuprophilic Cu(I)···Cu(I) interaction to form rod-shaped aggregates for restricting nonradiative transitions to obviously improve the ECL response. As a result, the ECL intensity of the aggregative Cu NCs was 3.5 times higher than that of the monodispersed Cu NCs. With the aid of the cascade strand displacement amplification (SDA) strategy, an outstanding ECL biosensor was developed to achieve the ultrasensitive detection of miR-141, whose linear range varied from 10 aM to 1 nM with a detection limit of 1.2 aM. This approach opened an avenue to prepare non-noble metal nanomaterials as robust ECL emitters and provided a new idea for detection of biomolecules for diagnosis of disease.

Ultrasensitive Electrochemiluminescence Biosensor Based on 2D Co3O4 Nanosheets as a Coreaction Accelerator and Highly Ordered Rolling DNA Nanomachine as a Signal Amplifier for the Detection of MicroRNA.

A novel ultrasensitive electrochemiluminescence (ECL) biosensor was constructed using two-dimensional (2D) Co3O4 nanosheets as a novel coreaction accelerator of the luminol/H2O2 ECL system for the detection of microRNA-21 (miRNA-21). Impressively, coreaction accelerator 2D Co3O4 nanosheets with effective mutual conversion of the Co2+/Co3+ redox pair and abundant active sites could promote the decomposition of coreactant H2O2 to generate more superoxide anion radicals (O2•-), which reacted with luminol for significantly enhancing ECL signals. Furthermore, the trace target miRNA-21 was transformed into a large number of G-wires through the strand displacement amplification (SDA) process to self-assemble the highly ordered rolling DNA nanomachine (HORDNM), which could tremendously improve the detection sensitivity of biosensors. Hence, on the basis of the novel luminol/H2O2/2D Co3O4 nanosheet ternary ECL system, the biosensor implemented ultrasensitive detection of miRNA-21 with a detection limit as low as 4.1 aM, which provided a novel strategy to design an effective ECL emitter for ultrasensitive detection of biomarkers for early disease diagnosis.

A light-up fluorescence platform based DNA: RNA hybrid G-quadruplet for detecting single nucleotide variant of ctDNA and miRNA-21

The nucleic acid assay is an area of great concern in the diagnosis and treatment of breast cancer. Here, we developed a DNA: RNA hybrid G-quadruplet (HQ) detection platform based on strand displacement amplification (SDA) and Baby Spinach RNA aptamer for single nucleotide variant (SNV) of circulating tumor DNA (ctDNA) and miRNA-21. This was the first in vitro construction of HQ for the biosensor. It found that HQ had much stronger ability to switch on fluorescence of DFHBI-1T than Baby Spinach RNA alone. Taking advantage of the platform and the FspI enzyme with high specificity, the biosensor achieved ultra-sensitive detection of SNV of the ctDNA (PIK3CA H1047R gene) and miRNA-21. The light-up biosensor had high anti-interference ability in complex actual samples. Hence, the label-free biosensor provided a sensitive and accurate method for early diagnosis of breast cancer. Moreover, it opened a new application model for RNA aptamers.

Ultrasensitive and colorimetric small extracellular vesicles (sEVs) analysis via dual-cycle signal tool

For the clinical diagnosis of diseases and for basic biological research, it is crucial to develop a trustworthy and efficient method for detecting small extracellular vesicles (sEVs) in multiple experimental conditions. Here, we create a colorimetric assay that enables sensitive and precise sEVs identification without the need for pricey equipment. In this assay, the exonuclease III (Exo III)-assisted signal recycle is activated by the released single-strand DNA (ssDNA) from SMBs (streptavidin magnetic beads)-aptamer-ssDNA complex after identification of sEVs. By integrating with the strand displacement amplification (SDA) process, a significant amount of double-strand DNA products with G-rich tails is produced. The G-rich tails fold to G-quadruplex under the assistance of hemin to catalyze the oxidation of TMB, yielding a color change. The approach offers a broad detection range of 5 orders of magnitudes based on the signal recycles and SDA. In addition, single-stranded DNA binding protein (SSB) is exploited in this method to minimize the background signal from non-specific digestion of Exo-III, making the method a robust tool for sEVs detection and disease diagnosis.

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.

Quadruple cascade strand displacement amplification reaction mediated polyfluorene derivative-based multiple FRET for synchronous determination of multiple biomarkers

Currently reported detections for biomarkers of Alzheimer’s disease (AD) were usually single-target detections, which were prone to generate misdiagnosis. This work developed quadruple cascade strand displacement amplification (QC-SDA)-mediated polyfluorene derivative-based multiple fluorescence resonance energy transfer (FRET) for simultaneous determination of AD biomarkers miRNA-16 and amyloid-beta oligomer species (AβOs). Carboxyl-functionalized poly [9,9-bis(3′-(N,N-dimethylamino)propyl)− 2,7-fluorene]-alt-2,7-(9,9-dioctylfluorene)] polymer nanoparticles (c-PFN PNPs) served as fluorescent donor and two fluorescent dyes served as fluorescent acceptors. When the targets combined with templates to continuously trigger QC-SDA, multiple infinite cycles occurred to generate numerous triggers. The resulting triggers initiated rolling circle amplification (RCA) to generate repeated sequences, which further hybridized with helper probes and reporter probes to achieve substantial immobilization of fluorescent acceptors on c-PFN PNPs, thereby realizing multiple FRET from c-PFN PNPs to acceptors with only single excitation. As FAM and Texas Red corresponded to miRNA-16 and AβOs, the high-throughput detections of biomarkers were achieved with limit of detection of 110 aM and 1.3 fM, respectively. The c-PFN PNPs coupling FAM and Texas Red provided an ideal multiplex FRET platform for synchronous detection of multi-target. What's more, the creative incorporation of amplification strategies QC-SDA and RCA endowed multiplex FRET with high sensitivity and ease of operation for bioanalysis.

A dual-amplification strategy-intergated SERS biosensor for ultrasensitive hepatocellular carcinoma-related telomerase activity detection.

Telomerase has been considered as a biomarker for early diagnosis and prognosis assessment of hepatocellular carcinoma (HCC), while the highly sensitive and specific methods remain challenging. To detect telomerase, a novel surface-enhanced Raman scattering (SERS) biosensor was constructed using the dual DNA-catalyzed amplification strategy composed of strand displacement amplification (SDA) and catalytic hairpin assembly (CHA). This strategy relies on the extension reaction of telomerase primer induced by telomerase, forming long-stranded DNAs with repetitive sequence to catalyze the follow-up SDA event. Subsequently, the SDA products can trigger the CHA reaction between the SERS probes (Au-Ag nanocages (Au-AgNCs) modified with hairpin DNA1 and Raman reporters) and capture substrate (Au@SiO2 array labeled with hairpin DNA2), resulting in the formation of numerous "hot spots" to significantly enhance the SERS signal. Results are promising that the established biosensor presented excellent reproducibility, specificity and sensitivity. Moreover, ELISA was applied as the golden standard to verify the application of the proposed biosensor in real samples and the results confirmed the satisfactory accuracy of our method. Therefore, the proposed SERS biosensor has the potential to be an ideal tool for the early screening of HCC.

Construction of bidirectional strand displacement-driven three-dimensional DNA walkers for single-molecule monitoring of multiple DNA glycosylases

Human single-stranded-selective monofunctional uracil glycosylase (hSMUG1) and alkyladenine glycosylase (hAAG) are important base-excision repair (BER) glycosylases responsible for deamination and alkylation repair. Their dysfunctional activities have strong association with various multifactorial diseases and cancers. Herein, by integrating three-dimensional (3D) DNA walker with single-molecule technique, we construct bidirectional strand displacement-driven 3D DNA walkers for single-molecule monitoring of multiple DNA glycosylases. In the presence of hSMUG1 and hAAG, the damaged U and I in bifunctional dumbbell probe are removed by APE1, activating bidirectional self-priming strand displacement amplification (SP-SDA) to produce two trigger DNAs. Trigger DNAs hybridize with Cy5/Alexa Fluor 488 signal probes and subsequently serve as the walker DNAs to induce cyclic APE1-powered cleavage of two signal probes, liberating abundant Cy5 and Alexa Fluor 488 from AuNPs. Due to high precision of intracellular BER mechanisms, high efficiency of bidirectional SP-SDA-directed 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging, this nanosensor exhibits a detection limit of 8.14 × 10−10 U/μL for hSMUG1 and 4.50 × 10−9 U/μL for hAAG. Importantly, it can measure kinetic parameters, identify potential inhibitors, discriminate cancer from normal cells, and quantify glycosylases activities in various cancer cells at single-cell level, with promising potentials in biomedical and clinical applications.

Simple Isothermal and Label-Free Strategy for Colorectal Cancer Potential Biomarker miR-625-5p Detection.

miRNA is considered a novel biomarker for cancer diagnosis and due to its low level in vivo, the development of new detection methods for it has become a research hotspot in recent years. Here, we firstly found that miR-625-5p was significantly upregulated in colorectal cancer tissues by means of differential expression analysis of the dbDEMC database and clinical validation. Subsequently, it was found that miR-625-5p promoted cell proliferation and migration but inhibited apoptosis through phenotypic experiments; thus, we initially identified miR-625-5p as a potential biomarker for colorectal cancer. Moreover, in order to monitor slight changes in the miR-625-5p level, we developed a novel detection method for it based on strand displacement amplification (SDA). In this system, a hairpin was designed to recognize and pair with miR-625-5p, which was used as a primer to initiate SDA, and a large number of complementary DNAs were generated via cyclic amplification, followed by the addition of SYBR Gold to achieve quantitative analysis of miR-625-5p. Moreover, this method showed a good response to miR-625-5p with a detection limit of 8.6 pM and a dynamic range of 0.01 to 200 nM, and the specificity of it was verified using a set of other miRNAs as an interference. Finally, we set up different concentrations of biologic samples for detection to verify the practicability of the method. The results of this study indicate that this detection method has great potential in clinical diagnosis.

An immobilization-free electrochemical aptamer-based assay for zearalenone based on target-triggered dissociation of DNA from polydopamine nanospheres with strand displacement amplification.

Zearalenone (ZEN), a widespread mycotoxin, can cause great harm to people's health. In order to assay ZEN, an immobilization-free electrochemical sensor has been developed. A multifunctional hairpin DNA has been carefully designed, including three functions: the aptamer for zearalenone (ZEN), primer, and template sequence. This hairpin DNA can anchor on polydopamine nanospheres (PDANSs), which can protect DNA against the digestion of enzymes and prevent the occurrence of strand displacement amplification (SDA). In the presence of ZEN, the hairpin DNA is dissociated from PDANSs due to the interaction between ZEN and the aptamer, and the SDA reaction is initiated with the help of endonuclease and polymerase. During the SDA process, substantial amounts of negatively charged dsDNA are generated. The MB molecules are embedded into the dsDNA grooves to obtain the complex with a negative charge. The confined MB is repelled on the surface of the negatively charged ITO electrode, leading to the decline of the current. This immobilization-free method possesses high sensitivity (LOD of 0.18 pg mL-1) and good selectivity and can be applied to assay ZEN in corn flour.

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.

Target-initiated fluorescent aptasensor based on multisite strand displacement amplification for label-free detection of ochratoxin A.

We developed a fluorescent aptasensor for label-free detection of ochratoxin A (OTA) based on TdT- and DNA polymerase-assisted multisite strand displacement amplification. This aptasensor exhibits good specificity and high sensitivity with a limit of detection (LOD) of 0.18 ng mL-1, and it can be further applied for the accurate quantification of OTA in complex real samples, holding promising applications in the field of food safety.

Programmable T-Junction Structure-Assisted CRISPR/Cas12a Electrochemiluminescence Biosensor for Detection of Sa-16S rDNA

Herein, a strand displacement amplification (SDA)-assisted CRISPR/Cas12a (LbCpf1) electrochemiluminescence (ECL) biosensor was fabricated for ultrasensitive identification of Staphylococcus aureus (Sa)-16S rDNA. A porphyrinic Zr metal-organic framework (MOF) (PCN-224) nanomaterial was prepared as the coreactant accelerator, which promoted the conversion of S2O82- and SO4*-, thus enhancing the reaction with CdS quantum dots (QDs) and amplifying the ECL emission signal. Meanwhile, with the presence of Sa-16S rDNA, the auxiliary probes and primers stimulated the SDA reaction under the action of Klenow fragment (3'-5' exo-) and Nt. BbvCI specifically recognized Sa-16S rDNA to form a defective T-junction structure and generated second primers to initiate the cycles. Such a structure transformed the input signal (Sa-16S rDNA) into substantial single-stranded DNA products (SP) through SDA. SP acted as activators and activated arbitrary side chain cleavage of CRISPR/Cas12a (trans-cleavage) and further realized effective annihilation of ECL signals. This ECL platform demonstrated desirable assay performance for Sa-16S rDNA with a wide response range of 1 fM to 10 nM, and the limit of detection was 0.437 fM (S/N = 3), showing good sensitivity and specificity. Therefore, the method not only expanded the applications of CRISPR/Cas12a but also opened up a novel strategy for clinical diagnosis.