Recycling Amplification Research Articles

Portable electrochemical biosensor based on laser-induced graphene and MnO2 switch-bridged DNA signal amplification for sensitive detection of pesticide

Developing portable, quantitative, and user-friendly analytical tools for sensitive pesticide assay is of significant importance for guaranteeing food safety. Herein, a novel electrochemical biosensor was constructed by integrating laser-induced graphene (LIG) electrode on polyimide (PI) foil and MnO2 nanosheets loaded on the paper for point-of-care test (POCT) of organophosphorus (OPs) residues. The principle of this biosensor relied on acetylcholinesterase (AChE)-catalyzed hydrolytic product-triggered disintegration of MnO2 nanosheets for releasing assistant DNA to initiate nicking enzyme-aided recycling amplification. In the presence of OPs, the activity of AChE was inhibited and could not initiate the cleavage of the electroactive molecules-labeled hairpin probe on the electrode, resulting in the maintenance of the electrochemical response to realize a “sign-on” determination of OPs. The proposed biosensor exhibited satisfactory analytical performance for OPs assay with a linear range from 3 to 4000 ng/mL and a limit of detection down to 1.2 ng/mL. Moreover, the biosensor was useful for evaluating the residual level of pesticides in the vegetables. Therefore, this novel biosensor holds great promise for OPs assay and opens a new avenue on the development of higher-performance POCT device for sensing applications in the environment and food safety fields.

Dual-labeling ratiometric electrochemical strategy initiated with ISDPR for accurate screening MecA gene

An outstanding dual-labeling ratiometric electrochemical biosensor based on isothermal strand displacement polymerization reaction (ISDPR) for highly sensitive and selective detection of mecA gene has been proposed. Concretely, in the presence of mecA gene, the addition of methylene blue (MB)-labeled primer and polymerase induced recycling amplification to change the structure of the ferrocene (Fc)-labeled hairpin probe, thereby releasing abundant target gene to realize the signal amplification and dual-signal output. Through this process, the electrochemical responses of Fc (IFc) and MB (IMB) were both substantially reduced and increased proportionally, ensuring that the value of IMB/IFc can accurately reflect the true detection level of mecA gene. Benefiting from the “signal-on/off” strategy, the fabricated biosensor exhibited outstanding sequence specificity to discriminate mismatched mecA gene, which verified to be 2.72 times that of single-label detection for perfect match/single base mismatch (PM/MM) discrimination ratio. This strategy effectively integrated the advantages of signal amplification and ratiometric modes, making the biosensor exhibit a broad working range with 10 fM - 3000 pM and a limit of detection (LOD) with 3.33 fM (S/N = 3). Moreover, the proposed biosensor has good feasibility for mecA gene determination in water samples due to acceptable recoveries (95–115%) and repeatability relative standard deviations (RSD) value of 4%. This will provide a powerful sensing platform for improving accuracy and decreasing background signal of sensor for ARGs screening in environmental monitoring.

A Homogeneous Label-Free Electrochemical microRNA Biosensor Coupling With G-Triplex/Methylene Blue Complex and λ-Exonuclease-Assisted Recycling Amplification.

A novel homogeneous label-free electrochemical biosensor using G-triplex/methylene blue (G3/MB) complex as the signal generator together with an amplification assisted by the λ-exonuclease (λ-Exo) has been successfully constructed for ultrasensitive microRNA (miRNA) detection. An integrated microelectrode was designed to realize the miniaturization of the homogeneous electrochemical assay. Taking advantage of G3, that can specifically bind with MB and decrease its diffusion current, a single-stranded functional DNA hairpin structure was designed as the bio-recognition probe. The probe consisted of G3, eight bases to block G3, and the complementary sequences of the target miRNA. Here we chose miRNA141—a potentially diagnostic biomarker of prostate cancer as the model target. The presence of miRNA141 could hybridize with the probe DNA to form a double-stranded structure with a 5′-phosphorylated terminus. Then λ-Exo was adopted to digest mononucleotides from the 5′-end, leading to the release of G3 part and miRNA141. The released miRNA could hybridize with another probe to trigger the cycling process, while the released G3 could therefore interact with MB to cause a detectable decrease of diffusion current. The proposed strategy showed a low detection limit of 16 fM and an excellent specificity to discriminate single-base mismatches. Furthermore, this sensor was applied to detect miRNA141 from diluted human serum samples, indicating that it has great potential in the application of nucleic acid detection in real samples.

An ultrasensitive electrochemiluminescence assay for nucleic acid detection based on carboxyl functionalized polymer dots

An electrochemiluminescence (ECL) sensing platform for ultrasensitive detection of miRNA-122 was developed by integrating novel emitters of polymer dots (Pdots) and signal amplification strategies. Firstly, we synthesized a novel polymer with carboxyl group which consisted of fluorene derivate and benzothiadiazole, corresponding Pdots materials were also prepared by nanoprecipitation method. Subsequently, an ECL biosensor based on the Pdots for miRNA-122 detection was constructed, which combines the amplification of duplex-specific nuclease (DSN)-assisted target recycling and rolling circle amplification (RCA) in-situ on the electrode. After the two-step amplification, amounts of ferrocene modified DNA (Fc-DNA) were captured on the electrode, which could quench the ECL signal of the Pdots. Ultimately, the proposed sensor presented a wide linear range of 0.1 fM to 100 pM and a low detection limit of 36 aM to miRNA-122. This work not only propose a universal paradigm for nucleic acid detection with good specificity and high sensitivity, but also provide an approach for designing novel Pdots to construct ECL sensor conveniently.

Construction of a Polarity-Switchable Photoelectrochemical Biosensor for Ultrasensitive Detection of miRNA-141.

As an early-stage tumor biomarker, microRNA (miRNA) has clinical application potential and its sensitive and accurate detection is significant for early tumor diagnosis. In this study, a photoelectrochemical (PEC) biosensing platform was fabricated for ultrasensitive miRNA-141 detection, which is based on a photocurrent polarity-switchable system using CdS quantum dots (QDs) in the presence of a 5,10,15,20-tetrakis (4-aminophenyl)-21H,23H-porphine (Tph-2H)-coated glassy carbon electrode (GCE). As an excellent photoactive material, Tph-2H has a narrow band gap that effectively gathers photoelectrons under visible light irradiation and improves the transfer ability of photogenerated electrons. Further, the detection sensitivity of miRNA-141 could be significantly improved by combining an enzyme-assisted recycle amplification reaction and a magnetic bead-based separation strategy. The proposed photocurrent polarity-switchable PEC biosensor could efficiently eliminate the false-positive or false-negative signals and achieve a wide linear response range from 1 fM to 1 nM with a low detection limit of 0.33 fM for miRNA-141, providing a potentially alternative solution for detecting other biomarkers in bioanalysis and clinical diagnosis.

Ultrasensitive colorimetric strategy for Hg2+ detection based on T-Hg2+-T configuration and target recycling amplification.

A novelty aptasensor for ultrasensitive detection of Hg2+ is developed, exploiting the combination of plasmonic properties of gold nanoparticles (AuNPs) and exonuclease III (Exo III)-assisted target recycling for signal amplification. In the presence of Hg2+, a DNA duplex can be formed due to the strong coordination of Hg2+ and T bases of single-stranded DNA (ssDNA) probe. Exo III digests the DNA duplex from the 3' to 5' direction, resulting in the releasing of Hg2+. Then, the released Hg2+ binds with another ssDNA probe through T-Hg2+-T coordination. After Exo III-assisted Hg2+ cycles, numerous ssDNA probes are exhausted, which promotes poly(diallyldimethylammonium chloride) (PDDA)-induced AuNP aggregation, leading to an obvious color change and aggregation-induced plasmon red shift of AuNPs (from 520 to 610nm). Therefore, this biosensor is ultrasensitive, which is applicable to the detection of trace level of Hg2+ with a linear range from 5 pM to 0.6nM and an ultralow detection limit of 0.2 pM. Furthermore, it enables visual detection of Hg2+ as low as 50 pM by the naked eye. More importantly, the assay can be applied to the reliable determination of spiked Hg2+ in sea water samples with good recovery.

A dual-signaling electrochemical ratiometric strategy combining “signal-off” and “signal-on” approaches for detection of MicroRNAs

A dual-signaling electrochemical ratio metric strategy was developed for detection microRNA-18a based on the duplex-specific nuclease-assisted target recycling and electrochemical atom transfer radical polymerization signal amplification. In the presence of target microRNA, RNA/DNA duplexes are formed, which become the substrate of the duplex-specific nuclease-assisted target recycling. Hence only the DNA strand is cleaved by duplex-specific nuclease enzyme, resulting in the throw away of methylene blue (MB) from the electrode (signal off) accompanied by releasing of target microRNA, which can be recycled in the next hybridization. The remaining piece of capture DNAs on the electrode surface hybridize with the Azide labeled-signal DNAs. “Click reactions” were carried out between 3-Butynyl-2-bromoisobutyrate and Azide to initiate the electrochemical atom transfer radical polymerization reaction. This process could bring a great number of ferrocenylmethyl methacrylate (FMMA) on the surface of electrode (signal on). The IFMMA/IMB value was proportionate to the microRNA-18a concentration and measured by square wave voltammetry.The promising potential of the proposed biosensor in clinical analyses was exhibited by its remarkable features such as strong performance, high specificity, agreeable storage stability, and notable selectivity in real sample evaluation with no pretreatment or amplification. Finally, our biosensing method offers such an application to be used for the early clinical diagnosis of Pancreatic Cancer.

An ultrasensitive and specific electrochemical biosensor for DNA detection based on T7 exonuclease-assisted regulatory strand displacement amplification

An electrochemical biosensor for determination of DNA is developed based on T7 exonuclease-assisted regulatory strand displacement dual recycling signal amplification strategy. First, the hairpin probe recognized and bound the target DNA to form a double strand nucleotide structure, and then the T7 exonuclease was introduced. After be digested by T7 exonuclease, the target DNA was released and entered the next cycle of T7 exonuclease-assisted recycle amplification, while accompanied by a large number of mimic targets (output DNAs) into another cycle. Second, the mimic target reacted with double-chain substrated DNA (CP) by a regulated toehold exchange mechanism, yielding the product complex of detection probes with the help of assisted DNA (S). Finally, after many cycles, a large number of detection probes were produced for binding numerous streptavidin-alkaline phosphatases. The electrochemical biosensor showed very high sensitivity and selectivity with a dynamic response ranged from 0.1 fM to 10 pM with a detection limit of 31.6 aM. Furthermore, this proposed biosensor was successfully applied to the detection of target DNA in 20% diluted serum. The developed strategy has been demonstrated to have the potential for application in molecular diagnostics.

A FRET-based detection of N-acetylneuraminic acid using CdSe/ZnS quantum dot and exonuclease III-assisted recycling amplification strategy

N-acetylneuraminic acid (Neu5Ac) is widely spread in many biologically significant glycans of mammals, commonly as a terminal α-glycoside. It is of great significance to develop analytical techniques for detection of Neu5Ac. Herein, a high-sensitive fluorescent biosensor for Neu5Ac has been developed based on FRET between CdSe/ZnS quantum dots (QDs) and BHQ2, as well as exonuclease III (Exo III)-assisted recycling amplification strategy. Employing the specially designed three-level FRET systems and fluorescent signal recovery mechanism, together with five-step recycling signal amplification chain reactions, an ultralow detection limit of 24 fM was achieved. Meanwhile, good linear response ranges within 0.2–12.5 pM and 12.5–1000 pM were founded. The assay has excellent performance in real sample detection, and thus offers great potential for detection of sialic acids modified glycans/lipids in the fields of medical diagnosis and food testing.

DNA Structure-Stabilized Liquid-Liquid Self-Assembled Ordered Au Nanoparticle Interface for Sensitive Detection of MiRNA 155.

Au nanoparticles (Au NPs) can be self-assembled in a bottom-up orderly manner at the oil-water interface, which is widely used as SERS platforms, but the stability of the Au NP interface needs to be improved due to shaking or shifting and the Brownian motion. The DNA structure with unique sequence specificity, excellent programmability, and flexible end-group modification capability owns good potential to precisely control the plasmonic structure's distance. In this study, a large area of the SERS substrate is obtained from the DNA structure-stabilized self-assembled ordered Au NPs on the cyclohexane-water interface. Combining with the exonuclease III (exo III)-assisted DNA recycling amplification strategy, we construct a liquid-phase SERS biosensor for efficient detection of microRNA 155 (miRNA 155). Compared with the traditional randomly assembled Au NPs on the two-phase interface, the SERS signal is significantly enhanced and more stable. The detection limit of the SERS biosensor for miRNA 155 reached 1.45 fmol/L, which has a very wide linear range (100 fmol/L-5 nmol/L). This work gives an efficient approach to stabilize the self-assembly Au NPs on the liquid-liquid interface, which can broaden the application of SERS analysis.

Graphene-based fluorometric determination of agrD gene transcription in methicillin-resistant Staphylococcus aureus using exonuclease III-aided target recycling and DNA walker cascade amplification.

A graphene-based bioassay is describedfor the fluorometric determination of agrD gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method includes exonuclease III (Exo III)-assisted target recycling and DNA walker cascade amplification. Hairpin1 (HP1) consists of a capture probe (CP) and DNA walker sequence. In the absence of the target, 5'-amino modified hairpin2 (HP2) labeled with carboxyfluorescein (FAM) at its 3' terminus is covalently linked to graphene via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, resulting in the quenching of the FAM signal. The stem-loop structure of HP1 opens when the target is added to form partially complementary DNA/RNA hybrids. Exo III then initiates the target recycling process by cleaving the CP and DNA walker cascade reaction by automatic walking. This iterative reaction causes the FAM to dissociate from the graphene, and the fluorescence can be measured at excitation/emission wavelengths of 480/514nm. Therefore, the target can be assayed by fluorescence. This method has a linear relationship with the concentration of target within the range 1fM to 100pM with a detection limit of 1fM. The developed bioassay was used to monitor biofilm formation and investigate the mechanism of drug action with satisfactory results. Schematic representation of the graphene-based fluorescent bioassay for agrD gene transcription in methicillin-resistant Staphylococcus aureus by using exonuclease III-aided target recycling and DNA walker cascade amplification.

Construction of a Sensing Platform Based on DNA-Encoded Magnetic Beads and Copper Nanoclusters for Viral Gene Analysis with Target Recycling Amplification.

The rapid and accurate monitoring of viral genes plays an important role in the area of disease diagnosis, biomedical research, and food safety. Herein, we successfully designed a sensing system that combined the technologies of target DNA recycling amplification, magnetic separation, and in situ formation of fluorescent copper nanoclusters (CuNCs) for viral DNA analysis. In the presence of target viral DNA (tDNA), a large quantity of output DNA (oDNA) was produced from hairpin DNA (hDNA) through an exonuclease III-assisted target recycling amplification strategy. Magnetic beads (MBs) labeled with capture DNA (cDNA) were hybridized with oDNA, and the partially complementary oDNA served as a bridge that could link AT-rich dsDNA on the surface of MBs, which led to a decrease of AT-rich dsDNA in solution after magnetic separation. On account of the lack of AT-rich dsDNA as a template in solution, in situ formation of fluorescent CuNCs was blocked, which resulted in a decrease in the fluorescence intensity at 590 nm. Therefore, taking advantage of one-step magnetic separation and in situ formation of CuNCs, the target viral DNA was sensitively and specifically detected in a linear range from 5 pM to 5 nM with a detection limit of 1 pM. The MB-based platform was not only reusable but also achieved magnetic separation, which could eliminate interferences in complex samples. The assay combining the MB-based probe with fluorescent CuNCs provided a universal, label-free, and reusable platform for viral DNA detection.

Functionalized europium-porphyrin coordination polymer: Rational design of high performance electrochemiluminescence emitter for mucin 1 sensing

The excellent characteristics of porphyrins have inspired widespread interest in electrochemiluminescence (ECL). However, the limited ECL intensity and poor stability of porphyrins in aqueous solution are still severely restricted further biological application. Here, we subtly synthesized a functionalized europium and 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) coordination polymer (Eu-PCP) by a one-step solvothermal method. In sharp contrast to the pristine TCPP, Eu-PCP showed a higher and more stable ECL red-light emission (673 nm) at low potential (−1.1 V, vs Ag/AgCl), which was 7.7-fold higher ECL intensity and 4.6-fold efficiency. In view of the crucial role of mucin 1 (MUC1) in tumor overexpression, it was selected as the target molecule. Combined with exonuclease III (Exo III)-assisted recycling amplification strategy, a ternary ECL biosensor was constructed for the MUC1 detection based on Eu-PCP as a satisfied ECL emitter, gold nanoparticles capped CeO2 (CeO2@Au) as the coreactant accelerator and peroxydisulfate as coreactant. Meanwhile, gold nanoparticles capped MnO2 (MnO2@Au) was used as the quenching probe to achieve a highly sensitive detection of MUC1. The proposed biosensor exhibited a wide linear range from 1 fg mL−1 to 10 ng mL−1 with a low limit of detection (0.32 fg mL−1). By changing the corresponding target recognition DNA, this strategy could be expanded to detect other biomarkers.

Ultrasensitive detection of trace Hg2+ by SERS aptasensor based on dual recycling amplification in water environment

Due to the nonbiodegradability and accumulation of mercury ion, even in extremely small amount, it will cause varying degrees of harm to environment and human health. Although researchers have developed many strategies to detect and monitor trace Hg2+, only a few provide sensitivities of less than 1.0 pM. Surface Enhanced Raman Spectroscopy (SERS) is a common method to detect mercury ion due to its high sensitivity, rapid detection and easy operation. In this work, we report a new SERS aptasensor based on dual recycling amplification for the detection of trace mercury ion, which combines SERS with nucleic acid signal amplification through functional aptamer and elaborately designed hairpin DNA. Under the optimal experimental conditions, this SERS aptasensor exhibits excellent selectivity and high sensitivity. A linear range (0.2–125 fM) and a low detection limit (0.11 fM) are obtained. By using specific aptamers, the strategy will provide a new idea for the trace detection of toxic contaminants in water environment.

Ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of p53 gene based on nicking endonuclease-assisted target recycling and rolling circle amplification

Tumor suppressor p53 gene is one vital anticancer gene that plays critical role in the prevention, diagnosis, and prognosis evaluation of cancers. Specific recognition and sensitive detection of p53 gene is important for early cancer screening and diagnosis. A ratiometric electrochemical biosensor is developed for ultrasensitive detection of tumor suppressor p53 gene in this work. Hairpin capture probe (Fc-HP-SH) is labeled with ferrocene (Fc) at the 3′ termini and thiol group at the 5′ termini. Fc-HP-SH was self-assembled on the surface of gold electrode through Au-S binding, while 6-mercaptohexanol was used to block the unoccupied sites on electrode surface. At the presents of p53 gene and nicking endonuclease, p53 gene specifically hybridizes with the loop sequences of Fc-HP-SH to unwind the hairpin structure. Nicking endonuclease subsequently cleaves DNA duplex to release p53 gene for target recycling. Residual single-stranded oligonucleotide is prolonged with the help of phi29 DNA polymerase via rolling circle amplification, leading to the increased gathering of electroactive probe methylene blue (MB) on long polynucleotide. Such variations cause the alterations of the differential pulse voltammetry peak currents of Fc at + 0.32 V (vs. Ag/AgCl) and MB at − 0.25 V (vs. Ag/AgCl), resulting in an obvious enhancement of the ratiometric signal of two peak currents. Under optimum experimental conditions, this biosensor has a response that covers the 1 fM to 10 pM p53 gene concentration range, and the detection limit is 0.23 fM (for S/N = 3). This biosensor highly recognized p53 gene over its mutants and was successfully applied in human serum samples. This approach shows excellent reproducibility, acceptable stability, good selectivity, and ultrahigh sensitivity toward p53 gene detection.

Streptavidin-functionalized terahertz metamaterials for attomolar exosomal microRNA assay in pancreatic cancer based on duplex-specific nuclease-triggered rolling circle amplification

Exosomal microRNA (miRNA) is a promising non-invasive biomarker for liquid biopsies. Herein, we fabricated a terahertz (THz) metamaterial biosensor that comprises an array of gold (Au) discs surrounded by annular grooves for exosomal miRNA assays based on duplex-specific nuclease (DSN)-triggered rolling circle amplification (RCA). In this strategy, the target miRNA is captured by a probe P0 immobilized on magnetic beads (MBs); it then repeatedly releases a primer P1 under the action of DSN, which acts as a highly specific initiator of the subsequent RCA step utilizing biotin-dUTP. After target recycling and nucleic acid amplification, the biotinylated amplification products were captured by the streptavidin (SA)-functionalized THz metamaterials, and further conjugated to SA-modified AuNPs that permit formation of a trimeric complex of SA-biotinylated RCA products-AuNP. The complex population scales with the starting concentration of the target miR-21, resulting in a red shift of the resonance peak of the THz metamaterials. This biosensor can lead to highly specific and sensitive detection with one-base mismatch discrimination and a limit of detection (LOD) down to 84 aM. Significant distinctions are seen in the frequency shifts for exosomal miR-21 quantitation in clinical plasma samples between pancreatic cancer patients and healthy controls. The frequency shifts of the THz metamaterials are consistent versus the reverse transcription-polymerase chain reaction (RT-PCR) results, illustrating the applicability and accuracy of our assay in real clinical samples.

Serine and histidine-functionalized graphene quantum dot with unique double fluorescence emission as a fluorescent probe for highly sensitive detection of carbendazim

The application of graphene quantum dots has been focused on fluorescence detection, but poor functionality and low luminous efficiency limit its many applications. The study reports synthesis of serine and histidine-functionalized graphene quantum dot (Ser-GQD-His) via one-step simple pyrolysis. The resulting Ser-GQD-His offers an unique double fluorescence emission. Ultraviolet-light excitation at 360 nm produces one strong blue fluorescence with the quantum yield of 28.6 %. While visible-light excitation at 485 nm produces one stronger yellow fluorescence with the quantum yield of 51.2 %. The Ser-GQD-His was used as an optical probe for fluorescence detection of carbendazim coupled with DNA recycling amplification. By target-induced DNA recycling, one carbendazim molecule can lead to the formation of many G-quadruplex/hemin DNAzymes. The formed DNAzymes catalyze oxidation of phenylenediamine into 2,3-diaminophenazine (DAP) and achieves to a significant signal amplification. Meanwhile, the interaction of DAP with Ser-GQD-His leads to a fluorescence quenching process of Ser-GQD-His and fluorescence enhancement of DAP. This further enhances the detection signal. The ratiometric fluorescence signal increases linearly with increasing carbendazim in the range from 1.0 × 10−16 to 1.0 × 10−9 M with the limit of detection of 6.1 × 10−17 M. The proposed method exhibits a higher sensitivity and selectivity compared with reported methods.

Sensitive and Label-free Detection of Bacteria in Osteomyelitis through Exo III-Assisted Cascade Signal Amplification.

Rapid and sensitive pathogenic bacterial identification and isolation from complicated clinical specimens are of great importance for the early diagnosis and prevention of osteomyelitis. Herein, we proposed a novel methicillin-resistant Staphylococcus aureus (MRSA) detection strategy through two specially designed streptavidin magnetic bead-based probes, including a capture probe and a report probe. In detail, the capture probe takes the responsibility to specially bind with the surface protein of MRSA and leads to the liberation of the promoter which could subsequently initiate report probe-based signal amplification. Afterward, the hybridization of the promoter probe with the report probe could then transform the protruding 3′ terminus of template DNA in the report probe into a blunt end. With the assistance of Exo III, the template could be digested to liberate the promoter to form a recycle and to liberate the biprobe to induce the following rolling circle amplification (RCA)-based signal amplification. Through the integration of the Exo III-assisted recycle and RCA-based signal amplification, the proposed method exhibited a favorable detection performance.

Orderly Assembled, Self-Powered FRET Flares for MicroRNA Imaging in Live Cells.

Signal amplification provides unparalleled opportunities for visualizing low-abundance targets in living cells. However, self-powered signal amplification has not been achieved because of the lack of "fuel" in living cells. Thus, the aim of this work was to develop an integrated amplification platform for the detection of intracellular miRNA by itself. This system, termed self-powered FRET flares (SPF), was first established by self-assembly to form a DNA nanostructure, and then the FRET flares and fuel DNA as the driving force were precisely and orderly loaded on it, which was able to power target recycle and realize signal amplification without any auxiliary additives under the trigger of intracellular miRNA-21. In addition, it employed AS1411 aptamer to target specific cancer cells and facilitated cell internalization of assembly DNA nanostructures. As a proof of concept, we demonstrated that SPF enabled rapid response to miRNA-21 and improvement of the detection sensitivity compared to previously proposed FRET flares without amplification. This strategy is promising for advancing integrated and self-powered nanomachines to execute diverse tasks, facilitating their biological and medical application.

An enzyme-free amplification strategy for sensitive assay of circulating tumor DNA based on wheel-like catalytic hairpin assembly and frame hybridization chain reaction

As an ideal liquid biopsy biomarker candidate, circulating tumor DNA (ctDNA) plays an important role in the non-invasive diagnosis of cancer. Sensitive and precise analysis of ctDNA plays a vital role in promoting clinical applications. Herein, we have developed a novel electrochemical biosensor for ctDNA assay coupling wheel-like catalytic hairpin assembly (WCHA) and frame hybridization chain reaction (FHCR). The ctDNA bound to the DNA transducer by the toehold strand displacement and released the initiator to promote the recycle amplification of WCHA, which lead to the formation of a great number of wheel-like DNA products hybridized with the capture probes immobilized on the gold electrode surface. In addition, the metastable dumbbell probes (DSH1 and DSH2) are designed for hybridization chain reaction. Compared with the traditional HCR, this novel form of HCR (FHCR) can fold up more compact and dense DNA nanostructures, which greatly reduce the space potential resistance and enhance the electronic transmission ability. The combination of dual signal amplification strategies (WCHA/FHCR) significantly improve the sensitivity of ctDNA detection, creating a limit of detection (LOD) as low as 8.3 fM. More importantly, the proposed method is successfully applied in the analysis of clinical samples, and has an effective classifier with an AUC of 0.909, showing a sensitivity of 86.7 % and a specificity of 80.0 % for the diagnosis of breast tumors. Therefore, this platform has great potential in bioanalysis and clinical diagnostics.