Target-triggered Catalytic Hairpin Assembly Research Articles

Target-driven cascade amplified assembly of covalent organic frameworks on tetrahedral DNA nanostructure with multiplex recognition domains for ultrasensitive detection of microRNA

BackgroundMicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. ResultsIn this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the “freeze-drying-reduction” method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. Significance and NoveltyTaking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening.

An enzyme-free and label-free ratiometric fluorescence signal amplification biosensor based on DNA-silver nanoclusters and catalytic hairpin assembly for tetracycline detection

Sensitive and accurate in antibiotic detection is crucial for food security, environmental conservation, public health, and environmental protection. In this study, we developed an enzyme-free and label-free ratiometric fluorescence biosensor for highly sensitive detection of tetracycline (TET). This biosensor utilized a combination of target-triggered catalytic hairpin assembly (CHA) and DNA-silver nanocluster (DNA-AgNCs) dual-color fluorescent signal probes. Upon TET binding to the aptamer, it initiated the leakage of CHA initiator, resulting in the formation of numerous double-stranded DNA complexes. This process exposed the foothold sequence of hairpin-structured DNA-AgNCs (H3-AgNCs) that exhibit yellow fluorescence. Subsequently, the conformational changes in H3-AgNCs significantly reduced the yellow fluorescence intensity (FI) and reveal the auxiliary DNA-AgNCs (a-DNA-AgNCs) linkage sequence. The binding of a-DNA-AgNCs brought two dark AgNCs close, forming nanocluster dimers emitting red fluorescence. The results demonstrate that the variation in ratiometric signal is linear with TET concentration between 0.1 and 50 ng/mL, with a detection limit as low as 0.086 ng/mL. Additionally, this approach exhibited good recovery results with spiked milk samples. This enzyme-free and label-free combined signal amplification method with proportional signal output based on DNA-AgNCs provides valuable empirical reference for creating low-cost, stable, and highly-sensitive biosensors.

Mass Nanotags Mediate Parallel Amplifications on Nanointerfaces for Multiplexed Profiling of RNAs.

Multiplexed profiling of RNAs aids in a comprehensive understanding of multiparameter-defined cellular processes and pathological states. We herein present a mass nanotags-enabled interfacial assembly system (MNTs-AS) with parallel amplification motors for simultaneous assaying of multiple RNAs in biosystems by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Four kinds of MNTs encoding corresponding RNA can be cyclically assembled on magnetic beads by target-triggered catalytic hairpin assembly (CHA) machineries on nanointerfaces, generating multiplexed and amplified characteristic ion signals assigned to target RNAs upon MALDI MS interrogation. By virtue of high sensitivity and multiplexing capability, the MNTs-AS-based MS assay allows precision subtyping of diverse breast cancer cells and their exosomes by multiplexed profiling of miRNA-21, miRNA-373, miRNA-155, and manganese superoxide dismutase mRNA via a single MS inquiry. This method provides a promising tool for unraveling multiple RNA-involved biological events in fundamental research and distinguishing different cancer subtypes in clinical practice.

Photocurrent-Polarity-Switching Photoelectrochemical Biosensor for Switching Spatial Distance Electroactive Tags.

This work presents a photocurrent-polarity-switching-based photoelectrochemical (PEC) biosensing platform for ultrasensitive detection of microRNA-21 (miR-21) through target-triggered catalytic hairpin assembly (CHA) for modulation of methylene blue (MB) and ferrocene (Fc) positional configurations using double-shelled Cu-doped ZnS nanocages (NCs)-Au nanoparticles (NPs) as photoactive materials. In the presence of miR-21, the assembly of MB-labeled HP1 and Fc-labeled HP2 leads to the generation of a large amount of double-stranded DNA (HP1-HP2), which pushes MB away from the electrode surface and brings Fc close to the electrode surface, resulting in effectively quenching the enhanced PEC signal to activate the photocurrent-polarity-switching system. Benefiting from the distance-controllable strategy, the designed PEC bioanalysis can effectively eliminate false-positive and false-negative signals due to the change of different signal expression patterns (from traditional the "signal-on" mode to the photocurrent-polarity-switching mode), thereby significantly improving the sensing specificity and sensitivity. The proposed PEC sensing system exhibited satisfying photocurrent responses toward target miR-21 within the working range from 1.0 fM to 1 nM at a low limit of detection (LOD) of 0.58 fM. More importantly, we demonstrated the successful integration of the proposed PEC biosensor with a handheld wireless device for instant detection of miR-21 concentrations in practical samples.

Ultrasensitive detection of microRNA-10b through target-triggered catalytic hairpin assembly and upconversion nanoparticles-based luminescence resonance energy transfer

The overexpression of microRNA-10b (miRNA-10b) is closely associated with hepatocellular carcinoma (HCC), which may act as an advance biomarker to diagnose HCC early. Here, a novel luminescent nanosensor for the ultrasensitive detection of miRNA-10b was firstly developed by target-triggered catalytic hairpin assembly (CHA) and upconversion nanoparticles (UCNPs)-based luminescence resonance energy transfer (LRET). The hybridization process of DNA1-modified UCNPs with DNA2-modified gold nanoparticles (AuNPs) makes the energy donor (UCNPs) and acceptor (AuNPs) into close proximity, resulting in luminescence quenching of UCNPs. The miRNA-10b can hybridize with the hairpin structure of DNA2, releasing the recognition domain site and activate the CHA reaction with its signal amplification chain (H3). At the same time, the LRET was inhibited accompany with the AuNPs moved away from the UCNPs, which resulted in the luminescence recovery of UCNPs. The miRNA-10b can be quantified by measurement of the luminescence recovery rate of UCNPs. The sensor has high sensitivity with a detection limit of 8.96 fM. In addition, the sensor can be used to the discrimination of miRNA-10b expression levels in human hepatocarcinoma and hepatocyte cell lines with satisfactory results. The technique shows great promise for early diagnosis of HCC because of its high sensitivity and selectivity.

Target-triggered catalytic hairpin assembly activation of CRISPR/Cas12a for amplified detection of therapeutic monoclonal antibody

It is of great significance to monitor therapeutic antibodies in human serum to obtain the pharmacokinetic data for assessing their therapeutic efficacy at the dosage of administration. Here, based on the target-triggered proximity binding approach, as well as the catalytic hairpin assembly (CHA) and CRISPR/Cas12a cleavage dual signal amplification strategy, we describe the establishment of a highly sensitive method for detecting panitumumab, a cancer therapeutic monoclonal antibody, in diluted human serums. The two proximity probes bind the target panitumumab simultaneously to form initiation sequences for subsequent CHA for the cyclic yield of many dsDNAs, which further activate the specific trans-cleavage activity of Cas12a to digest lots of fluorescently quenched ssDNAs to restore drastically enhanced fluorescence for detecting panitumumab with a 0.62 pM detection limit. Besides, the sensing approach can selectively discriminate panitumumab against other monoclonal antibodies and realize low levels of panitumumab detection in diluted serums. Moreover, by simple replacement of the proximity recognition probes, the established method can be easily expanded for the development of versatile sensing platforms for different biomolecules in addition to antibodies.

Target-induced tripedal G-quadruplex DNAzyme for multicolor visual point-of-care testing of biomarkers using Au nanorods-decorated electrospun nanofibrous films

A target-induced tripedal G-quadruplex DNAzyme (TGD) for visual point-of-care testing (POCT) of PSA proteins by using solid Au nanorods (Au NRs)-decorated electrospun nanofibrous films was constructed. The TGD was formed by the recycling amplification cascades of target-triggered catalytic hairpin assembly (CHA), which exhibited superior horseradish peroxidase (HRP) mimicking activity to facilitate the degradation of H2O2 into hydroxyl radicals (·OH). The ·OH oxidized the 3,3′,5,5′-tetramethylbenzidine (TMB) into TMB+. Then, the TMB+ was converted into TMB2+ in the acidic environment, which can etch the Au NRs for further promoting the blue shift of their longitudinal surface plasmon resonance (LSPR) absorption peak. Thereby, with the increasing PSA concentrations, the proposed strategy presented palpable color variations from green, cyan, violet, and red by naked eyes. Significantly, the cutoff values near 4.0 and 10.0 ng/mL of PSA in human blood serum could be discriminated with naked eyes, which could provide a semi-quantitative process for the early diagnosis and prognosis evaluation. In addition, this portable, and label-free TDG biosensor exhibited remarkable improvements in sensitivity with a low detection limit of 61.1 fg/mL for PSA protein. This strategy showed the great versatility and potential for the visual biomedical detection with portable type and early diagnosis of diseases.

CRISPR/Cas12a-based photoelectrochemical sensing of microRNA on reduced graphene oxide-anchored Bi2WO6 coupling with catalytic hairpin assembly

An innovative and generic CRISPR/Cas12a-driven photoelectrochemical (PEC) biosensing platform was developed for screening of microRNA-21 (miR-21) by coupling with target-triggered catalytic hairpin assembly (CHA) and reduced graphene oxide-anchored Bi2WO6 (rGO-BWO) as the photoactive material. CHA isothermal amplification involved two programmable hairpin DNA modules and miR-21 as an activator. In the presence of miR-21, the products of target-triggered CHA circuit were inserted into the Cas12a-crRNA duplex to initial trans-cleavage capacity of CRISPR/Cas12a nuclease, accompanying the digestion on alkaline phosphatase (ALP)-labeled single-stranded DNA (ssDNA)-encoded magnetic bead (MB) through the activated CRISPR system. The ALPs were detached from magnetic beads and promoted the generation of ascorbic acid (AA), which increased the photocurrent of rGO-BWO-modified electrode. The value of photocurrent was positively proportional to the level of AA, which was also linearly correlated with target concentrations. Under optimum conditions, the CRISPR-based PEC sensing system displayed satisfying photocurrent responses toward miR-21 within the range from 1.0 fM to 1.0 nM with a limit of detection of 0.47 fM. In addition, the biosensor exhibited acceptable stability and excellent selectivity. Impressively, CHA-mediated CRISPR-based PEC biosensing platform provides a universal and sensitive method for clinical cancer diagnostics and biomolecular research.

A rapid “cusp-covering” to Au nanostar as plasmonic sensor in a single-drop microreactor for the determination of kanamycin in biosamples

For complicated real sample detection, the metal nanomaterial-based plasmonic sensing strategy always presents great challenge since the reaction was easily affected by the matrix effect. In this study, a single-drop (9 μL) microreactor was designed for a plasmonic “cusp-covering” process to detect residual antibiotic (kanamycin, Kana) in biological samples. A target-triggered catalytic hairpin assembly and hybridization chain reaction (CHA-HCR) amplification cascades strategy was applied to recognize and amplify the signal of Kana. Then, a magnetic three-phase single-drop microextraction was introduced and provided a microreactor for the reaction of covering various thicknesses of Ag on the cusp of Au nanostar, the change in composition and morphology of the materials resulted in a significant shift of localized surface plasmon resonance (LSPR) signal. Moreover, a computational simulation was conducted to prove the advancement of the strategy. Taking advantage of the LSPR effect, the single-drop microreaction system, and the CHA-HCR amplification cascades, an excellent analytical performance was novelly achieved and the detection sensitivity was significantly enhanced. This strategy exhibited a low limit of detection of 33 aM for the determination of Kana with relative standard deviations < 5% (n = 3) and achieved a wide linearity ranging from 0.1 fM to 1 nM. The proposed strategy eliminated the matrix effect of the real sample and provided a stable environment for the minimized detection system, which is capable of sensitively detecting Kana in milk or other real biosamples.

Rapid and enzyme-free signal amplification for fluorescent detection of microRNA via localized catalytic hairpin assembly on gold nanoparticles.

Various signal amplification strategies have been developed for microRNA (miRNA) detection, but most of these amplification strategies always need some enzymes. In this work, we have constructed an enzyme-free signal amplification method for miRNA determination via target-triggered catalytic hairpin assembly (CHA). Two hairpin probes (H1 and H2) were ingeniously designed, and fluorescein (FAM)-labeled H1 (as a signal reporter) was conjugated on the gold nanoparticles (AuNPs) surface. In the presence of target miRNA, the cyclic self-assembly took place between H1 and H2 on the AuNPs, resulting in amplification of the fluorescence signal. Using miRNA-21 as a model analyte, the linear concentration range for miRNA-21 detection was from 0.1nM-10nM under the optimized experimental conditions (25μL AuNPs (3nM), 100nM H2, 25°C, pH 7.4). The fluorescence method exhibited high sensitivity to with a 10 pM detection limit. The recoveries in 2% normal human serum were in the range of 96.4%-103.6%. What's more, it was found that the addition of ten thymine bases (T10) as spacer between H1 and AuNPs could significantly accelerate the CHA reaction. A complete analysis for the determination of miRNA could be accomplished within 45min, which is faster than a number of previous reports.

A label-free and enzyme-free fluorescent aptasensor for amplified detection of kanamycin in milk sample based on target-triggered catalytic hairpin assembly

Kanamycin residues in foods can cause serious adverse reactions in human body, and the accurate and sensitive detection of kanamycin is of great significance to ensure human health. In this work, we constructed a label-free and enzyme-free fluorescent aptasensor for quantifying kanamycin in milk based on target-triggered catalytic hairpin assembly. First, we designed an aptamer probe that contained an aptamer unit for recognizing kanamycin, a trigger unit for triggering catalytic hairpin assembly and a suppression unit for ensuring the stability of aptamer probe. In the initial state, the trigger unit was closed by suppression unit and inactive. After the aptamer unit recognized and bound to kanamycin, a free trigger unit was released to trigger the cycle hybridization reactions between hairpin probe 1 and hairpin probe 2, generating G-rich complexes and realizing enzyme-free amplification detection. Then, N-methylmesoporphyrin IX as signal molecules were combined with G-rich complexes to generate a significant fluorescent signal, which realized label-free detection. Thus, the aptasensor provided a well sensitivity for kanamycin with a detection limit of 0.26 ng/mL. And it possessed well selectivity and could distinguish kanamycin from its analogues, which stemmed from the specific recognition between kanamycin and aptamer. The aptasensor had great potential application value in the field of ensuring food safety.

Highly Sensitive Detection and Intracellular Imaging of Micrornas Based on Target-Triggered Cascade Catalytic Hairpin Assembly

Highly Sensitive Detection and Intracellular Imaging of Micrornas Based on Target-Triggered Cascade Catalytic Hairpin Assembly

Smartphone-assisted colorimetric detection of BRCA-1 gene based on catalytic hairpin assembly amplification and G-quadruplex DNAzyme

• An enzyme-free colorimetric biosensor for sensitive detection of BRCA-1 gene is developed. • The target-triggered CHA and the peroxidase-like activity of G-quadruplex DNAzyme are used for signal amplification. • The colorimetric biosensor exhibits a high sensitivity toward target DNA with a detection limit of 10 pM. • The proposed strategy has a great potential application in genetic analysis and clinical diagnosis. In this study, we develop an enzyme-free colorimetric biosensor for the determination of breast cancer susceptibility gene (BRCA-1), which integrates catalytic hairpin assembly (CHA) signal amplification with the peroxidase-like activity of G-quadruplex DNAzyme. With the addition of BRCA-1 gene, the hairpin probes (H1 and H2) are able to generate numerous duplex DNA assemblies with liberated G-quadruplex forming sequences. After binding with cofactor hemin, the G-quadruplex forming sequences fold into G-quadruplex DNAzymes, which effectively catalyze the H 2 O 2 -mediated oxidation of 2,2’-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) to free-radical cation (ABTS •+ ), accompanied by a color transition from colorless to green. The colorimetric readout can be recorded using a smartphone camera or measured by UV-vis spectrophotometer. The developed colorimetric biosensor can detect target DNA as low as 10 pM with a linear range from 50 pM to 200 nM. Meanwhile, the colorimetric biosensing platform allows the determination of target DNA at a concentration as low as 0.2 nM through smartphone applications. In addition, the colorimetric biosensor shows excellent selectivity and acceptable applicability for the detection of BRCA-1 gene in human serum samples with satisfactory recoveries of 93.47–104.14% and RSDs of 6.89–8.24%, which might hold great application potential in genetic analysis and clinical diagnosis.

Metal-Mediated Polydopamine Nanoparticles-DNA Nanomachine Coupling Electrochemical Conversion of Metal-Organic Frameworks for Ultrasensitive MicroRNA Sensing.

The development of a robust sensing platform with an efficient probe assembly, and ingenious signal conversion is of great significance for bioanalytical application. In this work, a multipedal polydopamine nanoparticles-DNA (PDANs-DNA) nanomachine coupling electrochemical-driven metal-organic frameworks (MOFs) conversion-enabled biosensing platform was constructed. The PDANs-DNA nanomachine was designed based on Ca2+-mediated DNA adsorption and target-triggered catalytic hairpin assembly on PDANs, which not only maintained the DNA immobilization simplicity but also possessed a high walking efficiency. PDANs-DNA nanomachine could walk fast on the electrode via multiple legs under exonuclease III driving, resulting in the formation of DNA dendrimers through two hairpins assembly. The MOFs (Fe-MIL-88-NH2) probe was decorated on the DNA dendrimers to act as a porous metal precursor and converted into electroactive Prussian Blue by a controlled electrochemical approach, which was a facile, simple, and room-temperature approach compared with the commonly employed MOFs conversion methods. Using microRNA-21 (miRNA-21) as the model target, the proposed biosensor achieved miRNA-21 detection ranging from 10 aM to 10 pM with the detection limit of 5.8 aM. The proposed strategy presented a highly efficient walking platform with the ingenious electrochemical conversion of MOFs, providing more options for the design of an electrochemical platform and holding potential applications in clinical analysis and disease diagnosis.

Colorimetric aptasensor for sensitive detection of kanamycin based on target-triggered catalytic hairpin assembly amplification and DNA-gold nanoparticle probes

In this work, an enzyme-free colorimetric aptasensor has been constructed for the sensitive detection of kanamycin in complex samples, which integrates the target-triggered catalytic hairpin assembly (CHA) amplification with the aggregation of DNA-gold nanoparticle probes (AuNPs-DNA). In the presence of kanamycin, the hairpin probes (H1, H2 and H3) are able to form branched Y-shaped DNA nanostructures that possess three single-strand DNA (ssDNA) tails, which induce the assembly of AuNPs-DNA, resulting in a concomitant color change from red to blue-purple. The colorimetric readout can be visualized by naked eye or measured by UV–vis spectroscopy. The proposed aptasensor shows high sensitivity and selectivity for kanamycin detection with a broad linear range from 20 pM to 5 nM, and the limit of detection (LOD) is calculated to be 10 pM. Moreover, the colorimetric aptasensor exhibits excellent acceptable applicability for detection of kanamycin in complex samples (milk, honey, serum and river water) with satisfactory recoveries of 96.0–106% and RSDs of 1.92–4.04%, and the results are in accord with those of the ELISA method, indicating that the proposed sensing strategy has great potential applications in the fields of food safety, environmental monitoring and drug analysis.

Simple Tripedal DNA Walker Prepared by Target-Triggered Catalytic Hairpin Assembly for Ultrasensitive Electrochemiluminescence Detection of MicroRNA.

In contrast to common DNA walkers, multipedal DNA walkers exhibit larger walking area and faster walking kinetics and provide increased amplification efficiency. Consequently, they have received a considerable amount of attention in biosensing. However, most of them are synthesized by immobilizing multiple DNA walking strands on the surface of Au nanoparticles, which is tedious and time-consuming. Simple preparation of multipedal DNA walkers remains a challenge. Herein, we adopted a simple enzyme-free target-triggered catalytic hairpin assembly (CHA) circuit to synthesize a tripedal DNA walker. By walking on a DNA track-functionalized electrode, a sensitive electrochemiluminescence DNA nanomachine biosensor was constructed for sensing miRNA-21. The DNA walker was powered by toehold-mediated strand displacement; the whole process did not need the assistance of enzymes, thus avoiding tedious procedures and enzyme degradation under unfavorable environmental conditions. Specifically, a superior detection limit of 4 aM and a broad linear range of 10 aM to 1 pM were achieved. This CHA-tripedal DNA walker biosensor was then applied for the detection of miRNA-21 in human serum and showed high selectivity and excellent reproducibility, demonstrating its practical application in bioanalysis. In particular, the Y-shaped tripedal DNA walker comes from the DNA circuit, which makes the approach ideally suited for biosensing of small nucleic acid targets.

Enzyme-free signal amplified Au nanoparticle fluorescence detection of thrombin via target-triggered catalytic hairpin assembly

Herein, we proposed an enzyme-free target-triggered catalytic hairpin assembly (CHA) for signal amplified fluorescence thrombin detection. In the detection system, two hairpin oligonucleotides (HP1, HP2) were used. The HP1 containing complimentary units forms a closed stem structure which brings the fluorescein (FAM) near the gold nanoparticle (AuNP) surface where they will be quenched. Upon addition of thrombin, the CHA could be initiated to form HP1-HP2-thrombin complex, which then through strand displacement process, the complex transforms into double-strand HP1-HP2 with a rod-like structure with the release of thrombin and also sequestering fluorophore from quencher surface, resulting in fluorescence recovery. The released thrombin further participated in subsequent reaction cycles, hence, the signal of the detection system significantly amplified. The method showed a limit of detection (LOD) of 165 pM with a linear range from 0.2 to 100 nM. The proposed method could be used as a potential application in complex biological matrices.

A homogeneous strategy of target-triggered catalytic hairpin assembly for thrombin signal amplification

Sensitive and rapid detection of thrombin is play an important role in biomedical and biological analysis. In this work, tetraferrocene was synthesized and used for the construction of a homogeneous electrochemical aptasensor for sensitive and rapid detection of thrombin in human serum via coupling host-guest recognition of tetraferrocene and β-CD with catalytic hairpin assembly (CHA) signal amplification. The presence of target thrombin causes unfoldment of a large number of double hairpin probes (H1). Subsequently, two tetraferrocenes modified on H1 enter the β-CD’s cavity modified on a gold electrode (AuE) based on host-guest recognition of tetraferrocene and β-CD, which drastically amplifies the current response for thrombin detection. The prepared aptasensor shows a wide linear relationship between 1 pM and 3.125 nM, and detection limit of 0.28 pM. More importantly, this homogeneous strategy provides essential information for the manufacturing of sensors for sensitive and rapid analysis of other biomarkers.

A novel ultrasensitive photoelectrochemical biosensor for detecting microRNA 21 based on cosensitization strategy and p-n heterojunction quenching mode

Highly sensitive detection of biomarkers has a vital role in clinical diagnosis and disease treatment. Integrating cosensitization strategy and p-n heterojunction quenching mode, we have established a novel ultrasensitive “signal off” photoelectrochemical (PEC) biosensor for detecting microRNA 21 (miRNA 21) via target-triggered catalytic hairpin assembly (CHA) cycling amplification. The n-type nano-hybrid (Bi2S3-ZnS) wrapped by toluidine blue O dye (TBO@Bi2S3-ZnS composite) has been synthesized, in which TBO and Bi2S3 as photosensitizers for photoactive matrix of ZnS. Since the cosensitization effect of Bi2S3 and TBO, 136-fold enhancement in PEC signal of TBO@Bi2S3-ZnS has been acquired compared with ZnS. Trace miRNA 21 can be transformed to abundant p-type copper sulfide (CuS) labeled signal tags to form p-n heterojunction with TBO@Bi2S3-ZnS, leading to obvious quenching effect. Thus, the PEC signals decrease with the increasing concentration of miRNA 21 and achieve the quantitative detection. The proposed PEC biosensor exhibits good linear relationship to miRNA 21 ranging from 1.0 × 10−17 to 1.0 × 10−11 mol·L−1 with a limit of detection of 3.3 × 10−18 mol·L−1. This biosensor based on TBO used as photosensitizer for the first time may offer new opportunities for detecting other biomarkers and facilitate the application of organic dyes in PEC sensing field.

Target-Catalyzed Assembly of Pyrene-Labeled Hairpins for Exponentially Amplified Biosensing.

Rapid and sensitive detection of nucleic acids is vital for disease diagnosis. This work designed an enzyme-free isothermal strategy for rapid exponential signal amplification through target-triggered catalytic hairpin assembly (CHA) to induce the spatially sensitive fluorescent signal of the pyrene excimer. Functionally, this system consisted of three pyrene labelled hairpins (H1, H2, and H3) and one catalyst DNA C. In the presence of C, the CHA was activated to generate intermediate I, which contained a single-stranded region identical to the C sequence for initiating the second cycle of CHA to obtain 2I and thus achieved the exponential formation of I along with the switching of pyrene excimer. The fluorescent signal of the pyrene excimer could be further enhanced via the inclusion of γ-cyclodextrin and showed a linear increase upon increasing logarithm of C concentration. Through the introduction of a helping hairpin H4-containing C sequence and a region specific to the target, this strategy could be extended to realize the quick and sensitive detection of different analytes. Using dengue virus RNA as an analyte model, the proposed fluorescent method showed a linear range from 0.1 to 50 nM with a limit of detection of 0.048 nM at 3σ and good selectivity. The excellent performance and convenient operation demonstrated its promising application in clinical disease diagnosis.