Target Recycling Research Articles

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.

Electrochemical Aptasensor Based on PEI‐rGO/AuNWs and Zr‐MOF for Determination of Adenosine Triphosphate via Exonuclease I‐assisted Target Recycling Strategy

AbstractIn this study, a hierarchically porous Zr‐MOF‐labeled electrochemical biosensor for the detection of Adenosine Triphosphate (ATP) was constructed using polyethylenimine‐reduced graphene oxide/Gold nanowire nanocomposite (PEI‐rGO/AuNWs) and exonuclease I (Exo I) assisted target recycling strategy. PEI‐rGO/AuNWs nanocomposite not only favor the immobilization of c‐DNA but also facilitate the electron transfer and elevate the electrode surface area. In addition, hierarchically porous Zr‐MOFs (HP‐UiO‐66), high stability and large mesoporous property, could load more signal molecules. In the presence of ATP, ATP aptamer was released from the c‐DNA, thus introducing MB@Zr‐MOF‐s‐DNA onto the electrode surface. Meanwhile, Exo I selectively digested the aptamer which bound with ATP, the released ATP participated new binding with the rest aptamer. Therefore, a significant increase in MB current intensity was observed. Under optimal conditions, the proposed aptasensor showed excellent performance with a linear range from 10−6 μM to 10 μM and a detection limit of 3.76×10−8 μM by differential pulse voltammetry. In addition, the proposed biosensor showed an outstanding performance with reproducibility, stability and anti‐interference ability, and was applied to the determination of ATP in real samples.

Ultrasensitive PEC aptasensor based on one dimensional hierarchical SnS2|oxygen vacancy-WO3 co-sensitized by formation of a cascade structure for signal amplification

Herein, we developed a photoelectrochemical (PEC) aptasensing platform that relies on a dual-signal amplification strategy. The proposed PEC system consists of a one dimensional (1D) hierarchical SnS2|OV (oxygen vacancy)-WO3 nanorods (NRs) sensitized by CdS quantum dots (QDs)/TCPP (TCPP: meso-tetra(4-carboxyphenyl)-porphine), and exonuclease III-assisted target recycling. As a photoactive heterojunction, SnS2|OV-WO3 nanocomposite exhibits a PEC signal ~11 times higher than pure WO3 NRs due to the enhanced separation efficiency of photo-generated carriers. Moreover, it was also used to immobilize a hairpin DNA3 probe labeled with CdS QDs/TCPP, which formed a co-sensitization cascade structure with SnS2|OV-WO3 NRs on the sensor surface due to the well-matched energy levels. This special cascade structure effectively shortened the electron-transfer path and inhibited charge recombination, thereby improving the PEC performance. The addition of the target vascular endothelial growth factor 165 (VEGF165) triggered the exonuclease III-assisted target recycling to generate plentiful DNA sequences (S1). S1 was specifically hybridized with HP3 to unfold its hairpin structure. As a result, CdS QDs/TCPP detached from the sensor surface and SnS2|OV-WO3 NRs, which destructed the co-sensitization cascade structure and minimized the PEC signals. The proposed PEC aptasensor exhibited a dynamic determination range of 0.5 fM-10 nM, and a detection limit of 0.34 fM for VEGF165.

Dual CHA-mediated high-efficient formation of a tripedal DNA walker for constructing a novel proteinase-free dual-mode biosensing strategy

DNA walkers have been recognized as a type of powerful signal amplification tool for biosensors, but how to adopt a proper strategy to increase their amplification efficiency is still highly desirable. Herein we design a dual-catalytic hairpin assembly (CHA)-mediated strategy for the high-efficient formation of a tripedal Mg2+-dependent DNAzyme (MNAzyme)-DNA walker, and thus develop a novel proteinase-free dual-mode biosensing method for the kanamycin (Kana) antibiotic assay. The first CHA is initiated by a target-biorecognition reaction, which can produce the DNA walker and also induce the target recycling. The second CHA is initiated by a special base sequence designed as a one-half substrate of the MNAzyme. Upon the first CHA-triggered DNA walking at a magnetic bead (MB) track, this “pseudo-target” sequence can be released to induce another CHA-cycle for the formation of the same DNA walker. Meanwhile, the other one-half substrate strand exposed on the MB surface will trigger the quantitative hybridization chain reaction (HCR)-assembly of a G-quadruplex DNAzyme (G-DNAzyme)-enriched double-stranded DNA polymer. So the enzymatic reaction of G-DNAzymes enabled the convenient colorimetric and photoelectrochemical dual-mode signal transduction of the method. Due to the dual-CHA facilitation to the tripedal and three-dimensional DNA walking and synergetic signal amplification of HCR, this method exhibits very low detection limits of 9.4 and 0.55 fg mL−1, respectively. In combination with its wide linear range, automated manipulation, and excellent selectivity, repeatability and reliability, the proposed method is expected to be used for the convenient semiquantitative screening and accurate determination of possible antibiotic residues in complicated matrices.

MXene-MoS2 heterostructure collaborated with catalyzed hairpin assembly for label-free electrochemical detection of microRNA-21

Abnormal expression of microRNAs is greatly associated with the occurrence of various cancer types, revealing great potential of microRNA as biomarkers for cancer diagnosis and prognosis. Herein, a MXene-MoS2 heterostructure enhancing electrochemical biosensor coupled with catalytic hairpin assembly (CHA) amplification approach for label-free determination of microRNA-21 (miR-21) was successfully assembled. In particular, the unique micro-nano heterostructure with large specific area and favorable electroconductivity exhibited the ability of excellent confinement effect. Thus, rendered the MXene-MoS2 heterostructure the ability to trigger more target recycling reaction, giving new vitality to the traditional CHA amplification method. Meanwhile, thionine (Thi) and gold nanoparticles (AuNPs) were anchoring at the surface of MXene-MoS2 heterostructure, respectively, empowered the sensor the capability of capture probes fixation and miR-21 label-free determination. When numerous electronegative double-stranded DNA generated, the electron transfer was greatly hindered, resulting in signal decrease. Accordingly, the design denoted a broad dynamic range from 100 fM to 100 nM and a detection limit of about 26 fM, comparable or lower than previous reported methods for miR-21 detection. Furthermore, the sensing platform supplied satisfactory selectivity, reproducibility and stability towards the miR-21 detection. The real sample determination also showed a promising performance under clinical circumstance. Finally, from the clinical standpoint, the proposed biosensor is a considerable platform toward early disease detection and monitoring.

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.

Contemporary electrochemical sensing and affinity biosensing to assist traces metal ions determination in clinical samples

AbstractThe determination of metals in clinical samples is an important challenge since their levels are relevant to ensure personal exposure to safe environments, as well as to assist in the prevention and treatment of disease. The low concentrations of metal ions in clinical samples and their coexistence with other metals and/or molecules of diverse nature implies the need for sensitive and selective analytical methodologies, which can be provided by conventional techniques. However, most of these techniques do not satisfy other highly demanded requirements such as high sampling frequency, multiplexed ability, and in‐field analysis. In this context, the simplicity, speed of response, and compatibility with single or multiplexed point‐of‐care determinations have led to the successful incursion of electrochemical (bio)sensing into the clinical field for the determination of these analytes. Electrochemical (bio)devices have come on strong by exploiting and combining the unique attributes provided by electrochemical techniques (primarily voltammetry but also giving way to impedimetry in electrochemical affinity biosensing), electrode materials (Hg, Bi, Sb, Au, GCE, CPE, ITO, FTO), and substrates (conventional, screen‐printed, flexible, paper, wearable or tattoo‐type), suitable modifiers (nanomaterials, polymers, mesoporous films), bioreceptors (aptamers, peptides, biomolecular switches, DNAzymes, and DNA molecules), and nucleic acid amplification strategies (rolling circle and exonucleases‐assisted target recycling). Therefore, the purpose of this review is to provide a bird's eye view of the state of the art, discussing the remarkable opportunities and potentialities demonstrated in the last decade by electrochemical sensors and electrochemical affinity biosensors for the analysis of metal ions in challenging clinical samples and forecasting future directions in this exciting field.

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.

Sensitive electrochemical aptasensor for determination of sulfaquinoxaline based on AuPd NPs@UiO-66-NH2/CoSe2 and RecJf exonuclease-assisted signal amplification

The authors designed a sensitive label-free electrochemical aptasensor for the detection of sulfaquinoxaline (SQX), including the AuPd NPs@UiO-66-NH2/CoSe2 nanocomposites and RecJf exonuclease-assisted target recycle signal amplification strategy. AuPd NPs@UiO-66-NH2/CoSe2 nanocomposite with excellent conductivity and numerous active sites was successfully synthesized to provide a favorable sensing platform and load more double-strand DNA (dsDNA) on the electrode surface. The negatively charged phosphate group of the oligonucleotide and [Fe (CN)6] 3-/4- repel each other electrostatically, resulting in very low electrical signals. In the presence of SQX, its corresponding aptamer will be released from the double-stranded structure and then digested by RecJf exonuclease, which resulted in the SQX being released to initiate the next recycling to help amplify the DPV signal. Under the optimal conditions, the peak current has a linear relationship with the logarithmic of SQX concentration in the range of 1 pg/mL∼100 ng/mL and the obtained detection limit was 0.547 pg/mL. Furthermore, the contrasted aptasensor possess reliable specificity, reproducibility and stability toward SQX, and has been applied to detect SQX in pork samples with a satisfied recovery varied from 94.40% to 95.98%.

Ultrasensitive Electrochemical Biosensor for HPV16 Oncogene Based on Y‐shaped DNA Catalytic Hairpin Assembly and Template‐free DNA Extension Reaction

AbstractAn ultrasensitive electrochemical biosensor for HPV16 oncogene was explored. Hairpin DNA‐1, which can specifically bind with HPV16 oncogene, was fixed on the surface of gold electrode. Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA‐1 to construct Y‐shaped DNA nanostructure, liberating HPV16 oncogene for target recycling. The 3’ terminus of Y‐shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase. Methylene blue was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal. This signal was increased with the concentration of HPV16 oncogene, and the detection limit of HPV16 oncogene was as low as 0.19 fM.

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.

Novel label-free fluorescence aptasensor for chloramphenicol detection based on a DNA four-arm junction-assisted signal amplification strategy

A novel label-free fluorescence aptasensor was established for chloramphenicol (CAP) detection by DNA four-arm junction-assisted target recycling and SYBR Green I dye-aided fluorescence-signal amplification. The CAP aptamer was hybridized to its complementary strand (primer) to form a double-stranded primer/aptamer complex. In the presence of CAP, aptamers can specifically bind with CAP to dissociate primers, which can trigger the self-assembly of four hairpins to continuously generate DNA four-arm junctions. After digesting the excess hairpins using T7 exonuclease, SYBR Green I was inserted into the base pair-rich DNA four-arm junctions, which led to a significant increase in fluorescence intensity. Under optimal conditions, the developed aptasensor can detect CAP in a linear range of 1.0 pg mL−1 to 10 ng mL−1 with a detection limit of 0.72 pg mL−1. The recovery rates in milk and honey ranged from 90.3% to 106.6%. Thus, the method shows substantial potential for CAP detection in food products.

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.

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.

A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing.

The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice.

Target Recycling Transcription of Lighting-Up RNA Aptamers for Highly Sensitive and Label-Free Detection of ATP

Target Recycling Transcription of Lighting-Up RNA Aptamers for Highly Sensitive and Label-Free Detection of ATP

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.

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.

Engineering a high-efficient DNA amplifier for biosensing application based on perylene decorated Ag microflowers as novel electrochemiluminescence indicators

DNA-based amplifiers with high programmability and accurate molecular recognition ability have become a versatile platform for target amplification. However, the random diffusion of capture probes (CPs) in most DNA amplifiers limits the target recognition efficiency, affecting the limit of detection. Herein, a high-efficient DNA amplifier was developed by localizing the CPs consisted of the unique palindromic tails and target recognition sequences on Au nanoparticle modified magnetic beads (Au@MBs). In the presence of target K-ras gene, the CPs with high local concentration and orientation could capture the target efficiently to expose their palindromic tails, which could act as primers to trigger the polymerization for target recycling. More importantly, the polymerization products could involve in the next recycle and produce abundant mimic targets (MTs) continuously, thereby achieving the detection of trace K-ras gene. Meanwhile, a novel electrochemiluminescence (ECL) indicator of a thin-layer of perylene (Pe) molecules decorated Ag microflowers (Pe@Ag MFs) was obtained based on the reaction between the perylene cation radical (Pe•+) and Ag atoms. The obtained Pe@Ag MFs exhibited desirable ECL performance because (i) a thin-layer of Pe molecules could reduce the inner filter effect and inactive emitters, (ii) the Ag MFs as coreaction accelerator could react with S2O82− to produce more SO4•− and shorten the distance between Pe•− and SO4•− to significantly enhance the ECL intensity of Pe with less energy loss. This work paves the way for the development of efficient amplification strategy and offers a paradigm for the preparation of high-efficiency ECL indicators.

Ratiometric Electrochemical Detection of MicroRNA Based on Construction of A Hierarchical C@SnS2 Nanoflower Sensing Interface

Abstract Building a precise, sensitive, and selective sensing platform is highly significant for microRNA (miRNA) detection. Herein, an innovative ratiometric electrochemical biosensor sensitized with carbon nanoparticles-functionalized SnS2 nanoflowers (C@SnS2 NFs) and enzyme-assisted target recycling was explored for analysis of miRNA-21. In this strategy, the application of hierarchical C@SnS2 NFs structure as electrode material not only enhanced the surface loading capability for subsequent reactions but also improved the interfacial electron transfer. After introducing enzyme-assisted target recycling, single-target miRNA-21 could initiate the cleavage of multiple capture DNA strands by duplex-specific nuclease (DSN), leading to numerous rest single-strand DNAs on the electrode. Two probes, methylene blue-labeled probe 1 (probe 1-MB) and ferrocene-labeled probe 2 (probe 2-Fc), could selectively and competitively hybridize with the capture DNA and the rest single-strand DNA, depending on the completeness of DNA hybridization. By measuring the peak current intensity ratio of Fc and MB, miRNA-21 was quantified in a wide concentration range from 1 fmol/L to 10 pmol/L, with a detection limit of 300 amol/L. Moreover, this sensing system showed good analytical performance in the presence of interfering sequences and complex matrices, illuminating excellent selectivity and practical application potential of the hierarchical C@SnS2 NF sensing interface.