Duplex-specific Nuclease Research Articles

Label-free fluorescence detection of circulating microRNAs based on duplex-specific nuclease-assisted target recycling coupled with rolling circle amplification

A simpler, more rapid and selective biosensor has been developed for the sensitive detection of circulating miRNAs (c-miRNAs) based on duplex-specific nuclease (DSN)-assisted target recycling coupled with rolling circle amplification (RCA) using SYBR Gold Dye as a signal indicator. This biosensor exhibits a tremendously low detection limit of 0.3 fM of c-miRNAs and shows high selectivity for one-base mismatched c-miRNA. In addition, most importantly, the biosensor could accurately determine the amounts of target c-miRNA in total c-miRNAs isolated from human serums and human cancer cell lines, which confirms that the biosensor could be used to quantify c-miRNAs in complex biological samples and for the clinical early diagnosis of cancers based on c-miRNAs detection.

Signal amplification method for miR-205 assay through combining graphene oxide with duplex-specific nuclease.

Since microRNA-205 (miR-205) is a predictive biomarker for anti-radiation of nasopharyngeal carcinoma (NPC), quantitative detection of miR-205 is important for developing personalized strategies for the treatment of NPC. In this investigation, based on the graphene oxide sensor and duplex specific nuclease (DSN) for fluorescence signal amplification, a highly sensitive detection method for miR-205 was designed. A target-recycling mechanism is employed, where a single miR-205 target triggers the cleavage of many DNA signal probes. The method shows the ability to analyze miR-205 in solution, and it can detect miR-205 at concentrations as low as 132 pmol L−1 with a linear range of 5–40 nmol L−1. Furthermore, the method is specific in that it distinguishes between a target miRNA and a sequence with single base, double base and three base mismatches, as well as other miRNAs. Considering simplicity and excellent sensitivity/specificity, it is promising for applications in radioresistance studies as well as the early clinical diagnosis of NPC.

G-triplex based molecular beacon with duplex-specific nuclease amplification for the specific detection of microRNA.

High sequence homology among miRNA members challenges miRNA analysis. In this paper, we developed a simple and highly selective method based on a novel G-triplex molecular beacon (MBG3) and duplex-specific nuclease signal amplification (DSNSA) for miRNA detection. Herein, this MBG3 is a label-free molecular beacon with a special G-triplex probe. The excellent controllablity of the G-triplex probe allowed our MBG3 to have a short stem, which protected it from DSN digestion. Therefore, DSN cleavage induced false positive signals in most DSN signal amplification strategies have been minimised efficiently without special modifications. Importantly, the improved recognition ability of MBG3, together with the sensitive substrate selectivity of DSN, makes our novel method suitable for miRNA detection with high selectivity. The signal response of similar miRNA sequences with one-base difference has been reduced from 37% to 8% compared to the traditional linear ssDNA probe-DSN-based method.

An innovative "unlocked mechanism" by a double key avenue for one-pot detection of microRNA-21 and microRNA-141.

The accurate and quantitative detection of microRNAs (miRNAs) as next-generation, reliable biomarkers will provide vital information for cancer research and treatment. However, their unique, intrinsic features pose quite a challenge for miRNA profiling, especially for multiplexed detection. Thus, there is a strong and an ever-growing need to develop an accurate, simple, sensitive and specific miRNA sensing method.Methods: In this study, a simple and novel sensor is presented that uses a flow cytometry (FCM) method based on the double key “unlocked mechanism” and a fluorescence enrichment signal amplification strategy. The “unlocked mechanism” was cleverly designed via using hairpin DNA probes (HDs) labeled by fluorescent particles (FS) as the lock to block part of them, which can specifically hybridize with the probe on polystyrene microparticles (PS). The target miRNA and duplex-specific nuclease (DSN) forming the double key can specifically open the HDs and cleave a single-stranded DNA (ssDNA) into DNA/RNA dimers circularly in order to unlock the special part of the HDs to be specially enriched further on the PS.Results: The designed sensor with a hairpin structure and DSN special performance was found to have a high specificity. The circularly unlocking fluorescent probes and fluorescent signal enrichment can be beneficial for achieving a high sensitivity with a detection limit of 3.39 fM for miRNA-21. Meanwhile, the performance of multiplexing was estimated by simultaneous detection of miR-21 and miR-141, and the method also allowed for miR-21 detection in breast cancer blood samples.Conclusion: The designed sensor based on an “unlocked mechanism” and a signal enrichment strategy resulted in a one-pot, highly specific and sensitive detection of multiplex miRNAs. The whole detection without the need for a complex purification process is based on a FCM and is expected to have a great value in cancer diagnosis and biomedical research.

Highly specific and sensitive detection of microRNAs by tandem signal amplification based on duplex-specific nuclease and strand displacement.

Based on the full understanding of the hydrolysis patterns of duplex-specific nuclease (DSN) against the probe DNAs in DNA/microRNA heteroduplexes, a simple and generic platform for highly specific and sensitive detection of microRNAs was developed by seamlessly integrating DSN-assisted target recycling amplification and strand displacement amplification in tandem.

Colorimetric and fluorescent dual-mode detection of microRNA based on duplex-specific nuclease assisted gold nanoparticle amplification.

MicroRNAs (miRNAs) are attractive candidates for biomarkers for early cancer diagnosis, and play vital roles in physiological and pathological processes. In this work, we developed a colorimetric and fluorescent dual-mode sensor for miRNA detection based on the optical properties of gold nanoparticles (AuNPs) and the duplex-specific nuclease (DSN)-assisted signal amplification technique. In brief, FAM labelled hairpin probes (HPs) were immobilized on AuNPs, and fluorescence was efficiently quenched by the vicinity of the fluorophores to the AuNPs surface. In the presence of target miRNAs, the HPs could specifically hybridize with miRNAs and the DNA strand in the DNA/RNA heteroduplexes could be subsequently hydrolyzed by DSN. As a result, numbers of fluorophores were released into the solution, resulting in obvious fluorescence signal recovery. Meanwhile, the target miRNAs were able to participate in other hybridization reactions. With the DSN-assisted signal amplification technique, lots of gold nanoparticles were produced with short-chain DNA on their surface, which could aggregate in salt solution and result in a colorimetric detection. The proposed dual-mode strategy offers a sensitive, accurate and selective detection method for miRNAs. One reason is that the stem of the HPs was elaborately designed to avoid hydrolyzation by DSN under optimal conditions, which ensures a relatively low background and high sensitivity. The other is that the dual-mode strategy is more beneficial for enhancing the accuracy and reproducibility of the measurements. Moreover, the unique selective-cutting ability and single-base mismatch differentiation capability of the DSN also give rise to a satisfactory selectivity. This demonstrated that the developed method could quantitatively detect miR-21 down to 50 pM with a linear calibration range from 50 pM to 1 nM, and the analytical assay of target miRNAs in cell lysate samples revealed its great potential for application in biomedical research and clinical diagnostics.

Label-Free and Multiplexed Quantification of microRNAs by Mass Spectrometry Based on Duplex-Specific-Nuclease-Assisted Recycling Amplification.

MicroRNAs (miRNAs) are important biomarker candidates for cancer screening and early detection research. Generally, miRNAs undergo synergistic adjustments in tumor cells. Herein, a mass-spectrometric method based on a duplex-specific-nuclease (DSN)-enzyme-assisted signal-amplification technique was proposed for label-free and multiplexed detection of multiple miRNAs, and applied to the quantification of three miRNAs (i.e., miRNA-141, miRNA-21, and let-7a) in samples of HeLa and MDA-MB231 cell extracts. Experimental results showed that the digestion modes of DSN against three different DNAs complementary to miRNA-141, miRNA-21, and let-7a in their DNA-miRNA heteroduplexes were quite different, verifying the multiplexed-detection capability of the proposed method. Moreover, an advanced calibration model was derived for the quantitative analysis of the complex mass-spectral data measured during the label-free and multiplexed detection of miRNA-141, miRNA-21, and let-7a by the proposed mass-spectrometric method. With the aid of the advanced calibration model, the proposed mass-spectrometric method achieved quite reliable quantitative results for miRNA-141, miRNA-21, and let-7a in samples of HeLa and MDA-MB231 cell extracts, with recovery rates within the range of 89.2 to 111.6%. The limits of detection (LODs) of the proposed mass-spectrometric method for miRNA-141, miRNA-21, and let-7a in standard samples were estimated to be 42, 41, and 95 pM, respectively. Therefore, it is reasonable to expect that the proposed mass-spectrometric method can be a competitive alternative for the label-free and multiplexed detection of multiple miRNAs in clinical diagnosis.

MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification

DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

RETRACTED: A graphene oxide-based hairpin probe coupling duplex-specific nuclease signal amplification for detection and imaging of mRNA in living cells

In situ imaging of mRNA in living cells can help to monitor the real time mRNA expression and also useful for diagnosis and prognosis of the diseases. In this study, a new strategy was designed for simple, sensitive, and selective platform to detect the mRNA levels by combining a hairpin probe-graphene oxide (HP1/GO) and duplex-specific nuclease signal amplification (DSNSA). Initially, the DNA probe was adsorbed on the surface of GO to protect it from enzymatic digestion. Then, the target mRNA (T1) was hybridized with a partial hairpin probe which formed a duplex. Finally, under the action of DSN nuclease, the ssDNA in the DNA/RNA hybrid was selectively cleaved and produced small fragments. Then, T1 triggered the next reaction cycle, constituting a new circular exponential amplification. Here, we conclude that this assay is highly sensitive for the detection of target mRNA with the lower detection limit of 1 fM under optimal conditions. Furthermore, this strategy was successfully used for imaging of mRNA in living cells.

An improved and robust method to efficiently deplete repetitive elements from complex plant genomes

Genome size and complexity often present major challenges to genome-based approaches in crop plants and other agricultural species. For instance, repetitive sequences comprise 80% to 90% of the genome of hexaploid wheat, which has a haploid genome size of approximately 17 Gb. In this study, we developed an improved design and procedure for short-read library preparation that uses a modified adaptor and duplex-specific nuclease (DSN) for the efficient elimination of highly repeated sequence elements within genomes. The improved adapter, which has a hairpin-like form for stability, was constructed from truncated sequences adjacent to the original Illumina TruSeq adapter and can be converted to a full-length adapter structure during PCR amplification. Using the hairpin-structured adaptor, we prepared randomly sheared genomic libraries from rice and diploid, tetraploid, and hexaploid wheat cultivars and evaluated the efficiency of DSN for the enzymatic depletion of repetitive elements. According to real-time quantitative PCR analysis, the relative abundances of 18S and 25S ribosomal DNA decreased respectively to 1.15% and 3.54% in rice and 1.70%–1.95% and 14.71%–20.01% in the three wheat cultivars. Whole-genome sequencing analysis of a diploid wheat cultivar, KU104-1, indicated that DSN treatment with the designed hairpin-structured adapter dramatically reduced highly repetitive elements, such as Ty1-Copia and Ty3-Gypsy retrotransposons and DNA transposons, within the genome, while sequencing reads derived from low-copy genes and protein coding sequences increased more than 50%. Our new procedure should be useful not only for wheat genomes but also for other agricultural plant species with relatively large and complex genomes.

Electrochemiluminescence Resonance Energy Transfer System for Dual-Wavelength Ratiometric miRNA Detection.

In this study, we proposed a dual-wavelength electrochemiluminescence resonance energy transfer (ECL-RET) ratiometric sensor combined with duplex-specific nuclease (DSN)-assisted target recycling amplification to detect microRNAs (miRNAs). Due to the perfect overlapping of spectra, the gold-nanoparticle-luminol-layered-double-hydroxides (Au NP-luminol-LDH) nanocomposite and gold nanoclusters (Au NCs) exhibited excellent ECL-RET effect with high efficiency. The Au NP-luminol-LDH donor exhibits a strong and stable ECL emission at the wavelength peak of 440 nm, while the Au NC acceptor has an emission peak at 620 nm. Upon the introduction of DSN and target miRNAs, the specific DNA-RNA binding and nuclease cleaving could trigger the detachment of capture Au NCs-DNA from the surface of Au NP-luminol-LDH, resulting in an increased ECL signal of Au NP-luminol-LDH and a decreased fluorescence signal of Au NCs. By measuring the ratio of optical signals at 440 and 620 nm, the designed sensor provided a quantitative readout proportional to the target miRNAs concentration in the range of 10 aM to 100 pM with a lower detection limit (LOD) of 9.4 aM.

Quantification of MicroRNAs by Coupling Cyclic Enzymatic Amplification with Microfluidic Voltage-Assisted Liquid Desorption Electrospray Ionization Mass Spectrometry.

Quantitative assay of microRNAs (miRNAs) with mass spectrometric detection currently suffers from two major disadvantages, i.e., being insufficient in sensitivity and requiring an extraction or chromatographic separation prior to MS detection. In this work, we developed a facile and sensitive assay of targeted miRNAs based on the combination of cyclic enzymatic amplification (CEA) with microfluidic voltage-assisted liquid desorption electrospray ionization tandem mass spectrometry (VAL-DESI-MS/MS). The single-stranded DNA (ssDNA) probe was designed to have a sequence complementary to the miRNA target with an extension of a two-base nucleotide fragment (i.e., CpC) at the 3'-position as MS signal reporter, thus being easy to prepare and high in stability. In the proposed CEA-VAL-DESI-MS/MS assay, an ssDNA probe was added to a sample solution, forming a DNA-miRNA hybrid. Duplex-specific nuclease (DSN) was then added to cleave specifically the DNA probe in the heteroduplex strands. As the hybridization-cleavage cycle repeated itself for many rounds, a large quantity of CpC molecules was produced that was quantified by VAL-DESI-MS/MS with accuracy and specificity. miRNA-21 was tested as the model target. The assay had a linear calibration equation in the range from 2.5 pM to 1.0 nM with a limit of detection of 0.25 pM. Determination of miRNA-21 in cellular samples was demonstrated. miRNA-21 was found to be 95.3 ± 13.95 amol ( n = 3) in 100 mouse peritoneal macrophages with a recovery of 94.2 ± 2.6% ( n = 3). Interestingly, analysis of exosomes secreted from these cells revealed that exposure of the cells to chemical stimuli caused a 3-fold increase in exosomal level of miRNA-21. The results suggest that the proposed assay may provide an accurate and cost-effective means for quantification of targeted miRNAs in biomedical samples.

DNA-Functionalized Porous Fe3O4 Nanoparticles for the Construction of Self-Powered miRNA Biosensor with Target Recycling Amplification.

Herein, we have developed an ultrasensitive self-powered biosensor for miRNA assay based on biofuel cells. The system is composed of indium tin oxide cathode and graphene oxide/gold nanoparticle/glucose oxidase anode. Redox probe of [Fe(CN)6]3- is entrapped inside porous Fe3O4 nanoparticles by DNA. However, in the presence of target miRNA, hybridization reaction occurs between miRNA and DNA, which initiates the release of [Fe(CN)6]3-. Moreover, duplex specific nuclease is further employed to trigger target recycling amplification. As a result, much more redox probes are released and the open circuit voltage is significantly increased. A "signal-on" self-powered biosensor for miRNA quantification is thus developed. The detection range is from 10 aM to 10 fM; meanwhile, the limit of detection is as low as 1.4 aM, which is superior to that in most reported methods. Therefore, the proposed biosensor is expected to be a powerful point-of-care tool for miRNA diagnostics, which may have wide applications in the future.

Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection.

DNA-functionalized layered two-dimensional transition-metal dichalcogenides have attracted tremendous interest for constructing biosensors in recent years. In this work, we report diblock molecular beacons with poly-cytosine (polyC) tails anchored on molybdenum disulfide (MoS2) nanosheets as probes for microRNA detection. The polyC block is adsorbed on MoS2 and the molecular beacon block is available for hybridization to the target; duplex-specific nuclease provides signal amplification by target recycling. By changing the length of polyC, we regulate the density of probes on MoS2 and inhibit the adsorption of enzyme-cleaved oligonucleotides, thereby leading to higher quenching efficiency. PolyC-mediated molecular beacons on MoS2 have very low background signal, ultrahigh sensitivity (limit of detection ∼3.4 fM), specificity to detect a single nucleotide mismatch, and selectivity to detect target microRNA from serum samples. This detection platform holds great potential for quantitative analysis of miRNAs in clinical diagnosis and biomedical research.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL−1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot-DNA Conjugates.

Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.

A novel recyclable surface-enhanced Raman spectroscopy platform with duplex-specific nuclease signal amplification for ultrasensitive analysis of microRNA 155

In this work, a recyclable surface-enhanced Raman spectroscopy (SERS) biosensor was fabricated for ultrasensitive detection of miRNA 155 by using magnetic substrate (Fe3O4@PDA/Pt) and duplex-specific nuclease signal amplification (DSNSA) strategy. Here, the Fe3O4@PDA/Pt was used as SERS platform to immobilize toluidine blue Raman reporter and capturing DNA (S1), in which polydopamine (PDA) could avoid the aggregation of Fe3O4 and enhance the biocompatible of platform. When Au nanoflowers (AuNFs) modified probe DNA (S2) was partly hybridized with S1, an initial strong Raman signal (“on” status) was obtained by the approaching between AuNFs and TB. In the presence of target microRNA 155, they could completely hybridize with S2, which led to the isolation of S2 from substrate. Under the assistance of DSN enzyme, the S2 in the DNA/RNA duplex was hydrolyzed to release target miRNA 155, which could trigger another cycle. Thus the AuNFs were departed from TB with an obviously decreased signal. More important, with the application of AuNFs-modified S2, the Raman reporter was continuously assembled on the substrate throughout the detection. After the adding of S2, the S1 could re-hybridize with S2, which led the SERS biosensor return to “on” status, therefore the regeneration of biosensor was easily realized. From this principle, the recyclable biosensor could achieve a wide linear range from 1 fM to 10 μM and the detection limit was 0.28 fM. Herein, we proposed a novel method to construct the ultrasensitive and recyclable SERS platform, which was expected to be used in the clinical applications.

Purification-Free MicroRNA Detection by Using Magnetically Immobilized Nanopores on Liposome Membrane.

MicroRNAs have critical roles in a number of serious diseases and, as a result, are of major interest as clinical diagnostic targets. Conventionally, microRNAs are collected from blood and urine samples and are measured by either quantitative reverse-transcription polymerase chain reaction or microarray. Recently, nanopore sensing techniques have been applied for measuring microRNAs at the single-molecule level. However, existing techniques are technically complex, needing several tools and requiring purification and/or labeling of microRNA samples prior to use. Here we report a method for microRNA detection in a simple procedure requiring neither purification nor labeling. This system utilizes magnetic beads anchored with DNA and nanopores on a liposome membrane. In the presence of the target microRNA, it forms a duplex with complementary DNA, which is then cleaved by a duplex-specific nuclease (DSN). The cleaved DNA, which harbors a liposome on its terminus, is subsequently released from the magnetic bead, fuses to the lipid bilayer on chip, and emits an electrical signal derived from the formation of a nanopore. Because of a property of the DSN, the signals derived from microRNAs are expected to be amplified in an isothermal reaction. Our system possesses the specificity to detect target microRNAs from mixtures containing >106 different microRNA sequences and readily uses blood or urine samples. Although the limit of detection is above 10 nM and needs to be improved for practical diagnosis, because purification and labeling are not required, the presented system proposes a possible schematic for the development of easy and on-site diagnosis.

Construction of a Cytosine-Adjusted Electrochemiluminescence Resonance Energy Transfer System for MicroRNA Detection.

The cytosines in cluster-nucleation sequences play a vital role in the formation of silver nanoclusters (Ag NCs). Here, an innovative electrochemiluminescence (ECL) resonance energy transfer (RET) sensing system was developed using CdS quantum dots (QDs) as ECL donor and Ag NCs as ECL acceptor. Modulation of the number of cytosines in the cluster-nucleation sequences allowed tuning of Ag NCs absorption bands to match with the ECL emission spectrum of CdS QDs, yielding effective ECL-RET. The sensitivity of detection was improved by dual-target recycling amplification based on duplex-specific nuclease (DSN) and catalytic hairpin assembly. In the presence of target microRNA-21 (miRNA-21), DSN selectively cleaved the complementary DNA section (S1), resulting in the release of the transduction section (S2) and the reuse of miRNA-21 in the next recycling amplification. Interaction of the stem-loop structure of the DNA1 segment (H1) on CdS QDs-modified electrode with S2 led to the opening of the hairpin structure of H1 and the formation of H1:S2 duplex. Then, hairpin DNA2 encapsulated Ag NCs hybridized with the remaining single-stranded DNA segment of H1, and the S2 strand was replaced. Finally, the dissociated S2 participated in subsequent reaction cycles, introducing Ag NCs to the electrode surface and leading to ECL signal quenching of the CdS QDs. The proposed sensor showed excellent performance in detecting miRNA-21 at a wide linear range from 1 fM to 100 pM. The practical application ability of the strategy was tested in HeLa cells with acceptable results, suggesting that the detection platform is a promising approach for disease diagnosis and molecular biology research.

Ultrasensitive Photoelectrochemical Biosensor Based on DNA Tetrahedron as Nanocarrier for Efficient Immobilization of CdTe QDs-Methylene Blue as Signal Probe with Near-Zero Background Noise.

Usually, photoelectrochemical (PEC) assays were devoted to the direct modification of photoactive materials on sensing interface, thereby producing high initial signal and unneglected background noise, which could further result in lowsensitivity and restricted detection limit during the detection of targets. In this work, a PEC biosensor with near-zero background noise was established for ultrasensitive microRNA-141 (miRNA-141) detection based on DNA tetrahedron (TET) as nanocarrier for efficient immobilization of CdTe quantum dots (QDs)-Methylene Blue (MB) (TET-QDs-MB complex) as signal probe. First, CdTe QDs as PEC signal indicator was bound to the TET through DNA hybridizations. Then, massive MB as PEC signal enhancer was attached to DNA duplex of the TET immobilized with CdTe QDs via intercalation. Thereafter, the as-prepared TET-QDs-MB complex was considered as an efficient PEC signal probe owing to its excellent photovoltaic properties, thereby avoiding direct modification of photoactive materials on sensing interface and producing a near-zero background noise to improve the sensitivity of this PEC biosensor. Besides, the detection sensitivity could be further improved with the help of the duplex specific nuclease (DSN) enzyme-assisted target cycling amplification strategy. The proposed PEC biosensor performs a wide linear range from 50 aM to 50 pM with a low detection limit of 17 aM for miRNA-141, paving a new and promising horizon for highly accurate and ultrasensitive monitoring of multifarious analytes such as proteins, DNAs, and miRNAs in bioanalysis and disease diagnosis.