Hairpin Probe Research Articles

Assembling G-wire-induced pyrene excimer switching for microRNA amplified detection based on exonuclease III-assisted target recycling and Fe3O4@PDA nanoparticles

We reported a novel assembled G-wire-induced pyrene excimer switching probe for microRNA (miRNA) amplified detection on the basis of exonuclease III (Exo III)-assisted target recycling and Fe3O4@PDA nanoparticles. The multifunctional hairpin (HP) probe recognized and effectively bound to target miRNA, which formed a 3′-blunt terminal for Exo-III digestion and miRNA regeneration to initiate another Exo III-mediated cycling cleavage. After digestion, numerous HP-caged c-myc sequences were released, which were positively related to target miRNA and folded to the parallel G-quadruplex with K+. Additionally, Fe3O4 polydopamine nanoparticles (Fe3O4@PDA NPs)-based magnet separation was conducted to reduce background fluorescence signal, and parallel G-quadruplexes self-asembled into filamentous G-wires with Mg2+. G-wires generated from 5′-terminal pyrene-labeled c-myc sequences made close distance between two labeled pyrene molecules, and induced the “turn-on” fluorescence signal of pyrene excimer switching. Moreover, gamma-cyclodextrin (γ-CD) was added, which promoted pyrene moiety, causing signal amplification in pyrene excimer fluorescence. Therefore, miRNA was efficiently measured within the homogeneous solution. This method detected miRNA within the dynamic range of 1 pM to 5 nM, with the miR-141 limit of detection (LOD) being 99 fM. It distinguished perfect-matched and mismatched target miRNAs, and showed an accurate detection of miRNA levels in different cell lines and favorable reproducibility when analyzing miRNAs within spiked serum samples. We propose a promising method for G-wire-based biosensing in disease-related biomarkers detection.

Target-induced site-specific cleavage of phosphorothioate-modified hairpin DNA for amplified and label-free sensitive electrochemical myeloperoxidase assay

Abnormal myeloperoxidase (MPO) expression is associated with inflammation and neurodegenerative diseases. By using a new phosphorothioate (PT)-modified DNA hairpin probe, we fabricate a label-free and highly sensitive electrochemical sensor for monitoring MPO via integration of DNAzyme-assisted cleavage recycling with DNA polymerization reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). The target MPO mediates the specific cleavage of PT-modified hairpin probes with assistance of hydrogen peroxide and chloride ion to initiate DNAzyme-based cyclic cleavage of substrate hairpin probes for the release of numerous trigger strands. Subsequent hybridizations of the trigger strands with the thiolated-hairpin probes on the electrode surface expose the 3′-OH termini of hairpin probes for triggering TdT–catalyzed in situ DNA polymerization formation of long ssDNAs containing multiple G-quadruplex repeats. Hemin is confined on the electrode surface via the formation of G-quadruplex/hemin complexes to produce highly enhanced current signals for the determination of MPO in a sensitive and label-free way. The proposed sensor exhibits a linear range of 0.1–100 ng mL−1 and achieves a low detection limit of 0.046 ng mL−1 for MPO. Such sensor also shows high selectivity against other interferents and shows the capability to detect MPO in diluted human serum samples.

Hairpin probe assisted saturation current ratio method to determine plasma electronegativity

The saturation current ratio (SCR) method is considered to be one of the simplest methods to determine plasma electronegativity in electronegative discharges using a Langmuir probe (LP). However, its accuracy is susceptible to errors incurred in the estimation of electron and positive ion saturation currents from the ampere–voltage characteristics obtained by a cylindrical LP and partly due to errors in estimating the positive ion flux at the sheath boundary. In spite of its wide use, these underlying limitations and their remedies have not been adequately investigated. In this paper, we address the above problems by involving a DC biased hairpin resonator probe to determine the plasma potential and sheath area correction factor for a cylindrical LP. These measurements are further integrated with the standard SCR method to deduce the plasma electronegativity in an oxygen plasma.

Plasma density determination from ion current to cylindrical Langmuir probe with validation on hairpin probe measurements

Numerical and analytical approaches to plasma density determination from the ion current to cylindrical Langmuir probe are validated on hairpin probe measurements. An argon inductively coupled plasma discharge in a pressure range from 4.5 mTorr to 27 mTorr is studied. The discharge input power is varied in the range from 200 to 800 W, giving a plasma density in the range from 109 to 1011 cm−3. The approaches used for plasma density determination are analytical collisionless orbital motion limit theory, fluid semianalytical model of ion radial motion with ion collisions and particle-in-cell with a Monte Carlo collisions model of ion current collection by the cylindrical Langmuir probe. The relative error of different models is shown. The ion collisions should be taken into account, even at relatively low pressures, in order to get a reliable plasma density value from the ion current to the Langmuir probe.

Intramolecular Interactions in the Fluorophore–Quencher System in Linear and Hairpin Probes for Real-Time PCR

Intramolecular Interactions in the Fluorophore–Quencher System in Linear and Hairpin Probes for Real-Time PCR

Ultrasensitive detection of carcinoembryonic antigen based on exonuclease Ⅲ-assisted recycling and hybridization chain reaction strategies

In this work, we developed a novel ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen (CEA). Upon the addition of CEA, the specific binding between CEA and hairpin probe 1 (HP1) could trigger the Exo Ⅲ cleavage reaction and release large amounts of single stranded DNA (S1), which then hybridized with thiolated ferrocene-labeled hairpin probe 3 (Fc-HP3) on the electrode surface. Exo Ⅲ then cyclically digested the resulting duplexes to generate Fc-HP3 fragments on the electrode, accompanying the Fc molecules far away from the electrode surface and the Fc oxidation peak current signal (IFc) decreased. The HCR process was triggered by the Fc-HP3 fragment as an initiator, producing many long DNA sequences for electroactive probe methylene blue (MB) intercalation; thus, the MB oxidation peak current signal (IMB) increased. The IMB/IFc ratio was proportional to the logarithm value of CEA concentration from 100 fg mL−1 to 50 ng mL−1, with a lower detection limit of 30.5 fg mL−1. Furthermore, the aptasensor exhibited good selectivity and stability, with satisfactory reproducibility. Additionally, the developed aptasensor effectively measured CEA in real serum samples, verifying its practical application in biological samples.

Sensitive and reliable analysis of endometrial cancer related microRNA using ternary hybridization hairpin probe

MicroRNA (miRNA), as a kind of small non-coding ribonucleic acid (RNA) that plays a crucial role in regulating transcriptional activities, is a potential biomarker for EC diagnosis. However, reliable detection of miRNA remains a huge challenge, especially for these methods that require multiple probes for signal amplifications, due to the detective deviation caused by variation of probe concentrations. Herein, we present a novel approach for miRNA-205 identification and quantification by employing simply a ternary hairpin probe (TH probe). The ternary hybridization of three sequences results in the construction of the TH probe, which combines high-efficient signal amplification and specific target recognition. A significant number of G-rich sequences have been produced as a result of the enzymes assisted signal amplification process. The G-rich sequences can fold into G-quadruplexes, which can then be detected in a label-free manner by a common fluorescent dye (thioflavin T). Eventually, the approach exhibits a low limit of detection of 278 aM with a wide detection range of 7 orders of magnitude. In summary, the proposed approach possesses a great potential for both clinical diagnosis of EC and fundamental biomedical researches.

An electrochemical aptasensor for detection of streptomycin based on signal amplification assisted by functionalized gold nanoparticles and hybridization chain reaction.

A ratiometric electrochemical aptasensor based on gold nanoparticles (AuNPs) functionalization and hybridization chain reaction (HCR) assisted signal amplification has been for thefirsttime designed for the detection of streptomycin (STR). The double-stranded DNA (dsDNA) formed by the hybridization of ferrocene (Fc)-labeled STR aptamer (Apt) and capture probe (CP) is first immobilized on the gold electrode (GE) surface via Au-S reaction. The specific binding of the target and Apt results in numerous Fc detachment from the sensing interface. Then, the remaining single-stranded CP is combined with AuNPs modified with initiator DNA (iDNA) by auxiliary DNA (aDNA). Among them, the iDNA triggers HCR between two hairpin probes (H1/H2), thus capturing a large number of methylene blue (MB) electrochemical probe, which generates a strong electrochemical signal of MB and a weak electrochemical signal of Fc. Signals are collected by square wave voltammetry (the potential window ranging from -0.5V to 0.6V, vs.Ag/AgCl ), and the oxidation peak currents at -0.200V (MB) and 0.416V (Fc) are recorded. The use of the ratiometric method has effectively improved the accuracy and reliability of the analysis. The successful application of AuNPs and HCR greatly improves the sensitivity of the sensor, and the detection limit is as low as 0.08pM. It can sensitively determine STR in the range 0.1pM to 10nM. In addition, the designed aptasensor has been successfully applied to the detection of STR in milk and honey samples.

Target-activated multivalent sensing platform for improving the sensitivity and selectivity of Hg2+ detection

Sensitivity and selectivity are critical parameters to evaluate the performance of sensors. For trace detection, it remains a challenge to design a new sensor that achieves high sensitivity and selectivity simultaneously. Here, we present a target-activated dual Mg2+-dependent DNAzyme (MNAzyme) that served as a simple sensing model to explore the multivalency in improving the analytical sensitivity and selectivity for target detection. Mercury ion (Hg2+), a notorious toxic metal ion, was selected as a model target. In the presence of Hg2+, the thymine-rich regions of the hairpin probe and primer could hybridize to form a stable duplex via the thymine-Hg2+-thymine structure. Then, an intact enzyme sequence was exposed and two separate enzyme fragments were close to each other, generating a dual MNAzyme. Benefiting from the localized high-concentration of the enzyme strand, the dual MNAzyme showed a remarkable improvement in binding stability. The catalytic rate constant of the dual MNAzyme was theoretically 1.60 times higher than that of the monomeric counterpart, and the sensitivity and selectivity had 4.50 and 1.44-fold enhancement, respectively. When the dual MNAzyme was used for sensor applications, the limit of detection was determined to be 0.04 and 0.2 nM via UV–vis spectrophotometer and naked eye, respectively. Meanwhile, the method offered desirable selectivity toward Hg2+ against other metal ions. With the advantages of simple operation, high sensitivity, and desirable selectivity, the developed multivalent sensing platform could be easily expanded in the future for the on-site detection of other low-abundance analytes.

Spatial Confinement of Single-Drop System to Enhance Aggregation-Induced Emission for Detection of MicroRNAs

Due to high incidence, poor prognosis, and easy transformation into pancreatic cancer (PC) with high mortality, early diagnosis and prevention of acute pancreatitis (AP) have become significant research focuses. In this work, we proposed a magnetic single-drop microextraction (SDME) system with spatial confinement to enhance the aggregation-induced emission (AIE) effect for simultaneous fluorescence detection of miRNA-155 (associated with AP) and miRNA-196a (associated with PC). The target miRNAs were selectively recognized by the hairpin probe and triggered the DNA amplification reaction; then, the DNA strands with two independent probes of G-quadruplex/TAIN and Cy5 were constructed on the surfaces of the magnetic beads. The SDME process, in which a drop containing the fluorescence probes was formed at the tip of the magnetic microextraction rod rapidly within 10 s, was performed by magnetic extraction. In this way, G-quadruplex/TAIN was enriched owing to the spatial confinement of the single-drop system, and the fluorescence signal given off (by G-quadruplex/TAIN) was highly enhanced (AIE effect). This was detected directly by fluorescence spectrophotometry. The approach achieved low limits of detection of 2.1 aM for miRNA-196a and 8.1 aM for miRNA-155 and wide linear ranges from 10 aM to 10 nM for miRNA-196a and from 25 aM to 10 nM for miRNA-155. This novel method was applied to the fluorescence detection of miRNAs in human serum samples. High relative recoveries from 95.6% to 104.8% were obtained.

Addition of dNTPs can improve the detection sensitivity of catalytic hairpin assembly

Ever since the catalytic hairpin assembly (CHA) circuit has been highlighted as a powerful nucleic acid detection tool, nucleic acid detection methods based on CHA have been widely studied. However, the detection sensitivity of CHA-based methods is insufficient. The relatively high background signals resulting from the spontaneous reaction between the two hairpin probes is one of the major reasons for limiting the sensitivity of CHA. In this study, we established that the addition of deoxynucleotide triphosphates (dNTPs) to the reaction system can significantly reduce the background leakage of CHA. The dNTPs-CHA, coupled with a fluorescence lateral flow assay strip, is used for the rapid and highly sensitive detection of miRNA. It is capable of reliably detecting miRNA in serum samples down to a limit of 100 aM, which is an improvement in the lower detection limit by nearly five orders of magnitude compared to that of the pure CHA.

Site-specific cleavage-based chemiluminescent optical fiber biosensor for highly sensitive assay of myeloperoxidase activity and inhibitor screening

Nucleic acid site-specific cleavage (NSC) has been an attractive theme for detecting various disease markers. In this study, for the first time, we introduce NSC as the recognition mechanism for developing chemiluminescent optical fiber biosensor (CFOS). The NSC-CFOS is constructed by modifying the hairpin probe containing phosphorothioate site on the surface of an optical fiber, which acts as a specific cleavage site to recognize HOCl catalyzed by myeloperoxidase (MPO). An isothermal enzyme-free hybridization chain reaction amplification is introduced to the proposed NSC-CFOS, which can significantly improve the detection sensitivity. With the MPO-induced NSC as the sensing model, the sensor is employed for the ultrasensitive detection of circulating MPO activity and inhibitors screening. An extremely low detection limit of 0.0017 mU/mL and a wide detection range of 0.005–1 mU/mL have been achieved for MPO detection. Our study shows that combining the site-specific recognition-based sensing mechanism to the optical fiber sensor provides a very promising approach for the point-of-care testing of disease markers in biological samples.

Primer Exchange Reaction Coupled with DNA-Templated Silver Nanoclusters for Label-Free and Sensitive Detection of MicroRNA.

MicroRNAs (MiRNAs) play pivotal roles in regulating gene expression, and serve as crucial biomarkers for diagnosis of a variety of disease. However, label-free and sensitive miRNA detection remains a huge challenge due to the low abundance. Herein, we developed an approach through integrating primer exchange reaction (PER) with DNA-templated silver nanoclusters (AgNCs) for label-free and sensitive miRNA detection. In this method, PER was used to amplify miRNA signals and produce single-strand DNA (ssDNA) sequences. The produced ssDNA sequences mediated DNA-templated AgNCs based signal generation by unfolding the designed hairpin probe (HP). The generated AgNCs signal was correlated with the dosage of target miRNA. Eventually, the established approach exhibited a low detection of limit of 47 fM with a great dynamic range of more than five orders of magnitude. In addition, the method was also utilized to detect the miRNA-31 expression in collected clinical samples from pancreatitis patients and demonstrated that miRNA-31 was upregulated in patients, showing a great promising of the method in clinical application.

Bidirectional motivated bimodal isothermal strand displacement amplifier with a table tennis-like movement for the ultrasensitive fluorescent and colorimetric detection of depression-related microRNA.

An increasing number of studies have highlighted the potential of microRNAs (miRNAs) as physiological indicators of major depressive disorder (MDD). Herein, we developed a bidirectional-motivated bimodal isothermal strand displacement amplifier (BB-ISDA) for the ultrasensitive fluorescent and colorimetric detection of MDD-related miRNA-132. In the BB-ISDA system, a pair of functionalized hairpin probes (HP1 and HP2) with nicking recognition sites are designed to recognize target miRNA. The recognition of target miRNA by HP1 (or HP2) generates copious numbers of nicked triggers by HP1 (or HP2)-based ISDA to recognize HP2 (or HP1) by autonomous strand polymerization, cleavage, and displacement, which in turn induces the subsequent generation of copious numbers of nicked G-quadruplex triggers by HP2 (or HP1)-based ISDA to recognize HP1 (or HP2) along a same line. After many cycles, this bidirectional motivated table-tennis-like movement amplifies the fluorescent signal from HP1 and the colorimetric signal from HP2, simultaneously. The dual-signal output pattern was cross-validated for sensing miRNA-132. Each of the detection modal shows the capability for qualitative and quantitative detection of miRNA-132 with high sensitivity and specificity. The adaptability of the bimodal mechanism was verified via the detection of target miRNA-132 from clinical human blood samples. We envision that this BB-ISDA with dual-signal output for accurate and reliable analysis of miRNA is promising for the molecular diagnosis of human mental diseases.

Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is regarded as an ideal candidate biomarker for the non-invasive diagnosis of cancer. However, the lack of convenient and reliable detection methods for ctDNA restricts its clinical application. Herein, we developed a dual signal amplification strategy for sensitive detection of ctDNA based on hybridization chain reaction (HCR) and proximity hybridization-regulated CRISPR/Cas12a. The ctDNA initiates HCR through the continuous hybridization of two hairpin probes (H1 and H2), yielding long nicked double-stranded DNA nanowires composed of numerous split segments, which are successively connected to activate the trans-cleavage activity of CRISPR/Cas12a. In this case, the doubly labeled single-stranded DNA reporter can be cleaved to produce a strong fluorescent signal. Owing to the dual amplification of HCR and CRISPR/Cas12a, this strategy exhibits high sensitivity toward ctDNA with a low detection limit of 5.43 fM. Moreover, the proposed method was successfully applied for ctDNA detection in serum samples with satisfactory results, which has great potential in the clinical diagnosis of cancer.

Versatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high “on” signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.

Self-Generation of Distinguishable Fluorescent Probes via a One-Pot Process for Multiple MicroRNA Detection by Liquid Chromatography.

To address the challenge of signal production and separation for multiple microRNA (miRNA) detection, in this work, a "one-pot" process to self-generate distinguishable fluorescent probes was developed. Based on a long and short probe amplification strategy, the generated G-quadruplex fluorescent dye-free probes can be separated and detected by a high-performance liquid chromatography-fluorescence platform. The free hairpin probes enriched in guanine with different lengths and base sequences were designed and could be opened by the target miRNAs (miRNA-10b, miRNA-21, and miRNA-210). Cleaved G-quadruplex probes with fluorescent signal could be generated in a one-pot process after a duplex-specific nuclease-based cleavage, and the detection of multiple miRNAs could be realized in one run. No solid nanomaterials were applied in the assay, which avoided the blocking of the column. Moreover, without modification of expensive fluorescein, the experimental cost was greatly reduced. The one-pot reaction process also eliminated tedious preparation steps and suggested feasibility of automation. The limits of detection of miRNA-10b, miRNA-21, and miRNA-210 were 2.19, 2.20, and 2.75 fM, respectively. Notably, this method was successfully applied to multiplex detection of miRNAs in serum samples from breast cancer patients within 30 min.

Spatial confinement-based Figure-of-Eight nanoknots accelerated simultaneous detection and imaging of intracellular microRNAs

Developing highly efficient and reliable methods for simultaneous imaging of microRNAs in living cells is often appealed to understanding their synergistic functions and guiding the diagnosis and treatment of human diseases, such as cancers. In this work, we rationally engineered a four-arm shaped nanoprobe that can be stimuli-responsively tied into a Figure-of-Eight nanoknot via spatial confinement-based dual-catalytic hairpin assembly (SPACIAL-CHA) reaction and applied for accelerated simultaneous detection and imaging of different miRNAs in living cells. The four-arm nanoprobe was facilely assembled from a cross-shaped DNA scaffold and two pairs of CHA hairpin probes (21HP-a and 21HP-b for miR-21, while 155HP-a and 155HP-b for miR-155) via the "one-pot" annealing method. The DNA scaffold structurally provided a well-known spatial-confinement effect to improve the localized concentration of CHA probes and shorten their physical distance, resulting in an enhanced intramolecular collision probability and accelerating the enzyme-free reaction. The miRNA-mediated strand displacement reactions can rapidly tie numerous four-arm nanoprobes into Figure-of-Eight nanoknots, yielding remarkably dual-channel fluorescence proportional to the different miRNA expression levels. Moreover, benefiting from the nuclease-resistant DNA structure based on the unique arched DNA protrusions makes the system ideal for operating in complicated intracellular environments. We have demonstrated that the four-arm-shaped nanoprobe is superior to the common catalytic hairpin assembly (COM-CHA) in stability, reaction speed, and amplification sensitivity in vitro and living cells. Final applications in cell imaging have also revealed the capacity of the proposed system for reliable identification of cancer cells (e.g., HeLa and MCF-7) from normal cells. The four-arm nanoprobe shows great potential in molecular biology and biomedical imaging with the above advantages.

Aptamer recognition-promoted hybridization chain reaction for amplified label-free and enzyme-free fluorescence analysis of pesticide

Development of high-performance fluorescence sensors for pesticide is highly urgent but remains a grand challenge. It is due to that most of known fluorescence sensors detect pesticides based on enzyme-inhibited strategy, which requires high-price cholinesterase, suffers from serious interference of reductive materials, and can’t difference pesticides with each other; the known aptamer-based fluorescence ones entail tool enzymes or nanomaterials to transducer/amplify the signal and demand signalers to be tagged in nucleic acid, which are expensive and intricate. Herein, we develop a novel aptamer-based fluorescence system for label-free, enzyme-free and highly sensitive detection of pesticide (profenofos) based on target-initiated hybridization chain reaction (HCR)-assisted signal amplification and specific intercalation of N-methylmesoporphyrin IX (NMM) in G-quadruplex DNA. Hairpin probe ON1 recognizes profenofos to generate profenofos@ON1 complex, which switches the HCR to yield multiple G-quadruplex DNA, consequently making large numbers of NMM be locked. In comparison with profenofos absence, a sharply improved fluorescence signal was recorded and it was dependent on profenofos dose. Hence, label-free, enzyme-free and highly sensitive detection of profenofos is achieved with limit of detection of 0.085 nM, which compared favorably with or superior to those of known fluorescence methods. Furthermore, the present method was applied to determine the profenofos residue in rice with agreeable result, and will provide more valuable information for guaranteeing the pesticide-related food safety.

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.