Catalytic Hairpin Assembly Signal Amplification Research Articles

Portable SERS biosensor based on aptamer-assisted catalytic hairpin assembly signal amplification for ultrasensitive detection of Staphylococcus aureus

Bacteria infections pose a serious threat to public health, and it is urgent to develop facile and accurate detection methods. To meet the important need, a potable and high-sensitive surface enhanced Raman scattering (SERS) biosensor based on aptamer recognition and catalytic hairpin assembly (CHA) signal amplification was proposed for point-of-care detection of Staphylococcus aureus (S. aureus). The SERS biosensor contains three parts: recognition probes, SERS sensing chip, and SERS tags. The feasibility of the strategy was verified by gel electrophoresis, and the one-step test route was optimized. The bacteria SERS biosensor has a good linear relationship ranging from 10 to 107 CFU mL−1 with high sensitivity low to 5 CFU mL−1, and shows excellent specificity, uniformity, and repeatability on S. aureus identification and enumeration, which can distinguish S. aureus from other bacteria. The SERS biosensor shows a good recovery rate (95.73 %–109.65 %) for testing S. aureus spiked in milk, and has good practicability for detecting S. aureus infected mouse wound, which provides a facile and reliable approach for detection of trace bacteria in the real samples.

Z-Scheme Heterojunction Excited by DNA-Programmed Upconversion Nanotransducers for a Near-Infrared Light-Actuated Lab-on-Paper Device.

Herein, a flexible near-infrared (NIR) light-actuated photoelectrochemical (PEC) lab-on-paper device was constructed toward miRNA-122 detection, utilizing the combination of DNA-programmed NaYF4/Yb,Tm upconversion nanoparticles (UCNPs) and the Z-scheme AgI/WO3 heterojunction grown in situ on gold nanoparticle-decorated 3D cellulose fibers. The UCNPs were employed as light transducers for converting NIR light into ultraviolet/visible (UV/vis) light to excite the nanojunction. The multiple diffraction of NaYF4/Yb,Tm matched the absorption band of the Z-scheme AgI/WO3 heterojunction, resulting in enhanced PEC photocurrent output. This prepared Z-scheme heterojunction effectively directed charge migration and highly facilitated the electron-hole pair separation. Target miRNA-122 activated the nonenzyme catalytic hairpin assembly signal amplification strategy, generating duplexes which caused the exfoliation of NaYF4/Yb,Tm UCNPs from the biosensor electrode and lowered the photocurrent under 980 nm irradiation. Under optimized circumstances, the proposed NIR-actuated PEC lab-on-paper device presented accurate miRNA-122 detection within a wide linear range of 10 fM-100 nM with a low limit of detection of 2.32 fM, providing a reliable strategy in the exploration of NIR-actuated PEC biosensors for low-cost, high-performance bioassay in clinical applications.

Engineering DNA tetrahedron as a sensing surface of lateral flow test strips and ratiometric visual detection of exosomal microRNA-150–5p

We here report that DNA tetrahedral probe-based sensing surfaces are capable of efficiently capturing gold nanoparticles (AuNPs) on lateral flow test strips, and we have developed a ratiometric biosensor for exosomal microRNAs by integrating a tetrahedral probe and a catalytic hairpin assembly (CHA). DNA tetrahedrons were devised with barcodes and labeled with biotin as capture probes on the test and control lines of the strip. The CHA system consists of two hairpin substrates that can form stable duplexes via a programmable assembly reaction triggered by exosomal miRNAs. The duplexes hybridized with barcoded tetrahedra via complementary sequences and bound to streptavidin-modified AuNPs (SA-AuNPs) via biotin. Thus, a red line was observed on the test line owing to the immobilized SA-AuNPs. The remaining SA-AuNPs were captured by using a biotinylated tetrahedron on the control line. Because the barcoded and biotinylated tetrahedra consume SA-AuNPs simultaneously, a stronger signal in the test line results in a weaker signal in the control line, thus forming a ratiometric result. Tetrahedral probes avoid movement along with sample flow on sensing zones like single-stranded probes. The strip can have a detection limit of 58.90 fM with high selectivity due to the use of ratiometric color changing response, CHA signal amplification and tetrahedral capture probes. This system is able to rapidly detect exosomal microRNA-150–5p and may have a potential utility in point-of-care diagnosis of incipient diabetic nephropathy.

Simple and Ultrasensitive Detection of Glioma-Related ctDNAs in Mice Serum by SERS-Based Catalytic Hairpin Assembly Signal Amplification Coupled with Magnetic Aggregation.

Circulating tumor DNA (ctDNA) is more representative and accurate than biopsy and is also conducive to dynamic monitoring, facilitating accurate diagnosis and prognosis of glioma. Therefore, the present study aimed to establish and validate a novel amplified method for the detection of IDH1 R132H and BRAF V600E, which were associated with the genetic diagnosis of glioma. A dual-signal amplification method based on magnetic aggregation and catalytic hairpin assembly (CHA) was constructed for the simultaneous detection of ctDNAs. When target ctDNAs are present, the CHA reaction is initiated and leads to the assembly of Au-Ag nanoshuttles (Au-Ag NSs) onto magnetic beads (MBs). Further enrichment of MBs under an external magnetic field facilitated the dual-signal amplification of SERS. The limit of detection (LOD) for IDH1 R132H and BRAF V600E in serum was as low as 6.01 aM and 5.48 aM. The reproducibility and selectivity of the proposed SERS analysis platform was satisfactory. Finally, the platform was applied to quantify IDH1 R132H and BRAF V600E in the serum of subcutaneous-tumor‑bearing nude mice, and the results obtained by SERS were consistent with those from quantitative real-time polymerase chain reaction (qRT-PCR). The present study showed that the dual-signal amplification method is a simple and ultrasensitive strategy for gliomas-associated ctDNAs detection, which is crucial for early diagnosis and dynamic monitoring.

Versatile Electrochemiluminescence Biosensing Platform Based on DNA Nanostructures and Catalytic Hairpin Assembly Signal Amplification.

Achieving rapid and highly sensitive detection of biomarkers is crucial for disease diagnosis and treatment. Here, a highly sensitive and versatile dual-amplification electrochemiluminescence (ECL) biosensing platform was constructed for target detection based on DNA nanostructures and catalyzed hairpin assembly (CHA). Specifically, when the target DNA was present, it would hybridize with the auxiliary strands (D1 and D2) to form an I-shaped nanostructure, which in turn triggered the subsequent catalytic hairpin assembly reaction to generate plenty of double-stranded DNA complexes (H1-H2). The resulting double-stranded complex could be trapped on the electrode surface and adsorbed the ECL signal probe Ru(phen)32+.We found that the I-shaped nanostructure-triggered CHA reaction had higher amplification efficiency compared with traditional CHA amplification. Thus, a sensitive "signal-on" ECL biosensor was constructed for target DNA detection with a detection limit of 1.09 fM. Additionally, by combining the binding properties of C-Ag+-C with an elaborately designed "Ag+-helper" probe, the proposed strategy could be immediately utilized for the highly sensitive and selective detection of silver ions, demonstrating the versatility of the developed biosensing platform. This strategy provided a new approach with potential applications in disease diagnosis and environmental monitoring.

A Sample and Detection Microneedle Patch for Psoriasis MicroRNA Biomarker Analysis in Interstitial Fluid

Skin interstitial fluid (ISF) containing a great variety of molecular biomarkers derived from cells and subcutaneous blood capillaries has recently emerged as a clinically potential component for early diagnosis of a wide range of diseases; however, the minimally invasive sampling and detection of cell-free biomarkers in ISF is still a key challenge. Herein, we developed microneedles (MNs) that consist of gelatin methacryloyl (GelMA) and graphene oxide (GO) for the enrichment and sensitive detection of multiple microRNA (miRNA) biomarkers from skin ISF. The GO-GelMA MNs exhibited robust mechanical properties, fast sampling kinetics, and large swelling capacity, which enabled collecting ISF volume high to 21.34 μL in 30 min, facilitating effective miRNA analysis. It preliminarily realized the sensitive detection of three types of psoriasis-related miRNAs biomarkers either on the patch itself or in solution after release from the hydrogel by combining catalytic hairpin assembly signal amplification reaction. The automated and minimally invasive ISF miRNA detection technology of GO-GelMA MNs has great potential to monitor cell-free clinically informative biomarkers for personalized diagnosis and prognosis.

Rapid and sensitive detection of dual lung cancer-associated miRNA biomarkers by a novel SERS-LFA strip coupling with catalytic hairpin assembly signal amplification

A novel surface-enhanced Raman scattering-lateral flow assay strip in combination with catalytic hairpin assembly signal amplification has been developed for rapid and sensitive detection of miR-196a-5p and miR-31-5p associated with lung cancer.

Ultrasensitive detection of ochratoxin A based on biomimetic nanochannel and catalytic hairpin assembly signal amplification

In this paper, an ultrasensitive nanochannel sensor has been proposed for label-free Ochratoxin A (OTA) assay in combination with graphene oxide (GO) and catalyzed hairpin assembly (CHA). The high-performance sensor is segmented into two parts. One is composed of graphene oxide (GO) and DNA probes. In the presence of target OTA, OTA works as a catalyst to trigger the self-assembly pathway of the two probes and initiate the cycling of CHA circuits, which results in numerous double-stranded DNAs (dsDNA) in solution. The excess ssDNA probes are removed by GO. The other part is composed of biomimetic nanochannel coated with polyethyleneimine (PEI) and Zr4+, which can quantify the concentration of OTA by detecting the dsDNA in solution. The nanofluidic device has a detection limit of as low as 6.2 pM with an excellent selectivity. The nanochannel based assay was used to analyse food samples (red wine) with satisfied results. Thus, the proposed analytical method will provide a new approach the detection of OTA and can be applied for quality control to ensure food safety.

Highly sensitive detection of ochratoxin A based on bio-barcode immunoassay and catalytic hairpin assembly signal amplification

Herein, a highly sensitive ochratoxin A (OTA) detection strategy was developed based on a bio-barcode immunoassay with catalytic hairpin assembly (CHA). Two nanoprobes were designed for the assay: one was a gold nanoparticle (AuNP) harbouring numerous bio-barcode DNA and antibodies, and the other was an antigen-functionalized magnetic nanoparticle (MNP). In the presence of target OTA, the antigens of the MNPs competed with OTA for binding to the antibodies on the AuNPs. After magnetic separation, the unbound AuNPs and target OTA were washed away. Dithiothreitol (DTT) was then added to the MNP-bound AuNPs to elute the bio-barcode DNAs of AuNPs, which triggered the CHA reaction. Under optimal conditions, the proposed method could sensitively detect target OTA ranging from 0.001 to 10000 ng/mL. The limit of detection (LOD, 3 N/S) and limit of quantification (LOQ, 10 N/S) for OTA were 0.54 pg/mL and 1.80 pg/mL, respectively. The bio-barcode immunoassay was used to analyse food samples (corn, wheat, and peanut), and the recovery and relative standard deviations (RSD) ranged from 93.30% to 108.80% and from 3.2% to 6.9%, respectively. The total assay time was 6 h. Therefore, the proposed strategy will provide a new approach for the detection of mycotoxins and other small molecule analytes and can be applied for quality control to ensure food safety.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

Label-free impedimetric sensing platform for microRNA-21 based on ZrO2-reduced graphene oxide nanohybrids coupled with catalytic hairpin assembly amplification.

Herein, a sensitive electrochemical impedance sensor was constructed based on ZrO2-reduced graphene oxide (RGO)-modified electrode coupled with the catalytic hairpin assembly signal amplification strategy. Electrochemical impedance spectroscopy (EIS) was used to detect microRNA (miRNA) using the change in electron transfer resistance (ΔRet) originated from nucleic acid hybridization on the electrode surface. MiRNA-21 was used as a model to verify this strategy. The results indicated that ΔRet exhibited a good linear relationship with the concentration of miRNA-21 in the range from 1.0 × 10−14 mol L−1 to 1.0 × 10−10 mol L−1 with a detection limit of 4.3 × 10−15 mol L−1 (S/N = 3). Additionally, this sensor exhibited good selectivity, and it could be applied to detect miRNA-21 in human serum samples and measure the expression levels of miRNA-21 in human breast cancer cell lines (MCF-7); thus, this sensor has great potential in cancer diagnosis.

Target-induced catalytic hairpin assembly formation of functional Y-junction DNA structures for label-free and sensitive electrochemical detection of human serum proteins

The construction of simple and highly sensitive aptasensors will significantly advance the monitoring of proteins. In this work, we have developed a convenient and label-free protein-induced catalytic hairpin assembly signal amplification approach for aptamer-based sensitive electrochemical detection of thrombin in human serums. The target thrombin binds the aptamer-containing hairpin probes immobilized on the sensor electrodes and triggers catalytic assembly of other two hairpins to form many G-quadruplex Y-junction DNA structures in situ. These G-quadruplex structures further associate with hemin to form G-quadruplex/hemin complexes, which are then subject to electrochemical measurement and generate substantially amplified current output for label-free and sensitive detection of thrombin. The developed aptasensor shows a dynamic range of 0.01–1.0nM and a detection limit of 6.0p.m for thrombin detection. The sensor can also discriminate the target thrombin against other non-target proteins and exhibits promising results for detecting thrombin in human serums. With the successful construction of the thrombin aptasensor, our signal amplified method can be potentially applied to detect other target molecules by choosing appropriate aptamer/ligand pairs.