Catalytic Hairpin Assembly Amplification Research Articles

A Label-Free Fluorescent Amplification Strategy for High-Sensitive Detection of Pseudomonas aeruginosa based on Protective-EXPAR (p-EXPAR) and Catalytic Hairpin Assembly.

This study presents a fluorescent mechanism for two-step amplification by combining two widely used techniques, exponential amplification reaction (EXPAR) and catalytic hairpin assembly (CHA). Pseudomonas aeruginosa (P. aeruginosa) engaged in competition with the complementary DNA in order to attach to the aptamer that had been fixed on the magnetic beads. The unbound complementary strand in the liquid above was utilized as a trigger sequence to initiate the protective-EXPAR (p-EXPAR) process, resulting in the generation of a substantial quantity of short single-stranded DNA (ssDNA). The amplified ssDNA can initiate the second CHA amplification process, resulting in the generation of many double-stranded DNA (dsDNA) products. The CHA reaction was initiated by the target/trigger DNA, resulting in the release of G-quadruplex sequences. These sequences have the ability to bond with the fluorescent amyloid dye thioflavin T (ThT), generating fluorescence signals. The method employed in this study demonstrated a detection limit of 16 CFU/ml and exhibited a strong linear correlation within the concentration range of 50 CFU/ml to 105 CFU/ml. This method of signal amplification has been effectively utilized to create a fluorescent sensing platform without the need for labels, enabling the detection of P. aeruginosa with high sensitivity.

Quantification of MicroRNA in a Single Living Cell via Ionic Current Rectification-Based Nanopore for Triple Negative Breast Cancer Diagnosis.

Accurate analysis of microRNAs (miRNAs) at the single-cell level is extremely important for deeply understanding their multiple and intricate biological functions. Despite some advancements in analyzing single-cell miRNAs, challenges such as intracellular interferences and insufficient detection limits still remain. In this work, an ultrasensitive nanopore sensor for quantitative single-cell miRNA-155 detection is constructed based on ionic current rectification (ICR) coupled with enzyme-free catalytic hairpin assembly (CHA). Benefiting from the enzyme-free CHA amplification strategy, the detection limit of the nanopore sensor for miRNA-155 reaches 10 fM and the nanopore sensor is more adaptable to complex intracellular environments. With the nanopore sensor, the concentration of miRNA-155 in living single cells is quantified to realize the early diagnosis of triple-negative breast cancer (TNBC). Furthermore, the nanopore sensor can be applied in screening anticancer drugs by tracking the expression level of miRNA-155. This work provides an adaptive and universal method for quantitatively analyzing intracellular miRNAs, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.

Dual-Signal Amplification Strategy Based on Catalytic Hairpin Assembly and APE1-Assisted Amplification for High-Contrast miRNA Imaging in Living Cells.

Early tumor diagnosis is crucial to successful treatment. Earlier studies have shown that microRNA is a biomarker for early tumor diagnosis. The development of highly sensitive miRNA detection methods, especially in living cells, plays an indispensable role for early diagnosis and treatment of tumor. Although the catalytic hairpin assembly (CHA)-based miRNA analysis strategy is commonly used for disease diagnosis, further application of CHA is hindered due to its low amplification efficiency and low tumor recognition contrast. To address these limitations, we propose a dual-signal amplification strategy based on CHA and APE1-assisted amplification, enabling highly sensitive and high-contrast miRNA imaging. The miR-221 was selected as a target model. This dual-signal amplification strategy has exhibited high amplification efficiency, which could analyze miRNA as low as 21 fM. This strategy also exhibited high specificity, which could distinguish target miRNA and nontarget with single-base differences. Moreover, this method showed significant potential for practical application, as it could successfully distinguish the expression difference of miR-221 in the plasma samples of normal people and patients. Most importantly, the expression level of the APE1 enzyme in tumor cells is higher than that in normal cells, allowing this strategy to sensitively and specifically image miRNA within tumor cells. This proposed method has also been successfully used to indicate fluctuations of intracellular miRNA and to distinguish miRNA expression between normal cells and cancer cells with high contrast. We anticipate that this method will provide fresh insights and can be a powerful tool for tumor diagnosis and treatment based on miRNA analysis.

All-in-one nanoflare biosensor combined with catalyzed hairpin assembly amplification for in situ and sensitive exosomal miRNA detection and cancer classification

Exosomal miRNAs can reflect tumor progression and metastasis, and are effective biomarkers for cancer diagnosis. However, the accuracy of exosomal miRNA-based cancer diagnosis is limited by the low sensitivity and complicated RNA extraction of traditional approaches. Herein, a novel biosensor is developed for in situ, extraction-free, and highly sensitive analysis of exosomal miRNAs via nanoflare combined with catalyzed hairpin assembly (CHA) amplification. Without cumbersome and costly miRNA extraction or transfection agents, nanoflare can directly enter the exosomes to bind target miRNAs and generate a fluorescence signal that can be amplified by the CHA reaction to achieve the in situ and highly sensitive detection of exosomal miRNAs. Under the optimal conditions, the detection limit of 5 aM is obtained for three exosomal miRNAs, which is an order of magnitude lower than quantitative real time polymerase chain reaction (qRT-PCR). In combination with the linear discriminant analysis algorithm, five exosomes are distinguished with 100% accuracy. Importantly, five cancers including breast, lung, liver, cervical, and colon cancer from 64 patients are distinguished with 99% accuracy by testing exosomal miRNAs in clinical plasma. This simple, accurate, and sensitive biosensor holds the potential to be expanded into clinical non-invasive cancer diagnostic tests.

A highly sensitive and robust electrochemical biosensor for microRNA detection based on PNA-DNA hetero-three-way junction formation and target-recycling catalytic hairpin assembly amplification

Robust and sensitive methods for the detection of microRNAs (miRNAs) are crucial in the clinical diagnosis of cancers. In this study, a novel electrochemical biosensor with high sensitivity for miRNA-21 detection is developed, which relies on the formation of a peptide nucleic acid (PNA)-DNA hetero-three-way junction (H3WJ) and target-recycling catalytic hairpin assembly (CHA) amplification. The electroneutral PNA probes are initially immobilized onto a gold electrode to construct the sensor. Upon introduction of miRNA-21, target-recycling CHA is initiated, resulting in abundant double-stranded CHA products. Subsequently, association between the PNA probes and these products leads to the formation of PNA-DNA H3WJs. Consequently, the electrode surface is densely populated with numerous electroactive Ferrocene (Fc) groups, resulting in a significantly amplified current response for highly sensitive detection of miRNA-21 at concentrations as low as 0.15 fM. This approach demonstrates remarkable specificity towards target miRNAs and can be utilized for quantitative monitoring of miRNA-21 expression in human cancer cells. More importantly, the sensor exhibits exceptional stability and shows a significant reduction in background noise during miRNA detection, making this method a highly promising sensing platform for monitoring various miRNA biomarkers to facilitate the diagnosis of diverse cancers.

Electrochemiluminescence biosensing of spatially conjugated ester groups triggered by smart bridging between nano-silica and poly (ethylene maleate)

Organic luminescent materials with unique photoelectric properties are as a kind of emerging luminophore at the forefront of research in biological detection on account of their facilely varied functional groups. However, most organic luminophores are aromatic compounds with certain biological toxicity to limit their application. Herein, we envisioned a green organic electrochemiluminescencence (ECL) luminophore without aromatic ring structure but electron-rich functional groups via in-situ growth of poly (ethylene maleate) (PEM) on nano-silica (NS). The experimental and theoretical modelling results indicate that ECL signal of PEM-NSs was enhanced by the spatial conjugation of ester groups (ester clusters), due to the contribution of smart bridging of NS with different sizes. The smaller the NS size is, the more growth of ester groups on the NS surface is, and the closer individual ester groups are linked via “through-space interaction” to form stable ester clusters with smaller energy gaps, thus achieving high ECL performance. For further application, PEM-NS-1 was selected as meaningful ECL luminophore, and integrated with exonuclease III-assisted catalytic hairpin assembly amplification strategy, an efficient platform for biomarkers (microRNA-21) detection was constructed with the detection limit of 2.73 fM (S/N = 3). This work sets the stage for strengthening ECL of electron-rich functional groups, and provides a way of thinking for low-toxicity organic luminophores.

An all-in-one assay based on CRISPR/Cas13a and a DNA circuit for rapid and ultrasensitive detection of Echovirus 11

Echovirus 11 (ECHO 11) is a positive-stranded RNA virus. The infections of ECHO 11 are associated with many clinical syndromes, thus the deployment of rapid point-of-care diagnostics is necessary. This study reported a new diagnostic assay based on All-In-One CRISPR/Cas13a and a DNA circuit (termed AIOC) for ECHO 11 detection. By combination of CRISPR/Cas13a and catalytic hairpin assembly (CHA) circuit in one-pot reaction, multiple handling steps and uncapping contamination could be effectively avoided. After recognition of target RNA, the activation of CRISPR/Cas13a induced the RNase activity of Cas13a to cleave hairpin probes, resulting in subsequent CHA amplification. This simplified assay was able to detect ECHO 11 RNA in the range of 500 × 10-18 M to 5 × 10-9 M for VP1 gene and 100 × 10-18 M to 5 × 10-9 M for RdRP gene. And the limit of detection was 152 × 10-18 M and 24 × 10-18 M, respectively. More importantly, the practicability of AIOC assay was validated by clinical ECHO samples. Taking together, the AIOC assay is rapid (∼30 min), isothermal (37 ℃), ultrasensitive, specific, user-friendly and does not need the step of RNA reverse-transcription, making it an excellent detection performance in one-pot point-of-care assay.

A novel Aptamer-induced CHA amplification strategy for ultrasensitive detection of Staphylococcus aureus and NIR-triggered photothermal bactericidal Activity based on aptamer-modified magnetic Fe3O4@AuNRs

The development of multifunctional biosensors integrating recognition and therapeutic functions is critical for ensuring food safety and preventing foodborne diseases. Herein, an integrated design strategy was demonstrated to establish an innovative Fe3O4 @AuNRs-aptasensor for efficient detecting Staphylococcus aureus (S. aureus), which could enrich and photothermally eliminate bacteria under the radiation of near-infrared light irradiation (NIR). The detection assay relies on the binding of aptamer to Fe3O4 @AuNRs (Fe3O4 @ AuNRs-aptasensor) to induce the release of c-aptamer (c-Apt) in the presence of S. aureus, thereby triggering catalytic hairpin assembly (CHA) reaction and restoring the fluorescence of the reporter. The developed biosensor achieved direct, ultrasensitive, and specificity detection of S. aureus with a linear response ranging from 102 to 106 CFU/mL and a detection limit of 101 CFU/mL under optimal conditions. Furthermore, the aptasensor can be applied for the detection of S. aureus in milk with recoveries of 90.3–95.6% at the concentration between 1.0 × 102 and 1.0 × 104 CFU/mL. Additionally, Fe3O4 @AuNRs-aptasensor shows high photothermal sterilization performance under NIR irradiation. The study emphasizes the combination of high sensitivity base on molecular detection and photothermal antibacterial activity of novel nanomaterials activated by NIR light to design multifunctional probe, which show great potential for application in food analysis and early foodborne disease diagnosis.

Robust nontarget DNA-triggered catalytic hairpin assembly amplification strategy for the improved sensing of microRNA in complex biological matrices.

A simple but robust fluorescence strategy based on a nontarget DNA-triggered catalytic hairpin assembly (CHA) was constructed to probe microRNA-21 (miR-21). A short ssDNA rather than degradable target miRNA was employed as an initiator. Two molecular beacons needed to assist the CHA process were simplified to avoid unfavorable nonspecific interactions. In the presence of the target, the initiator was released from a partially duplex and triggered the cyclic CHA reaction, resulting in a significantly amplified optical readout. A wide linear range from 0.1 pM to 1000 pM for the sensing of miR-21 in buffer was achieved with a low detection limit of 0.76 pM. Fortunately, this strategy demonstrated an obviously improved performance for miR-21 detection in diluted serum. The fluorescence signals were enhanced remarkably and the sensitivity was further improved to 0.12 pM in 10% serum. The stability for miR-21 quantification and the capability for the analysis of single nucleotide polymorphisms (SNPs) were also improved greatly. More importantly, the biosensor could be applied to image miR-21 in different living tumor cells with high resolution, illustrating its promising potential for the assay of miRNAs in various complex situations for early-stage disease diagnosis and biological studies in cells.

Dual-target nucleic acid sequences responsive electrochemiluminescence biosensor using single type carbon dots as probe for SARS-CoV-2 detection based on series catalytic hairpin assembly amplification

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is rampant all over the world, and rapid and effective virus detection is the best auxiliary to curb the spread of the epidemic. A diagnosis can only be made if two or more different nucleic acid sequences are confirmed at the same time, and in most of traditional detection technologies, these target sequences have been detected separately. In this work, an electrochemiluminescent (ECL) biosensor employing a single ECL probe as signal output and responding to dual-target simultaneously is proposed for the first time. Taking the two sequences located in ORF 1ab region and N region of SARS-CoV-2 gene sequence as the model target and nitrogen doped carbon quantum dots (CDs) as ECL beacon, supplemented with catalytic hairpin assembly (CHA) reaction for signal amplification, the presented strategy has been successfully applied to the rapid detection of SARS-CoV-2. The developed SARS-CoV-2 biosensor based on the series CHA systems can realize the quantitative determination of SARS-CoV-2 in the range of 50 fM to 200 pM within 40 min. Moreover, the clinical validity of this method has been verified by the high consistency between the detection results of using this method and those using RT-qPCR for seven clinical pharyngeal swab samples.

Novel and sensitive electrochemical/fluorescent dual-mode biosensing platform based on the cascaded cyclic amplification of enzyme-free DDSA and functional nucleic acids

Herein, we present a novel electrochemical (EC)/fluorescent (FL) dual-mode biosensor for sensitive and accurate detection of target nucleic acids, which was based on the functional nucleic acids-involved enzyme-free dynamic DNA self-assembly of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for cascaded cyclic amplification. Originally, the CHA reaction of three well-designed hairpin probes were initiated by target sequence, forming abundant Mg2+-dependent three-way DNAzyme junctions (MTWDJ) which could recognize and cleave the methylene blue-labeled substrate hairpin (MB-Hs) to generate the MB-labeled fragments s1 (MB-s1) and the HCR initiator s2. Then, s2 triggered the HCR of four hairpins to produce long DNA nanowires which contained numerous G-quadruplex sequences and the same Mg2+-dependent DNAzyme (MNAzyme) sequences as MTWDJ. Therefore, the HCR copolymer could not only emerge the fluorescent signals through combining thioflavin T with G-quadruplex, but also generate MB-s1 and s2 via MNAzyme cleavage of MB-Hs to continue initiating the HCR. Meanwhile, MB-s1, the cleavage product of MTWDJ and MNAzyme, was captured on the DNA tetrahedron nanostructure modified electrode surface to bring electrochemical signals. Benefiting from integrating the efficient cyclic cleavage of MTWDJ and MNAzyme, the concatenated CHA and HCR amplification circuit, and the dual-mode detection, the sensitivity and accuracy of this biosensor were significantly improved. Under the optimal conditions, the proposed EC/FL dual-mode sensing strategy exhibited a superior analytical performance toward target nucleic acids, showing the promising application in bioanalysis and early disease diagnosis.

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.

Simultaneous detection of circulating tumor DNAs using a SERS-based lateral flow assay biosensor for point-of-care diagnostics of head and neck cancer.

Circulating tumor DNA (ctDNA) has recently emerged as an ideal target for biomarker analytes. Thus, the development of rapid and ultrasensitive ctDNA detection methods is essential. In this study, a high-throughput surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip is proposed. The aim of this method is to achieve accurate quantification of TP53 and PIK3CA E545K, two types of ctDNAs associated with head and neck squamous cell carcinoma (HNSCC), particularly for point-of-care testing (POCT). Raman reporters and hairpin DNAs are used to functionalize the Pd-Au core-shell nanorods (Pd-AuNRs), which serve as the SERS probes. During the detection process, the existence of targets could open the hairpins on the surface of Pd-AuNRs and trigger the first step of catalytic hairpin assembly (CHA) amplification. The next stage of CHA amplification is initiated by the hairpins prefixed on the test lines, generating numerous "hot spots" to enhance the SERS signal significantly. By the combination of high-performing SERS probes and a target-specific signal amplification strategy, TP53 and PIK3CA E545K are directly quantified in the range of 100 aM-1 nM, with the respective limits of detection (LOD) calculated as 33.1 aM and 20.0 aM in the PBS buffer and 37.8 aM and 23.1 aM in human serum, which are significantly lower than for traditional colorimetric LFA methods. The entire detection process is completed within 45 min, and the multichannel design realizes the parallel detection of multiple groups of samples. Moreover, the analytical performance is validated, including reproducibility, uniformity, and specificity. Finally, the SERS-LFA biosensor is employed to analyze the expression levels of TP53 and PIK3CA E545K in the serum of patients with HNSCC. The results are verified as consistent with those of qRT-PCR. Thus, the SERS-LFA biosensor can be considered as a noninvasive liquid biopsy assay for clinical cancer diagnosis.

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

A microfluidic chemiluminescence biosensor based on multiple signal amplification for rapid and sensitive detection of E.coli O157:H7

E. coli O157:H7 has become a crucial foodborne pathogen with certain morbidity and mortality. However, the available methods still face the significant challenges including the improvement of detection specificity and sensitivity for real sample analysis. Herein, a microfluidic chemiluminescence biosensor based on a multiple signal amplification strategy was developed for rapid and ultrasensitive detection of E.coli O157:H7. The microfluidic device was consisted of an upstream snake-shape mixing channel and a downstream ship-shape microcavity with micropillar array for high efficient mixing of CL reagents, sufficient CHA amplification reaction and CL reaction. The multiple signal amplification was achieved by bacteria competitively bind to trigger catalytic hairpin assembly (CHA) and HRP-Au NPs catalyzed CL reaction. Based on the platform, the detection of target bacteria is converted to the detection of nucleic acid with a limit detection down to 130 CFU/mL, which is better or comparable to previously reported biosensors. Moreover, the whole analysis time was about 1.5 h with only 10 μL of sample. The biosensor exhibited an excellent recovery rate ranged from 90.0% to 110.0% and good selectivity to the target bacteria. Overall, this developed biosensor is a promising tool for ultrahigh sensitive and rapid detection of foodborne pathogens.

Pump-free microfluidic chip based laryngeal squamous cell carcinoma-related microRNAs detection through the combination of surface-enhanced Raman scattering techniques and catalytic hairpin assembly amplification

MicroRNA (miRNA), as one of the ideal target biomarker analytes, plays an essential role in biological processes; thus, the development of rapidly sensitive detection methods is imperative. Herein, we proposed a pump-free surface-enhanced Raman scatting (SERS) microfluidic chip for the rapid and ultrasensitive detection of miR-106b and miR-196b, laryngeal squamous cell carcinoma (LSCC)-related miRNAs. Ag–Au core-shell nanorods (Ag-AuNRs) were applied to prepare SERS tags by modifying Raman reporters and hairpin DNAs. The capture probes were synthesized by labeling hairpin DNAs onto the magnetic beads (MBs) surface. In the presence of targets, the catalytic hairpin assembly (CHA) reactions between SERS tags and capture probes could be triggered, causing the aggregation of Ag-AuNRs. The tiny magnets installed under the rectangular chamber could magnetically gather the CHA products, leading to the further aggregation of Ag-AuNRs. Thus, this strategy could achieve the double aggregation of Ag-AuNRs, resulting in the significant amplification of the SERS signal. The proposed strategy achieved simultaneous and sensitive detection of miR-106b and miR-196b, with limits of detection low to aM level. The whole detection process could be completed within 5 min. Moreover, this microfluidic chip exhibited excellent reproducibility, stability, and specificity. The high accuracy of this SERS microfluidic chip was proved by practical analysis in LSCC patients’ serum. The results demonstrated that SERS could be a promising alternative clinical diagnosis tool and exhibited potential application for the dynamic monitoring of cancer staging.

A highly sensitive fluorometric biosensor for Fumonisin B1 detection based on upconversion nanoparticles-graphene oxide and catalytic hairpin assembly

Fumonisin (FB) is a common mycotoxin in corn, wheat, oats, and their related products. FB1 is the most predominant among fumonisins and is responsible for severe food contamination that may have deleterious effects on public health. Therefore, the demand for achieving sensitive detection of FB1 is becoming more and more pressing. In the present study, a creative biosensor for FB1 detection was developed based on fluorescence resonance energy transfer between upconversion nanoparticles (UCNPs) and graphene oxide (GO) with catalytic hairpin assembly (CHA) target recycling and amplification. In the presence of FB1, UCNPs were bound to the CHA amplification products away from the GO surface. Simultaneously, a strong fluorescence signal was produced at 980 nm (near-infrared light). The correlation between the concentration of FB1 and the fluorescence intensity exhibited a high relevance (R2 = 0.9971) ranging from 0.032 to 500 ng mL−1, and the method reached a considerably low limit of detection (0.0121 ng mL−1). It could be applied for the detection of FB1 in corn, oats, and infant supplements. The sensitive detection of FB1 proves the application potential of this method on food safety detection. This biosensor can enable high-throughput fungal toxin detection by allowing the use of various aptamers.

Highly sensitive and reliable detection of microRNA for clinically disease surveillance using SERS biosensor integrated with catalytic hairpin assembly amplification technology

MicroRNAs (miRNAs) play an important regulatory role in several diseases, especially as a class of promising biomarkers for cancer diagnosis and prognosis. Here, a biosensor based on surface enhanced Raman spectroscopy (SERS) combined with catalytic hairpin assembly (CHA) amplification technology was developed for ultra-sensitive detection of miRNA-21 and miRNA-155 in breast cancer serum. By using CHA strategy, the extremely low concentration of target microRNA in human serum can be significantly amplified through the re-hybridization with thousands of hairpin probes to trigger amplification cycles. Besides, a sandwich SERS sensing chip with numerous hot spots and signal self-calibration was built through the linkage between two-dimensional Au–Si substrate and upper Ag@4-MBA@Au core-shell nanoparticles. Using this specially-designed biosensing platform, a low detection limit of 0.398 fM and 0.215 fM with a dynamic range from 1 fM to 10 nM can be achieved for the detection of miRNA-21 and miRNA-155, respectively. Additionally, the analysis of these two miRNAs in serum samples is capable of identifying the breast cancer subjects from normal ones with 100% of accuracy, as well as potentially evaluating the molecular types and prognosis for breast cancer. These results demonstrate that the proposed SERS with CHA technology would be an alternative method for highly sensitive and reliable detection of miRNA biomarkers contributing to breast cancer diagnosis and prognosis.

Selective and sensitive detection of miRNA-198 using single polymeric microfiber waveguide platform with heterogeneous CHA amplification strategy.

Pancreatic cancer (PC), which has a high fatality rate, is a kind of cancer with poor diagnosis and poor prognosis. Development of selective and sensitive detection platform to diagnose and prognostic of PC has attracted considerable attention. The miRNA-198 has been reported a potential prognostic and early diagnostic marker signature of PC. Herein, we report a novel sensitive detection of miRNA-198 in buffer and serum based on one dimensional chitosan/fluorescein isothiocyanate (CS/FITC) fluorescent microfiber waveguide system combined with the catalytic hairpin assembly amplification strategy. By combination with condensing enrichment effect, the proposed detection platform exhibited high specificity and sensitivity to miRNA-198 target, giving a detection limit as low as 2fM. More importantly, the proposed detection platform can be applied directly to distinguish the expression of miRNA-198 in clinical serum, affording the ability to distinguish pancreatic cancer patients from those of healthy human beings, and quantify the expression variation of miRNA-198 for the pancreatic cancer patients before and after resection, which may pave the way to develop novel clinical diagnostic equipment for cancer diagnosis and therapeutic evaluation.

A target-initiated autocatalytic 3D DNA nanomachine for high-efficiency amplified detection of MicroRNA.

Considering the challenges of generating simple and efficient DNA (deoxyribonucleic acid) nanomachines for sensitive bioassays and the great potential of target-induced self-cycling catalytic systems, herein, a novel autocatalytic three-dimensional (3D) DNA nanomachine was constructed based on cross-catalytic hairpin assembly on gold nanoparticles (AuNPs) to generate self-powered efficient cyclic amplification. Typically, the DNA hairpins H1, H2, H3 and H4 were immobilized onto AuNPs first. In the presence of target microRNA-203a, the 3D DNA nanomachines were triggered to activate a series of CHA (catalytic hairpin assembly) reactions. Based on the rational design of the system, the products of the CHA 1 reaction were the trigger of the CHA 2 reaction, which could trigger the CHA 1 reaction in turn, generating an efficient self-powered CHA amplification strategy without adding fuel DNA strands or protein enzymes externally and producing high-efficiency fluorescence signal amplification. More importantly, the proposed autocatalytic 3D DNA nanomachines outperformed conventional 3D DNA nanomachines combined with the single-directional cyclic amplification strategy to maximize the amplification efficiency. This strategy not only achieves high-efficiency analysis of microRNAs (microribonucleic acids) in vitro and intracellularly but also provides a new pathway for highly processive DNA nanomachines, offering a new avenue for bioanalysis and early clinical diagnosis.