Search Results (30)

Co-infections pose significant challenges not only clinically, but also in terms of simultaneous diagnoses. The development of sensitive, multiplexed analytical platforms is critical for accurately detecting viral co-infections, particularly in complex biological environments. In this study, we present a mass spectrometry (MS)-based detection strategy employing a target-triggered hybridization chain reaction (HCR) to amplify signals and in situ photocleavable mass tags (PMTs) for the simultaneous detection of multiple targets. Hairpin DNAs modified with PMTs and immobilized loop structures on magnetic particles (Loop@MPs) were engineered for each target, and their hybridization and amplification efficiency was validated using native polyacrylamide gel electrophoresis (PAGE) and laser desorption/ionization MS (LDI-MS), with silica@gold core–shell hybrid (SiAu) nanoparticles being employed as an internal standard to ensure quantitative reliability. The system exhibited excellent sensitivity, with a detection limit of 415.12 amol for the hepatitis B virus (HBV) target and a dynamic range spanning from 1 fmol to 100 pmol. Quantitative analysis in fetal bovine serum confirmed high accuracy and precision, even under low-abundance conditions. Moreover, the system successfully and simultaneously detected multiple targets, i.e., HBV, human immunodeficiency virus (HIV), and hepatitis C virus (HCV), mixed in various ratios, demonstrating clear PMT signals for each. These findings establish our approach as a robust and reliable platform for ultrasensitive multiplexed detection, with strong potential for clinical and biomedical research. Full article

The accurate and sensitive detection of tumor-derived exosomes holds significant promise for the early diagnosis of cancer. In this study, an electrochemical aptasensor was developed for the high-performance detection of exosomes by integrating dual-probe recognition and hybridization chain reaction (HCR). A dual-probe recognition unit composed of a MUC1 aptamer (MUC1-Apt) probe and cholesterol probe was designed for capturing target exosomes and reducing the interference from free proteins, significantly improving the accuracy of exosome detection. It should be noted that the dual-probe recognition unit was formed in conjunction with the HCR. Moreover, a large number of biotins were also assembled on the HCR product, which were used to capture avidin–horseradish peroxidase (SA-HRP) for signal amplification. The CD63 aptamer (CD63-Apt) was immobilized on the surface of a gold electrode for specifically capturing exosomes to construct a classical sandwiched structure. The loaded SA-HRP can efficiently catalyze the reaction of 3, 3′, 5, 5′ tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) to generate a large electrochemical signal. According to this phenomenon, a linear relationship of this proposed aptasensor was achieved between the electrochemical response and 1 × 10²–1 × 10⁷ particles/mL exosomes, with a detection limit of 45 particles/mL. Moreover, the aptasensor exhibited accepted stability and potential clinical applicability. All results proved that this aptasensor has a promising application in exosome-based disease diagnostics. Full article

Changes in microRNA (miRNA) levels are closely associated with the pathological processes of many diseases. The sensitive and fast detection of miRNAs is critical for diagnosis and prognosis. Here, we report a platform employing CRISPR/Cas12a to recognize and report changes in miRNA levels while avoiding complex multi-thermal cycling procedures. A non-enzyme-dependent hybridization chain reaction (HCR) was used to convert the miRNA signal into double-stranded DNA, which contained a Cas12a activation sequence. The target sequence was amplified simply and isothermally, enabling the test to be executed at a constant temperature of 37 °C. The detection platform had the capacity to measure concentrations down to the picomolar level, and the target miRNA could be distinguished at the nanomolar level. By using photonic crystal microarrays with a stopband-matched emission spectrum of the fluorescent-quencher modified reporter, the fluorescence signal was moderately enhanced to increase the sensitivity. With this enhancement, analyzable fluorescence results were obtained in 15 min. The HCR and Cas12a cleavage processes could be conducted in a single tube by separating the two procedures into the bottom and the cap. We verified the sensitivity and specificity of this one-pot system, and both were comparable to those of the two-step method. Overall, our study produced a fast and sensitive miRNA detection platform based on a CRISPR/Cas12a system and enzyme-free HCR amplification. This platform may serve as a potential solution for miRNA detection in clinical practice. Full article

Butterfly larvae display intricate cognitive capacities and behaviors, but relatively little is known about how those behaviors alter their brains at the molecular level. Here, we optimized a hybridization chain reaction 3.0 (HCR v3.0) protocol to visualize the expression of multiple RNA molecules in fixed larval brains of the African butterfly Bicyclus anynana. We optimized the polyacrylamide gel mounting, fixation, and sample permeabilization steps, and mapped the expression domains of ten genes in whole larval brain tissue at single-cell resolution. The genes included optomotor blind (omb), yellow-like, zinc finger protein SNAI2-like (SNAI2), weary (wry), extradenticle (exd), Synapsin, Distal-less (Dll), bric-à-brac 1 (bab1), dachshund (dac), and acetyl coenzyme A acetyltransferase B (AcatB). This method can be used alongside single-cell sequencing to visualize the spatial location of brain cells that change in gene expression or splicing patterns in response to specific behaviors or cognitive experiences. Full article

Abnormal expression of miRNAs is associated with the occurrence and progression of cancer and other diseases, making miRNAs essential biomarkers for disease diagnosis and prognosis. However, the intrinsic properties of miRNAs, such as short length, low abundance, and high sequence homology, represent great challenges for fast and accurate miRNA detection in clinics. Herein, we developed a novel hybridization chain reaction (HCR)-based electrochemical miRNAs chip (e-miRchip), featured with gold nanostructured electrodes (GNEs) and silver nanoparticle reporters (AgNRs), for sensitive and multiplexed miRNA detection. AgNRs were synthesized and applied on the e-miRchip to generate strong redox signals in the presence of miRNA. The stem–loop capture probe was covalently immobilized on the GNEs, and was opened upon miRNA hybridization to consequently trigger the HCR for signal amplification. The multiple long-repeated DNA helix generated by HCR provides the binding sites for the AgNRs, contributing to the amplification of the electrochemical signals of miRNA hybridization. To optimize the detection sensitivity, GNEs with three distinct structures were electroplated, in which flower-like GNEs were found to be the best electrode morphology for miRNAs analysis. Under optimal conditions, the HCR-based e-miRchip showed an excellent detection performance with an LOD of 0.9 fM and a linear detection range from 1 fM to 10 pM. Moreover, this HCR-based e-miRchip platform was able to effectively distinguish miRNAs from the one- or two-base mismatches. This HCR-based e-miRchip holds great potential as a highly efficient and promising miRNA detection platform for the diagnosis and prognosis of cancer and other diseases in the future. Full article

Hepatitis, most importantly hepatitis B and hepatitis C, is a significant global health concern, requiring an accurate and early diagnosis to prevent severe liver damage and ensure effective treatment. The currently employed diagnostic methods, while effective, are often limited in their sensitivity, specificity, and rapidity, and the quest for improved diagnostic tools is ongoing. This review explores the innovative application of Raman spectroscopy combined with a chemometric analysis as a powerful diagnostic tool for hepatitis. Raman spectroscopy offers a non-invasive, rapid, and detailed molecular fingerprint of biological samples, while chemometric techniques enhance the interpretation of complex spectral data, enabling precise differentiation between healthy and diseased states and moreover the severity/stage of disease. This review aims to provide a comprehensive overview of the current research, foster greater understanding, and stimulate further innovations in this burgeoning field. The Raman spectrum of blood plasma or serum provides fingerprints of biochemical changes in the blood profile and the occurrence of disease simultaneously, while Raman analyses of polymerase chain reaction/hybridization chain reaction (PCR/HCR)-amplified nucleic acids and extracted DNA/RNA as the test samples provide more accurate differentiation between healthy and diseased states. Chemometric tools enhance the diagnostic efficiency and allow for quantification of the viral loads, indicating the stage of disease. The incorporation of different methodologies like surface enhancement and centrifugal filtration using membranes provides the ability to target biochemical changes directly linked with the disease. Immunoassays and biosensors based on Raman spectroscopy offer accurate quantitative detection of viral antigens or the immune response in the body (antibodies). Microfluidic devices enhance the speed of detection through the continuous testing of flowing samples. Raman diagnostic studies with massive sample sizes of up to 1000 and multiple reports of achieving a greater than 90% differentiation accuracy, sensitivity, and specificity using advanced multivariate data analysis tools indicate that Raman spectroscopy is a promising tool for hepatitis detection. Its reproducibility and the identification of unique reference spectral features for each hepatic disease are still challenges in the translation of Raman spectroscopy as a clinical tool, however. The development of databases for automated comparison and the incorporation of automated chemometric processors into Raman diagnostic tools could pave the way for their clinical translation in the near future. Full article

Exosomes carry diverse tumor-associated molecular information that can reflect real-time tumor progression, making them a promising tool for liquid biopsy. However, traditional methods for exosome isolation and detection often rely on large, expensive equipment and are time-consuming, limiting their practical applicability in clinical settings. Microfluidic technology offers a versatile platform for exosome analysis, with advantages such as seamless integration, portability and reduced sample volumes. Aptamers, which are single-stranded oligonucleotides with high affinity and specificity for target molecules, have been frequently employed in the development of aptamer-based microfluidics for the isolation, signal amplification, and quantitative detection of exosomes. This review summarizes recent advances in aptamer-based microfluidic strategies for exosome analysis, including (1) strategies for on-chip exosome capture mediated by aptamers combined with nanomaterials or nanointerfaces; (2) aptamer-based on-chip signal amplification techniques, such as enzyme-free hybridization chain reaction (HCR), rolling circle amplification (RCA), and DNA machine-assisted amplification; and (3) various aptamer-assisted detection methods, such as fluorescence, electrochemistry, surface-enhanced Raman scattering (SERS), and magnetism. The limitations and advantages of these methods are also summarized. Finally, future challenges and directions for the clinical analysis of exosomes based on aptamer-based microfluidics are discussed. Full article

The association between microRNA (miRNA) and various diseases has been established; miRNAs have the potential to be biomarkers for these diseases. Nevertheless, the challenge of correctly quantifying an miRNA arises from its low abundance and a high degree of family homology. Therefore, in the present study, we devised a chemiluminescence (CL) detection method for miRNAs, known as the hybridization chain reaction (HCR)-CL, utilizing the enzyme-free signal amplification technology of HCR. The proposed methodology obviates the need for temperature conversion and offers a straightforward procedure owing to the absence of enzymatic participation, and the lumino-H₂O₂-mediated CL reaction occurs at a high rate. The technique successfully detected 2.5 amol of the target analyte and 50 amol of miR-146b in a 1% concentration of human serum. In summary, the method developed in this study is characterized by its ease of operation, cost-effectiveness, remarkable analytical prowess, and ability to detect miRNA without the need for total RNA extraction from serum samples. This method is expected to be widely used for biological sample testing in clinical settings. Full article

A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging. Full article

Conventional spherical nucleic acid enzymes (SNAzymes), made with gold nanoparticle (AuNPs) cores and DNA shells, are widely applied in bioanalysis owing to their excellent physicochemical properties. Albeit important, the crowded catalytic units (such as G-quadruplex, G4) on the limited AuNPs surface inevitably influence their catalytic activities. Herin, a hybridization chain reaction (HCR) is employed as a means to expand the quantity and spaces of G4 enzymes for their catalytic ability enhancement. Through systematic investigations, we found that when an incomplete G4 sequence was linked at the sticky ends of the hairpins with split modes (3:1 and 2:2), this would significantly decrease the HCR hybridization capability due to increased steric hindrance. In contrast, the HCR hybridization capability was remarkably enhanced after the complete G4 sequence was directly modified at the non-sticky end of the hairpins, ascribed to the steric hindrance avoided. Accordingly, the improved SNAzymes using HCR were applied for the determination of AFB1 in food samples as a proof-of-concept, which exhibited outstanding performance (detection limit, 0.08 ng/mL). Importantly, our strategy provided a new insight for the catalytic activity improvement in SNAzymes using G4 as a signaling molecule. Full article

Today, with the globalization of the food trade progressing, food safety continues to warrant widespread attention. Foodborne diseases caused by contaminated food, including foodborne pathogens, seriously threaten public health and the economy. This has led to the development of more sensitive and accurate methods for detecting pathogenic bacteria. Many signal amplification techniques have been used to improve the sensitivity of foodborne pathogen detection. Among them, hybridization chain reaction (HCR), an isothermal nucleic acid hybridization signal amplification technique, has received increasing attention due to its enzyme-free and isothermal characteristics, and pathogenic bacteria detection methods using HCR for signal amplification have experienced rapid development in the last five years. In this review, we first describe the development of detection technologies for food contaminants represented by pathogens and introduce the fundamental principles, classifications, and characteristics of HCR. Furthermore, we highlight the application of various biosensors based on HCR nucleic acid amplification technology in detecting foodborne pathogens. Lastly, we summarize and offer insights into the prospects of HCR technology and its application in pathogen detection. Full article

In this study, we developed a novel method for the detection of Vibrio alginolyticus, utilizing the specific recognition of an aptamer for V. alginolyticus and signal amplification via hybridization chain reaction (HCR) and horseradish-peroxidase-conjugated streptavidin. The proposed HCR-based multivalent aptamer (multi-Apt) amplifier allows for sensitive detection of V. alginolyticus in a linear range from 10 to 10⁷ CFU/mL. The linear equation is y = 747.5x + 126.2, R² = 0.986, and the limit of detection (LOD) is 3 CFU/mL. Seawater and freshwater samples were utilized in the spike recovery experiment, yieldng a recovery rates ranging from 94.3% to 108.8%. The relative standard deviation (RSD) for all samples is below 6.73%. Taken together, the proposed method has great potential for application in monitoring of V. alginolyticus in aquaculture environments. Full article

A high-throughput cell-based monitoring platform was fabricated to rapidly measure the specific toxicity of unknown waters, based on AuNPs@aptamer fluorescence bioassays. The aptamer is employed in the platform for capturing the toxicity indicator, wherein hybrid chain-reaction (HCR)-induced DNA functional gold nanoparticle (AuNPs) self-assembly was carried out for signal amplification, which is essential for sensitively measuring the sub-lethal effects caused by target compounds. Moreover, the excellent stability given by the synthesized DNA nanostructure provides mild conditions for the aptamer thus used to bind the analyte. Herein, ATP was treated as a toxicity indicator and verified using lead-caused cell damage as a model. Under optimized conditions, excellent performance for water sample measurement was observed, yielding satisfactory accuracy (recovery rate: 82.69–114.20%; CV, 2.57%–4.65%) and sensitivity (LOD, 0.26 µM) without sample pretreatment other than filtration, indicating the method’s simplicity, high efficiency, and reliability. Most importantly, this bioassay could be used as a universal platform to encourage its application in the rapid quantification of specific toxicity in varied sources of samples, ranging from drinking water to highly contaminated wastewater. Full article

Tumor cell-derived exosomes are considered a potential source of cancer biomarkers. Here, we developed an electrochemical sensing platform for the rapid and simple detection of exosomes, using the CCRF-CEM exosome as a model. The platform utilizes cyclic nicking enzyme cleavage and a hybridization chain reaction (HCR) for dual-signal amplification. A hairpin aptamer probe (HAP) containing an aptamer was designed for the assay. The specific binding between the aptamer and PTK7, present on the exosome surface, causes a conformational change in the HAP. This facilitates hybridization between the HAP and the linker DNA, which subsequently triggers cyclic cleavage of the nicking endonuclease towards the linker DNA. Therefore, exosome detection is transformed into DNA detection. By combining this approach with HCR signal amplification, we achieved high-sensitivity electrochemical detection of CCRF-CEM exosomes, down to 1.1 × 10⁴ particles/mL. Importantly, this assay effectively detected tumor exosomes in complex biological fluids, demonstrating the potential for clinical diagnosis. Full article

The conventional hybridization chain reaction (HCR)-based electrochemical biosensors usually require the immobilization of probes on the electrode surface. This will limit the applications of biosensors due to the shortcomings of complex immobilization processes and low HCR efficiency. In this work, we proposed astrategy for the design of HCR-based electrochemical biosensors by integrating the advantages of homogeneous reaction and heterogeneous detection. Specifically, the targets triggered the autonomous cross-opening and hybridization oftwobiotin-labeled hairpin probes to form long-nicked dsDNA polymers. The HCR products with many biotin tags were then captured by a streptavidin-covered electrode, thus allowing for the attachment of streptavidin-conjugated signal reporters through streptavidin–biotin interactions. By employing DNA and microRNA-21 as the model targets and glucose oxidase as the signal reporter, the analytical performances of the HCR-based electrochemical biosensors were investigated. The detection limits of this method were found to be 0.6 fM and 1 fM for DNA and microRNA-21, respectively. The proposed strategy exhibited good reliability for target analysis in serum and cellular lysates. The strategy can be used to develop various HCR-based biosensors for a wide range of applications because sequence-specific oligonucleotides exhibit high binding affinity to a series of targets. In light of the high stability and commercial availability of streptavidin-modified materials, the strategy can be used for the design of different biosensors by changing the signal reporter and/or the sequence of hairpin probes. Full article