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Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (Apt EpCAM , Apt HER2 ) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 10 3 -10 7 particles/μL, with a limit of detection (LOD) of 3.3 × 10 2 particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Accurate and reliable detection of Staphylococcus aureus (S. aureus) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus. Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a's nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL -1 in electrochemical mode and 133.7 CFU mL -1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The choice of fixation method significantly impacts tissue morphology and visualization of gene expression and proteins after in situ hybridization chain reaction (HCR) or immunohistochemistry (IHC), respectively. In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation techniques prior to HCR and IHC on chicken embryos. Our findings underscore the importance of optimizing fixation methods for accurate visualization and subsequent interpretation of HCR and IHC results, with implications for probe and antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei and neural tubes compared to PFA fixation. Additionally, TCA fixation altered the subcellular fluorescence signal intensity of various proteins, including transcription factors, cytoskeletal proteins, and cadherins. Notably, TCA fixation revealed protein signals in tissues that may be inaccessible with PFA fixation. In contrast, TCA fixation proved ineffective for mRNA visualization. These results highlight the need for optimization of fixation protocols depending on the target and model system, emphasizing the importance of methodological considerations in biological analyses., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

According to the characteristics of DNA programming, the cascaded nucleic acid amplification technology with larger output can overcome the problem of insufficient sensitivity of single nucleic acid amplification technology, and it combines the advantages of two or even multiple nucleic acid amplification technologies at the same time. In this work, a novel cascade signal amplification strategy with strand displacement amplification (SDA) and cascade hybridization chain reaction (HCR) was proposed for trace detection of hAAG and VEGF165. HAAG-induced SDA produced a large amount of S2 to open H2 on Polystyrene (PS) nanospheres, thereby triggering cascade HCR to form DNA dendritic nanostructures with rich fluorescence (FL) signal probes (565 nm). It could realize the amplification of FL signals for the detection of hAAG. Moreover, many doxorubicin (Dox) were loaded into the GC bases of DNA dendritic nanostructures, and its FL signal was effectively shielded. VEGF165 specifically bound to its aptamer to form G-quadruplex structures, which released Dox to produce a high FL signal (590 nm) for detection of VEGF165. This work developed a unique multifunctional DNA dendritic nanostructure fluorescence probe, and cleverly designed a new "On-off" switch strategy for sensitive trace detection of cancer markers., Competing Interests: Declaration of competing interest We declared that we have no conflicts of interest to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF 4 :Yb 3+ , Er 3+ ) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Palmitoylation plays an important role in modulating protein trafficking, stability, and activity. The major predicament in protein palmitoylation study is the lack of specific and sensitive tools to visualize protein-specific palmitoylation. Although FRET approach was explored by metabolically labeled palmitic acid and antibody recognized target protein. The trans-membrane strategy suffers from low FRET efficiency due to the donor and acceptor located at different sides of membrane. Herein, we proposed a cis-membrane multi-fluorescence resonance energy transfer (multi-FRET) for amplified visualization of specific palmitoylated proteins through metabolic labeling and targeted recognition. The azido-palmitic acid (azido-PA) was metabolically incorporated into cellular palmitoylated proteins, followed by reacting with dibenzylcylooctyne-modified Cy5 (DBCO-Cy5) through copper-free click chemistry. The protein probe was attached to targeted protein by specific peptide recognition, which initiates a hybridization chain reaction (HCR) amplification process. The cis-membrane labeling method enables effective intramolecular donor-acceptor distance and allow to increase FRET efficiency. Simultaneously, HCR amplification triggered multi-FRET phenomenon with significantly improved FRET efficiency. With the superiority, this strategy has achieved the enhanced FRET imaging of palmitoylated PD-L1 and visualizing the palmitoylation changes of on PD-L1 under drug treatment. Furthermore, the established method successfully amplified visualization of PD-L1 palmitoylation in vivo and mice tumor slice. We envision the approach would provide a useful platform to investigate the effects of palmitoylation on the protein structure and function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rapid, sensitive, and accurate identification of pathogens is vital for preventing and controlling fish disease, reducing economic losses in aquaculture, and interrupting the spread of food-borne diseases in human populations. Herein, we proposed a hybridization chain reaction (HCR) cascaded dual-signal amplification platform for the ultrasensitive and specific detection of pathogenic microorganisms. A couple of specific primers for target bacterial 16S rRNAs were used to obtain amplified target single-stranded DNAs (AT-ssDNA). Then, AT-ssDNA initiated HCR amplification along with the opening of fluorophore (FAM) and a quencher (BHQ1) labeled hairpin reporter probe (H1), and the FAM fluorescence signal recovered. The proposed strategy could achieve a detection limit down to 0.31 CFU/mL for Staphylococcus aureus (S. aureus), 0.49 CFU/mL for Escherichia coli (E. coli) in buffer, and a linear range from 1 to 1 × 10 6  CFU/mL for S. aureus, 1 to 1 × 10 7  CFU/mL for E. coli. Furthermore, this platform enabled sensitive and precise detection of pathogenic microorganisms in complex samples such as fish blood and different organ tissues (large intestine, gallbladder, heart, liver, ren, gill, skin), which shows great potential in disease prevention and control in aquatic products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Herein, a sensitive immunosensor for the rapid detection of Dibutyl phthalate (DBP) small molecules was developed. Its principle is based on Au@Pt/Polyethyleneimine (PEI)-rGO and hybridization chain reaction signal amplification system. First, Au@Pt/PEI-rGO modified electrode is used as a signal amplification platform and the antibody is fixed. In the presence of target DBP, it competes with DBP-bovine serum albumin-S 0 for binding antibody. Finally, the DNA concatemer carrying a large number of signal molecules methylene blue combines with its S 0 to produce a reduced electrochemical signal. Under the most reasonable conditions, this immunosensor showed a good linear range from1 pg mL -1 to 0.1 μg mL -1 with a low detection limit of 0.276 pg mL -1 . It has been verified that this immunosensor has good selectivity, reproducibility and stability. The designed biosensing solution will open up a new path for DBP monitoring and other small molecule targets in the food field., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this work, we designed an ECL ratiometric biosensor with a three-stranded Y-type DNA (Y-DNA) probe and induced a hybridization chain reaction (HCR) for the highly sensitive detection of SARS-CoV-2 nucleic acid. The important component of this system is the self-assembled Y-Shaped probe based on three nucleic acids. Y1, Y2, and Y3 can be linked by complementary base pairing to Hairpin1 (H1), Hairpin2 (H2), and Ru modified DNA (Ru1), respectively. H1 and H2 can trigger the HCR reaction when activated by the SARS-CoV-2 RdRp gene and the 5' end of Ru1. The 5' end of Ru1 is modified with the Ru complex, which can produce a strong electrochemiluminescence luminescence signal at 620 nm under an applied voltage. Through the amplification of Y-DNA-induced HCR reaction, Ru1 on the electrode surface gradually increased, the ECL signal at 460 nm was gradually quenched, and the signal at 620 nm was steadily generated. The SARS-CoV-2 RdRp gene can be quantified according to the degree of decrease of ECL signal at 460 nm and the increase of ECL signal at 620 nm. Combining the two signal amplification strategies, this ratiometric ECL biosensor can accurately and efficiently detect the target gene with a detection limit of 59 aM., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A comparative review of the most common isothermal methods is provided. In the last two decades, the challenge of using isothermal amplification systems as an alternate to the most extensive and long-standing nucleic acids-amplifying method-the polymerase chain reaction-has arisen. The main advantage of isothermal amplification is no requirement for expensive laboratory equipment for thermal cycling. Considerable efforts have been made to improve the current techniques of nucleic acid amplification and the development of new approaches based on the main drawbacks of each method. The most important and challenging goal was to achieve a low-cost, straightforward system that is rapid, specific, accurate, and sensitive., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An electrochemical platform has been developed to detect protein kinase activity through the combined actions of Zr 4+ mediated signal transition and hybridization chain reaction (HCR)-stimulated DNAzymes nanowires. First of all, protein kinase A (PKA) phosphorylates substrate peptides immobilized on gold electrode surface. Thereafter, the DNA1 containing 5'-phosphoryl ends is linked to the phosphorylated substrate peptide via the robust phosphate-Zr 4+ -phosphate linkages. By the introduction of molecular beacons (MBs), the DNA1 can open the hairpin structures of MBs through toehold mediated strand displacement (TMSDR), leading to an autonomous stem-opening process and subsequent assembly of G-quadruplex-containing DNA chains by HCR. After the addition of hemin, the formed HRP-mimicking DNAzymes can catalyze the hydroquinone-H 2 O 2 system to generate amplified electrochemical signals. As expected, this method can achieve ultrahigh analytical performance with a low detection limit of 0.02U/mL and exhibit high cost-savings potential without the need for antibody, protease and labeling. Therefore, this method can serve as a new tool for the assay of protein kinase A and its inhibitor screening in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Salmonella, a typical foodborne pathogen, always seriously threatens the health and even life of both humans and animals. However, highly sensitive and fast quantitative methods for its detection are remaining to be challenged. Herein, we presented an efficient method with dual signal amplification strategy by combining immune hybridization chain reaction (HCR) with surface enhanced Raman scattering (SERS) to high sensitively detect Salmonella typhimurium in food. After sample preparation, S. typhimurium were specifically captured by immunomagnetic beads (IMBs), then aptamers and hairpin-probes were added to trigger HCR to form nicked dsDNA, finally 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was incubated with HCR products and then the whole system was mixed with AgNP colloid to detect the SERS intensity at 1610 cm -1 . As a result, a good linear relationship was achieved between SERS intensities and corresponding concentrations of S. typhimurium ranging from 10 to 10 5  CFU/mL, with a limit of detection (LOD) of 6 CFU/mL in 3.5 h. The proposed method has been successfully applied to capture and detect the S. typhimurium in spiked milk samples, and the results were consistent with those of the traditional plate counting method. The method, with combination of HCR and SERS, achieves double amplification of the detection signal and significantly improves the detection sensitivity of S. typhimurium. And it also shows good application potential for the highly sensitive detection of other contaminants in food., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this paper, a rationally designed aptasensing platform based on Hybridization Chain Reaction (HCR) and G-quadruplex DNAzyme for the fluorescence detection of Carcinoembryonic Antigen (CEA) has been developed. In the presence of target CEA, the aptamer sequence in Aptamer Probe (AP) specifically bound to CEA, resulting in the AP conformation change and thus releasing initiator, which triggered the autonomous cross-opening of Hairpin 1 (H1) and Hairpin 2 (H2) that yielded extended nicked double-stranded DNA via HCR. Upon the addition of hemin, G-rich segments at the end of H1 and H2 self-assembled into the peroxidase-mimicking hemin/G-quadruplex DNAzymes, which catalyzed the hydrogen peroxide-mediated oxidation of thiamine to achieve fluorescence detection of CEA. The HCR product, and the formation and catalytic performance of DNAzyme were characterized by agarose gel electrophoresis, UV-vis spectroscopy and fluorescence spectroscopy, respectively. Under optimal conditions, the fluorescent aptasensor showed a linear relationship ranging from 0.25 to 1.5 nM toward CEA with a detection limit of 0.2 nM. In addition, this aptasensor exhibited high selectivity for CEA without being affected by other interfering proteins, such as IgG, AFP and PSA. Furthermore, this proposed aptasensor was successfully applied to CEA analysis in diluted human serum samples. It is believed that this strategy has a promising potential in biochemical analysis and clinic application., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dynamic changes of protein-glycosylation on cell surface act as an important indicator that reflects cellular physiological states and disease developments. The enhanced visualization of protein-specific glycosylation is of great value to interpret its functions and mechanisms. Hence, we present an intramolecular trigger remodeling-induced hybridization chain reaction (HCR) for imaging protein-specific glycosylation. This strategy relies on designing two DNA probes, protein and glycan probes, labeled respectively on protein by aptamer recognition and glycan through metabolic oligosaccharide engineering (MOE). Upon the same glycoprotein was labeled, the complementary domain of two probes induces hybridization and thus to remodel an intact trigger, followed by initiating HCR assembly. Applying this strategy, we successfully achieved imaging of specific protein-glycosylation on CEM cell surface and monitored dynamic changes of the glycosylation after treating with drugs. It provides a powerful tool with high flexibility, specificity and sensitivity in the research field of protein-specific glycosylation on living cells., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel label-free fluorescent sensor for histidine was developed based upon Cu 2+ -specific DNAzyme, hybridization chain reaction(HCR) and triplex DNA. Cu 2+ can bind to the histidine, in the presence of histidine, leading to the inhibition of the cleavage of substrate strand of Cu 2+ -dependent DNAzyme, then the intact substrate strand trigger the HCR between H1 and H2. The HCR product can be recognized by triplex-forming oligonucleotide (TFO) through triplex formation and reported by the fluorescence of berberine, the fluorescence intensity of the sensing system was proportional to the concentration of histidine during the range of 5.7-455 nmol L -1 , with a detection limit of 2.0 nmol L -1 ., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: With hybrid coronary revascularization (HCR), minimally invasive left internal mammary artery (LIMA) to left anterior descending coronary artery (LAD) grafting is combined with percutaneous coronary intervention (PCI) of non-LAD vessels. The purpose of this study was to examine the short-term clinical and angiographic results in one of the largest HCR series to date., Methods: From 2003 to 2012, 300 consecutive patients (aged 64±12 years, female 31.7%, predicted risk of mortality 1.6%±2.1%) underwent HCR on an intent-to-treat basis at a single institution. After robotic or thoracoscopic LIMA harvest, off-pump LIMA to LAD grafting was performed through a 3- to 4-cm sternal-sparing, non-rib-spreading thoracotomy. PCI was utilized to treat non-LAD lesions either before, after, or concomitant with the surgical procedure., Results: Of the 300 patients undergoing HCR on an intent-to-treat basis, HCR was performed with surgery first in 192 patients (64.0%), PCI first in 56 (18.7%), and as a concomitant procedure in 21 (7.0%). Of the 31 patients (10.1%) who did not undergo HCR, 24 patients (8.0%) did not have PCI and thus were incompletely revascularized. For all patients, 30-day mortality, stroke, and nonfatal myocardial infarction occurred in 4 (1.3%), 3 (1.0%), and 4 (1.3%), respectively. Angiographic LIMA evaluation was performed in 248 patients and revealed a FitzGibbon A LIMA patency rate of 97.6% (242 of 248 patients). Repeat revascularization was required in 13 of 300 patients (4.3%)., Conclusions: Hybrid coronary revascularization represents an alternative approach for patients with multivessel coronary disease with excellent short-term outcomes. It provides a minimally invasive alternative to traditional coronary artery bypass graft surgery and may prove more durable than multivessel PCI., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)