A simple and sensitive electrochemical biosensor for circulating tumor cell determination based on dual-toehold accelerated catalytic hairpin assembly.

Tumor cells in blood circulation (CTCs) are vital biomarkers for noninvasive cancer diagnosis. We developed a simple and sensitive electrochemical biosensor based on dual-toehold accelerated catalytic hairpin assembly (DCHA) to distinguish CTCs from blood cells. In the presence of CTCs, the aptamer probe initiates the DCHA process, which produces amplified electrochemical signals. Compared with conventional catalytic hairpin assembly (CHA), the proposed DCHA showed high sensitivity, which led to a broader working range of 10-1000 cells mL-1 with a limit of detection of 4 cells mL-1. Furthermore, our method exhibited an excellent capability of distinguishing malignant breast cancers from healthy people, with a sensitivity of 97.4%. In summary, we have established an enzyme-free, easy-to-operate, and nondisruptive method for detecting circulating tumor cells in blood circulation based on the DCHA strategy. Its versatility and simplicity will make it more widely used in clinical diagnosis and biomedical research.

Localized DNA tetrahedrons assisted catalytic hairpin assembly for the rapid and sensitive profiling of small extracellular vesicle-associated microRNAs.

The profiling of small extracellular vesicle-associated microRNAs (sEV-miRNAs) plays a vital role in cancer diagnosis and monitoring. However, detecting sEV-miRNAs with low expression in clinical samples remains challenging. Herein, we propose a novel electrochemical biosensor using localized DNA tetrahedron-assisted catalytic hairpin assembly (LDT-CHA) for sEV-miRNA determination. The LDT-CHA contained localized DNA tetrahedrons with CHA substrates, leveraging an efficient localized reaction to enable sensitive and rapid sEV-miRNA measurement. Based on the LDT-CHA, the proposed platform can quantitatively detect sEV-miRNA down to 25 aM in 30 min with outstanding specificity. For accurate diagnosis of gastric cancer patients, a combination of LDT-CHA and a panel of four sEV-miRNAs (sEV-miR-1246, sEV-miR-21, sEV-miR-183-5P, and sEV-miR-142-5P) was employed in a gastric cancer cohort. Compared with diagnosis with single sEV-miRNA, the proposed platform demonstrated a higher accuracy of 88.3% for early gastric tumor diagnoses with higher efficiency (AUC: 0.883) and great potential for treatment monitoring. Thus, this study provides a promising method for the bioanalysis and determination of the clinical applications of LDT-CHA.

Epidemiological and clinical characteristics of respiratory viruses in 4403 pediatric patients from multiple hospitals in Guangdong, China.

BACKGROUND: Acute respiratory infections (ARI) cause considerable morbidity and mortality worldwide, especially in children. Unfortunately, there are limited multi-center data on common viral respiratory infections in south China. METHODS: A total of 4403 nasal swabs were collected from children in 10 cities in Guangdong, China in 2019. Seven respiratory viruses, influenza A virus (IFA), influenza B virus (IFB), respiratory syncytial virus (RSV), adenoviruses (ADV) and parainfluenza virus types 1-3 (PIV1, PIV2 and PIV3), were detected by direct immunofluorescence antibody assay. The personal information and clinical characteristics were recorded and analyzed. RESULTS: The results showed that at least one virus was detected in 1099 (24.96 %) samples. The detection rates of RSV, IFA, ADV, PIV3, PIV1 and PIV2 were 7.13 % (314/4403), 5.31 % (234/4403), 4.02 % (177/4403), 3.04 % (134/4403), 1.70 % (75/4403) and 1.16 % (51/4403), respectively. The detection rate of RSV was highest in 0-6-month-old children at 18.18 % (106/583), while the detection rate of IFA was highest in 12-18-year-old children at 20.48 % (17/83). The total detection rates in winter and spring were 35.67 % (219/614) and 34.56 % (403/1166), higher than those in summer, 17.41 % (284/1631), and autumn, 19.46 % (193/992). CONCLUSIONS: RSV and IFA were the main respiratory viruses in children. With increasing age the detection rate of RSV decreased in children, but the trends for the detection rates of IFA and IFB were the opposite. This study provided the viral etiology and epidemiology of pediatric patients with ARI in Guangdong, China.

Metabolic classification of circulating tumor cells as a biomarker for metastasis and prognosis in breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells. METHODS: Oncomine, TCGA and Kaplan-Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers. RESULTS: PGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P < 0.001). Blood GM+CTCs (DAPI+CD45-PGK1/G6PD+) was detectable (range 0-54 cells/5 mL) in 61.8% of tCTCs > 0 patients. Increased GM+CTCs number and positive rate were correlated with tumor metastasis and progression (P < 0.05). The GM+CTCs ≥ 2/5 mL level presented superior AUC of ROC at 0.854 (95% CI 0.741-0.968) in the diagnosis of BC metastasis (sensitivity/specificity: 66.7%/91.3%), compared with that of tCTCs (0.779) and CTCs-EMT subtypes (E-CTCs 0.645, H-CTCs 0.727 and M-CTCs 0.697). Moreover, GM+CTCs+ group had inferior survival with decreased 2 years-PFS proportion (18.5%) than GM+CTCs- group (87.9%; P = 0.001). CONCLUSIONS: This work establishes a PGK1/G6PD-based method for CTCs metabolic classification to identify the aggressive CTCs subpopulation. Metabolically active GM+CTCs subtype is suggested a favorable biomarker of distant metastasis and prognosis in BC patients.

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2.

BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease 2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. METHODS: By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false-negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

A fluorescent immunosensor for determination and imaging of circulating tumor cells based on a bifunctional DNA nanomachine.

A fluorescent platform was developed for the determination and visualization of circulating tumor cells by a toehold-mediated bifunctional DNA nanomachine. In the presence of target tumor cells, the DNA nanomachine was activated. Multiple DNA products were formed, including dendritic DNA products and double-strand DNA products. Dendritic DNA products bound to their target cells for the visualization, while double-strand DNA products were released for the determination of tumor cells. At fluorescence excitation and emission wavelengths of 530 and 550 nm, this method could detect as low as 43 cells/mL (S/N = 3) with a linear range of 100 to 10,000 cells/mL. In clinical hydrothorax samples, this platform exhibited high reliability with a recovery of 93 to 116%. At the fluorescence excitation and emission wavelengths of 490 and 515 nm, the specificity and biocompatibility of this method were further verified by tumor cells imaging. Furthermore, the robustness of the toehold-mediated bifunctional DNA nanomachine was demonstrated by the specific gene mutation detection in single-cell analysis. Graphical abstract Schematic illustration of the fluorescent immunosensor for determination and imaging of circulating tumor cells. The method is based on aptamer-based recognition and toehold-mediated bifunctional DNA nanomachine.

An AIE-Active Conjugated Polymer with High ROS-Generation Ability and Biocompatibility for Efficient Photodynamic Therapy of Bacterial Infections.

New, biocompatible materials with favorable antibacterial activity are highly desirable. In this work, we develop a unique conjugated polymer featuring aggregation-induced emission (AIE) for reliable bacterial eradication. Thanks to the AIE and donor-π-acceptor structure, this polymer shows a high reactive oxygen species (ROS)-generation ability compared to a low-mass model compound and the common photosensitizer Chlorin E6. Moreover, the selective binding of pathogenic microorganisms over mammalian cells was found, demonstrating its biocompatibility. The effective growth inhibition of bacteria upon polymer treatment under light irradiation was validated in vitro and in vivo. Notably, the recovery from infection after treatment with our polymer is faster than that with cefalotin. Thus, this polymer holds great promise in fighting against bacteria-related infections in practical applications.

Unique Protein Profiles of Extracellular Vesicles as Diagnostic Biomarkers for Early and Advanced Non-Small Cell Lung Cancer.

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recently investigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non-small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC-MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane-receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty-two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV-associated biomarker screening.

Stereotactic Photodynamic Therapy Using a Two-Photon AIE Photosensitizer.

Two-photon photodynamic therapy (TP-PDT) is emerging as a powerful strategy for stereotactic targeting of diseased areas, but ideal photosensitizers (PSs) are currently lacking. This work reports a smart PS with aggregation-induced emission (AIE) feature, namely DPASP, for TP-PDT with excellent performances. DPASP exhibits high affinity to mitochondria, superior photostability, large two-photon absorption cross section as well as efficient reactive oxygen species generation, enabling it to achieve photosensitization both in vitro and in vivo under two-photon excitation. Moreover, its capability of stereotactic ablation of targeted cells with high-precision is also successfully demonstrated. All these merits make DPASP a promising TP-PDT candidate for accurate ablation of abnormal tissues with minimal damages to surrounding areas in the treatment of various diseases.

Single-Exosome-Counting Immunoassays for Cancer Diagnostics.

Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for noninvasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic microbeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome complexes per microliter (∼10-17 M). We demonstrated the application of the droplet digital ExoELISA platform in quantitative detection of exosomes in plasma samples directly from breast cancer patients. We believe our approach may have the potential for early diagnosis of cancer and accelerate the discovery of cancer exosomal biomarkers for clinical diagnosis.

Isothermal Amplification on a Structure-Switchable Symmetric Toehold Dumbbell-Template: A Strategy Enabling MicroRNA Analysis at the Single-Cell Level with Ultrahigh Specificity and Accuracy.

Accurate analysis of microRNAs (miRNAs) at the single-cell level seriously requires analytical methods possessing extremely high sensitivity, specificity and precision. By rational engineering of a structure-switchable symmetric toehold dumbbell-template (STD-template), we propose a novel isothermal symmetric exponential amplification reaction (SEXPAR) method. The sealed and symmetric structure of the STD-template allows exponential amplification reaction (EXPAR) to occur upon every annealing of target miRNA without loss of amplification efficiency. In addition, the rigid and compact structure of the STD-template with an appropriate standard free energy ensures SEXPAR only be activated by target miRNA. As a result, the SEXPAR method isothermally quantified let-7a down to 0.01 zmol (6.02 copies per 10 µL) with an ultrahigh specificity which is efficient enough to discriminate one-base-mismatched miRNAs, and a remarkably high precision even for the determination of 6.02 copies let-7a (the standard deviation was reduced from >60% down to 23%). The dynamic range was also extended to 10 orders of magnitude. The method was successfully applied for the determination of let-7a in human tissues, sera and even single-cell lysate, with obviously better precision than quantitative reverse transcription polymerase chain reaction (RT-qPCR) and other EXPAR-based methods. The SEXPAR method may serve as a powerful technique for the biological research and biomedical studies of miRNAs and other short nucleic acids.

An enzyme-free homogenous electrochemical assay for sensitive detection of the plasmid-mediated colistin resistance gene mcr-1.

Antibiotic resistance associated with the mcr-1 gene of Gram-negative bacteria, which confers resistance to drugs of last resort and has the potential to spread via plasmids, is one of the most pressing issues facing global health today. Point-of-care testing for the mcr-1 gene is needed to aid in the identification of colistin resistance in the field and to control its horizontal transmission. Here, we report the successful development of an enzyme-free homogenous electrochemical strategy for sensitive detection of the antibiotic resistance gene mcr-1 using the hybridization chain reaction and mcr-1-specific toehold probe. The long double-stranded DNA polymer produced using this strategy could be detected by assessing the diffusion of methylene blue towards the surface of a screen-printed gold electrode. Under optimized conditions, a linear relationship was observed between the variation of peak current and the natural logarithm of the mcr-1 gene concentration in the range of 1 nM to 1 µM with a detection limit of 0.78 nM (S/N = 3). This enzyme-free, isothermal platform is a rapid, portable, disposable, and sensitive method for detection of plasmid-mediated colistin resistance.

Optimization of the Original TRIzol-Based Technique Improves the Extraction of Circulating MicroRNA from Serum Samples.

BACKGROUND: MicroRNAs (miRNAs) are a group of small endogenous, non-coding RNA molecules that have been demonstrated to be essential regulators of many critical biological and pathological processes. Because of their high stability in blood and the strong implication of miRNA expression profiles for human diseases, miRNAs are currently emerging as promising circulating biomarkers for the diagnosis or prognosis of a variety of human diseases. The TRIzol-based technique has been widely used for cell or tissue RNA extraction because of its economy and reliability. However, the original TRIzol-based RNA isolation protocol was not specifically designed for microRNA extraction from serum samples. When it was used to extract serum microRNAs, due to the short sequence and low level of microRNAs, the isolation efficiency in most cases could not meet the requirement. To address this issue, in this study, an improved TRIzol-based protocol was established by modifying the extraction procedure of the original TRIzol-based protocol. The performance of the improved TRIzol-based protocol was evaluated by comparison with other methods. METHODS: Total RNA was isolated by the improved TRIzol-based method, the original method, and two other commonly used methods. RT-qPCR and spectrophotometry were used to examine the quality and yield of total RNA. RESULTS: The improved method was more efficient than the original protocol and more suitable for real-time PCR-based profiling experiments. CONCLUSIONS: The optimized TRIzol-based method described in this report is suitable for the extraction of serum microRNAs and useful for the development of microRNAs as diagnostic biomarkers.

Isolation and Enrichment of Extracellular Vesicles with Double-Positive Membrane Protein for Subsequent Biological Studies.

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.

Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

An adhesion-based method for rapid and low-cost isolation of circulating tumor cells.

BACKGROUND: Noninvasive monitoring of cancer through circulating tumor cells (CTCs) is hampered long by unsatisfactory CTCs testing techniques. Efficient isolation of CTCs in a rapid and price-favorable way from billions of leukocytes is crucial for testing. METHODS: We developed a new method based on the stronger adhesive power of CTCs versus leukocytes to sensitively isolate CTCs. Using a BSA-coated microplate and low-speed centrifuge, this method could easily separate cancer cells within 20 min at a very low cost. RESULT: The capture ratio can reach 70.7-86.6% in various cancer cell lines (breast/lung/liver/cervical/colorectal cancer) covering different epithelial-mesenchymal transformation (EMT) phenotypes and cell sizes, demonstrating the potential for efficient pan-cancer CTCs detection. Moreover, the label-free process can well preserve cell viability (∼99%) to fit downstream DNA/RNA sequencing. CONCLUSIONS: A novel technique for non-destructive and rapid enrichment of CTCs has been devised. It has enabled the successful isolation of rare tumor cells in the patient blood sample and pleural effusion, highlighting a promising future of this method in clinical translation.

Quantification of placental extracellular vesicles in different pregnancy status via single particle analysis method.

BACKGROUND: The nano-sized, lipid bilayer-delimited placental extracellular vesicles (PEVs) released by the placenta are now regarded as important mediators involved in various physiological and pathological processes of pregnant women. The number and contents of PEVs are significantly altered in preeclampsia and are considered as potential biomarkers. However, the distribution pattern of PEVs in the maternal circulation in different pregnancy status is still unclear for the limitation of the traditional method with low sensitivity. METHODS: In this work, we recruited 561 pregnant women with different pregnancy status and investigated the distribution pattern of PEVs in the maternal circulation based on a single extracellular vesicle analysis method and placental alkaline phosphatase (PLAP), a placenta-specific marker. RESULTS: The concentration of PEVs in pregnant women increased with the progression of gestational age, while the ratio of PEVs decreased to about 10% in the third trimester. Surprisingly, the PLAP+ EVs also presented in the plasma of non-pregnant women and normal male about 5%. The change in the ratio of PEVs can reflect the pregnancy status and also had a better diagnostic value in severe preeclampsia (AUC = 0.7811). CONCLUSIONS: Our study not only reveals the distribution pattern of PEVs, but also identifies the diagnostic potential of PEVs as biomarkers.

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs. This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg. Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

In Situ Generation of Red-to-NIR Emissive Radical Cations in the Stomach for Gastrointestinal Imaging.

Red-to-near-infrared (NIR) fluorescent probes, with advantages such as high spatiotemporal resolution and in situ sensing abilities, are highly attractive for diagnosis of gastrointestinal diseases and targeted drug development. However, conventional red-to-NIR fluorophores with electron closed-shell structures require tedious synthetic procedures for preparation, and it is difficult to further decorate them with sensing groups. In this study, a series of easily prepared pyrroles with simple structures that can quickly be transformed into red-to-NIR emissive radical cations in acidic buffer solution and in vivo stomachs is developed. The in-situ-generated red-to-NIR emissive pyrrole radical cations in the stomach have excellent biocompatibility and stability and can be used not only for intravital gastrointestinal imaging with high spatiotemporal resolution, but also for dynamic monitoring of the gastric emptying process and assessment of anti-gastric-acid therapy. The acidity-induced generation of pyrrole radical cations is believed to provide a facile strategy for developing red-to-NIR fluorophores and studying gastrointestinal diseases.

Design strategies and advanced applications of primer exchange reactions in biosensing: A review.

Early disease diagnosis relies on the sensitive detection and imaging of biomarkers. Signal amplification is one of the most commonly used methods to improve detection sensitivity. Primer exchange reaction (PER) is a novel signal amplification technique that has garnered attention because of its simple and sensitive features. The classical PER involves a single catalytic hairpin, which enables the attachment of custom sequences to the primer chain, generating a long repeat sequence that can bind numerous signaling molecules and achieve powerful signal amplification. Currently, numerous PER-based signal amplification strategies are available that can improve detection sensitivity and promote the development of the signal amplification field. This review focuses on the mechanism of typical PER, the diversification of PER, and PER-based biosensors for various targets. Finally, the challenges and prospects of PER development are discussed.