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.

Single-particle analysis of circulating bacterial extracellular vesicles reveals their biogenesis, changes in blood and links to intestinal barrier.

Bacterial extracellular vesicles (BEVs) are nano-size particles secreted by bacteria that carry various bioactive components. These vesicles are thought to provide a new window into the mechanisms by which bacteria affect their hosts, but their fundamental proprieties within human remain poorly understood. Here, we developed a single-vesicle analytical platform that enabled BEV detection in complex biological samples of host. Using this platform, we found the presence of BEVs in the host circulation and they were mainly derived from gut microbes. We showed that the levels of circulating BEVs in humans significantly increased with aging due to an age-related increase in intestinal permeability. Significantly different levels of BEVs in blood were also found in patients with colorectal cancer and colitis. Together, our study provides new insights into circulating BEV biology and reveals their potential as a new class of biomarkers.

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.

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.

Rapid and efficient isolation platform for plasma extracellular vesicles: EV-FISHER.

Extracellular vesicles (EVs) have found diverse applications in clinical theranostics. However, the current techniques to isolate plasma EVs suffer from burdensome procedures and limited yield. Herein, we report a rapid and efficient EV isolation platform, namely, EV-FISHER, constructed from the metal-organic framework featuring cleavable lipid probes (PO4 3- -spacer-DNA-cholesterol, PSDC). The EV-FISHER baits EVs from plasma by cholesterol and separates them with an ordinary centrifuge. The captured EVs could be released and collected upon subsequent cleavage of PSDC by deoxyribonuclease I. We conclude that EV-FISHER dramatically outperforms the ultracentrifugation (UC) in terms of time (∼40 min vs. 240 min), isolation efficiency (74.2% vs. 18.1%), and isolation requirement (12,800 g vs. 135,000 g). In addition to the stable performance in plasma, EV-FISHER also exhibited excellent compatibility with downstream single-EV flow cytometry, enabling the identification of glypican-1 (GPC-1) EVs for early diagnosis, clinical stages differentiation, and therapeutic efficacy evaluation in breast cancer cohorts. This work portrays an efficient strategy to isolate EVs from complicated biological fluids with promising potential to facilitate EVs-based theranostics.

A portable smartphone-based hemoglobin point-of-care testing platform for accurate anemia diagnostics.

Anemia affects over 2 billion people worldwide, with the heaviest burden borne by women and children. At present, anemia is diagnosed by measuring hemoglobin (Hb) levels, which must be done in hospitals or commercial laboratories by skilled operators. In this work, we report a portable, affordable ($3), easy-to-operate (1 min) and accurate smartphone-based Hb analyzer (SHbA) that uses a drop of finger-pricked blood for anemia point-of-care test (POCT) applications. POCT of Hb was achieved using a smartphone ambient light sensor (ALS) to accurately measure the absorbance of colorimetric Hb biochemical analysis reagents in a microcuvette, as well as an Android-based application for results analysis. SHbA validation results agreed well with those reported by a hematology analyzer, and the SHbA has an anemia diagnosis sensitivity of 95.4% and specificity of 96.3% for venous blood (n = 360) and a sensitivity of 96.39% and specificity of 95.58% for fingertip blood (n = 475). In addition, SHbA exhibits excellent performance in the diagnosis and treatment guidance of anemia high-risk populations, including tumor chemotherapy patients (n = 424), pregnant women (n = 214) and thalassemia patients (n = 208). Importantly, volunteer self-testing results (n = 20) indicate that SHbA can be used for home-based anemia diagnosis and monitoring. SHbA has the advantages of high sensitivity and specificity while being cheap and easy to operate, making it widely applicable for the diagnosis and treatment of anemia, especially for high-risk patients in areas with poor medical resources.

A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction.

Existing detection methods for pathogen nucleic acid detection, such as polymerase chain reaction (PCR), are complicated and expensive to perform. Here, we report a simple and versatile strategy for highly sensitive detection of pathogen nucleic acid based on toehold-mediated strand displacement initiated primer exchange amplification (t-PER). In the presence of the target, the blocked hairpin substrate is released by toehold-mediated strand displacement, which triggers the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER open the molecular beacon to recover the fluorescence signal. The t-PER protocol also successfully directly detected human papilloma virus from clinical cervical swab samples, with consistent results compared to real time-polymerase chain reaction (RT-PCR). Moreover, the versatility and clinical feasibility of this method was further confirmed by measuring Epstein-Barr virus, hepatitis B virus, and Ureaplasma urealyticum from different clinical samples (serum samples and urine samples). This simple platform enabled specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.

Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection.

The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a "one to more" amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL-1. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.

DNA Nanowire Guided-Catalyzed Hairpin Assembly Nanoprobe for In Situ Profiling of Circulating Extracellular Vesicle-Associated MicroRNAs.

Extracellular vesicle-associated miRNAs (EV-miRNAs) are emerging as a new type of noninvasive biomarker for disease diagnosis. Their relatively low abundance, however, makes accurate detection challenging. Here, we designed a DNA nanowire guided-catalyzed hairpin assembly (NgCHA) nanoprobe for profiling EV-miRNAs. NgCHA showed high penetrability to EVs, which allowed rapid delivery of the probes into EVs. In the presence of targeted miRNAs within EVs, a fluorescent signal could be generated and amplified by confining the catalytic hairpin assembly system within the nanowires, thus greatly enhancing the analytical sensitivity. We showed that EV-miRNAs from various cell lines could be accurately quantified by NgCHA in situ. By using a four-EV-miRNA panel, this platform can identify patients with breast cancer at an early stage with 95.2% sensitivity and 86.7% specificity. Its applications for risk assessment as well as cancer type prediction were also successfully demonstrated. This platform is sensitive, low-cost, and simple compared with current methods. It may thus serve as a promising tool for the noninvasive diagnosis and monitoring of cancers and other diseases through EV-miRNA profiling.

Aggregation-Induced Emission Luminogen-Based Dual-Mode Enzyme-Linked Immunosorbent Assay for Ultrasensitive Detection of Cancer Biomarkers in a Broad Concentration Range.

The enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods for measuring antibodies and antigens in biological samples. However, developing new ELISAs with high detection sensitivity and broad detection dynamic ranges without resorting to complicated signal processing and equipment setups remains a challenge. In this work, we report a strategy to simultaneously improve the detection sensitivity and broaden the dynamic range by replacing the chromogenic reagents used in traditional ELISAs with an aggregation-induced emission luminogen (AIEgen). The developed AIE-ELISA could generate complementary absorbance and fluorescence signals with a linear detection range of 1.6-25,000 pg/mL. The application of this dual-mode AIE-ELISA in the detection of the prostate-specific antigen (PSA) realized a limit of detection of 1.3 pg/mL (3.78 × 10-14 M) and dynamic range improvement of approximately 2 orders of magnitude compared to a single-mode ELISA, which enabled it to discriminate a minor PSA difference in a patient's serum. The simpler experimental operation, faster enzyme response speed, and better photostability of AIEgen than the traditional chromogenic reagents used in ELISAs showed that our developed AIE-ELISA holds great potential in the fields of immunoassay, immunohistochemistry, and immunocytochemistry.

Synergistic Enhancement of Fluorescence and Magnetic Resonance Signals Assisted by Albumin Aggregate for Dual-Modal Imaging.

Dual-modal fluorescence and magnetic resonance imaging (FLI/MRI) is important for the early diagnosis of malignant tumors. However, facile and opportune strategies to synergistically enhance fluorescence intensity and magnetic resonance (MR) contrast have rarely been reported. Herein, we present a facile strategy using albumin aggregates (AAs) to synergistically enhance the fluorescence intensity by aggregation-induced emission (AIE) and MR contrast with prolonged rotational correlation time (τR) of Gd(III) chelates and the diffusion correlation time (τD) of surrounding water molecules. The amphiphilic dual-modal FLI/MRI probe of NGd was facilely loaded into albumin pockets and then formed AAs to generate a supramolecular structure of NGd-albumin aggregates (NGd-AAs), which show excellent biocompatibility and biosafety, and exhibit superior fluorescence quantum yield and r1 over NGd with 6- and 8-fold enhancement, respectively. Moreover, compared with the clinical MRI contrast agent Gd-DOTA, r1 of NGd-AAs showed a 17-fold enhancement. Therefore, NGd-AAs successfully elicited high-performance dual-modal FLI/MRI in vitro and in vivo and high contrast MR signals were observed in the liver and tumor after intravenous injection of NGd-AAs at a dosage of 6 µmol Gd(III)/kg body weight. This generic and feasible strategy successfully realized a synergistic effect for dual-modal FLI/MRI.

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.

Rapid electrochemical biosensor for sensitive profiling of exosomal microRNA based on multifunctional DNA tetrahedron assisted catalytic hairpin assembly.

Profiling of exosomal microRNA (exo-miRNA) is very important for cancer diagnosis and treatment. However, rapid and sensitive determination the trace of exo-miRNA in clinical samples has not been developed. Herein, a robust electrochemical biosensor was proposed using multifunctional DNA tetrahedrons assisted catalytic hairpin assembly (MDTs-CHA) for exo-miRNA analysis. The MDTs-CHA, contained two multifunctional tetrahedrons (T1 and T2), leverage localized reaction and cascade amplification to enable rapid and ultrasensitive exo-miRNA analysis. Employing the MDTs-CHA, the electrochemical platform allowed quantitative measurement of exo-miRNA down to 7.2 aM in 30 min with good specificity. Furthermore, by profiling four tumor-associated exo-miRNAs (miR-1246, miR-221, miR-375, and miR-21) in a breast cancer cohort, this platform showed high efficiency (AUC: 0.989) and high sensitivity of 90.5% for breast tumors diagnoses, with 80% sensitivity for early diagnoses (stage I-IIa). Therefore, this platform has great potential in bioanalysis and clinical diagnostics.

Identification of Transcriptional Variation in Aortic Remodeling Using a Murine Transverse Aortic Constriction (TAC) Model.

Arterial remodeling is a major pathological consequence of hypertension, which is recognized as the most common chronic non-communicable disease. However, the detailed mechanism of how arterial remodeling is induced by hypertension has not yet been fully elucidated. Evaluating the transcriptional changes in arterial tissue in response to elevated blood pressure at an early stage may provide new insights and identify novel therapeutic candidates in preventing arterial remodeling. Here, we used the ascending aorta of the transverse aortic constriction (TAC) model to induce arterial remodeling in C57BL/6 male mice. Age-matched mice were subjected to sham surgery as controls. The TAC model was only considered successful if the mice conformed to the criteria (RC/LC blood flow velocity with 5-10-fold change) 1 week after the surgery. Two weeks after surgery, the ascending aorta developed severe remodeling in TAC mice as compared to the sham group. High throughput sequencing was then applied to identify differentially expressed (DE) transcripts. In silicon analysis were then performed to systematically network transcriptional changes. A total of 1,019 mRNAs were significantly changed between TAC and the sham group at the transcriptional level. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that stress/stimulus/immune-related biological processes played a crucial role during arterial remodeling. Our data provide a comprehensive understanding of global gene expression changes in the TAC model, which suggests that targeting inflammation and vascular smooth cell transformation are potential therapeutic strategies to interfere with the aortic remodeling at an early stage in the development of hypertension.

Facile fluorescent aptasensor using aggregation-induced emission luminogens for exosomal proteins profiling towards liquid biopsy.

Surface protein patterns of tumor-derived exosomes could be promising noninvasive diagnostic biomarkers for liquid biopsy. However, a convenient and cost-effective platform for exosomal protein profiling is still lacking. Herein, a facile fluorescent aptasensor is developed to assess exosomal tumor-associated proteins, combining aptamers, aggregation-induced emission luminogens (AIEgens), and graphene oxide (GO) as recognition elements, fluorescent dye, and the quencher, respectively. Specifically, numberous TPE-TAs could bind one aptamer and form aggregates rapidly, resulting in an amplified fluorescence signal. In the absence of tumor-derived exosomes, GO absorbs the TPE-TAs/aptamer complex, allowing fluorescence quenching. When the target exosomes are introduced, the aptamer preferentially binds with its target. Thus the TPE-TAs/aptamer complexes detach from GO surface, followed by the appearance of a "turn-on" fluorescent signal. Under the optimized conditions, the linear range of target exosomes is estimated to be 4.07 × 105 to 1.83 × 107 particles/µL (0.68-30.4 pM) with a detection limit of 3.43 × 105 particles/µL (0.57 pM). This strategy demonstrated great performance in differentiating prostate cancer from healthy individuals (AUC: 0.9790). Furthermore, by profiling three tumor-associated protein markers including epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2) on exosomes in a breast tumor cohort, this sensing platform diagnoses breast tumors with high efficiency (AUC: 0.9845) and exhibits a high sensitivity of 97.37% for distinguishing malignant breast cancers, where the stage I cases were detected with 92.31% sensitivity. Therefore, this aptasensor provides a promising strategy to profile tumor-derived exosomal proteins for early diagnosis in liquid biopsy.

Homogenous Magneto-Fluorescent Nanosensor for Tumor-Derived Exosome Isolation and Analysis.

Tumor-derived exosomes carrying unique surface proteins have shown great promise as novel biomarkers for liquid biopsies. However, point-of-care analysis for tumor-derived exosomes in the blood with low-cost and easy processing is still challenging. Herein, we develop an integrated approach, homogenous magneto-fluorescent exosome (hMFEX) nanosensor, for rapid and on-site tumor-derived exosomes analysis. Tumor-derived exosomes are captured immunomagnetically, which further initiates the aptamer-triggered assembly of DNA three-way junctions in homogenous solution containing aggregation-induced emission luminogens and graphene oxide, resulting in an amplified fluorescence signal. By integrating magnetic isolation and enhanced fluorescence measurement, the hMFEX nanosensor detects tumor-derived exosomes in the dynamic range spanning 5 orders of magnitude with high specificity, and the limit of detection is 6.56 × 104 particles/µL. Analyzing tumor-derived exosomes in limited volume plasma from breast cancer patients demonstrates the excellent clinical diagnostic efficacy of the hMFEX nanosensor. This study provides new insights into the point-of-care testing of tumor-derived exosomes for cancer diagnostics.

Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases.

Noninvasive prenatal detection of monogenic diseases based on cell-free DNA is hampered by challenges in obtaining a sufficient fraction and adequate quality of fetal DNA. Analyzing rare trophoblastic cells from Papanicolaou smears carrying the entire fetal genome provides an alternative method for noninvasive detection of monogenic diseases. However, intracellular labeling for identification of target cells can affect the quality of DNA in varying degrees. Here, a new approach is developed for nondestructive identification of rare fetal cells from abundant maternal cells based on endoplasmic reticulum staining and linear discriminant analysis (ER-LDA). Compared with traditional methods, ER-LDA has little effect on cell quality, allowing trophoblastic cells to be analyzed on the single-cell level. Using ER-LDA, high-purity of trophoblastic cells can be identified and isolated at single cell resolution from 60 pregnancies between 4 and 38 weeks of gestation. Pathogenic variants, including -SEA/ deletion mutation and point mutations, in 11 fetuses at risk for α- or ß-thalassemia can be accurately detected by this test. The detection platform can also be extended to analyze the mutational profiles of other monogenic diseases. This simple, low-cost, and noninvasive test can provide valuable fetal cells for fetal genotyping and holds promise for prenatal detection of monogenic diseases.

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.

A novel nest hybridization chain reaction based electrochemical assay for sensitive detection of circulating tumor DNA.

As an ideal biomarker candidate, circulating tumor DNA (ctDNA) plays a vital role in noninvasive diagnosis of cancer. However, most traditional approaches for quantifying ctDNA are cumbersome and expensive. In the present work, a novel electrochemical biosensor based on nest hybridization chain reaction was proposed for the sensitive and specific detection of PIK3CA E545K ctDNA with a simple process. The nest hybridization chain reaction was initiated by the hybridization of two dumbbell-shaped DNA units which were assembled by two classes of well-designed DNA probes respectively, leading to the formation of a complex DNA structure. In the presence of target ctDNA, the amplified hybridization chain reaction products were captured by target ctDNA, resulting in a significant increase of electrochemical signal. Under the optimal conditions, the developed biosensor exhibited good analytical performance for the detection of target ctDNA with the linear range from 5 pM to 0.5 nM and the detection limit of 3 pM. Furthermore, this assay was successfully applied to the detection of ctDNA in spiked-in samples, pleural effusion and serum samples of malignant tumor patients. This simple and cost-effective sensing system holds great potentials for ctDNA detection and cancer diagnosis.