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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., 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.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., 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 © 2022. Published by Elsevier B.V.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 × 10 5 to 1.83 × 10 7 particles/μL (0.68-30.4 pM) with a detection limit of 3.43 × 10 5 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., 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 © 2020 Elsevier B.V. All rights reserved.)

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.

Rare cancer cells in body fluid could be useful biomarkers for noninvasive diagnosis of cancer. However, detection of these rare cells is currently challenging. In this work, a binding-induced dual catalytic hairpin assembly (DCHA) electrochemical cytosensor was developed for highly selective and sensitive detection of cancer cells. The fuel probe, released by hybridization between the capture probe and catalytic hairpin assembly (CHA) products of target cell-responsive reaction, initiated dual CHA recycling, leading to multiple CHA products. Furthermore, the hybridization between fuel probe and capture probe decreased non-specific CHA products, improving the signal-to-noise ratio and detection sensitivity. Under the optimal conditions, the developed cytosensor was able to detect cells down to 30 cells mL -1 (S/N = 3) with a linear range from 50 to 100,000 cells mL -1 and was capable of distinguishing target cells from normal cells in clinical blood samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel electrochemical sensing assay for sensitive determination of HIV-1 in a homogeneous solution has been developed using an electrochemical molecular beacon combined with a nafion-graphene composite film modified screen-printed carbon electrode (nafion-graphene/SPCE). The electrochemical molecular beacon (CAs-MB), comprising a special recognition sequence for the conserved region of the HIV-1 gag gene and a pair of carminic acid molecules as a marker, can indicate the presence of the HIV-1 target by its on/off electrochemical signal behavior. It is suitable for direct, electrochemical determination of HIV-1, thereby simplifying the detection procedure and improving the signal-to-noise (S/N) ratio. To further improve the sensitivity, the nafion-graphene/SPCE was used to monitor changes in the CAs-MB, which has notable advantages, such as being ultrasensitive, inexpensive, and disposable. Under optimized conditions, the peak currents showed a linear relationship with the logarithm of target oligonucleotide concentrations ranging from 40 nM to 2.56 μM, with a detection limit of 5 nM (S/N=3). This sensing assay also displays a good stability, with a recovery of 88-106.8% and RSD<7% (n=5) in real serum samples. This work may lead to the development of an effective method for early point-of-care diagnosis of HIV-1 infection., (© 2013 Elsevier B.V. All rights reserved.)

A novel electrochemical biosensor was developed for the analysis of BRAF V600E mutation in colorectal cancer cell samples based on a dual amplification strategy of amplification-refractory mutation system (ARMS) PCR and multiple enzyme labels. The labeled amplicons were conjugated on Fe3O4/Au nanoparticles using Au-S linkages. Alkaline phosphatases were then loaded onto the nanoparticles through biotin-streptavidin interactions. The resultant composite nanoparticles were characterized by transmission electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. In the presence of 2-phospho-l-ascorbic acid, the mutant alleles were quantified on a screen-printed carbon electrode (SPCE) from the anodic current of the enzymatic product, ascorbic acid. BRAF V600E mutant alleles concentrations as low as 0.8% were successfully determined in an excess of wild-type background. In a cell-line dilution model, the proposed method was more sensitive than were DNA sequencing and agarose gel electrophoresis. This work demonstrates a new strategy for sensitivly detecting BRAF V600E variations. It can pave the way for analyzing other rare mutations in complex cancer samples because of its high sensitivity, simplicity, low cost, and easy validation of assay procedures., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Acquired immune deficiency syndrome (AIDS) is a severe communicable immune deficiency disease caused by the human immune deficiency virus (HIV). The analysis laboratory diagnosis of HIV infection is a crucial aspect of controlling AIDS. The p24 antigen, the HIV-1 capsid protein, is of considerable diagnostic interest because it is detectable several days earlier than host-generated HIV antibodies following HIV exposure. We present herein a new sandwich HIV p24 immunosensor based on directly electroplating an electrode surface with gold nanoparticles using chronoamperometry, which greatly increased the conductivity and reversibility of the electrode. Under optimum conditions, the electrochemical signal showed a linear relationship with the concentration of p24, ranging from 0.01 ng/mL to 100 ng/mL (R > 0.99), and the detection limit was 0.008 ng/mL. Compared with ELISA, this method increased the sensitivity by more than two orders of magnitude (the sensitivity of ELISA for p24 is about 1 ng/mL). This immunosensor may be broadly applied to clinical samples, being distinguished by its ease of use, mild reaction conditions, guaranteed reproducibility, and good anti-interference ability.