Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 × 106 particles mL-1), 1.1 times higher purity (1.66 × 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 × 103 particles μL-1 (linear range from 5.0 × 103 to 5.0 × 107 μL-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.

Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 & times; 106 particles mL-1), 1.1 times higher purity (1.66 & times; 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 & times; 103 particles mu L-1 (linear range from 5.0 & times; 103 to 5.0 & times; 107 mu L-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.

Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA N 6-methyladenosine (m6A) modification regulation and function. However, challenges still remain due to the high context complexities, which call for a versatile toolbox for accurate live-cell monitoring of effectors. Here, we propose a demethylation-switchable aptamer sensor engineered with a site-specific m6A (DSA-m6A) for lag-free monitoring of the m6A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m6A model, as the "universal energy currency" role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m6A sensor upon demethylation and ATP binding in living organisms, thus enabling sensitive monitoring of FTO activity with neither time delay nor recourse to extra supply of substances. This ATP-driven DSA-m6A design facilitates biomedical research, including live-cell imaging, inhibitor screening, single-cell tracking of dynamic FTO nuclear translocation upon starvation stimuli, FTO characterization in a biomimetic heterotypic three-dimensional (3D) multicellular spheroid model, as well as the first report on the in vivo imaging of FTO activity. This strategy provides a simple yet versatile toolbox for clinical diagnosis, drug discovery, therapeutic evaluation, and biological study of RNA demethylation.

This article illustrated a novel method combining loop-mediated isothermal amplification (LAMP) with molec-ular beacon (MB) to specifically detect Mycobacterium tuberculosis (MTB) IS6110 gene. In this method, we designed a double-stranded DNA tail (S and Sc) at the 5' ends of the loop primers (LF and LB). When the target was present, the Sc would be continually generated by strand displacement reactions to hybridize with MB, then fluorescence signals were detected. Negligible background fluorescence would be detected in the absence of target due to the unparalleled advantage of MB. Under the optimized conditions (6 mM Mg2+, 0.48 U/mu L Bst 2.0 polymerase, 0.2 mu M MB and 2:1 annealing ratio of S-LF/LB to Sc), the detection limit of this assay was 4.3 x 104 copies mu L-1. The whole detection time was reduced to less than 30 min with a low cost of $2.37 per sample. We also successfully applied this method in real samples detection, which meant that this specific, fast and cost-effective method has the potential in clinical diagnosis of Tuberculosis (TB).

Cost-effective methods for DNA genotyping were needed because single nucleotide polymorphisms (SNPs) were essential biomarkers associated with many diseases. Allele-specific PCR (AS-PCR) has the advantages of mature instruments and high sensitivity. But conventional AS-PCR needs to multiply the number of reactions or primers for multiple targets, which complicates the operation and increases the cost. Herein, we proposed a novel AS-PCR method for multiple SNP genotyping in a single run. Wild-type allele-specific primer (WT primer) was designed for each target gene. The sample and WT primers only needed to undergo multiplexed AS-PCR once simulta-neously. After AS-PCR, the concentration of remaining primers varied among the samples of each genotype combination, due to the different matching performance between template and WT primers. The remaining primers then triggered multiplexed molecular beacon-rolling circle amplification, and the molecular beacons labelled with different fluorescent dyes corresponded to different targets. The fluorescence ratios of the sample to the positive control were used as the genotyping indexes. This method was able to detect samples with con-centrations as low as 10 fM. We successfully applied the method to the multiple genotyping of 23 hair root samples for ADH1B and ALDH2 genes, obtaining completely consistent results with sequencing. The reagent cost was 0.6 dollar for one sample, showing a good cost performance. This proposed approach had a great application prospect in simultaneously rapid and accurate genotyping of multi-SNPs, and provided a new method for personalized health management.

Protein p53 induces cell growth arrest, differentiation, apoptosis and DNA repair. More than 50% of malignant tumors produce mutations in the p53 gene. Hence, p53 sequence-specific analysis helps in early cancer diagnosis, increasing the treatment success rate. The single nucleic acid amplification method limits sensitivity, so we innovatively integrated exonuclease III (ExoIII)-assisted target recycling amplification with multi-site enzyme polymerization labeling. The integration dramatically improved the biosensor's sensitivity. Given the nonspecific reaction of ExoIII, we optimized the ExoIII amplification system to reduce false positives and background current. The single-strand DNA produced in the ExoIII system served as the bridge between the electrode and signal DNA tetrahedron (sTDN). Moreover, sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. Compared with single-stranded signal probes, sTDN modified by terminal transferase provided higher catalytic efficiency. Under the optimal experimental conditions, the cathodic current exhibited a logarithmic relation over 1.0-5.0 x 10(6) fM p53, with a detection limit of 0.47 fM. Applying this biosensor has successfully detected p53 in serum samples, providing a potential tool for cancer diagnosis and treatment.

High-cost viral nucleic acid detection devices (e.g., qPCR system) are limited resources for developing counties and rural areas, leading to underdiagnosis or even pandemics of viral infectious diseases. Herein, a novel virus detection strategy is reported. Such detection method is enabled by TR512-peptide-based biorthogonal capture and enrichment of commercially available Texas red fluorophore labeled nucleic acid on the functionalized paper. The GST-TR512 fusion protein electrostatically immobilized on the paper is constructed to retain the binding affinity of TR512-peptide toward Texas red fluorophore labeled nucleic acid released in the preamplification process, then the enrichment of analytes enhances fluorescence signal for rapid detection as volume of sample filters through the paper. The method is generally applicable to different nucleic acid preamplification strategies (PCR, RAA, CRISPR) and different virus types (Hepatitis B virus (HBV), African swine fever virus (ASFV), human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 or 2019 nCoV)). Finally, a full-set virus detection device is developed in house to detect the presence of Hepatitis B virus (HBV) viral gene in patients' blood samples. Taken together, we first apply TR512-peptide in the signal enrichment and the novel detection strategy may offer an inexpensive, rapid, and portable solution for areas with limited access to a standard diagnosis laboratory.

Cost-effective methods for DNA genotyping were needed because single nucleotide polymorphisms (SNPs) were essential biomarkers associated with many diseases. Herein, we proposed a single nucleotide polymorphism (SNP) genotyping method termed remaining allele-specific PCR (AS-PCR) primer-triggered molecular beacon rolling circle amplification (MB-RCA) to detect mutations in ADH1B and ALDH2 simultaneously. The genotyping strategy relied on the sharing primers whose 3’-terminal bases were specific to wild type alleles of ADH1B and ALDH2 respectively. The more sharing primers were consumed in AS-PCR, the fewer primers triggered the MB-RCA. The fluorescent intensities of MB-RCA were decided by the genotype of samples. The fluorescent intensity ratios of sample to positive control were used to discriminate allele genotypes: ratio threshold for ADH1B (0 ~ <0.36: wild type homozygote; 0.36 ~ <0.70: heterozygote; 0.70 ~ 1: mutant homozygote), ratio threshold for ALDH2 (0 ~ <0.36: wild type homozygote; 0.36 ~ <0.60: heterozygote; 0.60~ 1: mutant homozygote). The genotypes of 23 samples were determined by our method and obtained identical results with commercial sequencing. Our strategy only took 0.6 dollar for a sample, which showed great potentials in the field of alcohol metabolism capacity evaluation, disease predisposition and personalized treatment.

The great challenge for loop-mediated isothermal amplification (LAMP) is target-specific detection. However, the most popular fluorogenic probe method needs complicated sequence design and large reagent consumption. Rolling circle amplification (RCA), known for its simplicity, is presumably compatible with LAMP in single-tube reaction detection based on Bst DNA polymerase to realize universality. Therefore, this work combined LAMP with RCA through strand displacement strategy to determine pathogens. Specifically, pathogen gene initiated LAMP process, displacing two single strands (S1 and S2) hybridized with LAMP loop primers for dual-primer rolling circle extension. Then, Molecular beacon (MB) probe paired to RCA amplicons emitting fluorescence signal for real-time detection. For detecting different pathogens, LAMP primers were specifically designed without changing the detection strategies through same RCA set. The established LAMP-RCA technique quantitatively detected invA and malB ranging from 10(2) to 10 (6) copies mu L-1, also demonstrating the universality. Meanwhile, S. typhimurium without genomic DNA extraction was directly quantified ranging from 10(2) to 3.16 x 10(4) CFU mu L-1, with a detection limit of 32 CFU mu L -1 . With good selectivity, this work was successfully applied for S. typhimurium determination in 10 % milk, indicating that the established method had considerable potential in complex samples.

Single nucleotide polymorphism (SNP) analysis based on allele-specific polymerase chain reaction (AS-PCR) is a relatively effective and economical method compared with other genotyping technologies such as DNA sequencing, DNA hybridization and isothermal amplification strategies. But AS-PCR is limited by its labor-intensive optimization of reaction parameters and time-consuming result assessment. In this study, we put forward a novel idea of data processing to address this problem. SNP analysis was accomplished by AS-PCR with endpoint electrochemical detection. For each sample, two separate reactions were run simultaneously with two sets of allele-specific primers (wild-type primers for W system and mutant primers for M system). We measured their redox current signals on screen-printed electrodes once AS-PCR finished and calculated the difference value of current signals between two systems to determine the genotyping result. Based on the difference value of fluorescent signals, real-time fluorescent PCR was used to study reaction parameters in AS-PCR. With screened parameters, we obtained the genotyping results within 50 min. 36 hair-root samples from volunteers were analyzed by our method and their genotypes of ALDH2 gene (encoding aldehyde dehydrogenase 2) were totally identical with data from commercialized sequencing. Our work first employed difference value between two reaction systems to differentiate allele and provided a novel idea of data processing in AS-PCR method. It is able to promote the quick analysis of SNP in the fields of health monitor, disease precaution, and personalized diagnosis and treatment.