MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA.

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64-106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification.

Prokaryotic Argonautes (pAgos) have been recently used in many nucleic acid biosensing applications but have rarely been used for regulating the isothermal amplification system. Herein, we reported Thermus thermophilus Argonaute (TtAgo)-mediated background-suppressed exponential isothermal amplification (EXPAR) as the first example to explore the binding activity of pAgos toward regulation of the amplification template. It was demonstrated that thermophilic pAgos efficiently eliminated nonspecific hybridization between templates by their binding affinity with the template, resulting in greatly enhancing the specificity of EXPAR. TtAgo-mediated, background-suppressed EXPAR was employed to detect miRNA with a detection limit of 10-15 M, which was 1000 times and 100 times more sensitive than that of traditional RT-PCR and EXPAR, respectively. This method further showed good performance in discriminating cancer patients from healthy individuals, indicating its potential for practical clinical applications.

Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease Activity of Prokaryotic DNA Polymerase I.

In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs.

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Hybridized Chain Reaction-Amplified Alkaline Phosphatase-Induced Ag-Shell Nanostructure for the Sensitive and Rapid Surface-Enhanced Raman Scattering Immunoassay of Exosomes.

Exosomes are small extracellular vesicles that can be utilized as noninvasive biomarkers for diagnosis and treatment of cancer and other diseases. This study reports on a strategy involving a hybridized chain reaction-amplified chain coupled with an alkaline phosphatase-induced Ag-shell nanostructure for the ultrasensitive and rapid surface-enhanced Raman scattering immunoassay of exosomes. Exosomes from prostate cancer were captured using prostate-specific membrane antigen aptamer-modified magnetic beads; then, the hybridized chain reaction-amplified chain was released, incorporating a large number of functional moieties with signal amplification effects. Moreover, the steps of traditional immunoassay were simplified using magnetic materials, and the rapid, sensitive, and accurate detection of exosomes was achieved. Results could be obtained within 40 min with a detection limit of 19 particles/µL. Furthermore, the sera of human prostate cancer patients could be easily distinguished from those of healthy controls, highlighting the potential use of exosome analysis in clinical diagnostics.

Artificial Nucleotide Aptamer-Based Field-Effect Transistor for Ultrasensitive Detection of Hepatoma Exosomes.

An aptamer-based field-effect transistor (Apta-FET) is a well-developed assay method with high selectivity and sensitivity. Due to the limited information density that natural nucleotide library holds, the Apta-FET faces fundamental restriction in universality to detect various types of analytes. Herein, we demonstrate a type of Apta-FET sensors based on an artificial nucleotide aptamer (AN-Apta-FET). The introduction of an artificial nucleotide increases the diversity of the potential aptamer structure and expands the analyte category of the Apta-FET. The AN-Apta-FET specifically detects hepatoma exosomes, which traditional Apta-FET fails to discriminate from other tumor-derived exosomes, with a limit of detection down to 242 particles mL-1. The AN-Apta-FET distinguishes serum samples of hepatocellular carcinoma patients within 9 min from those of healthy people, showing the potential as a comprehensive assay tool in future disease diagnosis.

Simultaneous Rapid Nucleic Acid and Protein Detection in a Lateral Chromatography Chip for COVID-19 Diagnosis.

In this work, we report a fast, portable, and economical microfluidic platform for the simultaneous detection of nucleic acid and proteins. Using SARS-CoV-2 as a target, this microfluidic chip enabled to simultaneously detect the SARS-CoV-2 RNA (N gene) antigen (or specific IgG antibody) with respective detection limits of 1 copy/µL for nucleic acid, 0.85 ng/mL for antigen, and 5.80 ng/mL for IgG within 30 min with high stability and anti-interference ability. The capability of this system in clinical applications was further evaluated using clinical samples, displaying 100% sensitivity and 100% specificity for COVID-19 diagnosis. These findings demonstrate the potential of this method to be used for the detection and subsequent control of pathogens.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM).

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Aptamer-Initiated Catalytic Hairpin Assembly Fluorescence Assay for Universal, Sensitive Exosome Detection.

Cancer-cell-derived exosomes are regarded as noninvasive biomarkers for early cancer diagnosis because of their critical roles in intercellular communication and molecular exchange. A robust aptamer-initiated catalytic hairpin assembly (AICHA) fluorescence assay is proposed for universal, sensitive detection of cancer-derived exosomes. The AICHA was verified with the specific detection of MCF-7 cell-derived exosomes with a wide calibration range of 8.4 particles/µL to 8.4 × 105 particles/µL and a low detection limit (LOD) of 0.5 particles/µL. The universality of the AICHA method was verified for PANC-1 cell-derived exosomes, the LOD of which was determined to be 0.1 particles/µL. The performances in serum samples were detected with a recovery rate range of 95.45-106.2%, which demonstrates its significant potential for protein biomarker analysis and cancer diagnosis.

Isothermal gene amplification coupled MALDI-TOF MS for SARS-CoV-2 detection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide for more than a year and has undergone several mutations and evolutions. Due to the lack of effective therapeutics and long-active vaccines, accurate and large-scale screening and early diagnosis of infected individuals are crucial to control the pandemic. Nevertheless, the current widely used RT-qPCR-based methods suffer from complicated temperature control, long processing time and the risk of false-negative results. Herein, we present a three-way junction induced exponential rolling circle amplification (3WJ-eRCA) combined MALDI-TOF MS assay for SARS-CoV-2 detection. The assay can detect simultaneously the target nucleocapsid (N) and open reading frame 1 ab (orf1ab) genes of SARS-CoV-2 in a single test within 30 min, with an isothermal process (55 °C). High specificity to discriminate SARS-CoV-2 from other coronaviruses, like SARS-CoV, MERS-CoV and bat SARS-like coronavirus (bat-SL-CoVZC45), was observed. We have further used the method to detect pseudovirus of SARS-CoV-2 in various matrices, e.g. water, saliva and urine. The results demonstrated a great potential of the method for large scale screening of COVID-19, which is an important part of the pandemic control.

Calix[4]pyrrole-based Crosslinked Polymer Networks for Highly Effective Iodine Adsorption from Water.

A series of calix[4]pyrrole-based crosslinked polymer networks designed for iodine capture is reported. These materials were prepared by Sonogashira coupling of α,α,α,α-tetra(4-alkynylphenyl)calix[4]pyrrole with bishalide building blocks with different electronic properties and molecular sizes. Despite their low Brunauer-Emmett-Teller surface areas, iodine vapor adsorption capacities of up to 3.38 g g-1 were seen, a finding ascribed to the presence of a large number of effective sorption sites including macrocyclic π-rich cavities, aryl units, and alkyne groups within the material. One particular system, C[4]P-BTP, was found to be highly effective at iodine capture from water (uptake capacity of 3.24 g g-1 from a concentrated aqueous KI/I2 solution at ambient temperature). Fast capture kinetics (kobs =7.814 g g-1 min-1 ) were seen. Flow-through adsorption experiments revealed that C[4]P-BTP is able to remove 93.2 % of iodine from an aqueous source phase at a flow rate of 1 mL min-1 .

Surface Plasmon Coupling Electrochemiluminescence Immunosensor Based on Polymer Dots and AuNPs for Ultrasensitive Detection of Pancreatic Cancer Exosomes.

Polymer dots (Pdots) have become attractive electrochemiluminescence (ECL) luminophores due to their facile synthesis, easy modification, and stable electrochemical and optical properties. However, their ECL efficiency is not high enough for practical applications. In this work, we proposed an ECL immunosensor based on localized surface plasmon resonance (LSPR) between AuNPs and Pdots for the determination of pancreatic cancer exosomes. Based on the finite-difference time-domain simulations and the band energy of Pdots and AuNPs, we proposed the possible LSPR mechanism. The hot electrons of plasmonic AuNPs were photoexcited to surface plasmon states by ECL emission of Pdots, and then the excited hot electrons were transferred to the conduction band of Pdots, which significantly improved the ECL efficiency of Pdots. The ECL immunosensor displayed a wide calibration range of 1.0 × 103 to 1.0 × 106 particles/mL with a detection limit of 400 particles/mL. Cancer-related protein profiling revealed high selectivity toward different expressions of exosomal surface proteins from PANC-01, HeLa, MCF-7, and HPDE6-C7 cell lines. The proposed ECL system exhibits a promising prospect for protein biomarker profiling and early cancer-related diagnosis.

Sandwich/competitive immuno-sensors on micro-interface for SARS-CoV-2 neutralizing antibodies.

A simple, rapid and robust method to quantify SARS-CoV-2 neutralizing antibodies (nAbs) is urgently needed for determining COVID-19 serodiagnosis, vaccine development and evaluation of vaccine efficacy. In this study, we report sandwich/competitive immuno-sensors based on lateral chromatography micro-interface for accurate quantification of SARS-CoV-2 nAbs. Fluorescent microspheres (FMS) labeled receptor binding domain (RBD) antigen was prepared for detection of nAbs with high sensitivity. Sandwich and competitive immunoassay were conducted on the microfluidic-based sensor within 10 min and the fluorescent signal of immunoassay was analyzed by a portable microfluidic immunoassay instrument. The nAbs detection range of sandwich immuno-sensor and competitive immuno-sensor was 4.0 ng/mL to 400 ng/mL and 2.13 ng/mL to 213 ng/mL, respectively. Furthermore, the sandwich immuno-sensor was demonstrated to be comparable with existing methods and used to detect 182 clinical serum samples from vaccinated individuals. Sandwich immuno-sensor based on lateral chromatography micro-interface allowed reliable, fast, and low-cost detection of nAbs, which holds considerable potential for nAbs testing.

Rapid differential diagnosis of the B.1.617.2 (delta) variant of SARS-CoV-2 using an automated Cas12a-microfluidic system.

An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.

Facile Synthesis Strategy from Sludge-Derived Extracellular Polymeric Substances to Nitrogen-Doped Graphene Oxide-Like Material and Quantum Dots.

Extracellular polymeric substances (EPS) are microbial aggregates derived from waste sewage sludge accumulated in sewage treatment plants, which provides natural, renewable, and abundant carbon, nitrogen, oxygen sources for the development of carbon materials to achieve the value-added utilization of waste sewage sludge resources. In this work, a nitrogen-doped graphene oxide (GO)-like material (N-GO) was simply produced using EPS as starting materials. A facile H2O2 oxidation-assisted method (room temperature) was developed to synthesize nitrogen-doped GO-like quantum dots (N-GOQDs) with strong tunable fluorescence from N-GO for the first time. This approach eliminates the conventional use of toxic chemicals, concentrated acids as well as expensive equipment, and strict condition requirements, which provides new insights into the synthesis of N-GO and N-GOQDs. In addition, this H2O2-assisted method was further demonstrated to prepare yellow fluorescent GO quantum dots (GOQDs) from GO successfully. The as-prepared N-GO shows excellent adsorption capacity for removing organic matters (malachite green, rhodamine B, and methylene blue) from water in 10 min. The water-soluble N-GOQDs were demonstrated to be a low toxicity and good biocompatibility fluorescence probe for bioimaging.

Dual-modality loop-mediated isothermal amplification for pretreatment-free detection of Septin9 methylated DNA in colorectal cancer.

Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is  proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/µL with a limit of detection of 0.02 ± 0.002 ng/µL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids. A dual-modality loop-mediated isothermal amplification (LAMP) integrated with immuno-magnetic beads (IMB) enrichment was proposed for the determination of methylated Septin9 gene in colorectal cancer (CRC).

Recent Progress in Detection and Profiling of Cancer Cell-Derived Exosomes.

Exosomes, known as nanometer-sized vesicles (30-200 nm), are secreted by many types of cells. Cancer-derived exosomes have great potential to be biomarkers for early clinical diagnosis and evaluation of cancer therapeutic efficacy. Conventional detection methods are limited to low sensitivity and reproducibility. There are hundreds of papers published with different detection methods in recent years to address these challenges. Therefore, in this review, pioneering researches about various detection strategies are comprehensively summarized and the analytical performance of these tests is evaluated. Furthermore, the exosome molecular composition (protein and nucleic acid) profiling, a single exosome profiling, and their application in clinical cancer diagnosis are reviewed. Finally, the principles and applications of machine learning method in exosomes researches are presented.

Lab in a tube: Isolation, extraction, and isothermal amplification detection of exosomal long noncoding RNA of gastric cancer.

Tumor-derived exosomes that inherit molecular information on parental cells hold great promise for cancer diagnostics. Currently, two main technical challenges, time-consuming and labor-intensive isolation of exosome and nucleic acid extraction with limited recovery that have restricted the detection of ultralow abundance exosomal nucleic acids. Here, we proposed a simple, efficient and "lab in a tube" system for the detection of exosomal nucleic acids, which fully integrated exosomes enrichment using immunomagnetic beads (IMB) (10 min), fast exosomes lysis based on NP-40 lysate (5 min) and sensitive loop-mediated isothermal amplification (LAMP) in a tube. This method was demonstrated by detecting two exosomal long noncoding RNA biomarkers of gastric cancer (HOTTIP and lncRNA-GC1) with a dynamic detection ranging from 300 ng/µL to 10 ng/µL, and the detection limit of LAMP was 10 ng/µL. Additionally, this platform exhibited good performance in the analysis of exosomal HOTTIP RNA directly in human serum samples, which has the potential for detection of low-abundance exosomal nucleic acid biomarkers from cancers.

Cascade signal amplification for sensitive detection of exosomes by integrating tyramide and surface-initiated enzymatic polymerization.

A novel cascade signal amplification based on tyramide signal amplification (TSA) and surface-initiated enzymatic polymerization (SIEP) was first reported for the sensitive and template-free detection of colorectal cancer (CRC) exosomes. This assay exhibited 20.9-fold signal amplification with a low detection limit of 12.8 particles per µL. Furthermore, accurate and reproducible results were obtained for detecting exosomes in serum samples, suggesting its potential application in exosomes analysis and clinical diagnostics.