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.

Extraction and Purification of Nicotine from Tobacco Rhizomes by Supercritical CO2.

Currently, in the ongoing development of the tobacco industry, a large amount of tobacco rhizomes is discarded as waste. These wastes are usually disposed of through incineration or burial. However, these tobacco wastes still have some economic value. High-purity nicotine has a promising market outlook as the primary raw material for electronic cigarette liquid. Nicotine is not only found in tobacco leaves but also in the rhizomes of tobacco plants. This study presents a method for treating tobacco waste and extracting high-purity nicotine from it. After mixing the raw material powder and entrainer in specific ratios, as much of the nicotine in tobacco roots can be extracted as possible using supercritical carbon dioxide extraction. The effects of temperature, the ratio of the entrainer, and the volume fraction of ethanol in the entrainer on the nicotine yield in supercritical fluid extraction (SFE) at 25 MPa for 120 min were discussed. By using 90% ethanol (a raw material mass-to-volume ratio of 1:5) as the entrainer, we obtained the highest nicotine yield of 0.49% at 65 °C. Meanwhile, the purity of the crude extract was 61.71%, and after purification, it increased to 97.57%. In this way, we can not only obtain nicotine with market value but also further reduce the harm to the environment caused by tobacco waste disposal.

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.

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

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.

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.

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.

Rapid quantification of thiocyanate in milk samples using a universal paper-based SERS sensor.

Sodium thiocyanate (NaSCN) is a naturally antibacterial component in milk, but excessive consumption of thiocyanate may cause potential risks to human health. Currently available methods for the detection of thiocyanate have some disadvantages such as poor sensitivity and high price. Here, we report a robust and cost-effective method to detect NaSCN based on paper substrates deposited with in situ reduced Ag nanoparticles by surface-enhanced Raman spectroscopy (SERS). Densely packed multilayer AgNPs provide uniform narrow nanogaps, which exponentially enhance the Raman signals. Moreover, these homogeneous narrow hotspots ensure that this method has high sensitivity (the limit of detection is 10-12 mol L-1), a wide linear range (from 10-9 mol L-1 to 10-4 mol L-1), and remarkable reproducibility (the coefficient of variation within a SERS sensor is 6.5%). Spiked milk samples were detected and the recovery rates of NaSCN were in the range of 95.1%-108.0%. This paper-based SERS sensor offers great potential for sensitive NaSCN detection and milk analysis.

Efficient Microfluidic-Based Air Sampling/Monitoring Platform for Detection of Aerosol SARS-CoV-2 On-site.

Airborne pathogens have been considered as highly infectious and transmittable between humans. With the pandemic outbreak of the coronavirus disease 2019 (COVID-19), an on-site diagnostic system-integrated airborne pathogen-monitoring machine is recommended by experts for preventing and controlling the early stage ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread. In this work, a small-volume rotating microfluidic fluorescence chip-integrated aerosol SARS-CoV-2 sampling system was constructed to satisfy the demand for rapid on-site sample collection and detection of SARS-CoV-2. The rotating microfluidic fluorescence system with small volume has very high sensitivity in the detection of SARS-CoV-2 (detection limit of 10 copies/µL with the shortest Ct value of 15 min), which is comparable to reverse transcription polymerase chain reaction (RT-PCR). The precision variation coefficients within/between batches are very low [coefficient of variation (CV) % ≤ 5.0%]. Our work has passed the comprehensive inspection of the microfluidic chip performance by the Shanghai Medical Device Testing Institute [National Medical Inspection (Design) no. 4408] and successfully tested 115 clinical samples. The integrated system exhibits 100% specificity, high sensitivity (10 copies/µL), and good precision (CV % ≤ 5.0%) in the rapid detection of SARS-CoV-2, thus realizing rapid monitoring and diagnostics of SARS-CoV-2 nucleic acid on-site.

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.

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).

Rapid Differential Diagnosis of Seven Human Respiratory Coronaviruses Based on Centrifugal Microfluidic Nucleic Acid Assay.

With the global outbreak of the coronavirus disease 2019 (COVID-19), the highly infective, highly pathogenic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has attracted great attention. Currently, a method to simultaneously diagnose the seven known types human coronaviruses remains lacking and is urgently needed. In this work, we successfully developed a portable microfluidic system for the rapid, accurate, and simultaneous detection of SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and four other human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. The disk-like microfluidic platform integrated with loop-mediated isothermal amplification provides highly accurate, sensitive, and specific results with a wide linear range within 40 min. The diagnostic tool achieved 100% consistency with the "gold standard" polymerase chain reaction in detecting 54 real clinical samples. The integrated system, with its simplicity, is urgently needed for the diagnosis of SARS-CoV-2 during the COVID-19 pandemic.

Microfluidic Immunoassays for Sensitive and Simultaneous Detection of IgG/IgM/Antigen of SARS-CoV-2 within 15 min.

The outbreak of SARS-CoV-2 is posing serious global public health problems. Facing the emergence of this pandemic, we established a portable microfluidic immunoassay system for easy-to-use, sensitive, rapid (<15 min), multiple, and on-site detection of IgG/IgM/Antigen of SARS-CoV-2 simultaneously. This integrated method was successfully applied for detecting SARS-CoV-2 IgM and IgG antibodies in clinical human serum as well as SARS-CoV-2 antigen in pharyngeal swabs from 26 patients with COVID-19 infection and 28 uninfected people. The assay demonstrated high sensitivity and specificity, which is promising for the diagnosis and monitoring as well as control of SARS-CoV-2 worldwide.

Rapid and simultaneous analysis of twelve virulence factor genes by a microfluidic-CFPA chip for identifying diarrheagenic Escherichia coli.

Diarrheagenic Escherichia coli as an enteropathogen has caused serious public safety problems, especially in children. A fast, easy-to-use, high-throughput method is urgently needed for diarrheagenic Escherichia coli strain identification and monitoring. In this study, we developed a portable microfluidic device based on circular fluorescent probe-mediated isothermal nucleic acid amplification for accurate and rapid detection of twelve virulence factor genes of five diarrheagenic Escherichia coli strains for point-of-care testing. This microfluidic system showed excellent performance in identifying five diarrheagenic Escherichia coli strains within 60 min, when applied in 67 clinical samples collected from hospitals for identifying diarrheagenic Escherichia coli, with a good clinical sensitivity (96.9%), specificity (97.1%) and stability (CV < 5.0%). This integrated microfluidic system is a promising diagnostic tool for applications in the identification and monitoring of diarrheagenic Escherichia coli epidemics worldwide, particularly in developing countries.

Sequence-Specific Probe-Mediated Isothermal Amplification for the Single-Copy Sensitive Detection of Nucleic Acid.

There is currently the lack of a method for precisely monitoring the progress of isothermal amplification reactions by means of sequence-specific fluorescent probes like the TaqMan probe used in the PCR system. Here, we created a circular fluorescent probe-mediated isothermal amplification (CFPA) method. This novel method uses two circular fluorescent probes and Bst DNA polymerase to construct an overlapping structure that can be cut off by flap structure-specific endonuclease 1, separating the fluorescence and quenching groups on the probes. The results showed single-copy sensitivity, ultrahigh specificity, stability (C.V. < 0.1), and anti-interference ability in detecting nucleic acid samples. A clinical trial demonstrated the perfect effectiveness of this method in the diagnosis of rotavirus infection and consistency with the gold standard method used in the clinic ( p > 0.05). In summary, we present a new, reliable, and precise isothermal amplification approach for applications in biomedical research and the clinical accurate diagnosis of pathogen infections.

Graphene Oxide-Based Suppression of Nonspecificity in Loop-Mediated Isothermal Amplification Enabling the Sensitive Detection of Cyclooxygenase-2 mRNA in Colorectal Cancer.

Cyclooxygenase-2 (COX2) mRNA represents a key biomarker for identifying subjects with colorectal cancer (CRC), while there is still no rapid and sensitive detection method for COX2 mRNA. Loop-mediated isothermal amplification (LAMP) is extensively developed for the amplification of nucleic acids; however, its application is frequently hindered by serious nonspecific amplification. Herein, this work reported a graphene oxide (GO)-based LAMP method to enable the one-step detection of COX2 mRNA in cancer cells and serum samples. We found that GO greatly enhanced the specificity of LAMP through decreasing nonspecific hybridization and the fluorescence background signal because of the simultaneous adsorption of single-stranded primers and DNA staining dyes on GO. The detection limit of developed GO-based LAMP was 2 orders of magnitude more sensitive compared to that of classical LAMP. Then a GO-based reverse transcription (RT)-LAMP strategy was further developed and applied to detect COX2 mRNA in CRC cancer cells and serum samples with high specificity. The GO-based LAMP platform with advantages of low cost, simplicity, high specificity, and sensitivity holds considerable potential for real-time fluorescence monitoring of nucleic acid amplification in a wide range of fields.

A hybridization chain reaction coupled with gold nanoparticles for allergen gene detection in peanut, soybean and sesame DNAs.

Food allergy is an abnormal immune response of the immune system to some foods, which has caused great harm to people's health. Therefore, it is particularly important to detect allergens in food. In this article, a hybridization chain reaction (HCR) was coupled with gold nanoparticles (AuNPs) to detect the allergen genes of peanut, soybean and sesame DNAs. Two hairpin probes (H1 and H2) were designed for the allergen target genes of peanut, soybean and sesame DNAs. In the presence of target DNA, the hybridization chain reaction was triggered by it producing long double-stranded DNA (dsDNA) products. In the gold nanoparticle system, long dsDNA couldn't be adsorbed on the surface of AuNPs. When the concentration of salt ions in the solution increased, gold nanoparticles accumulated and led to a decrease of ultraviolet absorption. In the absence of target DNA, no hybridization chain reaction occurred. The hairpin probes could be adsorbed on the surface of AuNPs and no accumulation happened for gold nanoparticles even if the concentration of salt ions in the solution was increased. This method required no enzymes and had a strong specificity, so it was very easy to distinguish target DNA from non-target DNA. The detection limit of three allergens detected by this method was as low as 0.5 nM. The feasibility of this method for the detection of commercial commodities had been demonstrated by the successful detection of the DNAs extracted from commercial commodities, which were treated with extreme thermostable single-stranded binding protein (ET SSB).

Real-time fluorescence loop-mediated isothermal amplification assay for rapid and sensitive detection of Streptococcus gallolyticus subsp. gallolyticus associated with colorectal cancer.

The increasing threat of Streptococcus gallolyticus subsp. gallolyticus (SGG) infections has gained considerable attention for its strong association with colorectal cancer (CRC). Herein, we proposed real-time fluorescence loop-mediated isothermal amplification (LAMP) as a novel, simple, rapid, and highly sensitive assay for identifying SGG for the first time. This assay was capable of detecting SGG with initial DNA concentrations ranging from 102 to 108 copies per microliter, under isothermal conditions within 30 min via real-time fluorescence monitoring. Our method was tested for specific identification of SGG strains without cross-reaction with other Streptococcus gallolyticus subspecies and Escherichia coli. The developed LAMP shows a superior performance with shorter time and higher sensitivity compared with conventional polymerase chain reaction (PCR). Significantly, this proposed approach was successfully applied for detecting SGG in clinical urine samples, which is non-invasive diagnosis, showing excellent accuracy and reliability to discriminate healthy controls and CRC patients. For comparison, these samples were also tested against PCR assay. These results yielded an analytical sensitivity of 100% and a specificity of 100% for SGG testing using LAMP. The findings suggest LAMP can be employed for detecting SGG infections which is useful for diagnosis and screening of CRC.