Long-term monitoring of ultratrace nucleic acids using tetrahedral nanostructure-based NgAgo on wearable microneedles.

Real-time and continuous monitoring of nucleic acid biomarkers with wearable devices holds potential for personal health management, especially in the context of pandemic surveillance or intensive care unit disease. However, achieving high sensitivity and long-term stability remains challenging. Here, we report a tetrahedral nanostructure-based Natronobacterium gregoryi Argonaute (NgAgo) for long-term stable monitoring of ultratrace unamplified nucleic acids (cell-free DNAs and RNAs) in vivo for sepsis on wearable device. This integrated wireless wearable consists of a flexible circuit board, a microneedle biosensor, and a stretchable epidermis patch with enrichment capability. We comprehensively investigate the recognition mechanism of nucleic acids by NgAgo/guide DNA and signal transformation within the Debye distance. In vivo experiments demonstrate the suitability for real-time monitoring of cell-free DNA and RNA with a sensitivity of 0.3 fM up to 14 days. These results provide a strategy for highly sensitive molecular recognition in vivo and for on-body detection of nucleic acid.

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.

Electro-Optical Multiclassification Platform for Minimizing Occasional Inaccuracy in Point-of-Care Biomarker Detection.

On-site diagnostic tests that accurately identify disease biomarkers lay the foundation for self-healthcare applications. However, these tests routinely rely on single-mode signals and suffer from insufficient accuracy, especially for multiplexed point-of-care tests (POCTs) within a few minutes. Here, this work develops a dual-mode multiclassification diagnostic platform that integrates an electrochemiluminescence sensor and a field-effect transistor sensor in a microfluidic chip. The microfluidic channel guides the testing samples to flow across electro-optical sensor units, which produce dual-mode readouts by detecting infectious biomarkers of tuberculosis (TB), human rhinovirus (HRV), and group B streptococcus (GBS). Then, machine-learning classifiers generate three-dimensional (3D) hyperplanes to diagnose different diseases. Dual-mode readouts derived from distinct mechanisms enhance the anti-interference ability physically, and machine-learning-aided diagnosis in high-dimensional space reduces the occasional inaccuracy mathematically. Clinical validation studies with 501 unprocessed samples indicate that the platform has an accuracy approaching 99%, higher than the 77%-93% accuracy of rapid point-of-care testing technologies at 100% statistical power (>150 clinical tests). Moreover, the diagnosis time is 5 min without a trade-off of accuracy. This work solves the occasional inaccuracy issue of rapid on-site diagnosis, endowing POCT systems with the same accuracy as laboratory tests and holding unique prospects for complicated scenes of personalized healthcare.

An Alternating Current Electroosmotic Flow-Based Ultrasensitive Electrochemiluminescence Microfluidic System for Ultrafast Monitoring, Detection of Proteins/miRNAs in Unprocessed Samples.

Early diagnosis of acute diseases is restricted by the sensitivity and complex process of sample treatment. Here, an ultrasensitive, rapid, and portable electrochemiluminescence-microfluidic (ECL-M) system is described via sandwich-type immunoassay and surface plasmonic resonance (SPR) assay. Using a sandwich immunoreaction approach, the ECL-M system employs cardiac troponin-I antigen (cTnI) as a detection model with a Ru@SiO2 NPs labeled antibody as the signal probe. For miR-499-5p detection, gold nanoparticles generate SPR effects to enhance Ru(bpy)3 2+ ECL signals. The system based on alternating current (AC) electroosmotic flow achieves an LOD of 2 fg mL-1 for cTnI in 5 min and 10 aM for miRNAs in 10 min at room temperature. The point-of-care testing (POCT) device demonstrated 100% sensitivity and 98% specificity for cTnI detection in 123 clinical serum samples. For miR-499-5p, it exhibited 100% sensitivity and 97% specificity in 55 clinical serum samples. Continuous monitoring of these biomarkers in rats' saliva, urine, and interstitial fluid samples for 48 hours revealed observations rarely documented in biotic fluids. The ECL-M POCT device stands as a top-performing system for ECL analysis, offering immense potential for ultrasensitive, rapid, highly accurate, and facile detection and monitoring of acute diseases in POC settings.

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.

Ultra-sensitive photoelectrochemical biosensor for determination of African swine fever virus based on surface plasmon resonance.

Sensitive and specific detection of African swine fever virus (ASFV) is crucial for agricultural production and economic development due to the mortality and infectivity. In this study, a bismuth induced enhanced photoelectrochemical (PEC) biosensor based on in-situ loop mediated isothermal amplification (LAMP) was constructed using deposited bismuth nanoparticles loaded bismuth oxycarbonate (Bi/(BiO)2CO3) as photoactive material, using primers designed according to LAMP as recognition elements, and using in-situ LAMP to achieve nucleic acid amplification of target genes. As the Bi induced surface plasmon resonance (SPR) effect, enhanced light captures and effective electron hole separation, it could effectively enhance the photoelectric activity, so the prepared Bi/(BiO)2CO3 nanohybrid had higher photocurrent intensity and good stability. The constructed PEC biosensor has realized the detection of ASFV in real samples with good sensitivity, specificity and repeatability. In the range from 1.0 × 10-13 to 1.0 × 10-7 g/L, the photoelectric current decreased with the increase of the concentration of ASFV, and the detection limit was 3.0 × 10-14 g/L (about 0.048 copies/µL). Combining the advantages of LAMP with the excellent performance of PEC, it provides a simple, economical and efficient method for nucleic acid diagnosis, and also provides a new idea for biosensor detection.

Real-Time Monitoring and Early Warning of a Cytokine Storm In Vivo Using a Wearable Noninvasive Skin Microneedle Patch.

A cytokine storm may be the last attack of various diseases, such as sepsis, cancer, and coronavirus disease 2019, that can be life threatening. Real-time monitoring of cytokines in vivo is helpful for assessing the immune status of patients and providing an early warning of a cytokine storm. In this study, a functional carbon nanotube biointerface-based wearable microneedle patches for real-time monitoring of a cytokine storm in vivo via electrochemical analysis are reported. This wearable system has sensitivity with a detection limit of 0.54 pg mL-1 , high specificity, and 5 days of stability with a coefficient of variation of 4.0%. The system also has a quick response of several hours (1-4 h) to increasing cytokines. This wearable microneedle patch may offer a promising route for real-time biomolecule wearables construction. The patch is also the first reported integrated capture and monitoring system that is capable of real-time measurement of protein markers in interstitial fluid.

Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Introduction: Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Methods: Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). Results and discussion: The newly developed system was shown to be sensitive with detection limits of 101 copies/µl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/µl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

Rapid identification and quantitation of single plant seed allergen using paper-based microfluidics.

Nucleic acid amplification is a sensitive and powerful tool for allergen detection. However, it is limited due to the relatively cumbersome methods required to extract nucleic acids from single plant seed allergen (e.g. peanut and soybean). In view of this, an approach of extracting nucleic acid with untreated glass-fiber paper (paper-based microfluidics) was applied for nucleic acid capture and purification from plant seed allergen and commercial products. After cut by hollow cylindrical cutter, a certain size the paper chip it used to absorb DNA. And this paper-based microfluidics with DNA was directly applied for amplification by loop-mediated isothermal amplification (LAMP). To evaluate the adsorption performance of paper chip to DNA, CTAB and SDS method were used as comparisons. From amplification results, the established technique has good specificity, high repeatability (C.V. values are 4.41% and 6.17% for peanut and soybean) and favorable sensitivity (7.39 ng/µL or peanut and 6.6 ng/µL for soybean), and successfully used for commercial products (2 kinds of candy and 2 kinds of cakes containing peanut, and 2 kinds of drinks, candy and 2 kinds of biscuits containing soybean). This speed and flexible detection method makes it suit for applications in point-of-care (POC) detection at different scenario, such as custom house and import port.

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.

Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA.

Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can extract analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an on-line wearable microneedle patch for extraction and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The experimental results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis.

Accurate identification of SARS-CoV-2 variant delta using graphene/CRISPR-dCas9 electrochemical biosensor.

Delta (B.1.617.2), a highly infectious variant of SARS-CoV-2, has been sweeping the world, and threatening the safety of human life seriously. It is urgent to develop a highly selective and sensitive assay to accurately identify the SARS-CoV-2 variant Delta. In this work, we constructed a graphene/CRISPR-dCas9 electrochemical biosensor to accurately identify SARS-CoV-2 variant Delta, where the signal was further amplified by embedded electrochemical probe [Ru(phen)2dppz]BF4. This detection assay could be finished within 47 min totally, with the detection limit of 1.2 pM and good reproducibility with a C·V.% of 2.48% (n = 5). And the biosensor could selectively identify Delta among SARS-CoV-2 and other variants, including Alpha, Beta, Gamma. This assay was further validated by 26 real clinical samples, showing 100% clinical sensitivity and 100% clinical specificity, which provides a new direction for identifying other SARS-CoV-2 variants in the future.

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.

Wearable chem-biosensing devices: from basic research to commercial market.

Wearable chem-biosensors have been garnering tremendous interest due to the significant potential in tailored healthcare diagnostics and therapeutics. With the development of the medical diagnostics revolution, wearable chem-biosensors as a rapidly emerging wave allow individuals to perform on-demand detection and obtain the required in-depth information. In contrast to commercial wearables, which tend to be miniaturized for measuring physical activities, the recent progressive wearable chem-biosensing device have mainly focused on non-invasive or minimally invasive monitoring biomarkers at the molecular level. Wearables is a multidisciplinary subject, and chem-biosensing is one of the most significant technologies. In this review, the currently basic academic research of wearable chem-biosensing devices and its commercial transformation were summarized and highlighted. Moreover, some representative wearable products on the market for individual health managements are presented. Strategies for the identification and sensing of biomarkers are discussed to further promote the development of wearable chem-biosensing devices. We also shared the limitations and breakthroughs of the next generation of chemo-biosensor wearables, from home use to clinical diagnosis.

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).

A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA polymerase.

The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics. We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which-through enzymatic engineering-has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe-primer pair than those of other isothermal amplifications. We tested the method's ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/µL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/µL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing. In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.

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.

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.