Search

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.

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 β-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.

Food allergy is an abnormal immune response of the human immune system to some foods, which can cause extremely serious harm to the human body. Therefore, the development of a sensitive and rapid detection approach for food allergens in various food matrices is essential to assist patients in managing their allergies. In this paper, a kind of circular fluorescence probe-mediated isothermal amplification technology (CFPA) was developed and used to detect the peanut allergen encoding gene (Ara h 6) and soybean allergen encoding gene (Gly m Bd 28 K). This novel assay could be finished within 60 min with high sensitivity (10 copies/μL~1 × 107 copies/μL), specificity (no cross-reaction with other allergens), and reproducibility (CV

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)

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.

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.

The group B Streptococcus (GBS) is a type of pathogen that seriously threatens the health of mothers and infants. Prompt and timely diagnosis is crucial for good patient outcomes. However, the traditional bacterial culture and polymerase chain reaction methods are limited by their speed and involve complex operating procedures. Herein, we successfully established an integrated microfluidic sample-to-answer system for nucleic acid-based detection of GBS directly in vaginal/anal swab samples. Meanwhile, we demonstrated a dynamical reaction mechanism of Bst/FEN1-based nucleic acid amplification, which differs from traditional Bst-based isothermal amplification strategies. The system integrates cell lysis and nucleic acid purification, separation, amplification, and detection, enabling rapid (about 45 min to the entire analysis) and highly accurate (98% accuracy) analysis in a clinical setting. Experimental results show that the system offers a good detection limit (500 CFU/mL), perfect specificity (no cross-reactivity with 25 other common pathogens), excellent stability (coefficient of variation less than 3%), and good anti-interference performance. This novel system holds great potential as a nucleic acid-based diagnostic tool in clinical applications for detecting not only GBS but also other types of pathogens.

Food safety is a major and enduring challenge and has a profound impact on the quality of human life. Loop-mediated isothermal amplification (LAMP) is a relatively novel gene amplification method under isothermal conditions with rapidity, simplicity and high specificity. This review will describe the principles and development of the LAMP technique along with its advantages and disadvantages, such as LAMP integrated on classical microfluidic chips, paper-chips, electrochemical devices, nanomaterials and digital devices. Moreover, we will systematically and comprehensively review its applications in the field of food safety, such as in the detection of foodborne pathogens, allergens and organophosphorus pesticides and genetically modified organisms; finally its development trends in food safety will be discussed.

Graphical abstract 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). Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-04979-8.

With the development of flexible advanced materials and microfluidic technology, wearable biosensors provide a new strategy for the continuous monitoring of health. In this study, a novel bandage-like wearable flexible microfluidic recombinase polymerase amplification (RPA) sensor was constructed for the rapid and visual detection of nucleic acids. This wearable sensor is triggered by human body heat (30°C-37 °C) and allows for visual nucleic acid (a conserved nucleic acid fragments of zika virus) detection within 10 min. The sensor displays good sensitivity and selectivity, with a detection limit of 10 copies/μL. The wearable sensor has exhibited well-defined accuracy when applied to testing clinical serum samples. In addition, the wearable RPA sensor was proved to be feasible by human trials under different daily activities. This wearable sensor of nucleic acids will probably be of great significance in the field of online pathogen detection for wounds, for tumour biomarker diagnosis, and for the detection of epidermal cell molecular lesions.

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.

Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles (MNs) can extract analytes of interest from interstitial fluid (ISF) in a minimally invasive fashion. However, some MNs are limited by their ability to perform high-effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Herein, we explored the synergetic effect of clustered regularly interspaced short palindromic repeats (CRISPR)-activated graphene biointerfaces, and reported an on-line CRISPR wearable microneedles patch for extraction and in vivo monitoring of Epstein-Barr virus cell-free DNA (EBV CfDNA) in ISF. This wearable system can orientally capture and directly quantify unamplified ISF CfDNA in vivo within 75 min, with anti-interference ability of 60%, and has good electrochemical stability within 3 days (RSD = 9.04%). 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 ISF CfDNA and could be used for early disease screening and prognosis.

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).

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future.

The use of paper-based methods for clinical diagnostics is a rapidly expanding research topic attracting a great deal of interest. Some groups have attempted to realize an integrated nucleic acid test on a single microfluidic paper chip, including extraction, amplification, and readout functions. However, these studies were not able to overcome complex modification and fabrication requirements, long turn-around times, or the need for sophisticated equipment like pumps, thermal cyclers, or centrifuges. Here, we report an extremely simple paper-based test for the point-of-care diagnosis of rotavirus A, one of the most common pathogens that causes pediatric gastroenteritis. This paper-based test could perform nucleic acid extraction within 5 min, then took 25 min to amplify the target sequence, and the result was visible to the naked eye immediately afterward or quantitative by the UV–Vis absorbance. This low-cost method does not require extra equipment and is easy to use either in a lab or at the point-of-care. The detection limit for rotavirus A was found to be 1 × 103 copies/mL. In addition, 100% sensitivity and specificity were achieved when testing 48 clinical stool samples. In conclusion, the present paper-based test fulfills the main requirements for a point-of-care diagnostic tool, and has the potential to be applied to disease prevention, control, and precision diagnosis.

Food allergies can greatly harm people’s health, and therefore detecting allergens in foods is extremely important. By integrating loop-mediated isothermal amplification (LAMP) with a microfluidic chip, we have developed a method for detecting the allergen genes of peanut (Arachis hypogaea), sesame (Sesamum indicum), and soybean (Glycine max) using a colorimetric method suitable for the naked eye, known as the colorimetric LAMP microfluidic chip. In the presence of peanut, sesame, or soybean in the samples, the corresponding reaction well of the microfluidic chip will appear pink, or otherwise remain light brown. This method of detection is specific and can easily distinguish these three allergens from others in foods. The detection limit for peanut, sesame and soybean allergens was 0.4 ng/μL using the LAMP-microfluidic chip. The accuracy of this novel and rapid method was validated using allergenic foods obtained commercially and was comparable with that of the typical TaqMan real-time PCR method.

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.

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.

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 (

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., Graphical abstract Image 1

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., Graphical abstract Image 1

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.

Simplifying the procedure of immunoassay is still a challenge due to problems such as multiple washing processes, complicated chemical modification and expensive cost. In this study, we developed a portable centrifugal microchip fluorescence immunoassay for washing-free, rapid, quantitative and point-of-care (POC) detection of protein. The designed microchip was fabricated by polycarbonate and assembled by double-sided adhesive tape using injecting molding with high scalability and low cost. The centrifugal strategy is capable of washing-out the bio-fluid and improving signal-to-noise ratio. Matrix nano-spotting method was employed to facilitate satisfactory immunological binding sites with the advantage of high capture efficiency and reproducibility. The proposed approach was capable of sensitively detecting procalcitonin (PCT) with a wide dynamic ranging from 0.10 ng/mL to 70.00 ng/mL within 10 min. Furthermore, this novel integrated diagnostic tool was successfully applied to detect PCT in 101 clinical samples with good consistency with Roche’s method, indicating its attractive practical application capability. With favorable simplicity, rapidity, low cost and excellent analytical performance, our method holds great promise for POC diagnostics of proteins.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 101 copies/μL, 102 copies/μL and 102 copies/μL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s, Graphical abstract Image 1, Highlights • In this research, we firstly integrated microfluidic chip with the RT-LAMP technology for the high-throughput and multi-target detection of three emerging and re-emerging swine coronaviruses. • The microfluidic-RT-LAMP chip system established greatly facilitated the rapid, specific and sensitive diagnosis of the PEDV, PDCoV and SADS-CoV. • The application of cost-effective microfluidic chip device is characterized with excellent prospects in swine enteropathogenic coronaviruses monitoring and control for remote livestock farms.

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.

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.

The outbreak of African swine fever has brought serious public safety problems to the world, and there is a need for reliable diagnostic tools to prevent and control the epidemic. Here, we successfully established a portable microfluidic-circular fluorescent probe-mediated isothermal nucleic acid amplification (CFPA) system for the rapid, high-throughput, accurate, and sensitive detection of African swine fever virus at point-of-care settings. This detection system has a detection limit of 10 copies/μL, good stability (C.V. < 5%), and 92.73% sensitivity, and 100% specificity when tested on 220 pig samples collected for the diagnosis of African swine fever virus, with a median time to threshold of 10.8 min. This novel integrated diagnostic tool is urgently needed and has promising applications for the monitoring and control of African swine fever epidemics worldwide and, in particular, the serious outbreak in China.

Using polymerase chain reaction and genotyping, Epstein-Barr virus cell-free DNA (EBV Cf DNA) was detectable in interstitial fluid (ISF). Microneedles offer a minimally invasive approach to capture such Cf DNA. However, a key challenge of microneedles lies in the ability to specifically isolate biomarkers within a short time. We introduced a hydrogel microneedle patch for rapid and easy capture of EBV Cf DNA from ISF in situ around 15 min, with a maximum capture efficiency of 93.6%. Then, quantification of EBV Cf DNA was achieved by electrochemical recombinase polymerase amplification wearable flexible microfluidics, with a detection limit of 3.7 × 102 copies/μL. Animal tests supported the performance of microneedles for EBV Cf DNA capture. Collectively, these data showed that the microneedle patch may have broad implications for patients with Cf DNA-related disease and cancer metastasis in minimally invasive manners.

With the development of proteomics and the continuous discovery of biomarkers of trace proteins, it is important to accurately quantify low abundance protein, especially in urine for clinical diagnostics. In this paper, we reported a novel nano-biotinylated liposome-based immuno-loop-mediated isothermal amplification (LI-LAMP) for the ultrasensitive detection of REG1A (a biomarker for pancreatic ductal adenocarcinoma (PDAC) in urine) with high specificity. The detection range was 1µg/mL to 1fg/mL, with a detection limit of 1fg/mL, and no cross-reactivity was observed to occur in this assay. Compared with the amount of REG1A added, REG1A recovery using this method was 130% and 89%. Detection of REG1A concentrations using the LI-LAMP assay from real samples were in good agreement with those determined using ELISA, and relative deviations were not more than 10%. LI-LAMP shows good potential as a clinical diagnostic assay.

Here, we introduced a novel exonuclease-assisted isothermal nucleic acid amplification (Exo-NAT) utilizing the full-length Bst DNA polymerase combined with a melting curve analysis. This method achieved an ultrahigh specificity with a good detection limit (102 copies) in both singleplex and multiplex detection, which was validated by detecting three diarrhea-inducing pathogens, rotavirus A, astrovirus, and adenovirus, in 42 clinical samples.

Sexually transmitted infections are a serious public health concern worldwide, especially in young people. More than 30 pathogens can cause sexually transmitted diseases and co-infection often occurs. Therefore, the development of fast, low-cost and easy-to-use diagnostic screening methods is urgently needed for disease prevention and control. Herein, we established an all-in-one microfluidic nucleic acid diagnosis system, which could simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum directly from genitourinary secretions with minimal manual manipulations. This system integrated nucleic acid extraction, amplification, and detection on a single microfluidic chip and could be automatically performed in an integrated detection device. This novel diagnosis tool showed good detection limits, stability (coefficient of variation6%), specificity (no cross-reaction with 23 other pathogens for each target) and resistance to interference by other substances and the diagnostic efficacy was similar to that of PCR. The turn-around time was reduced to 50 min from sample to answer with automated testing steps. This novel diagnosis tool has the advantages of highly integrated, automated, sample-to-answer detection, and could thus replace the traditional method. This could significantly improve the prevention and control of sexually transmitted diseases.

Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

A nanozyme composed of mesoporous silica and platinum nanoparticles (MS-PtNPs) was synthesized and is shown to display peroxidase-like activity. Its activity can be controlled by loading with single-stranded DNA. The PtNPs on the MS are homogeneously distributed and act as enzyme mimics. The adsorption of DNA probe on the MS blocks the nucleation sites of PtNPs. This leads to a decrease in the peroxidase-mimicking activity. After introduction of target DNA that is complementary to the DNA probe, the activity of the nanozyme is recovered. By using the 3,3,5,5-tetramethylbenzidine/H2O2 chromogenic system, a rapid method was developed for colorimetric determination of DNA. The assay, best performed at 450 nm, has a linear response in the 5 nM to 100 nM DNA concentration range and a 2.6 nM detection limit. It possesses high selectivity and can distinguish even a single-base mismatch.

Allergen genes of the peanut and soybean were selected as model targets. Four hairpin DNA probes, H1, H2, H3, H4 were designed. Cy3-labeled H1 and H2 were used to detect peanut DNA, while FAM-labeled H3 and H4 were used to detect soybean DNA. Graphene oxide (GO) was used as the adsorption material for capturing the hairpin probes, and as a selective fluorescence quencher to reduce the background signal. To develop an allergen gene detection system with a GO-based paper chip format, we integrated the hybridization chain reaction (HCR) with fluorescence resonance energy transfer (FRET) in our design. The results showed that in the absence of peanut DNA (TP) and soybean DNA (TS), the detection probes attached to the GO surface, which quenched their fluorescence. In the presence of TP or TS, however, complementary probe binding to the targets initiated HCR, producing long double-stranded DNA products that could not be absorbed onto the GO surface. Hence, a strong red or green fluorescent signal was generated. The detection limit for both peanut and soybean DNA was 1 nM using this method, indicating the high sensitivity of our approach. This method also exhibited good specificity and a single chip could be used to simultaneously detect two different targets.

Loop-mediated isothermal amplification is a promising method in the area of nucleic acid detection. However, it suffers from a high rate of false-positive amplifications that largely restrict its application. In this study, we observed gold nanoparticles (AuNP) absorbing single-stranded DNA primers and interacting with Bst DNA polymerase via electrostatic adsorption. As a result of these interactions, the presence of the gold nanoparticles exerted a hot-start effect on the loop-mediated isothermal amplification system. Based on these results, we developed a novel AuNP-mediated nucleic acid isothermal amplification assay. This assay displays significantly enhanced specificity—the proportion of false positive decreased from 76% to 0% and from 100% to 0% for the detection of rotavirus and the β-actin gene, respectively, with the hot-start temperature of 48 °C. Moreover, our AuNP-based assay maintained good sensitivity and a satisfactory detection limit (1 × 103copies/μL) compared with the conventional assay. This approach has the potential to solve the nonspecificity problem of loop-mediated isothermal amplification, thereby promoting its real-world application, particularly, in clinical settings.

In this study, a novel micro-system made of a fluid circuit board and various functional components, inspired by integrated microcircuit techniques, was developed. The circuit board was fabricated on a thermoplastic sheet using two-dimensional (2D) cutting technology. The functional components, including mixers, dilutors, reactors, pumps, retreaters, and detectors, were fabricated via paper (or membrane) cutting/folding and integrated into the system, similar to the way that electronic components are integrated into a micro-integrated circuit board. This system, validated through the rapid detection of human immunodeficiency virus (HIV) and the starch catabolism process, could potentially be another microfluidic system for analytical science.