Talanta

Digital polymerase chain reaction (PCR) is a fast-developed technology, which makes it possible to provide absolute quantitative results. However, this technology has not been widely used in research field or clinical diagnostics. Although digital PCR has been born for two decades, the products on this subject still suffer from either high cost or cumbersome user experience, hence very few labs have the willingness or budget to routinely use such product; On the other hand, the unique sensitivity of dPCR over traditional qPCR shows great potential applications. Here, a cost-effective digital PCR method based on a microfluidic printing system was introduced, trying to overcome those shortcomings. The microfluidic droplet printing technology was utilized in this study to directly generate droplet array containing PCR reaction solution onto the simple glass substrate for the subsequent PCR and imaging, which could be done with any regular flat-panel PCR machine and microscope. The method introduces a new perspective in droplet-based digital PCR in that the droplets generated with this method aligns well in an array without touch with each other, therefore the regular glass and oil could be used without any special surfactant. With simple analysis, the data generated with this method showed reliable quality, which followed the Poisson distribution trend. Compared with other expensive digital PCR methods, this system is more affordable and simpler to integrate, especially for those biological or medical labs which are in need for the digital PCR options but short in budget. Therefore, this method is believed to have the great potential in the future market application.

Tuberculosis is still one of the most serious infectious diseases resulting in lethal death worldwide. The traditional method is still not enough to meet the clinical requirements of rapid diagnosis, high specificity and sensitivity. Fast, sensitive and accurate detection of mycobacterium tuberculosis (MTB) is an urgent need for the treatment and control of tuberculosis disease. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas12a) exhibits strongly nonspecific degradation ability of exogenous single-strand nucleic acid (trans-cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA (gRNA) based on conserved sequences of MTB from gRNA library we designed. Then, we proposed a novel method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay based on fluorescence detection pattern showed 4.48 fM of limit of detection (LOD) and good linear correlation of concentration and fluorescence value (R2=0.9775). Also, it showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% confidence interval, respectively, compared with culture method. The CRISPR/Cas12a system showed good specificity, excellent sensitivity and accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Magnetic microbeads are small iron oxide nanoparticles coated with a bioaffinity material that selectively binds to specific biomolecules of interest, enabling their capture and isolation from complex biological samples. Magnetic microbeads are widely used for purification of specific biomolecules in various experiments in molecular biology. However, current methods of manual pipetting to separate supernatants from magnetic microbeads are often inefficient- time-consuming, labor intensive and inaccurate. Furthermore, the use of pipetting robots and liquid handlers specifically designed for multi-well plates can be a cost-prohibitive approach due to the high cost of equipment and disposable supplies. Here, we developed a centrifugation-based method for high-throughput separation of supernatant from magnetic microbeads. To facilitate the centrifugal separation process, we used the 384 transfer plate (Watson, Japan) and a magnetic stand equipped with a 384-well magnetic stand, allowing easy handling of several hundred samples and rapid separation of supernatant from magnetic microbeads. The centrifugal force was used to drive the separation of target molecules from the magnetic microbeads, and sample were successfully separated with relatively high recovery rates. Thus, this technology provides a simple, rapid, and cost- and labor-effective biomolecule separation method with potential applications in various fields, including molecular biology, clinical diagnostics, and biotechnology, and is a valuable addition to the existing toolbox of biomolecule separation methods.

Aptamers are widely used in various applications; however, their quantification methods remain underdeveloped. In this study, we established a universal RT-qPCR-based method for DNA aptamer quantification. By incorporating reverse transcription, primers and probes could be added to aptamers, enabling their detection via qPCR (Figure 1). We developed a software tool for primer and probe design and optimized the reverse transcription process. Two aptamers, CD9-aptamer and CD63-aptamer, were selected as model systems representing two distinct aptamer types: the CD9-aptamer contains an intrinsic primer-binding site within its sequence, whereas the CD63-aptamer does not. Using this method, the limit of detection (LOD) for CD9-aptamer reached 10-14 M, while the LOD for CD63-aptamer was 10-12 M. Compared to existing quantification methods, this approach significantly improves accuracy and cost-effectiveness. Additionally, the method supports SYBR Green, TaqMan, and one-step RT-qPCR assays, broadening its applicability and enhancing precision for various aptamer-based applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/662296v1_fig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@44ec4corg.highwire.dtl.DTLVardef@f59184org.highwire.dtl.DTLVardef@2480fdorg.highwire.dtl.DTLVardef@906845_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Schematic illustration of RT-qPCR-Based Aptamer Quantification. C_FIG

Seasonal flu and pandemics, which account for millions of infections and hundreds of thousands of deaths, require rapid and reliable detection mechanisms to implement preventive and therapeutic measures. Current detection methods of viral infections have limitations in speed, accuracy, accessibility, and usability. This project presents a novel, widely applicable viral diagnostic test that uses a modified version of rolling circle amplification (RCA) to be sensitive, specific, direct RNA targeted, colorimetric and operable at room temperature. We are specifically detecting the following high-impact viruses: SARS-CoV-2, Influenza A (H1N1pdm09), and Influenza B (Victoria Lineage), although our test can be adapted to any viral infection. Results using synthetic viral DNA and RNA sequences show that our diagnostic test takes approximately one hour, detects femtomolar concentrations of RNA strands, and differentiates between virus strains. We believe implementing our diagnostic test will provide faster responses to future viral-related outbreaks for quicker societal recovery.

Monitoring the effectiveness of statin therapy in patients with dyslipidemia is essential for ensuring optimal treatment outcomes. The current standard involves lipid profiling via blood tests to detect abnormalities in blood lipids. This study evaluated the feasibility of a non-invasive, breath-based approach to statin therapy monitoring using Nozes electronic nose (eNose) platform. A total of 35 participants were enrolled, 25 with elevated low-density lipoprotein cholesterol (LDL-C) levels and 10 healthy controls. The high LDL-C group provided breath specimens both before starting statin therapy and after 6 to 8 weeks of treatment. These breath specimens were digitized using Nozes eNose platform and analyzed using machine learning (ML) algorithms. Results showed a 91% sensitivity and 87% specificity in identifying high blood cholesterol cases, demonstrating the potential of Nozes eNose platform for non-invasive monitoring of statin therapy through exhaled breath.

In the recent years, microRNAs (miRNAs) have been discovered to play a very important role in biological processes such as development, differentiation, and apoptosis. The miRNA expression levels in cells are associated with diverse diseases including cancers. MiRNA inhibitors have been widely employed for studying the functions and targets of miRNAs by transfecting the inhibitors into cells. The concentrations of miRNA inhibitors used for such studies can vary depending on the types of miRNAs being tested, the cell lines under study, and the analysis methods. Therefore, in order to obtain accurate results, appropriate amounts of miRNA inhibitors have to be used in the experiments. Apart from amounts, the evaluation of inhibitors may also have to be conducted for functional studies.\n\nHere we developed capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for evaluating miRNA inhibitor and for optimizing miRNA inhibitor concentrations, in the A549 lung cancer cell line. The target miRNAs, miRNA-23a and miRNA-24 are biomarker candidates in lung cancer cell lines. Our results showed that miRNA-23a and miRNA-24 were effectively inhibited upon transfection with 20 nM miRNA inhibitors using CE-LIF method. Furthermore, these results demonstrated the potential of CE for fast, specific, sensitive and specific analyses for the evaluation and determination of the optimal concentration of miRNA inhibitors for functional studies.\n\nAbstract Graphics\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC=\"FIGDIR/small/566786v4_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@2bbfeeorg.highwire.dtl.DTLVardef@45b7e3org.highwire.dtl.DTLVardef@12ba79borg.highwire.dtl.DTLVardef@108ec2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.

Early diagnosis of bacterial causing the disease is important for treatment of patent and preventing the spread of pathogen. Utilizing of POCT devices to detect the pathogens on-site will accelerate the diagnosis of infectious disease. By using loop-mediated-amplification, we developed a microfluidic chip for multiplex detection of three bacterial, where the samples were driven by negative pressure were loaded quickly. The performance of the device was preliminarily evaluated. The specificities of the detections were demonstrated. And the LOD for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were measured as 17.15, 5.67 and 16.47 ng/L, respectively. The results demonstrated the feasibility of the method.

Volatile organic compounds (VOCs) are common constituents of fruits, vegetables, and crops, and are closely associated with their quality attributes, such as firmness, sugar level, ripeness, translucency, and pungency levels. While VOCs are vital for assessing vegetable quality, traditional detection methods, such as Gas Chromatography-Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS) are limited by expensive equipment, complex sample preparation, and slow turnaround time. Additionally, the transient nature of VOCs complicates their detection using these methods. Here, we developed a paper-based colorimetric sensor array combined with needles that could induce vegetable VOC release in a minimally invasive fashion and analyze VOCs in situ with a smartphone reader device. The colorimetric sensor array was optimized using sulfur compounds as main targets and classified fourteen different vegetable VOCs, including sulfoxides, sulfides, mercaptans, thiophenes, and aldehydes. By combining principal components analysis (PCA) analysis, the integrated sensor platform proficiently discriminated between four vegetable subtypes originating from two major categories within 2 min of testing time. This rapid and minimally invasive sensing technology holds great promise for conducting field-based vegetable quality monitoring. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/628229v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12bf541org.highwire.dtl.DTLVardef@f2a809org.highwire.dtl.DTLVardef@f5f5b7org.highwire.dtl.DTLVardef@1d7027f_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectTransdermal biosensors may provide an alternative to conventional blood-based biomarker measurement. Our purpose was to determine the binary correlation between transdermal (Infrasensor; RCE, Inc, Carlsbad, CA) and conventional blood-based measurements. MethodsThis was a secondary analysis from a previously published observational cardiac troponin I (cTnI) study performed to establish the upper reference level of cTnI, at 10 US hospitals. After obtaining informed consent, 2 cohorts of patients were enrolled: 1) those who completed a health assessment questionnaire and appeared healthy, and 2) those with a known elevated cTnI per the local hospital standard assay. All blood lab analyses were performed at the University of Maryland Medical Center, Baltimore, MD. Normal was defined as cTnI <53.48 ng/L (male) or 34.11 ng/L (female) using the Siemens Atellica IM assay (Siemens Medical Solutions, Mountain View, CA), NT-proBNP <450 pg/mL (>75 years) or <124 pg/mL (<75 years), creatinine >1.17 mg/dL (male) or >0.95 mg/dL (female), and HbA1c <6.4%. The Infrasensor was placed on the patients wrist for measurement and blood drawn for analysis at approximately the same time. ResultsOf 840 enrolled patients, the median (IQR) age was 46 (30,57), 416 (49.5%) were female, 10.36% Hispanic, 6.7% Asian, 12.9% African American, and 69.1% White. Elevated lab tests were 102 hscTnIs, 156 NTproBNPs, 37 HbA1Cs, and 163 creatinines. Significant binary correlations were found between all transdermal signals and the corresponding lab blood levels ConclusionInfrasensor transcutaneous measurement demonstrates similar results as that obtained from blood testing in the central laboratory. CapsuleThe Infrasensor (RCE, Inc, Carlsbad, CA, USA) is rapid point of care transcutaneous biomarker measurement device. This study evaluated its ability to provide qualitative results for troponin I, NTproBNP, creatinine, and HbA1c levels in 840 patients. Significant correlations were found between all transdermal signals and the corresponding binary lab blood levels.

Analysis of circulating cell-free DNA (cfDNA) methylation abnormalities has emerged as a powerful strategy for detecting various diseases, particularly cancers. This study demonstrates a process for developing a multiplex methylation-specific qPCR assay for cancer biomarker detection. AbstractAnalysis of circulating cell-free DNA (cfDNA) methylation abnormalities has emerged as a promising strategy for detecting various diseases, including cancer. While single-biomarker approaches may lack sensitivity, comprehensive next-generation sequencing (NGS) can be cumbersome and expensive. Multiplex methylation qPCR assays offer a practical intermediate solution by providing accurate, accessible, and affordable methylation biomarker detection. In this study, we demonstrated a process for developing a multiplex methylation-specific qPCR assay using colorectal cancer (CRC) methylation biomarkers as a case study. Starting with a set of CRC methylation biomarkers, we developed the Multiplex Methylation-specific qPCR (MMqPCR) algorithm to scan differentially methylated regions (DMRs) for methylation-specific PCR primer and probe design. We then established a systematic process to eliminate assay cross-reactions, reduce background noise, and assess multiplex compatibility. Using a low concentration of hypermethylated DNA (0.1%) in a digital analysis, we demonstrated a 95.8% detection rate with the multiplex strategy, significantly outperforming the singleplex approach (47.9%). The multiplex qPCR development principles and automated primer design algorithm presented here provide valuable tools for developing disease screening, detection or monitoring methods based on methylation analysis.

BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.

Nucleic acid sequences carry the most fundamental information about life, and gene detection plays a crucial role in studying and applying gene function. Here, we present a novel probe design that utilizes probe cleavage and capillary electrophoresis for gene detection, termed ProbeCE which detects genes by measuring the relative fluorescence signal intensity. Through the evaluation of probes with varying lengths and modifications, ProbeCE is believed to support the simultaneous detection of hundreds of genes. Additionally, the assessment of low-concentration samples has demonstrated the high sensitivity of the ProbeCE method. Compared to the conventional amplification product capillary electrophoresis (AmpCE) method, this new approach significantly increases detection throughput while simplifying primer design and enhancing specificity. In addition, ProbeCE is less expensive than current medium- or high-throughput sequencing for detecting large numbers of known genes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/643483v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@188decborg.highwire.dtl.DTLVardef@dae443org.highwire.dtl.DTLVardef@c01a55org.highwire.dtl.DTLVardef@d653_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65{degrees}C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.

In recent decades, nanopores have become a promising diagnostic tool. Protein and solid-state nanopores are increasingly used for both RNA/DNA sequencing and small molecule detection. The latter is of great importance because small molecules are difficult or expensive to detect using available methods such as HPLC or LC-MS. Moreover, DNA aptamers are an excellent detection element for sensitive and specific detection of small molecules. Here, we describe a method for the quantification of ethanolamine using Oxford Nanopores ready-to-use sequencing platform. To this end, we have developed a strand displacement assay using a binding ethanolamine aptamer and magnetic beads. The displaced aptamer can be detected using the MinION(R) nanopores and analysed/quantified using our in-house developed analysis software.

The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultra-low concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity. This process is termed the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). The proposed method was validated by detecting an inflammatory factor, human interleukin-6 (human IL-6), and a tumor marker, human vascular endothelial growth factor (human VEGF), which achieved limit of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/m (0.81 fM), respectively, demonstrating that CLISA is at least 102-fold more sensitive than conventional ELISA.

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E. coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.

1The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 105 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.

Oncogenesis is inevitably associated with microRNA expression deregulation. Thus, development of both miRNA targeting substances or small RNA for ant-cancer therapy have been reported. Specifically, repression of the miR-16-1-3p and miR-16-2-3p activities play pivotal roles in osteosarcoma and many other cancers. The majority of miRNA sensors use protein degradation to measure miRNA activities. Here we report miRNA sensors that use fluorescent protein synthesis rather than degradation to measure miRNA activity. Specifically, miR-16-1-3p and miR-16-2-3p sensors consist of the bidirectional tet-On system driving the expression of the Katusha2S protein that is regulated by the RNA interference and GFP as a reference. These sensors specifically detect mir16-1-3p and mir16-2-3p small RNA mimics in the osteosarcoma cell line after doxycycline induction. Kinetic measurements of the reporter responses to the miRNA mimics revealed that pre-induced sensors reach significant differences from the control faster, within 2h than the sensors that were induced after mimic transfection. Thus, kinetic measurements of the fluorescent protein synthesis during doxycycline induction of the tet-On system are feasible for determination of the small RNA activity.