Talanta

Cardiovascular disease (CVD) is a serious worldwide health concern that necessitates the development of novel diagnostic techniques for early identification and personalized healthcare management. Even before the insights provided by gut microbiota, current research has demonstrated the importance of circulating microbiome (CMB) in the evolution of cardiometabolic illness risk and progression. We developed a nanobiosensor that uses specific labeled capture probes with perovskite quantum dots (PQDs) to detect the targeted 16S rRNA sequences in the peripheral milieu. With ideal applicability, specificity, and sensitivity, this sensor delivers unique insights into the presence and characterization of circulating microbiota signatures. Developing a nanophotonic microbiome detection method in body fluids may pave the way for creating a distinctive tool for CVD risk prediction for population-based screening programs in low and middle-income countries.

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.

Developing rapid and highly sensitive methods for alkaline phosphatase (ALP) activity analysis is significant for the clinical diagnosis and treatment of diseases. Here, a Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for ALP activity detection in which the ALP-catalyzed hydrolysis of 3-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon -mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied in ALP activity analysis with a detection limit (LOD) of 0.0013 U L-1 (3{sigma}) and a detection range of 0.0025 U L-1 to 1 U L-1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of the potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid, highly sensitive, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

Foodborne bacteria are major causes that affect human health. Development of new method that could achieve Rapid, sensitive and quantitative detection of pathogen is urgently needed. In this research, a step emulsion microfluidic,combined with droplet-based digital-RPA, was developed to detect Vibrio parahaemolyticus, a major seafood-borne pathogenic bacteria. Specific and rapid detection of Vibrio parahaemolyticus in 30 min has been achieved by this new device, with a detection limit of 10 CFU/L, about 10-times lower than classical tube-based RPA. This device was demonstrated to be a promising tool for detection of pathogenic bacteria.

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.

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

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.

The postsynaptic density (PSD) is a complex, multi-layered protein network largely situated on the internal surface of the postsynaptic membrane. It is the first processing unit for incoming synaptic transmissions, and changes in its internal structure are associated with synaptic strength and plasticity. These structural changes are largely governed by multivalent interactions between its components. The in vitro characterization of such complexes requires unbiased methods that can be used to estimate the size of the emerging assemblies for systems with multiple possible stoichiometries. Here we present an experimental method for detecting specific PSD proteins as well as their complexes based on their diffusion in a microfluidic environment. The method requires a fluorescent labelling technique that does not disrupt the function of labelled proteins, a microfluidic device that can maintain laminar flow for protein solutions, a microscope that can record the fluorescent signal emitted by these solutions, and an analytic software package that can process the collected experimental data and convert them into approximate particle sizes. We demonstrate the applicability of our method on protein constructs of various postsynaptic proteins, including the multivalent assembly between GKAP and LC8.

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.

SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked at different concentrations into PBS and human serum, were rinsed through immunofiltration columns. Specific antibodies were retained within the IFC and labelled with an isotype specific biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. Measuring signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than ELISA. Further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment.

Cystatin-C is an important renal function biomarker, and conventional quantification requires centralized laboratory analyzers, which limits timely testing in primary care and resource-limited settings. To address this need, we developed and validated a simple, rapid, and quantitative smartphone-based (SP) lateral flow immunoassay (LFIA) for measuring serum Cystatin-C. The SP-LFIA platform consists of a colorimetric LFIA strip and a custom SP reader with uniform LED illumination and macro lens for image capture. Quantitative image analysis of the colorimetric signal is performed by a dedicated application using a pre-defined third order polynomial calibration model. Following systematic optimization, the assay demonstrated a wide quantitative range of 0.32-8.00 mg/L, with a limit of detection of 0.15 mg/L. Analytical validation conducted according to CLSI guidelines showed excellent precision, with intra- and inter-assay coefficients of variation below 10%, and no significant interference from bilirubin, triglycerides, hemoglobin, or rheumatoid factor. Accelerated stability testing confirmed robust strip performance after storage at 50 {degrees}C for 28 days. Method comparison using 100 clinical serum samples showed high agreement with a commercial PETIA reference method (R{superscript 2} = 0.993) and minimal bias. These results indicate that the developed smartphone-based LFIA provides a reliable, cost-effective, and practical tool for point-of-care Cystatin-C monitoring.

To overcome the technical bottlenecks in the precise quantification and molecular typing of viable foodborne pathogens, this study establishes a microfluidic droplet digital PCR (ddPCR) based method for rapid and accurate detection and quantification of viable Listeria monocytogenes with clonal specificity. In contrast to the time-consuming plate culture methods and unspecific rapid detection methods, the method in this study employs clonal complex (CC) specific primers and probe for strain-specificity and integrate the nucleic acid dye propidium monoazide (PMA) to effectively distinguish viable from dead bacteria. The rapid and precise quantification of viable bacteria is achieved through microdroplet counting. This method does not require DNA extraction, and the entire detection process takes only about 3 hours, with a quantitative detection limit of 3.3x102 CFU/mL, providing strong technical support for the risk monitoring of highly pathogenic specific types of L. monocytogenes.

Polymerase chain reaction (PCR) is a method widely used to amplify trace amount of nucleic acids. It needs a process of thermocycling (repeated alternation of temperature). Traditional thermocycler relies on bulk size of metal block to achieve thermocycling, which results in high cost and the lack of portability. Here, a PCR chip made of graphene Transparent Conductive Films (TCFs) was employed. The thermocycling of the chip was fulfilled by a temperature programed microcontroller and a cooling fan under a low driving voltage (12V). A 35 cycles PCR was accomplished within 13 minutes using the chip and the thermocycler. The transparency of the graphene PCR chip enables the PCR reaction to be visually monitored by naked eye for a color change. The PCR chip and the thermocycler have a low cost at $2.5 and $6 respectively, and thus are feasible for Point-of-care testing (POCT) of nucleic acids in a disposable manner. The whole platform makes it possible to perform a low-cost testing of nucleic acids for varieties of purposes outside of laboratories or at resource limited locations.

Hybridization of fluorescent molecular probes, such as molecular beacons or linear molecular probes, with their molecular targets can be confirmed through fluorescence spectra. In this study, we investigate the interaction between fluorescent probes, specifically linear molecular probes, and their targets in the presence of chaotropic agents. Our findings show that double-stranded structures containing mismatched nucleotides (e.g., single-nucleotide polymorphisms) occupy lower energy levels (red shifted) compared to those without mismatched nucleotides. Molecular duplexes formed in buffers without chaotropic agents do not exhibit significant differences, independently on whether they contain mismatched nucleotides or are formed with perfectly matching targets. This effect appears in the presence of a naturally occurring chaotropic agent, urea, and was confirmed in the concentration range of 1 mM to 4 mM. These findings suggest that urea and similar agents may play a significant role in the formation of mismatched nucleotide structures.

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 development of chemiluminescent immunoassays (CLIAs) is a complex and iterative process that relies on costly laboratory infrastructure, limiting its accessibility and application across healthcare settings and disease areas. Here, we detail the CLIA Mobile Development Kit (CLIAMDK) a modular, mobile, and inexpensive platform to assess image sensors, smartphones and data processing workflows for CLIA development. For its demonstration, we developed two CLIAs targeting renin and aldosterone, key biomarkers for diagnosing primary aldosteronism. The results from our performance study, including 50 patient samples, demonstrate the potential of our platform in a real-world scenario. We found that the performance of our mobile reader platform is comparable to that of a state-of-the-art plate reader, with a Lower Limit-of-Detection (LLoD) approaching 41 femtomolar. We envision that our platform will help accelerate CLIA development, make it more accessible, and lay the foundations for novel, distributed, yet highly sensitive diagnostic tests.

Luminescence is a powerful method for detecting trace analytes and monitoring biological processes. However, most bioluminescence reagents, including luciferase and its substrates, are sensitive to temperature, limiting their useable shelf lives, and resulting in inconsistent performance. Enhancing the stability of these reagents could improve data quality, simplify workflows, and address cold chain storage issues. In this study, we demonstrate the application of the platform stabilization technology, capillary-assisted vitrification (CAV), as a tool to stabilize different luciferases and their substrates, and the application of the stabilized reagents in both in vitro and in vivo bioluminescent assays. We demonstrate that CAV-stabilized reagents can be stored and shipped ambiently, maintain consistent performance over time, and are suitable for use in cell viability quantification, tumor monitoring, in vivo imaging, microbial detection, and immunoassays. Additionally, different reagents can be co-formulated to make ready-to-use assay kits that can also be shipped and stored ambiently. Our results demonstrate that CAV stabilization is a viable alternative to traditional storage methods, with broad potential to improve bioluminescence workflows.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

There are various techniques for detecting recombinant proteins such as western blot, SDS-PAGE, ELISA and fluorescence microscopy. However, these methods are difficult to handle, time-consuming and need special tools. We developed a rapid, inexpensive, sensitive, and straightforward approach to address these problems using an Antihistidine biosensor. Colloidal gold nanoparticles (GNPs) were synthesized by chitosan as reducer and stabilizer via the green synthesis method. Then, anti-His tag antibody was immobilized on the Chitosan-gold nanoparticles (CS-GNPs) surface to visually detect spotted target protein on nitrocellulose (NC) membrane. Our results showed that the designed dot-blot immunoassay can detect histidine-tagged recombinant proteins with the limit of detection (LOD) of 1{micro}g/ml without any signal enhancement directly from cell lysate within 5 minutes. In an extra step, we applied HAuCl4 and NH2OH\ HCl as a gold enhancement system and increased the detection sensitivity to 0.1 {micro}g/ml. The results showed that the developed assay can rapidly detect production of recombinant proteins, and it can be used as a screening method in low-resource laboratories.

BackgroundThe clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding specificity, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. ResultsWe have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R2 = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 minutes, a significant improvement over traditional methods. ConclusionsSE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The methods compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.