Enhanced Detection Sensitivity Research Articles

Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection

BackgroundHigh-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. ResultAutomatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 μL droplet on MALDI plate, high recovery (76–116%) and rapid evaporation in 2 min. The analytes were concentrated 4–8 times, forming even crystallization, reaching the detection limit at the concentration of 50 μg L−1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%–114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SignificanceThe ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

Advanced detection of cervical cancer biomarkers using engineered filamentous phage nanofibers.

Cervical cancer is a major global health concern, characterized by its high incidence and mortality rates. The detection of tumor markers is crucial for managing cancer, making treatment decisions, and monitoring disease progression. Vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PDL-1) are key targets in cervical cancer therapy and valuable biomarkers in predicting treatment response and prognosis. In this study, we found that combining the measurement of VEGF and soluble PDL-1 can be used for diagnosing and evaluating the progression of cervical cancer. To explore a more convenient approach for detecting and assessing cervical cancer, we designed and prepared an engineered fd bacteriophage, a human-safe viral nanofiber, equipped with two peptides targeting VEGF and PD-L1. The dual-display phage nanofiber specifically recognizes and binds to both proteins. Utilizing this nanofiber as a novel capture agent, we developed a new enzyme-linked immunosorbent assay (ELISA) method. This method shows significantly enhanced detection sensitivity compared to conventional ELISA methods, which use either anti-VEGF or anti-PD-L1 antibodies as capture agents. Therefore, the phage dual-display nanofiber presents significant potential in detecting cancer markers, evaluating medication efficacy, and advancing immunotherapy drug development. KEY POINTS: • The combined measurement of VEGF and soluble Programmed Death-Ligand 1(sPD-L1) demonstrates an additive effect in the diagnosis of cervical cancer. Fd phage nanofibers have been ingeniously engineered to display peptides that bind to VEGF and PD-L1, enabling the simultaneous detection of both proteins within a single assay • Genetically engineered phage nanofibers, adorned with two distinct peptides, can be utilized for the diagnosis and prognosis of cancer and can be mass-produced cost-effectively through bacterial infections • Employing dual-display fd phage nanofibers as capture probes, the phage ELISA method exhibited significantly enhanced detection sensitivity compared to traditional sandwich ELISA. Furthermore, phage ELISA facilitates the detection of a single protein or the simultaneous detection of multiple proteins, rendering them powerful tools for protein analysis and diagnosis across various fields, including cancer research.

16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.

Background and Objectives: Ascites, often associated with liver cirrhosis, poses diagnostic challenges, particularly in detecting bacterial infections. Traditional methods have limitations, prompting the exploration of advanced techniques such as 16S rDNA next-generation sequencing (NGS) for improved diagnostics in such low-biomass fluids. The aim of this study was to investigate whether the NGS method enhances detection sensitivity compared to a conventional ascites culture. Additionally, we aimed to explore the presence of a microbiome in the abdominal cavity and determine whether it has a sterile condition. Materials and Methods: Ten patients with clinically suspected spontaneous bacterial peritonitis (SBP) were included in this study. A traditional ascites culture was performed, and all ascites samples were subjected to 16S ribosomal RNA gene amplification and sequencing. 16S rRNA gene sequencing results were interpreted by comparing them to positive and negative controls for each sample. Results: Differential centrifugation was applied to all ascites samples, resulting in very small or no bacterial pellets being harvested. The examination of the 16S amplicon sequencing libraries indicated that the target amplicon products were either minimally visible or exhibited lower intensity than their corresponding negative controls. Contaminants present in the reagents were also identified in the ascites samples. Sequence analysis of the 16S rRNA gene of all samples showed microbial compositions that were akin to those found in the negative controls, without any bacteria isolated that were unique to the samples. Conclusions: The peritoneal cavity and ascites exhibit low bacterial biomass even in the presence of SBP, resulting in a very low positivity rate in 16S rRNA gene sequencing. Hence, the 16S RNA sequencing method does little to enhance the rate of positive samples compared to traditional culture methods, including in SBP cases.

Real-time and visual detection of viable Salmonella in milk from remote pasture via IMS-LAMP-NALFS

Salmonella is a prevalent foodborne pathogen commonly found in dairy products. Controlling the presence of Salmonella during dairy product production is of utmost concern due to the potential for contamination at any stage of the production chain, particularly at the source. Moreover, the unique location of pastures and limited testing resources further highlight the necessity of establishing a rapid and precise detection method to ensure milk quality control and enhance overall farm safety. To address these issues, in this study, we constructed a method as well as a portable toolbox for rapid on-site detection of Salmonella. The method involves immunomagnetic separation (IMS) using streptavidin-coupled magnetic nanoparticles and biotinylated antibodies. The reaction conditions for magnetic capture and enrichment of Salmonella were optimized to efficiently remove matrix interference from milk and enhance detection sensitivity. Additionally, an improved propidium monoazide (PMAxx) treatment was employed to eliminate false positive results caused by dead bacteria. This treatment was combined with loop-mediated isothermal amplification (LAMP) and nucleic acid lateral flow strip (NALFS) (LAMP-NALFS) for on-site detection of viable bacteria in milk. The resulting IMS-LAMP-NALFA assay achieved rapid and sensitive detection of viable Salmonella, with a detection sensitivity of 1 × 102 CFU/mL in milk samples, completed within 90 min under optimal conditions. Based on this achievement, a portable toolbox was designed for easy transportation and on-site testing in pastures. It is equally applicable for rapid detection of viable Salmonella contamination in food products in resource-limited areas such as pastures, where the observed detection rate of Salmonella in unpasteurized raw milk was 7.93 % (28 out of 353 samples tested). Consequently, the IMS-LAMP-NALFA method and the associated portable toolbox represent highly effective means of enhancing the detection of viable Salmonella in dairy products.

Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay

Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP-99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP-specific scFv antibodies and also propose a multi-amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.

Local Singularity Spectrum: An Innovative Graphical Approach for Analyzing Detrital Zircon Geochronology Data in Provenance Analysis

Detrital zircon geochronology plays a crucial role in provenance analysis, serving as one of the fundamental strategies. The age spectrum of detrital zircons collected from the sedimentary unit of interest is often compared or correlated with that of potential source terranes. However, biases in the age data can arise due to factors related to detrital sampling, analysis techniques, and nonlinear geological mechanisms. The current study reviewed two sets of detrital zircon datasets established in 2011 and 2021 to discuss the origins of the Tibetan Plateau. These datasets collected from different media effectively demonstrate a progressive understanding of provenance affinity among the main terranes on the Tibetan Plateau. This highlights issues regarding weak and unclear temporal connections identified through analyzing the age spectrum for provenance analysis. Within this context, a local singularity analysis approach is currently employed to address issues associated with unclear and weak provenance information by characterizing local variations in nonlinear behaviors and enhancing detection sensitivity towards subtle anomalies. This new graphical approach effectively quantifies temporal variations in detrital zircon age populations and enhances identification of weak provenance information that may not be readily apparent on conventional age spectra.

Synthesis of pyrochlore-type La2Sn2O7@GO for the in-situ profiling of organophosphate pesticides paraoxon ethyl in environmental samples

The detection and monitoring of organophosphate pesticides in environmental samples are of paramount importance due to their potentially harmful effects on human health and ecosystems. In this study, we present the detection of paraoxon ethyl (POE), a widely used organophosphate pesticide, utilizing an electrochemical method. We synthesized graphene oxide-incorporated pyrochlore-type lanthanum stannate (La2Sn2O7@GO) as a sensing material for enhanced detection sensitivity and selectivity. The electrochemical detection of POE was performed using a glassy carbon electrode modified with the synthesized nanocomposite. The modified electrode La2Sn2O7@GO/GCE exhibited a remarkable electrochemical response towards POE, showing a significant reduction potential and an increase in the peak current compared to the bare and other modified electrodes. The proposed electrochemical method showed excellent detection sensitivity and selectivity for POE, with a linear response range of concentrations from 0.01 to 360 μM and a low detection limit (LDL) of 2.73 nM. The sensor also exhibited good stability and reproducibility, making it a promising tool for practical pesticide detection applications. The developed sensor offers a cost-effective, sensitive, and reliable platform for the rapid monitoring of organophosphate pesticides in environmental samples, contributing to the advancement of pesticide detection technologies and environmental protection efforts.

Simultaneous detection of SARS-CoV-2 S1 protein by using flexible electrochemical and Raman enhancing biochip

Flexible laser-scribed graphene (LSG) substrates with gold nanoislands have been developed as biochips for in situ electrochemical (EC) and surface-enhanced Raman scattering (SERS) biodetection (biomolecules and viral proteins). A flexible biochip was fabricated using CO2 laser engraving polyimide (PI) films to form a 3D porous graphene-like nanostructure. Gold nanoislands were deposited on the LSG substrates to enhance the intensity of the Raman signals. Moreover, the addition of auxiliary and reference electrodes induced a dual-function EC-SERS biochip with significantly enhanced detection sensitivity. The biochip could selectively and easily capture SARS-CoV-2 S1 protein through the SARS-CoV-2 S1 antibody immobilized on EC-SERS substrates using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The grafted antibody specifically bound to SARS-CoV-2, resulting in a significant increase in the SERS signal of the target analyte. The limit of detection (LOD) of the SARS-CoV-2 S1 protein was 5 and 100 ng/mL by using EC and SERS detection, respectively. Although the LOD of the SARS-CoV-2 S1 protein detected using SERS is only 100 ng/mL, it can provide fingerprint information for identification. To improve the LOD, EC detection was integrated with SERS detection. The three-electrode detection chip enables the simultaneous detection of SERS and EC signals, which provides complementary information for target identification. The dual-functional detection technology demonstrated in this study has great potential for biomedical applications, such as the rapid and sensitive detection of SARS-CoV-2.

A magnetic-enhanced FRET biosensor for simultaneous detection of multiple antibodies

Accurate, rapid and sensitive detection of specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in human samples is crucial for preventing and assessing pandemics, especially in the case of recent COVID-19 outbreaks. However, simultaneous and efficient detection of IgG and IgM in a single system remains challenging. Herein, we developed a multicolor nanosystem capable of quantitatively analyzing anti-SARS-CoV-2 IgG and IgM with high sensitivity within 20 ​min. The detection system consists of core-shell upconversion nanoparticles (csUCNPs), secondary antibodies labeled with fluorescent dyes (sab), and magnetic nanocrystals (PMF). By leveraging the Förster resonance energy transfer (FRET) effect, the photoluminescence (PL) intensity of blue and green regions is restored for IgG and IgM detection, respectively. Inspiringly, owing to the introducing of PMF, the limits of detection (LODs) of IgG and IgM tested are improved to 89 ​fmol ​L−1 and 19.4 ​fmol ​L−1, representing about 416-folds and 487-folds improvement over only-dye dependent system, respectively. Mechanistic investigations reveal that the high collective effect and surface energy transfer efficiency from csUCNPs to PMF contribute to the enhanced detection sensitivity. The assay enables us to quantify clinical vaccinated samples with high specificity and precision, suggesting our multicolor platform can be a promising alternative for clinical point-of-care serological assay.

On-site detection of Sporothrix globosa using immunoglobulin Y, magnetic separation and loop-mediated isothermal amplification

BackgroundSporothrix globosa (S. globosa) is an agricultural activity-related but neglected pathogenic fungus responsible for sporotrichosis. Timely detection is crucial for managing and preventing its spread. However, due to the lack of efficient recognition elements for enriching S. globosa, the current approaches for detecting S. globosa are not simple and/or sensitive enough. This hinders their wider application of fast screening. ResultsHerein, we successfully prepared immunoglobulin Y (IgY) targeting S. globosa, and developed a rapid and accurate detection method, improving upon current limited and inadequate detection approaches. Our method combined the use of IgY and loop-mediated isothermal amplification (LAMP) to enhance detection sensitivity and specificity simultaneously. The IgY was fabricated on magnetic beads to specifically concentrate S. globosa in samples, while LAMP amplified the captured target after simple boiling DNA extraction. By using our method, as low as 4.66 × 102 Cells mL−1S. globosa was accurately detected in soil and corn straw samples. We further integrated this assay into a portable toolbox for sample-to-result detection in resource-limited areas. By using this toolbox, we have colorimetrically detected soil and corn straw samples contaminated by S. globosa, suggesting the promising on-site detection potential. Significance and noveltyA new IgY recognizing S. globosa was prepared. Through the combination of IgY enrichment and LAMP amplification, the detection sensitivity and specificity were improved simultaneously. This method eliminated thermal cycling, simplified the operation, and reduced the analysis time. Compared to existing methods, our approach is more suitable for on-site detection and can significantly improve public health responses to sporotrichosis outbreaks.

Au nanoparticles decorated β-Bi2O3 as highly-sensitive SERS substrate for detection of methylene blue and methyl orange.

In this work, Au/Bi2O3 was synthesized by loading Au nanoparticles (NPs) onto β-Bi2O3 by a simple solution reduction method. β-Bi2O3 was synthesized by a precipitation-thermal decomposition procedure, which results in significantly improved SERS detection limits down to 10-9 M for methylene blue (MB) and 10-7 M for methyl orange (MO) as probe molecules, comparable to those reported for the best semiconductor SERS substrates. In particular, further deposition of Au NPs (5.20% wt%) onto β-Bi2O3 results in a two-order-of-magnitude enhancement in detection sensitivity, achieving a detection limit of 10-11 M for MB and 10-9 M for MO. Under ultraviolet/visible irradiation, the Au/Bi2O3 hybrids substrate exhibits superior self-cleaning ability due to its photocatalytic degradation ability which can be applied repeatedly to the detection of pollutants. The advanced composite substrate simultaneously achieved ultra-low mass loading of Au NPs, outstanding detection performance, good reproducibility, high stability and self-cleaning ability. The development strategy of low load noble metal coupled high performance semiconductor β-Bi2O3 to obtain nano-hybrid materials provides a method to balance SERS sensitivity, cost effectiveness and operational stability, and can be synthesized in large quantities, which is a key step towards commercialization and has good reliability prospects.

Perovskite-based electrochemiluminescence analysis of H2O2.

The detection of hydrogen peroxide (H2O2) represents an extensive requirement across various domains, including food, environmental, and medical fields. This study introduces a highly sensitive technique for the quantification of H2O2, integrating the electrochemiluminescence properties of perovskite with bio-catalyzed precipitation. A water-soluble perovskite-based electrochemiluminescence (ECL) biosensing interface was constructed, wherein H2O2 catalyzes a precipitation reaction that leads to the formation of an insoluble precipitate on the electrode surface. This occurrence effectively quenches the electrochemiluminescence signal of the perovskite, thus facilitating the quantitative detection of H2O2. The modified perovskite demonstrated excellent ECL performance, offering a stable signal source, while the bio-catalyzed precipitation reaction significantly amplified the quenching effect, thereby enhancing detection sensitivity. This strategy exhibits excellent stability and sensitivity, presenting a promising method for the detection of hydrogen peroxide, which holds great potential for applications in various fields.

Towards the next generation of detectors for n-γ capture reactions at n_TOF (CERN)

Benzene-d6 has been the scintillation material of choice for performing most of the neutron-capture cross-section measurements at CERN n_TOF for more than 20 years. Recently a revamping of solidstate Stilbene scintillators has been justified by their good timing performances, lack of toxicity and excellent neutron-gamma discrimination by Pulse-Shape Analysis. Consequently, several measurements were recently performed at the n_TOF facility at CERN and INFN-Catania to characterize their performance. We benchmarked both regular and deuterated stilbene scintillation crystals in EAR2@CERN. Based on these results, we are developing and designing a new generation of detectors with enhanced detection sensitivity and improved safety and maintenance conditions.

A new colorimetric aptasensor for paraquat detection based on the designed aptamer with multiple paraquat binding sites in combination with gold nanoparticles.

The presence of paraquat in the environment poses a danger to human health, leading to a growing demand for an uncomplicated and highly responsive method to detect paraquat. This work reports a new, simple, and sensitive colorimetric aptasensor based on the designed aptamers containing 1-5 paraquat binding sites (R1-R5) in combination with gold nanoparticles (AuNPs). Although the aptamers with more binding sites exhibited greater paraquat interaction capability, the aptasensor based on the R3 aptamer showed the highest detection sensitivity for paraquat in a linear range of 5-50 nM with a limit of detection of 1.29 nM, meaning that it is 2.14 fold more sensitive than the R1-aptasensor. This R3-aptasensor selectively detected paraquat but not the other tested herbicides, including difenzoquat, 2,4-D, ametryn, atrazine, and glufosinate. Also, it efficiently detected paraquat spiked in water samples within the precision acceptance criterion of recovery rates (96.8-105.0%) and the relative standard deviations (1.50-3.81%). These results demonstrated the development of a new aptasensor for paraquat detection, in which the multiple paraquat binding sites of the aptamers could enhance detection sensitivity.

Cybersecurity and Risk Prediction Based on Machine Learning Algorithms

Abstract This paper focuses on the application of machine learning in cybersecurity and risk prediction. Considering the complexity and dynamics of the cyber environment, the role of plain Bayesian algorithms in improving the accuracy of cybersecurity prediction is explored. First, the limitations of traditional linear regression machine learning algorithms in cybersecurity are analyzed, pointing out that it is difficult for these algorithms to capture complex nonlinear relationships. For this reason, plain Bayes is introduced to improve machine learning for more accurate prediction and assessment of cybersecurity risks by capturing functions and probabilistic agent models. In terms of methodology, this paper combines various technical tools such as statistical analysis, feature extraction, and risk assessment. The research results show that the improved algorithm outperforms the traditional method in simulation experiments, which is reflected in the 10% improvement in risk assessment accuracy and 20% enhancement of detection sensitivity to network intrusion. In addition, through big data analysis, the algorithm successfully identifies key risk points in network security and effectively predicts potential security threats. The improved machine learning algorithm with plain Bayes has significant advantages in network security and risk prediction and can effectively enhance network security protection capability and prediction accuracy.

Multiple signal amplification strategy using porous materials and novel controlled release self-on mode for ultrasensitive immunoassay

The porous structure of the luminophore, catalyst and carrier, combined with multiple signal amplification and novel controlled release encapsulation strategies, achieving an efficient self-on ultrasensitive ECL immunoassay for PSA.

The evolution of immune profiling: will there be a role for nanoparticles?

Immune profiling provides insights into the functioning of the immune system, including the distribution, abundance, and activity of immune cells. This understanding is essential for deciphering how the immune system responds to pathogens, vaccines, tumors, and other stimuli. Analyzing diverse immune cell types facilitates the development of personalized medicine approaches by characterizing individual variations in immune responses. With detailed immune profiles, clinicians can tailor treatment strategies to the specific immune status and needs of each patient, maximizing therapeutic efficacy while minimizing adverse effects. In this review, we discuss the evolution of immune profiling, from interrogating bulk cell samples in solution to evaluating the spatially-rich molecular profiles across intact preserved tissue sections. We also review various multiplexed imaging platforms recently developed, based on immunofluorescence and imaging mass spectrometry, and their impact on the field of immune profiling. Identifying and localizing various immune cell types across a patient's sample has already provided important insights into understanding disease progression, the development of novel targeted therapies, and predicting treatment response. We also offer a new perspective by highlighting the unprecedented potential of nanoparticles (NPs) that can open new horizons in immune profiling. NPs are known to provide enhanced detection sensitivity, targeting specificity, biocompatibility, stability, multimodal imaging features, and multiplexing capabilities. Therefore, we summarize the recent developments and advantages of NPs, which can contribute to advancing our understanding of immune function to facilitate precision medicine. Overall, NPs have the potential to offer a versatile and robust approach to profile the immune system with improved efficiency and multiplexed imaging power.

Intelligent point of care testing for medicine diagnosis

AbstractIntelligent point‐of‐care testing (iPOCT) is a rapidly advancing technology that combines molecular detection and bio‐sensing techniques to achieve instant and accurate analysis through advanced sensors and intelligent algorithms. Molecular detection technologies, such as nucleic acid isothermal amplification, biochip and microfluidics, enable rapid analysis of the chemical composition and concentration of samples, thereby determining their presence. Bio‐sensing technology utilizes the recognition properties of biomolecules in conjunction with sensors to detect specific biomolecules or biological processes. iPOCT technology holds broad prospects for application in the field of healthcare and is expected to enhance detection sensitivity, accuracy, and intelligence through further development. In this review, we delineate the developmental journey of iPOCT, followed by a discussion on the principles of molecular detection and bio‐sensing technology, as well as the design of iPOCT devices for health monitoring and disease diagnosis. Furthermore, we showcased illustrative examples of iPOCT applications in disease diagnosis, highlighting the integration of iPOCT technology with wearable devices and smartphones for attaining more precise and personalized diagnostic results.

Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde.

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs in vitro using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.

Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction.

Urological cancers such as bladder or prostate cancer represent one of the most malignant tumors that accounts for an extremely high mortality. However, conventionally standard diagnostics for urological cancers are hardly available in low-resource settings. We developed herein a hand-held fluorescent imaging platform by integrating a multiplexed isothermal exponential amplification reaction (EXPAR) with a microgel-enriched methodology for sensitive profiling of quaternary microRNAs (miRNAs) in urine and quick diagnosis of urological cancers at the early stage. The target miRNA mixtures in the urine underwent four parallel EXPARs without cross-reactivity, followed by surface concentration and hybridization by the encoded polyacrylamide microgels. This mix-and-read strategy allowed for one-pot analysis of several key miRNAs simultaneously and provided 5-fold enhancement in fluorescent detection sensitivities compared to the individual EXPAR-based assays. Four urinary miRNAs (let-7a, miRNA-155, -223, and -143) could be quantitatively determined in a wide linear range from 50 fM to 30 nM, with the limits of detection at femtomolar levels. Using a smartphone-based imaging microreader, healthy and cancerous cohorts with prostate, bladder, and renal cell cancers could be discriminated in 30 min with the accuracy >83% using linear discriminant analysis. The developed detection platform has proven to be a portable, noninvasive, and useful complement to the toolbox for miRNA-based liquid biopsies, which holds immense potential and advantage for regular and large-scale applications in early cancer diagnosis.