From Shadows to Spotlight: Enhancing Bacterial DNA Detection in Blood Samples through Cutting-Edge Molecular Pre-Amplification.

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Comparative Analysis of Cytokine Profiles in Cerebrospinal Fluid and Blood Serum in Patients with Acute and Subacute Spinal Cord Injury.

BACKGROUND: Cytokines are actively involved in the regulation of the inflammatory and immune responses and have crucial importance in the outcome of spinal cord injuries (SCIs). Examining more objective and representative indicators of the patient's condition is still required to reveal the fundamental patterns of the abovementioned posttraumatic processes, including the identification of changes in the expression of cytokines. METHODS: We performed a dynamic (3, 7, and 14 days post-injury (dpi)) extended multiplex analysis of cytokine profiles in both CSF and blood serum of SCI patients with baseline American Spinal Injury Association Impairment Scale grades of A. RESULTS: The data obtained showed a large elevation of IL6 (>58 fold) in CSF and IFN-γ (>14 fold) in blood serum at 3 dpi with a downward trend as the post-traumatic period increases. The level of cytokine CCL26 was significantly elevated in both CSF and blood serum at 3 days post-SCI, while other cytokines did not show the same trend in the different biosamples. CONCLUSIONS: The dynamic changes in cytokine levels observed in our study can explore the relationships with the SCI region and injury severity, paving the way for a better understanding of the pathophysiology of SCI and potentially more targeted and personalized therapeutic interventions.

Neuroinflammatory Dysfunction of the Blood-Brain Barrier and Basement Membrane Dysplasia Play a Role in the Development of Drug-Resistant Epilepsy.

Drug-resistance epilepsy (DRE) is a key problem in neurology. It is possible that damage to the blood-brain barrier (BBB) may affect resistance in DRE. The aim of this work was to assess the damage and dysfunction in the BBB in the area of epileptic foci in patients with DRE under conditions of neuroinflammation. The changes to the BBB in temporal lobe epilepsy (by immunohistochemistry and transmission electron microscopy), levels of neuroinflammatory proteins, and cytokine levels in the blood (by multiplex analysis) were studied. Increased levels of vascular endothelial growth factor (VEGF) and growth-regulated protein (GRO), and decreased levels of epidermal growth factor (EGF) in plasma, combined with overexpression of the VEGF-A receptor by endotheliocytes were detected. Malformation-like growths of the basement membrane of the capillaries of the brain complicate the delivery of antiepileptic drugs (AEDs). Dysplasia of the basement membrane is the result of inadequate reparative processes in chronic inflammation. In conclusion, it should be noted that damage to the microcirculatory network of the brain should be considered one of the leading factors contributing to DRE.

Multicolor aptasensors for pesticide multiresidues detection in agricultural products using bioorthogonal surface-enhanced Raman scattering tags.

Realizing accurate pesticide multiresidue detection in a complex matrix is still a challenge for point-of-care sensing methods. Herein, we introduced background-free and multicolor aptasensors based on bioorthogonal surface-enhanced Raman scattering (SERS) tags and successfully applied them to analyze multiple pesticide residues. The excellent anti-interference and multiplex capability are due to the application of three bioorthogonal Raman reporters involving 4-ethenylbenzenamine (4-EBZM), Prussian blue (PB) and 2-amino-4-cyanopyridine (AMCP) with alkynyl and cyano groups, which demonstrated apparent Raman shift peaks at 1993 cm-1, 2160 cm-1, and 2264 cm-1 in the biologically Raman-silent region, respectively. Ultimately, a detection range of 1-50 nM for acetamiprid, atrazine and malathion was achieved with detection limits of 0.39, 0.57 and 0.16 nM, respectively. The developed aptasensors were successfully used to determine pesticide residues in real samples. These proposed multicolor aptasensors offer an effective strategy for pesticide multiresidue detection with advantages of anti-interference, high specificity and high sensitivity.

LC-MS/MS-based multiplex antibacterial platform for therapeutic drug monitoring in intensive care unit patients.

Empirically prescribed standard dosing regimens of antibacterial agents may result in insufficient or excess plasma concentrations with persistently poor clinical outcomes, especially for patients in intensive care units (ICUs). Therapeutic drug monitoring (TDM) of antibacterial agents can guide dose adjustments to benefit patients. In this study, we developed a robust, sensitive, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the quantification of 14 antibacterial and antifungal agents (beta-lactams piperacillin, cefoperazone, and meropenem; beta-lactamase inhibitors tazobactam and sulbactam; antifungal agents fluconazole, caspofungin, posaconazole, and voriconazole; and daptomycin, vancomycin, teicoplanin, linezolid, and tigecycline) that can be used for patients with severe infection. This assay requires only 100 µL of serum with rapid protein precipitation. Chromatographic analysis was performed using a Waters Acquity UPLC C8 column. Three stable isotope-labeled antibacterial agents and one analogue were used as internal standards. Calibration curves ranged from 0.1-100 µg/mL, 0.1-50 µg/mL, and 0.3-100 µg/mL for different drugs, and all correlation coefficients were greater than 0.9085. Intra- and inter-day imprecision and inaccuracy values were below 15%. After validation, this new method was successfully employed for TDM in routine practice.

One-step multiplex analysis of breast cancer exosomes using an electrochemical strategy assisted by gold nanoparticles.

Exosomes, as a non-invasive biomarker, perform an important role in breast cancer screening and prognosis monitoring. However, establishing a simple, sensitive, and reliable exosome analysis technique remains challenging. Herein, a one-step multiplex analysis electrochemical aptasensor based on a multi-probe recognition strategy was constructed to analyze breast cancer exosomes. HER2-positive breast cancer cell (SK-BR-3) exosomes were selected as the model targets and three aptamers including CD63, HER2 and EpCAM aptamers were used as the capture units. Methylene blue (MB) functionalized HER2 aptamer and ferrocene (Fc) functionalized EpCAM aptamer, which were modified on gold nanoparticles (Au NPs), i.e. MB-HER2-Au NPs and Fc-EpCAM-Au NPs, were used as signal units. When the mixture of target exosomes, MB-HER2-Au NPs and Fc-EpCAM-Au NPs were added on the CD63 aptamer modified gold electrode, two Au NPs modified by MB and Fc could be specifically captured on the electrode by the recognition of three aptamers with target exosomes. Then one-step multiplex analysis of exosomes was achieved by detecting two independent electrochemical signals. This strategy can not only distinguish breast cancer exosomes from other exosomes (including normal exosomes and other tumor exosomes) but also HER2-positive breast cancer exosomes and HER2-negative breast cancer exosomes. Besides, it had high sensitivity and can detect SK-BR-3 exosomes with a concentration as low as 3.4 × 103 particles mL-1. Crucially, this method can be applicable to the examination of exosomes in complicated samples, which is anticipated to afford assistance for the screening and prognosis of breast cancer.

Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

[Pleiomorphism of the cytokine profile in nasal polyp tissue depending on the phenotype of chronic rhinosinusitis with nasal polyps]. / Pleiomorfizm tsitokinovogo profilya v tkani polipov v zavisimosti ot fenotipa polipoznogo rinosinusita.

Clinical phenotypes of chronic rhinosinusitis with nasal polyps (CRSwNP) are characterized with different inflammation patterns of mRNA expression of cytokines and depend on presence of allergic rhinitis (AR), atopic bronchial asthma (aBA) or nonatopic bronchial asthma (nBA). OBJECTIVE: To compare inflammation response in patients with different phenotypes of CRSwNP according to level secretion of the key cytokines in nasal polyp tissue. MATERIAL AND METHODS: 292 patients with CRSwNP were divided into four phenotypes: group 1 - CRSwNP without respiratory allergy (RA) and without BA; group 2a - CRSwNP+ AR with aBA; group 2b - CRSwNP+AR without aBA; group 3 - CRSwNP+nBA. Control group (n=36) included patients with hypertrophic rhinitis without atopy or BA. Using multiplex assay we defined the level of IL-1ß, IL-4, IL-5, IL-6, IL-13, IFN-γ, TGF-ß1, TGF-ß2, TGF-ß3 in nasal polyp tissue. RESULTS: The evaluation of cytokines levels in nasal polyps in different CRSwNP phenotypes showed a pleiotropy of different cytokine secretion depending on different comorbid pathology. In control group we estimated the lowest levels of all detected cytokines in comparison with other CRS groups. High levels of local proteins IL-5 and IL-13 and low levels of all isoform of TGF-ß characterized CRSwNP without RA and BA. The combination of CRSwNP with AR showed high levels of proinflammatory cytokines IL-6 and IL-1ß, and high levels of TGF-ß1 and TGF-ß2. The combination of CRSwNP with aBA estimated low levels of proinflammatory cytokines IL-1ß, IFN-γ; in case of CRS+nBA we determined the highest levels of TGF-ß1, TGF-ß2 and TGF-ß3 in nasal polyp tissue. CONCLUSIONS: Each CRSwNP phenotype is characterized by different mechanism of local inflammation. This underlies the necessity to diagnose BA and respiratory allergy among these patients. The evaluation of local cytokine profile in different CRSwNP phenotypes can help to determine the target anticytokine therapy for patients who has low efficacy of basic corticosteroid therapy.

Point-of-care vertical flow immunoassay system for ultra-sensitive multiplex biothreat-agent detection in biological fluids.

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 µm to the current 0.45 µm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

Cytokine Profiling in Different SARS-CoV-2 Genetic Variants.

This study is a successor of our previous work concerning changes in the chemokine profile in infection that are associated with different SARS-CoV-2 genetic variants. The goal of our study was to take into account both the virus and the host immune system by assessing concentrations of cytokines in patients infected with different SARS-CoV-2 variants (ancestral Wuhan strain, Alpha, Delta and Omicron). Our study was performed on 340 biological samples taken from COVID-19 patients and healthy donors in the timespan between May 2020 and April 2022. We performed genotyping of the virus in nasopharyngeal swabs, which was followed by assessment of cytokines' concentration in blood plasma. We noted that out of nearly 30 cytokines, only four showed stable elevation independently of the variant (IL-6, IL-10, IL-18 and IL-27), and we believe them to be 'constant' markers for COVID-19 infection. Cytokines that were studied as potential biomarkers lose their diagnostic value as the virus evolves, and the specter of potential targets for predictive models is narrowing. So far, only four cytokines (IL-6, IL-10, IL-18, and IL-27) showed a consistent rise in concentrations independently of the genetic variant of the virus. Although we believe our findings to be of scientific interest, we still consider them inconclusive; further investigation and comparison of immune responses to different variants of SARS-CoV-2 is required.

Synthesis, Self-Assembly in Crystalline Phase and Anti-Tumor Activity of 2-(2-/4-Hydroxybenzylidene)thiazolo[3,2-a]pyrimidines.

A series of new thiazolo[3,2-a]pyrimidines different by aryl substituents in 2 and 5 positions are synthesized and characterized in solution as well as in the crystalline phase using 1H and 13C NMR-, IR-spectroscopies, mass-spectrometry methods, and single crystal X-ray diffraction (SCXRD). The SCXRD study revealed the role of intermolecular H-bonding in the formation of supramolecular architectures (racemic monomers, centrosymmetric racematic dimers, or homochiral 1D chains) of obtained thiazolo[3,2-a]pyrimidines derivatives depending on solvents (aprotic DMSO or protic EtOH) used upon the crystallization process. Moreover, the in vitro study of cytotoxicity toward different tumor cells showed their high or moderate efficiency with moderate cytotoxicity against normal liver cells which allows to consider the obtained thiazolo[3,2-a]pyrimidine derivatives as promising candidates for application as antitumor agents.

Enhancing of detection resolution via designing of a multi-functional 3D connector between sampling and detection zones in distance-based microfluidic paper-based analytical device: multi-channel design for multiplex analysis.

One of the problems in the distance-based microfluidic paper-based analytical device (DB-µPAD) that limits the detection resolution is the mixing of reagents from the detection to the sampling zone or vice versa due to spreading by capillary action. In the present paper, to overcome mixing of the reagents in the zones, a multi-functional connector using a three-dimensional (3D) design has been developed externally to connect the two zones. Using such a novel design, it is acertained that there is no any mixing due to the dispersion of the reagents in the two zones. Interestingly, the simple 3D connector has other functions, such as its potential to be used as a masking zone and/or reaction zone whenever is needed. Based on this proposed 3D connector-based DB-µPAD, three parallel microchannels were built as detection zones with one sampling zone for multiplex analysis for the detection of Fe2+, Ni2+, and hardness of water. In the Ni2+ channel, the connector piece worked as both masking part and connection part. In the Fe2+ line, the connector served as the connector and reducing pad (Fe3+ to Fe2+ ions). While in the third channel, the connector has connection function only. Satisfactory limit of detection (LoD), accuracy, and precision were obtained using this design. The LODs obtained with the proposed design were 1.13, 0.62, and 1.87 mg.L-1 for total hardness, Fe2+, and Ni2+, respectively.

Biomarkers in Liquid Biopsies for Prediction of Early Liver Metastases in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies with poor survival rates. Only 20% of the patients are eligible for R0-surgical resection, presenting with early relapses, mainly in the liver. PDAC patients with hepatic metastases have a worse outcome compared to patients with metastases at other sites. Early detection of hepatic spread bears the potential to improve patient outcomes. Thus, this study sought for serum-based perioperative biomarkers allowing discrimination of early (EHMS ≤ 12 months) and late hepatic metastatic spread (LHMS > 12 months). Serum samples from 83 resectable PDAC patients were divided into EHMS and LHMS and analyzed for levels of inflammatory mediators by LEGENDplexTM, which was validated and extended by Olink® analysis. CA19-9 serum levels served as control. Results were correlated with clinicopathological data. While serum CA19-9 levels were comparable, Olink® analysis confirmed distinct differences between both groups. It revealed significantly elevated levels of factors involved in chemotaxis and migration of immune cells, immune activity, and cell growth in serum of LHMS-patients. Overall, Olink® analysis identified a comprehensive biomarker panel in serum of PDAC patients that could provide the basis for predicting LHMS. However, further studies with larger cohorts are required for its clinical translation.

A battery of tandem mass spectrometry assays with stable isotope-dilution for the quantification of 15 anti-tuberculosis drugs and two metabolites in patients with susceptible-, multidrug-resistant- and extensively drug-resistant tuberculosis.

OBJECTIVE: Anti-tuberculosis (antiTB) drugs are characterized by an important inter-interindividual pharmacokinetic variability poorly predictable from individual patients' characteristics. Therapeutic drug monitoring (TDM) may therefore be beneficial for patients with Mycobacterium tuberculosis infection, especially for the management of multidrug/extensively drug resistant- (MDR/XDR)-TB. Our objective was to develop robust HPLC-MS/MS methods for plasma quantification of 15 antiTB drugs and 2 metabolites, namely rifampicin, isoniazid plus N-acetyl-isoniazid, pyrazinamide, ethambutol (the conventional quadritherapy for susceptible TB) as well as combination of agents against MDR/XDR-TB: bedaquiline, clofazimine, delamanid and its metabolite M1, levofloxacin, linezolid, moxifloxacin, pretomanid, rifabutin, rifapentine, sutezolid, and cycloserine. METHODS: Plasma protein precipitation was used for all analytes except cycloserine, which was analyzed separately after derivatization with benzoyl chloride. AntiTB quadritherapy drugs (Pool1) were separated by Hydrophilic Interaction Liquid Chromatography (column Xbridge BEH Amide, 2.1 × 150 mm, 2.5 µm, Waters®) while MDR/XDR-TB agents (Pool 2) and cycloserine (as benzoyl derivative) were analyzed by reverse phase chromatography on a column XSelect HSS T3, 2.1 × 75 mm, 3.5 µm (Waters®). All runs last <7 min. Quantification was performed by selected reaction monitoring electrospray tandem mass spectrometry, using stable isotopically labelled internal standards. RESULTS: The method covers the clinically relevant plasma levels and was extensively validated based on FDA recommendations, with intra- and inter-assay precision (CV) < 15% over the validated ranges. Application of the method is illustrated by examples of TDM for two patients treated for drug-susceptible- and MDR-TB. CONCLUSION: Such convenient extraction methods and the use of stable isotope-labelled drugs as internal standards provide an accurate and precise quantification of plasma concentrations of all major clinically-used antiTB drugs regimens and is optimally suited for clinically efficient TDM against tuberculosis.

Mollusk allergy in shrimp-allergic patients: Still a complex diagnosis. An Italian real-life cross-sectional multicenter study.

Introduction: Shellfish allergy is an important cause of food allergies worldwide. Both in vivo and in vitro diagnostics failure nowadays is caused by the poor quality of the extracts associated with the scarce availability of allergenic molecules in the market. It is known that not all patients with shellfish allergies experience adverse reactions to mollusks. It is still unclear how to detect and diagnose these patients correctly. Aim: To investigate the features of shrimp-allergic patients either reactive or tolerant to mollusks, with the currently available diagnostic methods. Methods: Nineteen centers, scattered throughout Italy, participated in the real-life study, enrolling patients allergic to shrimp with or without associated reactions to mollusks. Patients underwent skin tests using commercial extracts or fresh raw and cooked shrimp and mollusks, and IgE reactivity to currently available allergenic extracts and molecules was measured in vitro. Results: Two hundred and forty-seven individuals with a self reported adverse reactions to shrimp participated in the study; of these 47.8% reported an adverse reaction to mollusks ingestion (cephalopod and/or bivalve). Neither of the tests used, in vivo nor in vitro, was able to detect all selected patients. Accordingly, a great heterogeneity of results was observed: in vivo and in vitro tests agreed in 52% and 62% of cases. Skin tests were able to identify the mollusk reactors (p < 0.001), also using fresh cooked or raw food (p < 0.001). The reactivity profile of mollusk reactors was dominated by Pen m 1, over Pen m 2 and Pen m 4 compared to tolerant subjects, but 33% of patients were not detected by any of the available molecules. Overall, a higher frequency of IgE rectivity to shrimp was recorded in northern Italy, while mollusk reactivity was more frequent in the center-south. Conclusion: The current diagnostic methods are inadequate to predict the cross-reactivity between crustaceans and mollusks. The detection of mollusks hypersensitivity should still rely on skin tests with fresh material. The exclusion of mollusks from shrimp allergic patients' diets should occur when clinical history, available diagnostic instruments, and/or tolerance tests support such a decision.

A Rebuilding-Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N-in-1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens.

Sensitive, rapid and low-cost nucleic acid detection is critical for controlling infectious pathogens. Here, we develop a ready-to-use and multimodal detection based on a rebuilding-free, ultrasensitive and selective strategy named dual hairpin ligation-induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS-CoV, SARS-CoV-2 as model targets, we demonstrate DHLApro provides ≈zM level ultra-sensitivity, being equaling to 0.45 copy/µL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the "N pathogens-in-1" OR gate screening and accurate multi-channel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross-interference between the multiple primer sets is also avoided.

Evaluating the Effects of Curcumin on the Cytokine Storm in COVID-19 Using a Chip-Based Multiplex Analysis.

SARS-CoV-2 can stimulate the expression of various inflammatory cytokines and induce the cytokine storm in COVID-19 patients leading to multiple organ failure and death. Curcumin as a polyphenolic compound has been shown to have anti-inflammatory properties and inhibit the release of numerous pro-inflammatory cytokines. We present multiplex analysis using the Evidence Investigator biochip system to determine the effect of curcumin on serum level of cytokines which are typically elevated in cytokine storm events, including tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and IL-10.

Simple, Skin-Attachable, and Multifunctional Colorimetric Sweat Sensor.

In situ analysis of sweat provides a simple, convenient, cost-effective, and noninvasive approach for the early diagnosis of physical illness in humans and is particularly useful in family care. In this study, a flexible and skin-attachable colorimetric sweat sensor for multiplexed analysis is developed using a simple, cost-effective, and convenient method. The obtained sweat sensor can be used to simultaneously detect glucose, lactate, urea, and pH value in sweat, as well as sweat loss and skin temperature. Only 2.5 µL of sweat is enough for the whole test, and the sweat loss and chemical-sensing results can be read out conveniently by naked eyes or a smartphone. In addition, body temperature can also be detected with an additional electrical circuit. Our sweat sensor provides a new, cost-effective, and convenient approach for in vitro diagnosis of multiple components in sweat, and the easy fabrication and cost-effectiveness make our sensor commercializable in the near future.

Development of a streptavidin-bridged enhanced sandwich ELISA based on self-paired nanobodies for monitoring multiplex Salmonella serogroups.

Salmonella are major pathogens that cause foodborne diseases. In this work, a broad-spectrum Salmonella nanobody-01 (Nb-01) was isolated and applied in the development of a streptavidin-bridged sandwich ELISA (SAB-ELISA) for simultaneously identifying five Salmonella serovars, including Salmonella Enteritidis (S. Enteritidis), Salmonella Typhimurium (S. Typhimurium), Salmonella London (S. London), Salmonella Paratyphi B (S. Paratyphi B) and Salmonella Hadar (S. Hadar). In this work, streptavidin (SA) was utilized as a scaffold to directionally immobilize biotinylated nanobody (BiNb) and Salmonella was detected by phage-displayed nanobodies. The SAB-ELISA can be accomplished within 180 min with a limit of detection (LOD) of 6.31 × 103 colony forming units (CFU) mL-1, 9.15 × 103 CFU mL-1, 4.23 × 103 CFU mL-1, 7.31 × 103 CFU mL-1 and 7.25 × 103 CFU mL-1 towards S. Typhimurium, S. Enteritidis, S. London, S. Paratyphi B and S. Hadar, respectively. In comparison of sandwich ELISA by passive immobilization of Nb-01 on polystyrene plate, the sensitivity was increased by around 6-fold, which confirmed the enhanced immobilization efficacy of SAB-ELISA. Furthermore, the feasibility of the assay for S. Typhimurium determination in actual samples was evaluated, showing excellent recovery, inter-day and intra-day precision.

High-throughput multiplex analysis of mAb aggregates and charge variants by automated two-dimensional size exclusion-cation exchange chromatography coupled to mass spectrometry.

Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis.