Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity are challenging for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 13-quinone standard mixture, we optimized separation within a 14-minute HPLC gradient and achieved a sensitivity ranging from 55.2 to 0.3 femtomoles in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 quinones, revealing distinct quinone profiles at various wastewater treatment stages. This rapid and sensitive workflow enables quinone profiling in complex samples, and holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications.

This study reports the development of an integrated, portable analytical platform combining a micro-spectrometer with loop-mediated isothermal amplification (LAMP) for the rapid, on-site detection of Vibrio vulnificus. The system, controlled by LinkIt 7697 microprocessors, achieves precise thermal regulation within ±0.32 °C at 64 °C and features a customized optical path for real-time fluorescence monitoring. Utilizing calcein as a metal-sensitive fluorescent indicator, the device captures the spectral shift and intensity enhancement resulting from the displacement of manganese ions during DNA synthesis. Unlike traditional photodiode-based sensors, the integrated micro-spectrometer enables full-spectrum analysis at 1-min intervals, providing superior discrimination between true positive signals and background autofluorescence. Experimental results demonstrate successful detection of Vibrio vulnificus genomic fragments within 40 min, achieving a detection limit of 0.1 pg/μL, with data automatically synchronized to a cloud database for remote epidemiological surveillance. The combination of high-fidelity spectral acquisition and isothermal chemistry offers a robust, cost-effective solution for food safety monitoring and point-of-care testing in resource-limited environments.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and the gatekeeper of the cell for many antimicrobial peptides (AMPs). Indolicidin (ID) is a cationic AMP isolated from cytoplasmic granules of bovine neutrophils. ID, however, is too toxic and hemolytic for therapeutic use. ID45, a novel ID derivative, was developed through amino acid substitution and displays an increased antimicrobial potential and reduced hemolysis. LPS, located in the bacterial envelope, is the suspected target receptor for ID and ID45. The interaction between ID or the derivative ID45 with LPS was characterized using capillary electrophoresis (CE). The binding constant (Kb) for the interaction between ID and LPS as well as ID45 and LPS, was determined using a range of CE techniques. Preincubation CE (PI-CE) was utilized over a temperature range of 20 - 30 °C to establish a Kb with thermodynamic parameters. PI-CE Kb values for the ID-LPS interaction (3.13 ± 0.77 × 10[4] M[-1]) and ID45-LPS interaction (14.83 ± 1.67 × 10[4] M[-1]) were determined at pH 7.2. Both interactions were thermodynamically favorable and determined to be due to electrostatic forces. CE Front Analysis (CE-FA) was also utilized to provide support to the PI-CE methodology. The results show that ID45 binds five times more strongly to LPS at equilibrium, perhaps explaining the observed increase in antimicrobial activity and decreased toxicity compared to ID. ID45 does have the potential to be a novel drug candidate.

Alogliptin (ALO), as a dipeptidyl peptidase-4 (DPP-4) inhibitor is one of the commonly prescribed oral hypoglycemics for its potential effects on incretin and glucagon levels. For the first time silver nanoparticles (AgNPs) were introduced into the polymeric membrane cocktail of a newly developed ALO-selective electrode as ion-to-electron transducers. The optimized sensor achieved a near-Nernstian slope of 60.43 and 58.66 mV/decade as authentic and spiked plasma samples, respectively over a linear range of 8.60×10[-7]-1.55×10[-3] M with a limit of detection of 2.86×10[-7] M. The membrane blend involves also; tricresyl phosphate (TCP) as plasticizer and phosphotungestic acid (PT) as cationic exchanger with almost typical Nernstian slope. Testing water layer formation has proven no negative effects on the electrode performance with acceptable operational lifetime and fast dynamic response time. Validation of the experimental parameters has experienced mean percentage recoveries of 95.00 % and relative standard deviation values (%RSD) less than 5.00 %. An average recovery percentage of ≈ 99.00 % was obtained upon application to commercial tablets without significant differences with a previously reported method. The proposed sensor has been exploited as unique application in determining ALO - rabbit plasma binding interaction kinetics through constructing Hill plot, binding isotherms and Vant' Hoff plot in different temperatures. The calculated parameters have proven both spontaneity and positive cooperative binding. Additionally, this sensor has been used as a proof-of-concept demonstration to track the pharmacokinetic behavior of ALO in rabbit males, illustrating its in-vivo pathway. The method was assessed using AGREE, AGREEprep, Complex-GAPI, and BAGI greenness/blueness metrics (scores of 0.69, 0.74, 79, and 77.5, respectively), which are reported and discussed below rather than characterized only qualitatively.

Parasitic diseases continue to be an important public health problem and impact over one billion people worldwide; therefore, there is an urgent need for new, high-performance diagnostic methods that are sensitive, rapid, and accessible. Conventional parasitological and laboratory-based diagnosis can be time-consuming, require specialized equipment and highly trained personnel, and often has limited sensitivity at low parasite loads, all of which can hinder effective treatment, disease surveillance, and transmission control. This review critically analyzes the latest developments in immunological assays and new biosensor technologies for detecting parasitic infections. This review assesses established immunodiagnostic tests such as indirect immunofluorescence (IFA) assays, enzyme-linked immunosorbent assays (ELISA), and antigen-based detection methods for their analytical performance, clinical usefulness, and limitations. This review will focus on the development of electrochemical immunosensors, nucleic acid-based biosensors, and aptamer-based sensing platforms, which can offer enhanced sensitivity, selectivity, rapid response, miniaturization, and potential for point-of-care (POC) analysis. Conventional reviews discuss parasitological and/or immunological diagnostic approaches in isolation, whereas this review combines established immunodiagnostic approaches with next-generation biosensing approaches and critically evaluates the analytical transition to portable POC platforms. Lastly, we discuss opportunities for novel uses in signal amplification using nanomaterials, multiplexed detection of multiple biomarkers, microfluidic integration, and portable analytical devices. This holistic view can inform the design of rational platforms for sensitive, economical, and easy-to-deploy diagnostics for parasitic infections.

Multiplex quantitative real-time PCR (M-qPCR) is increasingly becoming a commonly used technique in molecular detection of pathogens due to its advantages in quantification and throughput. However, imbalanced amplification is still a potential problem affecting performance of M-qPCR assays. In this study, a novel universal primer multiplex quantitative real-time PCR (UP-M-qPCR) assay using EvaGreen and melting curve analysis was developed, using five swine diarrhea-related viruses as a case study. UPs and the chimeric primers (CPs) were introduced in this assay for sequential virus-specific and universal amplification to produce large amounts of amplicons for five target viruses with similar efficiency. Subsequent melting analysis of the amplicons displayed five distinguishable target-specific melting peaks with characteristic melting temperatures (Tms), while no non-specific amplification or melting peak was observed for five non-target viruses. The assay achieved a limit of detection (LOD) of 5 copies/μL of viral plasmids in various settings including high-background co-infections. Meanwhile, the coefficient of variation of Tm value and Ct values fell within 0-0.57% and 0.12-4.37% respectively, suggesting good repeatability of the assay. The UP-M-qPCR and corresponding singleplex qPCR were used in parallel to detect field samples for five viruses. The diagnostic sensitivity and specificity of the UP-M-qPCR were 90.9-100% and 99.4-100%, respectively, with a kappa value of 0.95-1, indicating good consistency. Overall, this study presents a melting curve analysis-based UP-M-qPCR assay, enabling the concurrent detection of multiple viruses with high sensitivity and specificity, which provides a technical platform easily adaptable for multiple pathogens detection.

Citrate synthase is a well-established substrate to measure the relative activity of molecular chaperones at low concentrations and with high precision. Under denaturing conditions, citrate synthase unfolds and forms monomeric intermediates prone to aggregate at elevated temperatures. Aggregation can be monitored by measuring an increase in light scattering. Chaperone binding will prevent its aggregation and mitigate light scattering depending on the molar excess of a chaperone added over citrate synthase. Here, we present a convenient and cost-efficient method to produce porcine citrate synthase from E. coli to circumvent the need for commercial citrate synthase purified from pig hearts. The recombinant citrate synthase is catalytically active and functions upon chemical denaturation as a substrate in the aggregation assay, aggregating with similar kinetics as commercial citrate synthase and resulting in reproducible measurements for the redox-regulated chaperone function of Get3. Aggregation strongly depends on substrate concentration, temperature and pH, whereas the addition of DTT or low amounts of salt barely affected light scattering. We further applied the citrate synthase aggregation assay to determine and compare relative chaperone activities of Get3 induced by hydrogen peroxide in its nucleotide-bound and -free conformation and showed that hypochlorous acid readily activated Get3's chaperone function independent of its nucleotide-binding state. Generating large amounts of recombinant citrate synthase which is stable over long time and can be used as a substrate in aggregation assays will facilitate thorough in vitro analyses of molecular chaperones, their activation mechanisms and functional regulation.

Aequorin is a Ca[2+]-binding photoprotein that emits blue light upon binding calcium ions and has been widely used as a calcium indicator. Although the metal ion specificity of aequorin was investigated in the early 1970s, discrepancies among previous studies have left its ion selectivity incompletely understood. In this study, we systematically reevaluated the metal ion specificity of recombinant aequorin using an FDSS-μCELL® v2 automated imaging system equipped with integrated dispensing and highly sensitive luminescence detection. Luminescence responses of recombinant aequorin to 29 metal ions (10[-8]-10[-3] M) were characterized by measuring maximum luminescence intensity (Imax), integrated luminescence over 60 s (Int. 60s), and residual luminescence activity following the addition of excess Ca[2+]. Based on luminescence kinetics and recovery of aequorin activity, the responses to metal ions were classified into six categories. Ca[2+] induced a concentration-dependent flash-type luminescence. Similar flash-type responses were observed with Sr[2+], Y[3+], Pb[2+], and several lanthanide ions, whereas Ba[2+], Ce[3+], Ga[3+], Mg[2+], Nd[3+], and V[3+] induced weak glow-type luminescence without significant loss of aequorin activity. In contrast, Cd[2+], Co[2+], Cu[2+], Fe[2+], Ni[2+], and Zn[2+] induced glow-type luminescence accompanied by reduced residual activity, whereas Cr[3+] and Zr[4+] produced no luminescence but also decreased aequorin activity. These findings provide a comprehensive quantitative reevaluation of the metal ion selectivity of aequorin and indicate that flash- and glow-type luminescence arise from distinct modes of metal ion interaction with the EF-hand motifs and the resulting conformational changes in aequorin.

We developed a plate-based luminescence assay for TEV protease using NanoLuc substrates site-specifically biotinylated through BirA co-expression in Escherichia coli. Substrates containing a TEV recognition sequence and AviTag, with or without a G4S linker, were immobilized on streptavidin-coated plates. Protease activity was quantified from the luminescence of NanoLuc released after cleavage. G4S insertion improved biotinylation efficiency and the luminescence response of immobilized substrates. Four-parameter logistic analysis showed lower EC20 and EC50 values and improved limits of detection and quantification. These findings demonstrate that linker insertion enhances substrate preparation and analytical sensitivity in this assay.

Free fatty acids (FFAs) are important targets in metabolic diseases. However, accurate quantification of a wide range of C2-C24 FFAs in the liver is challenging due to their structural diversity and large variations in abundance. To address this, a stepwise derivatization pretreatment was employed in this study: after liver homogenization, short-chain fatty acids were first extracted with diethyl ether-water (1:1, V/V). The ether layer was dehydrated with anhydrous Na2SO4 and then derivatized with BSTFA. The remaining pellet was extracted with chloroform-methanol (1:2, V/V), and after nitrogen evaporation, the extract was subjected to methyl esterification. A quantitative method for 39 FFAs was established on a Trace1600-TSQ9610 platform (TG-SQC column, MRM mode). Methodological validation showed that for all FFAs, the linear correlation coefficients R[2] were ≥ 0.991, intra- and inter-day accuracies ranged from 85.94% to 115.28%, precision RSD ≤ 13.16%, stability RSD ≤ 14.91%, and the LLOQ ranged from 0.03 to 10 μg/mL. When applied to the livers of spleen-deficiency SD rats, the method revealed significant decreases in C14:0, C15:0, and C18:0, and significant increases in C9:0, C18:1n9c, and C20:4n6 (VIP > 1, |Log2FC| > 1, P < 0.05). This method provides a reliable tool for hepatic FFA profiling and the discovery of preliminary candidate biomarkers. Limitations of this study include a small sample size (n = 3 per group), which may affect the robustness of the supervised OPLS-DA model and the interpretation of the differential findings, and the extraction recovery was not independently evaluated.

Measuring binding affinities of proteins to their specific sites in double-stranded (ds) DNA yields one of the fundamental parameters for characterizing protein-DNA interactions. Although many methods for determining binding affinities exist, all methods have limitations. Adding new approaches will enhance cross-validations and expand the range of targets that can be studied. We present inexpensive capillary electrophoresis (CE) methods for determining protein binding affinities to double-stranded DNA (dsDNA) in buffers with physiologically relevant salt concentrations, including divalent ions. We demonstrate the methods by monitoring the binding of the mismatch repair proteins Thermus aquaticus (Ta) MutS and Saccharomyces cerevisiae (Sc) MutSα to dsDNA containing a mismatch. We separate dsDNA and TaMutS-dsDNA complexes using bare fused silica (BFS) capillaries with pressure assistance. ScMutSα, like many proteins, sticks to BFS capillaries. Consequently, we developed a simple automated procedure to covalently coat BFS capillaries with commercially available methoxy polyethylene glycol silane (mPEG-silane), which minimizes protein sticking to capillary walls and allows separation of protein-dsDNA complexes using reverse polarity. The mPEG-silane coating remained sufficiently intact for over 24 h of continuous sample separation and was successfully regenerated. We show that the binding affinities of TaMutS and ScMutSα to dsDNA substrates determined by CE agree with those determined by fluorescence anisotropy or microscale thermophoresis.

A novel electrochemical immunosensor was designed for the ultrasensitive and selective detection of osteocalcin (OCN) normally overexpressed by osteosarcoma cells. The immunosensing was performed using anti-osteocalcin antibody (anti-OCN) modified probe along with a bio-conjugate, where the probe was fabricated by the covalent assembly of anti-OCN with conducting polymer poly-o-aminobenzoic acid (poly-o-ABA) formed over a layer of gold nanostar (AuNS). Bio-conjugates comprised of hydrazine (Hyd) along with anti-OCN was covalently immobilized on citrate capped AuNPs, reveals the immunoreaction at the sensor probe, where Hyd in bioconjugate was used to reduce hydrogen peroxide for direct signal amplification. The osteocalcin was quantitively analyzed using cyclic voltammetry. This technique exhibited an excellent diagnosis tool for the sensitive and selective detection of OCN, and shows broad dynamic range 2 to 64 ng mL[-1] with the detection limit of 0.19 ng mL[-1]. More importantly, it could distinguish osteocalcin from normal and other cancerous proteins, which indicated the promising applications in clinical diagnosis and treatment of osteosarcoma.