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.

Rapid and reliable identification of pathogens is of great importance in healthcare settings and in the control of agonistic and antagonistic disease outbreaks. Broad-range methods for molecular detection of parasites, bacteria and viruses in samples with unknown contents are thus highly desirable. Hyperplex PCR (hpPCR) is a recently invented analysis technique that allows for parallel detection of up to 100 DNA and RNA targets. We present the development and evaluation of a proof-of-concept 16-plex hpPCR method targeting RNA viruses, bacteria and a parasite to enable detection of various gastrointestinal pathogens relevant in food or waterborne disease outbreak scenarios. The panel also includes highly pathogenic microbes such as Bacillus anthracis and Yersinia pestis. The hpPCR method showed high selectivity with detection of all expected targets. None of the tested bacteria (11), parasites (1) or viruses (4) gave false positive signals or cross-reactions for any other target in the panel. The limit of detection was comparable to single-plex qPCR/RT-qPCR assays targeting the same pathogens. HpPCR tolerated high amounts of humic acid, with somewhat better results for the bacteria compared to the viruses. The 16-plex hpPCR method may be supplemented with additional assays to further widen the group of targeted pathogens. A hpPCR method simultaneously targeting a multitude of microbes including bacteria, parasites and viruses may thus provide timely and selective detection and identification of pathogens in an outbreak situation.

Bacterial infections, especially those triggered by bacterial biofilms, remain a thorny challenge in antibacterial treatment. Metal-organic frameworks (MOFs), as carriers of metal ions that can sustainably release antibacterial ingredients, provide a promising alternative strategy for combating bacterial infections. Although the introduction of metal ions into MOFs is a relatively uncomplicated process, it remains an arduous task to eradicate all pathogenic bacteria with a limited dosage. Herein, an economical and efficient hollow Co-doped ZIF-L-PDA (HZIF-L-PDA (Co)) nanomaterial was synthesized via a simple dopamine-mediated self-assembly reaction. The uniform incorporation of Co and Zn in the ZIF-L skeleton and the polymerization of dopamine were completed through a one-pot method, avoiding complexity and harsh synthesis conditions. Due to the abundant metal nodes of ZIF-L and the photothermal and adhesion ability of polydopamine (PDA), HZIF-L-PDA (Co) can adhere to the bacterial surface and rely on its chemical antibacterial action and photothermal effect to exhibit a synergistic antibacterial effect, causing cell membrane rupture and invagination in Escherichia coli O157:H7 (E. coli O157:H7) and Staphylococcus aureus (S. aureus), respectively. Antibacterial experiments displayed that the sterilization rates of HZIF-L-PDA (Co) driven by near-infrared (NIR) light at low concentration (75 μg/mL) against E. coli O157:H7 and S. aureus were 96.9% and 97.3%, respectively. The HZIF-L-PDA (Co) realized favorable eradication against E. coli O157:H7 mature biofilms, while the therapeutic effect against S. aureus biofilms was limited at the tested low concentration. Overall, we proposed an easily fabricated MOF-based nanocomposite that fought pathogenic bacteria through chemical and photothermal effects, promising as an alternative to antibiotics.

In this study, iron oxide nanoparticles were synthesized through a simple, cost-effective, and environmentally friendly green synthesis route using aqueous rose extract as a natural reducing and stabilizing agent. The synthesized nanoparticles were combined with graphene oxide (GO) and employed to modify a carbon paste electrode (CPE) for the simultaneous electrochemical detection of dopamine (DOP) and tryptophan (TRP). The fabricated Fe2O3/Fe3O4-GO/CPE sensor was characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and chronoamperometry. The modified electrode exhibited significantly enhanced electrocatalytic activity, sensitivity, and electron transfer capability compared with the bare electrode. Under optimized conditions, the sensor demonstrated wide linear dynamic ranges of 0.5-300 μM for dopamine and 1-300 μM for tryptophan, with low detection limits of 0.32 μM and 0.47 μM, respectively. The proposed sensor also showed excellent repeatability, good stability, high selectivity against common interfering species, and enhanced electroactive surface area due to the synergistic effect of iron oxide nanoparticles and graphene oxide. These results indicate that the developed nanocomposite-modified electrode is a promising, sensitive, and low-cost electrochemical platform for the simultaneous determination of biologically important analytes in pharmaceutical and biomedical applications. Future studies may focus on applying the proposed sensor to real biological and pharmaceutical samples and further improving its performance for portable and point-of-care electrochemical sensing applications.

To address the limitations of quercetin (Qu), including low bioavailability, limited targeting, and insufficient synergistic enhancement, we developed a ZIF-8-based nanodelivery system (Qu-ZIF-8-CMLA) coated with a carboxymethyl laminarin (CMLA) layer. The platform was designed for macrophage-targeted delivery with potential for oral administration. Qu-ZIF-8-CMLA exhibited a hydrodynamic diameter of 151.5 nm, a drug loading capacity of 15.2%, and colloidal stability. Characterization confirmed CMLA coating of the Qu-ZIF-8 surface through electrostatic interactions and hydrogen bonding. The resulting nanoparticles maintained structural integrity and exhibited pH-responsive, controlled-release properties. In simulated gastric fluid, Qu-ZIF-8-CMLA released 23.54% of the Qu, whereas release reached 70.08% under simulated intestinal conditions, indicating a more sustained release profile than free Qu. Release kinetic analysis suggested a non-Fickian mechanism involving polysaccharide erosion and drug diffusion. In RAW264.7 macrophages, CMLA modification increased uptake by 6.35-fold, while competitive inhibition with free CMLA significantly attenuated this enhancement, supporting the involvement of CMLA-sensitive receptor-associated recognition in macrophage uptake. At high concentrations, Qu-ZIF-8-CMLA maintained higher RAW264.7 cell viability than Qu-ZIF-8 under the tested conditions. Anti-inflammatory activity was evaluated in LPS-stimulated RAW264.7 macrophages by measuring TNF-α, IL-1β, and IL-6 secretion using ELISA. Qu-ZIF-8-CMLA significantly reduced three pro-inflammatory cytokines compared with the LPS model group, and no statistically significant differences were detected between the Qu-ZIF-8-CMLA and untreated control groups (p > 0.05). Qu-ZIF-8-CMLA showed significantly greater anti-inflammatory activity than free Qu and Qu-ZIF-8. Collectively, these findings provide in vitro proof of concept for CMLA-modified, macrophage-targeted Qu delivery and support further investigation of this platform for oral anti-inflammatory applications.

Acyl phosphate substrate availability is critical for applicable biochemical studies. Accordingly, various synthesis methods have been developed over the years typically focused on acetyl phosphate. One of these synthesis methods was proven versatile, capable of producing assorted primary and secondary acyl phosphates with high purity as measured by NMR spectroscopy, total carbon analysis and hydroxamate assays. Biochemical activity assays using synthesized acyl phosphate substrates generate results consistent with previously published kinetic constant data for Methanosarcina thermophila acetate kinase, while kinetic constant results for new substrates are reported for both M. thermophila acetate kinase and Desulfovibrio vulgaris str. Hildenborough butyrate kinase.

Glucose biosensors are pivotal to diabetes management; however, traditional fingerstick blood collection methods are often challenging due to their invasiveness, pain, high cost, and inability to provide continuous monitoring. Here, we constructed a paper-based colorimetric sensing platform using a multifunctional metal-organic framework (MOF) composite system to enable rapid, noninvasive detection of glucose in saliva. This platform utilized a CuCo-MOF framework, loaded with glucose oxidase (GOx) and ZIF-90 via a synergistic self-assembly mechanism, to catalyze the colorimetric reaction of H2O2 and tetramethylbenzidine (TMB). The synergistic effect of the composite significantly enhanced the overall catalytic efficiency, enabling a sensitive response to glucose levels in saliva. Using ImageJ, color images captured with a smartphone were analyzed using RGB color channels to quantitatively determine glucose levels in saliva. The detection time was 20 min, with a detection limit (LOD) of 13.5 μM and a linear range of 50-2000 μM. This platform offered advantages with the absence of blood sample collection, ease of operation, and visual response. It provided new material strategies and design ideas for developing portable, non-invasive blood glucose monitoring technology.

This study aimed to develop a CRISPR-dCas13a-based electrochemical biosensor as a proof-of-concept analytical platform for the sensitive and selective detection of miR-331-5p, which has been reported to be associated with Parkinson's disease. The biosensor was prepared by sequentially forming a self-assembled monolayer with 11-mercaptoundecanoic acid (MUA) on a gold electrode, functionalizing the surface with a generation-4 polyamidoamine (PAMAM-G4) dendrimer, immobilizing dCas13a, and assembling the target-specific sgRNA recognition complex. The electrode-modification steps were evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The impedance response increased linearly with miR-331-5p concentration over the experimentally evaluated range of 5-400 pM. The limit of detection and limit of quantification were 7.82 and 23.71 pM, respectively. Responses to the non-target miRNAs miR-146a-5p, miR-19 b-3p, miR-24-3p, and miR-7-5p were substantially lower than the response to miR-331-5p. Triplicate spike-recovery measurements in healthy human serum and storage-stability studies supported the analytical applicability and reproducibility of the platform. These findings demonstrate a CRISPR-dCas13a/EIS-based proof-of-concept analytical platform for miR-331-5p detection. Validation in well-characterized clinical cohorts will be required before any diagnostic application can be established.

Porcine rotavirus (PoRV) causes severe viral diarrhea in piglets. Owing to the lack of specific antiviral therapies, rapid on-site detection is urgently required for swine disease control. Herein, a lateral-flow immunoassay (LFIA) amplified by Fe-Co bimetallic nanozyme was established for PoRV detection using two screened high-affinity anti-PoRV monoclonal antibodies. The peroxidase-like catalytic activity of Fe-Co nanozyme was employed for signal amplification on the immunochromatographic strip. The limit of detection of this LFIA was 100 TCID50/mL, which was two-fold more sensitive than conventional commercial colloidal-gold-based test strips. No cross-reactivity was observed against other common porcine pathogens, including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine sapelovirus (PSV), transmissible gastroenteritis virus (TGEV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV). The intra-batch and inter-batch coefficients of variation were both below 10%, demonstrating favorable repeatability. The strip remained stable for at least 9 months under 4°C and ambient-temperature storage. When evaluated with 72 clinical pig fecal samples using RT-qPCR as the reference, the LFIA showed a sensitivity of 95.45%, specificity of 100%, and a total agreement rate of 98.61%, with a kappa value of 0.97 (95% CI: 0.891-1.000), indicating excellent agreement between the two methods. This Fe-Co nanozyme-amplified LFIA possesses high sensitivity, outstanding specificity, reliable stability and easy-to-operate characteristics. It can be applied for on-site PoRV screening in pig farms and facilitates early epidemic intervention of PoRV-associated diarrhea.

Sustainable chemistry is increasingly used in pharmaceutical analysis to reduce environmental impact while improving laboratory personnel safety. This study describes the first sustainable electrochemical method for determining flibanserin (FLB) and caffeine (CAF) in various matrices, including bulk powder, pharmaceutical dosage form, and human plasma samples. FLB is used for the treatment of Hypoactive sexual desire disorder, while CAF may be co-administered with FLB as part of the therapeutic regimen, highlighting the need for reliable methods for their simultaneous determination. The experimental parameters were optimized using a zinc oxide nanoparticle polyethylene glycol -modified carbon paste electrode as working electrode at scan rate of 100 mV s[-1], and Britton-Robinson buffer (pH 7) as the supporting electrolyte using square wave voltammetry. Both analytes showed linear responses from 5.00 × 10[-7] M to 100.00 × 10[-7] M. The limits of detection for FLB and CAF were 1.617 × 10[-7] M and 1.265 × 10[-7] M, respectively. The limits of quantification were 4.851 × 10[-7] M for FLB and 3.795 × 10[-7] M for CAF. The method demonstrated satisfactory analytical performance and was validated in accordance with ICH guidelines. Student's t-test and F-test showed no significant differences between the proposed and reported methods. Sustainability was comprehensively evaluated using the Carbon Footprint Reduction Index, Multi-Color Assessment Tool, Analytical Green Star Area, and Click Analytical Chemistry Index. Overall, the proposed method provides a rapid, sensitive, and sustainable alternative for the simultaneous determination of FLB and CAF.

Osteoclast fusion is an essential step in the acquisition of bone-resorbing activity; however, the molecular mechanisms underlying this process remain incompletely understood. In this study, we developed a novel quantitative monitoring system for osteoclast fusion using NanoBiT, a split-luciferase technology. This system enabled sensitive detection of receptor activator of NF-κB ligand (RANKL)-dependent osteoclast fusion in RAW264 cells and bone marrow-derived macrophages, as well as interleukin-4 (IL-4)-induced foreign body giant cell fusion. The time-course analysis of luciferase activity during osteoclast differentiation was closely correlated with osteoclast fusion observed by conventional microscopic analysis. In addition, luciferase activity was inhibited by cyclosporin A in a dose-dependent manner and completely abolished by Dcstamp knockout, demonstrating its validity for chemical screening and for evaluating genes involved in osteoclast fusion. Using this platform, we screened approximately 2300 known bioactive compounds and identified THZ-P1-2, a phosphatidylinositol-5-phosphate 4-kinase type 2 inhibitor, and Brequinar, a dihydroorotate dehydrogenase inhibitor, as novel modulators of osteoclast fusion. Compared with fluorescence-based assays, this luminescence-based system minimizes false-positive signals caused by cellular or compound autofluorescence and provides superior sensitivity and quantifiability. Furthermore, the assay is rapid, simple, and highly compatible with high-throughput screening, making it a useful platform for studying osteoclast fusion and for screening compounds that modulate this process.