N-hydroxysuccinimide Research Articles

Digital microfluidic chip with photopatterned reactive sites for direct biomolecules immobilization and magnetic beads-free immunoassay

Digital microfluidics (DMF), as an emerging liquid-handling technology, is widely recognized as an ideal platform for enzyme-linked immunosorbent assay (ELISA). However, current DMF-based ELISA methods are highly dependent on magnetic beads (MBs), which usually involves tedious modification of MBs, complex peripherals for MBs control, and impairs the parallel processing capability of DMF. To address these issues, we herein developed a photopatterned reactive sites-integrated DMF chip (PRS-DMF) for MBs-free immunoassay. By taking advantage of click chemistry between N-(allyloxycarbonyloxy) succinimide and thiol-ene substrate, the preparation of PRS is very simple, and leaves the reactive N-hydroxysuccinimide (NHS) ester groups on the surface. This enables direct biomolecules immobilization without any surface modification/activation processes. Moreover, the patterned micropillar array can notably increase the specific surface area, and promote the liquid mixing and immunoreaction. As a consequence, highly sensitive and MBs-free ELISA can be readily achieved on PRS-DMF, with a proof-of-concept H5N1 assay having been successfully demonstrated herein, which offers a low limit of detection (LOD) of 147 pg/mL. This also avoids the use of complex magnetic peripherals, and thus, contributing to the instrument miniaturization into a very compact form. It is therefore reasonably expected that the proposed PRS-DMF may serve as a promising platform for a variety of biological and biomedical applications, especially in the field of point-of-care testing (POCT).

CMC/Gel/GO 3D-printed cardiac patches: GO and CMC improve flexibility and promote H9C2 cell proliferation, while EDC/NHS enhances stability.

In this research, carboxymethyl cellulose (CMC)/gelatin (Gel)/graphene oxide (GO)-based scaffolds were produced by using extrusion-based 3D printing for cardiac tissue regeneration. Rheological studies were conducted to evaluate the printability of CMC/Gel/GO inks, which revealed that CMC increased viscosity and enhanced printability. The 3D-printed cardiac patches were crosslinked with N-(3-dimethylaminopropyl)-n'-ethylcarbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) (100:20 mM, 50:10 mM, 25:5 mM) and then characterized by mechanical analysis, electrical conductivity testing, contact angle measurements and degradation studies. Subsequently, cell culture studies were conducted to evaluate the viability of H9C2 cardiomyoblast cells by using the Alamar Blue assay and fluorescence imaging. A high concentration of EDC/NHS (100:20 mM) led to the stability of the patches; however, it drastically reduced the flexibility of the scaffolds. Conversely, a concentration of 25:5 mM resulted in flexible but unstable scaffolds in PBS solution. The suitable EDC/NHS concentration was found to be 50:10 mM, as it produced flexible, stable, and stiff cardiac scaffolds with high ultimate tensile strength (UTS). Mechanical characterization revealed that % the strain at break of C15/G7.5/GO1 exhibited a remarkable increase of 61.03% compared to C15/G7.5 samples. The improvement of flexibility was attributed to the hydrogen bonding between CMC, Gel and GO. The electrical conductivity of 3D printed CMC/Gel/GO cardiac patches was 7.0×10-3 S/cm, demonstrating suitability for mimicking the desired electrical conductivity of human myocardium. The incorporation of 1 wt% of GO and addition of CMC concentration from 7.5 wt % to 15 wt % significantly enhanced relative % cell viability. Overall, although this research is at its infancy, CMC/Gel/GO cardiac patches have potential to improve the physiological function of cardiac tissue.

Determining factor of enzyme conjugates, bridge heterology and analytical variables of immunogens in prednisolone ELISA

In this study, adipic acid dihydrazide (ADH), carbohydrazide (CH), ethylenediamine (EDA), and urea (U) were used as spacer molecules, covalently linking prednisolone (PSL) to carrier proteins for immunogen preparation, and PSL to enzymes for enzyme conjugate preparation using N-hydroxysuccinimide (NHS)-mediated carbodiimide reactions. The resulting immunogens were used to generate antiserum in New Zealand white rabbits. Antibodies produced against immunogens with various spacers were tested for immunoreactivity with enzyme conjugates, both with and without spacers, in a total of twenty different combinations. All combinations demonstrated binding and were subsequently evaluated through displacement studies. Sensitivity and specificity tests revealed that the combination of PSL-21-HS-U-BSA-antibody with PSL-21-HS-HRP enzyme conjugate exhibited the best sensitivity (0.032 ng/mL) and limited cross-reactivity with other steroids. This combination was further examined for analytical parameters, showing recovery rates of 93.87–101.19 % for PSL from spiked human serum samples, with intra- and inter-assay CVs of <8.88 %. The serum PSL values obtained by this method showed strong correlation with a commercially available ELISA kit (r2 = 0.97, n = 78).

Analytical Challenges in Novel Pentavalent Meningococcal Conjugate Vaccine (A, C, Y, W, X)

Multivalent meningococcal conjugate vaccines are a significant focus for the scientific community in light of the WHO’s mission to defeat meningitidis by 2030. Well-known meningococcal vaccines such as MenAfriVac, Nimenrix, Menveo, and MenQuadfi are licensed in various parts of the world and have been successful. Recently, the World Health Organization (WHO) qualified MenFive (meningococcal A, C, Y, W, and X) conjugate vaccine, further enhancing the battery of vaccines against meningitis. The antigenic nature of the current and new serogroups, the selection of carrier proteins, and the optimal formulation of these biomolecules are pivotal parameters for determining whether a biological preparation qualifies as a vaccine candidate. Creating appropriate quality control analytical tools for a complex biological formulation is challenging. A scoping review aims to identify the main challenges and gaps in analyzing multivalent vaccines, especially in the case of novel serogroups, such as X, as the limited literature addresses these analytical challenges. In summary, the similarities in polysaccharide backbones between meningococcal serogroups (C, Y, W sharing a sialic backbone and A, X sharing a phosphorous backbone) along with various conjugation chemistries (such as CNBr activation, reductive amination, CDAP, CPIP, thioether bond formation, N-hydroxy succinimide activation, and carbodiimide-mediated coupling) resulting into a wide variety of polysaccharide -protein conjugates. The challenge in analyzing carrier proteins used in conjugation (such as diphtheria toxoid, tetanus toxoid, CRM diphtheria protein, and recombinant CRM) is assessing their purity (whether they are monomeric or polymeric in nature as well as their polydispersity). Additional analytical challenges include the impact of excipients, potential interference from serogroups, selection and establishment of standards, age-dependent behavior of biomolecules indicated by molecular size distributions, and process-driven variations. This article explains the analytical insights gained (polysaccharide content, free saccharide, free proteins, MSD) during the development of the MenFive vaccine and highlights the crucial gaps and challenges in testing.

Europium-Infused Polystyrene Nanoparticles for Enhanced Bacterial Labeling in Microscopic Imaging

Luminescence microscopic imaging has become an indispensable tool in diagnostics and therapeutics. Lanthanide luminescent complexes (LLCs) have proven to be invaluable in this endeavor due to certain attractive characteristics such as long decay times, large Richardson shifts and narrow emission bands. However, the insolubility of these complexes and the negative effects of the aqueous environment on their photophysical properties still restrict their biological applications. By embedding luminescent Eu3+ complexes into polystyrene nanoparticles (NPs), these complexes can not only maintain their photophysical properties, but even enhance them. This leads to exceptionally high quantum yields (> 90 %) and decay times ( >800 μs). In addition, the surfaces of the NPs described here are covered with primary amine groups, which may be exploited for various interesting bioconjugation chemistries. In the present application, they were modified with N-hydroxysuccinimide (NHS) esters and then coupled either electrostatically or covalently to both, Gram-positive and Gram-negative bacteria, labeling them for luminescence microscopic imaging. This, to the best of our knowledge, is the first report of polymer NPs infused with LLCs for bacteria labeling.

A Facile Strategy for Preparing Flexible and Porous Hydrogel-Based Scaffolds from Silk Sericin/Wool Keratin by In Situ Bubble-Forming for Muscle Tissue Engineering Applications.

In the present study, it is aimed to fabricate a novel silk sericin (SS)/wool keratin (WK) hydrogel-based scaffolds using an in situ bubble-forming strategy containing an N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling reaction. During the rapid gelation process, CO2 bubbles are released by activating the carboxyl groups in sericin with EDC and NHS, entrapped within the gel, creating a porous cross-linked structure. With this approach, five different hydrogels (S2K1, S4K2, S2K4, S6K3, and S3K6) are constructed to investigate the impact of varying sericin and keratin ratios. Analyses reveal that more sericin in the proteinaceous mixture reinforced the hydrogel network. Additionally, the hydrogels' pore size distribution, swelling ratio, wettability, and in vitro biodegradation rate, which are crucial for the applications of biomaterials, are evaluated. Moreover, biocompatibility and proangiogenic properties are analyzed using an in-ovo chorioallantoic membrane assay. The findings suggest that the S4K2 hydrogel exhibited the most promising characteristics, featuring an adequately flexible and highly porous structure. The results obtained by in vitro assessments demonstrate the potential of S4K2 hydrogel in muscle tissue engineering. However, further work is necessary to improve hydrogels with an aligned structure to meet the features that can fully replace muscle tissue for volumetric muscle loss regeneration.

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy.

Single-molecule Förster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5'-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3'-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5'-EDC/NHS activation is of general interest to all nucleic acids with a 5'-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes. Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

Chemofunctionalization of knitted silk to improve interface connection in a nano/micro scaffold based on polycaprolactone-chitosan-multi-walled carbon nanotube/silk

Nano/micro hybrid scaffolds in long-term healing tissue engineering can simultaneously offer both mechanical and biological properties. In this study, a hybrid scaffold was fabricated through electrospinning of polycaprolactone (PCL)-chitosan (Cs)/ multi-walled carbon nanotubes (MWCNTs) based nanofibers onto a chemically functionalized knitted silk substrate (F-Silk) and the scaffold were evaluated with regard to morphology, chemical and crystalline structure, hydrophilicity, mechanical properties, bioactivity, biodegradability, and cellular behavior. Chemical functionalization of silk using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) resulted in greater integrity in the formation of nanofibers onto the microfibers. The presence of MWCNTs significantly reduced the contact angle of the scaffolds from 79.72° ± 2.72 to 68.92° ± 5.63. Chemical functionalization of silk, the presence of nanofiber coating, and the presence of MWCNTs increased the ultimate tensile strength of the hybrid scaffolds by 18 %, 20 %, and 30 % compared to raw silk fabric, respectively. The presence of MWCNTs and chemical functionalization of knitted silk increased the bioactivity and reduced the degradation rate of hybrid scaffolds. The increase in the amount of carboxyl groups as a result of adding 0.5 wt% of MWCNTs significantly improved the adhesion, growth and proliferation of chondrocyte cells on the hybrid scaffolds as observed through cell morphology. According to the obtained results, hybrid scaffold based on PCL-Cs-MWCNTs/F-silk can be a suitable option for further research in cartilage tissue engineering.

Enhanced SERS detection of the colorectal cancer biomarker utilizing a two-dimensional silver substrate

To improve the sensitivity, accuracy and specificity of the assay, a two-dimensional silver substrate with EF=5.85×108 was first synthesized as a SERS substrate, on the surface of which DSP molecules were modified to form a DSP-antibody coupling through the activation of two N-hydroxy succinimide (NHS) esters to capture TNF-α. Subsequently, aptamerized silver-coated gold nanospheres (Au@TFMBA@Ag) were synthesized as Surface-Enhanced Raman Scattering (SERS) recognition probes. These probes were employed to create a sandwich structure for the quantitative detection of Tumor Necrosis Factor-alpha (TNF-α), utilizing the SERS signal intensity at 1374 cm−1. Quantitative detection of TNF-α was successfully accomplished within the concentration range of 10−4 to 10−10 mg·mL−1. Clinical serum samples were collected and subjected to testing. Significance analysis, conducted through the T-test (p < 0.0001), unequivocally showed the method's ability to differentiate between sera from normal individuals and those diagnosed with colon cancer.

Voltammetric-based immunosensing of Newcastle disease virus on polyethylene glycol-containing self-assembled monolayer modified gold electrode

A voltammetric immunosensor for the detection of Newcastle disease virus (NDV) has been developed by employing polyclonal antibody targeting NDV (anti-NDV) as a bioreceptor. Anti-NDV was immobilized on polyethylene glycol (PEG)-containing self-assembled monolayer (SAM) which was activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) coupling on screen-printed gold electrode (SPGE). The introduction of PEG-containing SAM on the SPGE allowed the bioreceptor to covalently bound to the electrode surface whilst still providing a hydrophilic layer on the electrode which is important to greatly reduce non-specific bindings. The bioreceptor functionalized electrode was then allowed to be incubated with NDV-spiked samples. The electrode surface modification with PEG-containing SAM, immobilization of anti-NDV as bioreceptor, up to the detection of NDV were characterized electrochemically through differential pulse voltammetry (DPV) analysis in [Fe(CN)6]3- as the redox probe. Decrement of anodic current peak (Ipa) of [Fe(CN)6]3- was seen as the concentration of NDV increased from 0.156 to 20 HA μL-1 with the limit of detection (LoD) of 1.50 HA μL-1 at 3σ m-1. The detection of NDV in HA μL-1 unit in this study would ease interlaboratory interpretation as it was the same unit used in hemagglutination (HA) assay of conventional NDV diagnosis. The specificity of anti-NDV used as bioreceptor towards NDV was confirmed through western blot analysis, whilst the selectivity of the bioreceptor-functionalized electrode has been tested with allantoic fluid as the negative control in which no apparent changes of anodic peak (Ipa) has been seen. This simple, fast, and less laborious electrochemical detection method could become an alternative to the conventional method for NDV detection.

Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

An unusual impedimetric biosensor design based on 3-MPDS for highly sensitive detection of AFB1 in food samples

Aflatoxin B1 (AFB1), a mycotoxin produced by fungi of the genus Aspergillus, particularly Aspergillus flavus, is recognized as the aflatoxin with the highest carcinogenic and mutagenic potential. This study presents a cost-effective, disposable AFB1 biosensor system based on 3-mercaptopropyltrimethoxysilane (3-MPDS) and the highly sensitive impedance technique. The surfaces of the ITO-PET (indium tin oxide/polyethylene terephthalate) electrodes were modified with 3-MPDS, and N-hydroxysuccinimide (NHS) was used as a crosslinker. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were employed for immobilization, optimization, and analytical studies. Additionally, the surface morphology of the biosensor was analyzed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The biosensor demonstrated a linear range from 0.01 fg/mL to 200 fg/mL, with a limit of detection (LOD) of 0.01 fg/mL and a limit of quantification (LOQ) of 0.04 fg/mL. Finally, the proposed biosensor was tested and validated with real food samples, including rice, peanuts, milk, chili pepper, and corn.

Determination of degradation for sulfo-SIAB, SM(PEG)2, and sulfo-GMBS by RPLC-UV analysis

Bi-functional N-Hydroxysuccinimide (NHS) linkers are widely used in the conjugation processes linking an immunogen with a carrier protein capable of boosting immunity. A potential vaccine candidate against HIV-1, called fusion peptide (FP), is covalently linked to the recombinant tetanus toxoid heavy-chain fragment C (rTTHC) via this type of linker. A reversed-phase liquid chromatography (RPLC-UV) method was used to monitor the linker’s degradation kinetics in various buffers, mimicking the steps in the conjugation process. The kinetics of the reactivities of the linkers are revealed in this study and can provide a good guidance to help effective conjugation process before these linkers are completely hydrolyze to the inactive degradants. Three cross-linkers degradation pathways were evaluated: Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB), PEGylated SMCC (SM(PEG)2), and N-γ-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS). We have reported kinetics for Sulfo-SIAB.

Ultrafast and Chemoselective Biotinylation of Living Cell Surfaces for Time-Resolved Surfaceome Analysis.

Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with N-hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.

Activation of Inert Supports for Enzyme(s) Immobilization Harnessing Biocatalytic Sustainability for Perennial Utilization.

Although Nature's evolution and intelligence have gifted humankind with noteworthy enzyme candidates to simplify complex reactions with ultrafast, overselective, effortless, mild biological reactions for millions of years, their availability at minute-scale, short-range time-temperature stability, and purification costs hardly justify recycling/or reuse. Covalent immobilization, particularly via multipoint bonds, prevents denaturing, maintains activities for long-range time, pH, and temperature, and makes catalysts available for repetitive usages; which attracts researchers and industries to bring more immobilized enzyme contenders in science and commercial progressions. Inert-support activation, the most crucial step, needs appropriate activators; under mild conditions, the activator's functional group(s) still present on the activated support rapidly couples the enzyme, preventing unfolding and keeping the active site alive. This review summarizes exciting experimental advances, from the 1950s until today, in the activation strategies of various inert supports with five different surface activators, the cyanogen bromide, the isocyanate/isothiocyanate, the glutaraldehyde, the carbodiimide (with or without N-hydroxysuccinimide (NHS)), and the diazo group, for the immobilization of diverse enzymes for broader applications. These activators under mild pH (7.5 ± 0.5) and temperature (27 ± 3 °C) and ordinary stirring witnessed support activation and enzyme coupling and put off unfolding, harnessing addressable activities (CNBr: 40 ± 10%; -N═C═O/-N═C═S: 32 ± 7%; GA: 70 ± 15%; CDI: 60 ± 10%; -N+≡N: 80 ± 15%), while underprivileged stability, longevity, and reusabilities keep future investigations alive.

Electrochemical detection of anti-tissue transglutaminase antibody using quantum dots-doped polypyrrole-modified electrode

A nanohybrid-modified glassy carbon electrode based on conducting polypyrrole doped with carbon quantum dots (QDs) was developed and used for the electrochemical detection of anti-tissue transglutaminase (anti-tTG) antibodies. To improve the polypyrrole conductivity, carrier mobility, and carrier concentration, four types of carbon nanoparticles were tested. Furthermore, a polypyrrole-modified electrode doped with QDs was functionalized with a PAMAM dendrimer and transglutaminase 2 protein by cross-linking with N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC). The steps of electrode surface modification were surveyed via electrochemical measurements (differential pulse voltammetry (DPV), impedance spectroscopy, and X-ray photoelectron spectroscopy (XPS)). The surface characteristics were observed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and contact angle measurements. The obtained modified electrode exhibited good stability and repeatability. DPV between − 0.1 and 0.6 V (vs. Ag/AgCl 3 M KCl reference electrode) was used to evaluate the electrochemical alterations that occur after the antibody interacts with the antigen (transglutaminase 2 protein), for which the limit of detection was 0.79 U/mL. Without the use of a secondary label, (anti-tTG) antibodies may be detected at low concentrations because of these modified electrode features.Graphical

Effects of Cross-linker Chemistry on Bioelectrocatalytic Reactions in a Redox Cross-linked Network of Glucose Dehydrogenase and Thionine.

Enzyme-mediator bioconjugation is emerging as a building block for designing electrode platforms for the construction of biosensors and biofuel cells. Here, we report a one-pot bioconjugation technique for flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and thionine (TH) using a series of cross-linkers, including epoxy, N-hydroxysuccinimide (NHS), and aldehydes. In this technique, FAD-GDH and thionine are conjugated through an amine cross-linking reaction to generate a redox network, which has been successfully employed for the oxidation of glucose. The bioconjugation chemistry of cross-linkers with the amino groups on FAD-GDH and thionine plays a vital role in generating distinct network structures. The epoxy-type cross-linker reacts with the primary and secondary amines of thionine at room temperature, thereby producing an FAD-GDH-TH-FAD-GDH hyperbranched bioconjugate network, the aldehyde undergoes a rapid cross-linking reaction to produce a network of FAD-GDH-FAD-GDH, while the NHS-based cross-linker can react with the primary amines of both FAD-GDH and thionine, forming an FAD-GDH-cross-linker-TH polymeric network. This reaction has the potential to enable the conjugation of a redox mediator with a FAD-GDH network, which is particularly essential when designing an enzyme electrode platform. The data demonstrated that the polymeric cross-linked network based on the NHS cross-linker exhibited a considerable increase in electron transport while producing a catalytic current of 830 μA cm-2. The cross-linker spacer arm length also affects the overall electrochemical function of the network and its performance; an adequate spacer length containing a cross-linker is required, resulting in a faster electron transfer. Finally, a leaching test confirmed that the stability of the enzyme electrode was improved when the electrode was tested using the redox probe. This study elucidates the relationship between cross-linking chemistry and redox network structure and enhances the high performance of enzyme electrode platforms for the oxidation of glucose.

Coupling enterotoxigenic Escherichia coli heat-stable peptide toxin with 8-arm PEG enhances immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.

Developmentand validation of carbodiimide-activated ELISA plate for quantifying IgG levels against Streptococcus pneumoniae capsular polysaccharides.

Background:Conventional microtiter plates lack the surface strength needed for effective binding of pneumococcalpolysaccharide antigens. This study tackles the limitation by altering the surface of polystyrene plates through carbodiimide activation under acidic pH conditions.Method:The microtiter plates were activated with carbodiimide coupling agents, N,N'-Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS). They were subsequently coated with 13 pneumococcal antigens at a concentration of 5 μg/ml with a pH of 3.5. The IgG antibody titer was assessed utilizing the World Health Organization (WHO) ELISA protocol for 30 human serum samples. In addition, validation experiments were conducted to evaluate specificity and precision.Results: The modified plates exhibited two-times higher antibody titers compared to conventional plates across all 13 serotypes. Observations revealed elevated antibody levels, with geometric concentrations ranging between 0.96 μg/ml and 4.24 μg/ml.Conclusion:Carbodiimide activation and acidic pH modification of microtiter plates enhance sensitivity and specificity in detecting pneumococcal antibodies, critical for vaccination planning and immunity assessment.

Uricase biofunctionalized plasmonic sensor for uric acid detection with APTES-modified gold nanotopping

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL)−1, and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine.