Immobilization of HRP enzyme on polymeric microspheres and its use in decolourisation of organic dyes.

In this study, horseradish peroxidase (HRP) enzyme was immobilized on Pd(II) containing polymeric microspheres by adsorption method and used for the decolourisation of Methyl Orange (MO) and Rhodamine B (RB) dyes. The synthesized microspheres were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy-Energy Dispersive X-ray (SEM/EDX), Thermal Gravimetric Analysis (TGA). The effects of pH, dye concentration, temperature, and H2O2 concentration on the decolourisation of MO and RB were determined. According to the results of various parameters studied, when 2-AEPS-napht-HRP support was used, MO and RB were biodegraded to 69.72% and 80.65%, respectively, within 60 min. When 2-AEPS-napht-Pd-HRP support was used, MO and RB were biodegraded to 58.35% and 90.81%, respectively, under optimum conditions. When the reproducibility results of the immobilized supports were examined, it was observed that they remained efficient during the first five reusability cycles and even reached 65% decolourisation efficiency after the 9th reuse. The immobilized enzyme (2AEPS-npht-HRP and 2AEPS-npht-Pd-HRP) showed remarkable resistance to higher temperatures compared to the free enzyme.

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

Comparison of three rapid diagnostic tests for Plasmodium falciparum diagnosis in Ghana.

BACKGROUND: Accurate diagnosis and timely treatment are crucial in combating malaria. METHODS: A total of 449 samples were screened for Plasmodium falciparum infection by expert microscopy, qPCR, and three RDTs, namely Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and pLDH on separate bands), Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), and SD Bioline Malaria Ag Pf (detecting HRP2). hrp2/3 deletion typing was done by digital PCR. RESULTS: 45.7% (205/449) individuals tested positive by qPCR for P. falciparum with a mean parasite density of 12.5 parasites/µL. Using qPCR as reference, the sensitivity of microscopy was 28.3% (58/205), the Biocredit RDT was 52.2% (107/205), the NxTek RDT was 49.3% (101/205), and the Bioline RDT was 39.5% (81/205). When only samples with densities > 20 parasites/µL were included (n = 89), sensitivity of 62.9% (56/89) by microscopy, 88.8% (79/89) by Biocredit, 88.8% (79/89) by NxTek, and 78.7% (70/89) by Bioline were obtained. All three RDTs demonstrated specificities > 95%. The limits of detection (95% probability that a sample tested positive) was 4393 parasites/µL (microscopy), 56 parasites/µL (Biocredit, considering either HRP2 or pLDH), 84 parasites/µL (NxTek), and 331 parasites/µL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the pLDH target, or eight samples negative for all RDTs but qPCR-positive at densities > 20 parasites/µL carried hrp2/3 deletions. CONCLUSION: The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies. All three RDTs performed better than microscopy.

Injectable Dual Fenton/Enzymatically Cross-Linked Double-Network Hydrogels Based on Acrylic/Phenolic Polymers with Highly Reinforced and Tunable Mechanical Properties.

Injectable hydrogels have been extensively used as promising therapeutic scaffolds for a wide range of biomedical applications, such as tissue regeneration and drug delivery. However, their low fracture toughness and brittleness often limit their scope of application. Double-network (DN) hydrogel, which is composed of independently cross-linked rigid and ductile polymer networks, has been proposed as an alternative technique to compensate for the weak mechanical properties of hydrogels. Nevertheless, some challenges still remain, such as the complicated and time-consuming process for DN formation, and the difficulty in controlling the mechanical properties of DN hydrogels. In this study, we introduce a simple, rapid, and controllable method to prepare in situ cross-linkable injectable DN hydrogels composed of acrylamide (AAm) and 4-arm-PPO-PEO-tyramine (TTA) via dual Fenton- and enzyme-mediated reactions. By varying the concentration of Fenton's reagent, the DN hydrogels were rapidly formed with controllable gelation rate. Importantly, the DN hydrogels showed a 13-fold increase in compressive strength and a 14-fold increase in tensile strength, compared to the single network hydrogels. The mechanical properties, elasticity, and plasticity of DN hydrogels could also be modulated by simply varying the preparation conditions, including the cross-linking density and reagent concentrations. At low cross-linker concentration (<0.05 wt %), the plastic DN hydrogel stretched to over 6,500%, whereas high cross-linker concentration (≥0.05 wt %) induced fully elastic hydrogels, without hysteresis. Besides, DN hydrogels were endowed with rapid self-recovery and highly enhanced adhesion, which can be further applied to wearable devices. Moreover, human dermal fibroblasts treated with DN hydrogels retained viability, demonstrating the biocompatibility of the cross-linking system. Therefore, we expect that the dual Fenton-/enzyme-mediated cross-linkable DN hydrogels offer great potential as advanced biomaterials applied for hard tissue regeneration and replacement.

Dissecting the Distinct Tumor Microenvironments of HRD and HRP Ovarian Cancer: Implications for Targeted Therapies to Overcome PARPi Resistance in HRD Tumors and Refractoriness in HRP Tumors.

High-grade serous tubo-ovarian cancer (HGSTOC) is an aggressive gynecological malignancy including homologous recombination deficient (HRD) and homologous recombination proficient (HRP) groups. Despite the therapeutic potential of poly (ADP-ribose) polymerase inhibitors (PARPis) and anti-PDCD1 antibodies, acquired resistance in HRD and suboptimal response in HRP patients necessitate more precise treatment. Herein, single-cell RNA and single-cell T-cell receptor sequencing on 5 HRD and 3 HRP tumors are performed to decipher the heterogeneous tumor immune microenvironment (TIME), along with multiplex immunohistochemistry staining and animal experiments for validation. HRD tumors are enriched with immunogenic epithelial cells, FGFR1+PDGFRß+ myCAFs, M1 macrophages, tumor reactive CD8+/CD4+ Tregs, whereas HRP tumors are enriched with HDAC1-expressing epithelial cells, indolent CAFs, M2 macrophages, and bystander CD4+/CD8+ T cells. Significantly, customized therapies are proposed. For HRD patients, targeting FGFR1+PDGFRß+ myCAFs via tyrosine kinase inhibitors, targeting Tregs via anti-CCR8 antibodies/TNFRSF4 stimulation, and targeting CXCL13+ exhausted T cells by blocking PDCD1/CTLA-4/LAG-3/TIGIT are proposed. For HRP patients, targeting indolent CAFs, targeting M2 macrophages via CSF-1/CSF-1R inhibitors, targeting bystander T cells via tumor vaccines, and targeting epithelial cells via HDAC inhibitors. The study provides comprehensive insights into HRD and HRP TIME and tailored therapeutic approaches, addressing the challenges of PARPi-resistant HRD and refractory HRP tumors.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Using artificial intelligence to study atherosclerosis from computed tomography imaging: A state-of-the-art review of the current literature.

With the enormous progress in the field of cardiovascular imaging in recent years, computed tomography (CT) has become readily available to phenotype atherosclerotic coronary artery disease. New analytical methods using artificial intelligence (AI) enable the analysis of complex phenotypic information of atherosclerotic plaques. In particular, deep learning-based approaches using convolutional neural networks (CNNs) facilitate tasks such as lesion detection, segmentation, and classification. New radiotranscriptomic techniques even capture underlying bio-histochemical processes through higher-order structural analysis of voxels on CT images. In the near future, the international large-scale Oxford Risk Factors And Non-invasive Imaging (ORFAN) study will provide a powerful platform for testing and validating prognostic AI-based models. The goal is the transition of these new approaches from research settings into a clinical workflow. In this review, we present an overview of existing AI-based techniques with focus on imaging biomarkers to determine the degree of coronary inflammation, coronary plaques, and the associated risk. Further, current limitations using AI-based approaches as well as the priorities to address these challenges will be discussed. This will pave the way for an AI-enabled risk assessment tool to detect vulnerable atherosclerotic plaques and to guide treatment strategies for patients.

Development of a Phage-Displayed Nanobody-Based Competitive Immunoassay for the Sensitive Detection of Soybean Agglutinin.

Soybean agglutinin (SBA) is a primary antinutritional factor in soybeans that can inhibit the growth of humans and mammals, disrupt the intestinal environment, and cause pathological changes. Therefore, detecting and monitoring SBA in foods is essential for safeguarding human health. In this paper, M13 phage-displayed nanobodies against SBA were isolated from a naive nanobody library. An M13 phage-displayed nanobody-based competitive enzyme-linked immunosorbent assay (P-cELISA) was then established for SBA analysis using biotinylated anti-M13 phage antibody (biotin-anti-M13) and streptavidin poly-HRP conjugate (SA-poly-HRP). The biotin-anti-M13@SA-poly-HRP probe can easily amplify the detection signal without the chemical modifications of phage-displayed nanobodies. The established P-cELISA presented a linear detection range of 0.56-250.23 ng/mL and a limit of detection (LOD) of 0.20 ng/mL, which was 12.6-fold more sensitive than the traditional phage-ELISA. Moreover, the developed method showed good specificity for SBA and acceptable recoveries (78.21-121.11%) in spiked wheat flour, albumen powder, and whole milk powder. This study proposes that P-cELISA based on biotin-anti-M13@SA-poly-HRP may provide a convenient and effective strategy for the sensitive detection of SBA.

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies.

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection in pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and could play an important role in monitoring ASFV infections in pig farms.

Linker histone H1 regulates homeostasis of heterochromatin-associated cRNAs.

Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas.

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

Ultrasensitive cortisol electrochemical immunosensor amplifying by Au single-atom nanozymes and HRP enzymes.

Cortisol, a corticosteroid hormone as a primary stress hormone response to internal and external stress, has been regarded as a gold standard reliable biomarker to evaluate human mental stress. The double enzymes strategy, using nanozyme and enzyme amplifying the electrochemical signal, has been widely used to improve the performance of electrochemical biosensors. An ultra-sensitive electrochemical cortisol sensor based on Au single-atom nanozymes had been fabricated through HRP labeled anti-cortisol antibody binding with Au by Au-S bond. Based on the high catalytic activity of Au single-atom nanozymes and the high selectivity of HRP-labeled anti-cortisol antibodies, the cortisol electrochemical sensor-based Au single-atom nanozymes had an excellent response to cortisol, such as high electrochemical activity, high sensitivity, high selectivity, and wide linear range (0.15-300 ng mL-1) and low detection (0.48 pg mL-1) through the four-parameter logistic model with 95% confidence. The electrochemical cortisol sensor was used to determine the cortisol concentration of human saliva at different times.

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody.

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6-200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%-102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Targeted Combination of Poly(ADP-ribose) Polymerase Inhibitors and Immune Checkpoint Inhibitors Lacking Evidence of Benefit: Focus in Ovarian Cancer.

The emergence of targeted therapeutics in ovarian cancer, particularly poly (ADP-ribose) polymerase inhibitors (PARPi's), has created additional opportunities for patients seeking frontline and recurrent disease management options. In particular, PARPi's have shown clinical benefits in BRCA mutant and/or homologous recombination deficient (HRD) ovarian cancer. Until recently, response was thought to be limited in BRCA wild-type, homologous recombination proficient (HRP) cancers. Therefore, attempts have been made at combination therapy involving PARPi to improve patient outcomes. Additionally, immune checkpoint inhibitors (ICIs) have demonstrated underwhelming results involving ovarian cancer. Many are searching for reliable biomarkers of immune response to increase efficacy of ICI therapy involving ovarian cancer. In this review, we examine the evidence supporting the combination of PARPi and ICIs in ovarian cancer, which is still lacking.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Comparison of two peroxidases with high potential for biotechnology applications - HRP vs. APEX2.

Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.

Free Radical Inhibition Using a Water-Soluble Curcumin Complex, NDS27: Mechanism Study Using EPR, Chemiluminescence, and Docking.

There is a growing interest in the use of natural compounds to tackle inflammatory diseases and cancers. However, most of them face the bioavailability and solubility challenges to reaching cellular compartments and exert their potential biological effects. Polyphenols belong to that class of molecules, and numerous efforts have been made to improve and overcome these problems. Curcumin is widely studied for its antioxidant and anti-inflammatory properties as well as its use as an anticancer agent. However, its poor solubility and bioavailability are often a source of concern with disappointing or unexpected results in cellular models or in vivo, which limits the clinical use of curcumin as such. Beside nanoparticles and liposomes, cyclodextrins are one of the best candidates to improve the solubility of these molecules. We have used lysine and cyclodextrin to form a water-soluble curcumin complex, named NDS27, in which potential anti-inflammatory effects were demonstrated in cellular and in vivo models. Herein, we investigated for the first time its direct free radicals scavenging activity on DPPH/ABTS assays as well as on hydroxyl, superoxide anion, and peroxyl radical species. The ability of NDS27 to quench singlet oxygen, produced by rose bengal photosensitization, was studied, as was the inhibiting effect on the enzyme-catalyzed oxidation of the co-substrate, luminol analog (L012), using horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) system. Finally, docking was performed to study the behavior of NDS27 in the active site of the peroxidase enzyme.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus.

The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Nucleocapsid protein (N protein) is the primary antigen of PRRSV for development of sensitive diagnostic assays. Two high affinity nanobodies against N protein, Nb12 and Nb35, were selected and employed to develop a sandwich ELISA. Further we improved the ELISA method to obtain greater sensitivity, a trivalent nanobody (3 × Nb35) and a bivalent nanobody-HRP fusion protein (2 × Nb12-HRP) were expressed and used. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID50/ml (median tissue culture infectious dose), which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.