Search Results (281)

In addressing the challenges posed by high costs, low accuracy, and cumbersome operations in mycotoxin detection, a novel aptamer-antibody sandwich chemiluminescent biosensor for detecting aflatoxin B₁ (AFB₁) was developed. The indirect competition between AFB₁, aflatoxin B₁-ovomucoid complete antigen (AFB₁-OVA), and rabbit anti-ovomucoid (OVA) antibody results in the formation of a sandwich complex. This sandwich assay is linked to a horseradish peroxidase-labeled antibody, which catalyzes luminol chemiluminescence for the indirect detection of AFB₁. The biosensor was designed to operate with high precision, low cost, and a low detection limit for AFB₁, which is contingent upon experimental conditions such as pH, reagent concentration, temperature, and incubation time. The optimization of pH, aptamer concentration, competitive incubation time, competitive incubation temperature, and HRP-labeled antibody concentration was instrumental in achieving these objectives. Experimental findings demonstrated that the sensor’s detection limit was 0.067 ng/mL, exhibiting excellent linearity (R² = 0.99679) within the concentration range of 0.25–10 ng/mL. The recovery rate of spiked samples ranged from 94.4% to 108.05%. This sensor boasts a low detection limit, straightforward operation, and minimal cost, thus offering a novel solution for developing cost-effective, high-precision mycotoxin detection methods. Full article

Grouper (Epinephelus sp.) is one of the most economically important aquaculture species in China and Southeast Asian countries. Nervous necrosis virus (NNV) infection causes significant mortality in grouper aquaculture. Detection of anti-NNV antibodies using the ELISA method has been reported as effective in preventing vertical transmission of NNV in sea bass and flounder. In this study, IgM antibodies were purified from the serum of groupers and subsequently used to immunize rabbits to obtain a secondary antibody. Additionally, the NNV capsid protein (CP) was cloned, over-expressed, and purified in Escherichia coli. Consequently, an ELISA method was developed to detect NNV-specific antibodies in groupers by utilizing purified recombinant NNV capsid protein (CP) and horseradish peroxidase (HRP)-labeled rabbit anti-grouper antibody. This ELISA system was applied in three asymptomatic grouper hatcheries in China, revealing significant variations in antibody levels. Our ELISA method can effectively detect NNV-specific antibodies and is simpler than other methods, making it suitable for subsequent large-scale applications in grouper. Full article

Inflammatory bowel disease (IBD) is characterized by oxidative stress imbalance and intestinal barrier disruption. Reducing excessive ROS has become a promising therapeutic strategy. Compared with conventional polyphenols, nanomaterials offer greater stability and bioavailability for ROS scavenging. Here, ellagic acid (EA) was converted into uniform nanoparticles (EAs) with reactive oxygen scavenging capacity through horseradish peroxidase (HRP)-mediated oxidative polymerization and subsequently encapsulated in the anti-gastric acid hydrogel F-DP to obtain the hybrid system F-DP@EAs. EAs reduced ROS, MDA, NO, IL-1β, and TNF-α levels in vitro, while increasing IL-4 and IL-10 expression, thus alleviating inflammation. Herein, F-DP@EAs prolonged intestinal retention time and exerted superior protective effects in the DSS-induced colitis model. Oral F-DP@EAs lowered DAI, preserved colon length, increased glutathione (GSH) and superoxide dismutase (SOD), decreased NO and MDA, restored zonula occludens-1 (ZO-1), and reduced mucosal lesions. These findings demonstrate that combining nanoparticle and hydrogel technologies markedly enhances the preventive and protective efficacy of EA, highlighting F-DP@EAs as a promising candidate for future IBD therapy. Full article

This study discloses the noncovalent immobilization of a bienzyme cascade composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto magnetically responsive polyamide microparticles (PA MPs). Porous PA6, PA4, and PA12 MPs containing iron fillers were synthesized via activated anionic ring-opening polymerization in suspension, alongside neat PA6 MPs used as a reference. Four hybrid catalytic systems (GOx/HRP@PA) were prepared through sequential adsorption of HRP and GOx onto the various PA MP supports. The initial morphologies of the supports and the hybrid biocatalysts were characterized by SEM, followed by evaluation of the catalytic performance using a two-step glucose oxidation cascade process. Among all systems, the GOx/HRP@PA4-Fe complex exhibited the highest activity, being approximately 1.5 times greater than the native enzyme dyad, followed by the PA6-supported system with slightly inferior performance. All systems obeyed Michaelis–Menten kinetics, with the immobilized cascades displaying higher Kₘ and Vₘₐₓ values than the non-immobilized enzyme pair while maintaining comparable catalytic efficiencies, CE (CE = k_cat/Kₘ). Subsequently, the immobilized and native enzyme systems were employed for the polymerization of aniline. According to UV–VIS, complete monomer conversion was achieved within 24 h for selected catalysts, and FTIR analysis confirmed the formation of polyaniline in the emeraldine base form without the use of template molecules. These findings highlight the potential of Fe-containing polyamide microparticles as efficient supports for the sustainable, enzyme-mediated synthesis of intrinsically conductive aromatic polymers. Full article

Galactomannan (GM) is a polysaccharide secreted by opportunistic pathogenic fungi of the Aspergillus genus. It is prescribed as a diagnostic biomarker of invasive aspergillosis in immunocompromised patients by the guidelines for diagnosis and management of Aspergillus diseases. It has been shown previously that the measurement of soluble horseradish peroxidase (HRP) using surface-enhanced Raman scattering (SERS) of 2,3-diaminophenazine enzymatic reaction product on silver nanoparticles is largely superior in detection limit compared to colorimetric readout. In this study, a highly sensitive SERS-based HRP measurement protocol was applied to enzyme-linked immunosorbent assay (ELISA) for GM quantification in biological fluids. The detection limit for GM was 4.3 pg per sample, which is one and a half orders of magnitude lower compared to colorimetric detection with o-phenylenediamine as a substrate and five times more sensitive than ELISA using 3,3′,5,5′-tetramethylbenzidine. Full article

Traumatic brain injury (TBI) leads to severe neurological dysfunction, disability, and even death. Surgical intervention and neurorehabilitation represent the current clinical management methods, yet there remains no effective treatment for recovery after TBI. Post-traumatic hyperinflammation and vascular injury are the key therapeutic challenges. Therefore, a novel-designed multifunctional HT/SAA/HSYA hydrogel based on hyaluronic acid (HA) co-loaded with salvianolic acid A (SAA) and hydroxysafflor yellow A (HSYA) was developed in order to simultaneously target inflammation and vascular injury, addressing key pathological processes in TBI. The HT hydrogel was formed through covalent cross-linking of tyramine-modified HA catalyzed by horseradish peroxidase (HRP). Results demonstrated that the HT hydrogel possesses a porous structure, sustained release capabilities of loaded drugs, suitable biodegradability, and excellent biocompatibility both in vitro and in vivo. WB, immunofluorescence staining, and PCR results revealed that SAA and HSYA significantly reduced the expression level of pro-inflammatory cytokines (IL-1β and TNF-α) and inhibited M1 macrophage polarization through the suppression of the TLR4/NF-κB inflammatory pathway. In vivo experiments confirmed that the HT/SAA/HSYA hydrogel exhibited remarkable pro-angiogenic effects, as evidenced by increased expression of CD31 and α-SMA. Finally, H&E staining showed that the HT/SAA/HSYA hydrogel effectively reduced the lesion volume in a mouse TBI model, and demonstrated more pronounced effects in promoting brain repair at the injury site, compared to the control and single-drug-loaded hydrogel groups. In conclusion, the HT hydrogel co-loaded with SAA and HSYA demonstrates excellent anti-inflammatory and pro-angiogenic effects, offering a promising therapeutic approach for brain repair following TBI. Full article

With the rapid development of Internet of Things (IoT) and bioscience technology, wearable smart devices are developing toward advanced trends such as flexibility, convenience and environmental-friendliness. Poly (p-styrenesulfonic acid) (PSS), as a common template and dispersant, is indispensable in the polymerization of conductive polymers. However, the doping amount of PSS has a significant effect on the electrical conductivity of the polymer. Herein, different molar quantities of PSS were used to assist the polymerization of 3,4-ethylenedioxythiophene (EDOT) monomer in a horseradish peroxidase/hydrogen peroxide (HRP/H₂O₂) low-temperature system to obtain conductive finishing solutions with more excellent electrical properties. Then, the polyester nonwoven fabric was immersed in the conductive finishing solution, and when the addition ratio of EDOT and PSS was 1:2, the conductive performance was optimal (3.27 KΩ cm⁻¹). Finally, the conductive fabric was assembled into a pressure sensor and a temperature sensor, which can transmit Morse code in the form of single-parameter (pressure response or temperature response) or collaboration. Overall, this research has great potential for production of poly(3,4-ethylenedioxythiophene) (PEDOT)-based composites and their applications in smart wearable device. Full article

Gelatin, a biocompatible and biodegradable polymer, has garnered considerable attention in tissue engineering (TE) due to its diverse applications enabled by its tunable physical properties. Among the various strategies employed for the fabrication of gelatin-based hydrogels, the use of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂) as a catalytic system has been highlighted as an effective tool for producing hydrogels with highly modifiable properties. Herein, we explore recent progress in the utilization of the HRP/H₂O₂ catalytic system for the creation of gelatin-based hydrogels, with an emphasis on TE applications. Particular attention has been given to the interplay between variations in the concentration equilibrium of HRP and H₂O₂ and the fine-tuning of gel properties tailored for various TE applications. Emerging trends, such as in situ gelation and hybrid bioinks, have also been examined through the lens of their prospective applications, extrapolating from the findings in cell cultures and animal models. A comprehensive review of two databases (Scopus and Web of Science) was conducted. The data extracted from each study included the materials used for each application, methods used for material preparation, cells used in the TE application, laboratory animals used, and whether computational/simulation techniques were implemented. The applications included both homopolymeric hydrogels, using only gelatin as the backbone, and copolymeric hydrogels, with ≥2 polymers. Full article

The rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) are major foodborne pathogenic bacteria that pose significant public health risks, highlighting the need for effective detection methods. In this study, highly sensitive double-antibody sandwich-based enzyme-linked immunosorbent assays (ELISAs) were developed for the rapid detection of E. coli O157:H7 and S. Typhimurium, utilizing a streptavidin-polymerized horseradish peroxidase (SA-PolyHRP)-based signal enhancement system. Systematic optimization was performed on key parameters, including the capture antibody concentration, detection antibody, and blocking agent. Compared to the method using SA-HRP, substitution with SA-PolyHRP significantly improved detection sensitivity, achieving limits of detection (LODs) of 1.4 × 10⁴ CFU/mL for E. coli O157:H7 and 6.0 × 10³ CFU/mL for S. Typhimurium, with sensitivity enhancements of 7.86-fold and 1.83-fold, respectively. Specificity tests confirmed no cross-reactivity with non-target or closely related pathogenic strains. The matrix effect was effectively mitigated through 10-fold and 100-fold dilutions for E. coli O157:H7 and S. Typhimurium, respectively. Both pathogens were successfully detected in beef samples spiked with 5 CFU after 5 h of incubation. This study demonstrates the effectiveness of PolyHRP-based signal enhancement for the highly sensitive and specific detection of foodborne pathogens, offering a promising approach for rapid food safety monitoring and public health protection. Full article

The accurate and sensitive detection of tumor-derived exosomes holds significant promise for the early diagnosis of cancer. In this study, an electrochemical aptasensor was developed for the high-performance detection of exosomes by integrating dual-probe recognition and hybridization chain reaction (HCR). A dual-probe recognition unit composed of a MUC1 aptamer (MUC1-Apt) probe and cholesterol probe was designed for capturing target exosomes and reducing the interference from free proteins, significantly improving the accuracy of exosome detection. It should be noted that the dual-probe recognition unit was formed in conjunction with the HCR. Moreover, a large number of biotins were also assembled on the HCR product, which were used to capture avidin–horseradish peroxidase (SA-HRP) for signal amplification. The CD63 aptamer (CD63-Apt) was immobilized on the surface of a gold electrode for specifically capturing exosomes to construct a classical sandwiched structure. The loaded SA-HRP can efficiently catalyze the reaction of 3, 3′, 5, 5′ tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) to generate a large electrochemical signal. According to this phenomenon, a linear relationship of this proposed aptasensor was achieved between the electrochemical response and 1 × 10²–1 × 10⁷ particles/mL exosomes, with a detection limit of 45 particles/mL. Moreover, the aptasensor exhibited accepted stability and potential clinical applicability. All results proved that this aptasensor has a promising application in exosome-based disease diagnostics. Full article

In this study, the structure and functional and in vitro digestion properties of whey protein isolate (WPI) modified by ultrasonic pretreatment combined with a double oxidase system containing horseradish peroxidase (HRP), glucose oxidase and D-glucose were assessed. SDS-PAGE results confirmed the occurrence of crosslinking reactions. Ultrasonic treatment significantly increased HRP-mediated WPI crosslinking, as demonstrated by reductions in free amino and sulfhydryl groups. CD and FTIR spectroscopies indicated that the structure of the crosslinked WPI was more stable. The particle size of the modified WPI was significantly reduced, resulting in better colloidal stability. Compared with the untreated WPI, the crosslinked WPI possessed enhanced surface hydrophobicity, gelation properties, emulsion stability, and thermal stability but reduced digestibility. These findings provide new insights into ultrasonication combined with a double oxidase system to further improve the structure and functional properties of proteins and broaden their application range in the food industry. Full article

Systemic chemotherapy is still the first-line treatment for cancer, and it’s associated with toxic side effects, chemoresistance, and ultimately cancer recurrence. Rapid gelling hydrogels can overcome this limitation by providing localized delivery of anti-cancer agents to solid tumors. Silk hydrogels are extremely biocompatible and suitable for anti-cancer drug delivery, but faster gelling formulations are needed. In this study, we introduce a rapid gelling hydrogel formulation (<3 min gelling time) due to chemical crosslinking between silk fibroin and curcumin, initiated by the addition of minute quantities of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The novel observation in this study is that curcumin, while being a free-radical scavenger, also participates in accelerating silk di-tyrosine crosslinking in the presence of HRP and H₂O₂. Using UV-Vis, rheology, and time-lapse videos, we convincingly show that curcumin accelerates silk di-tyrosine crosslinking reaction in a concentration-dependent manner, and curcumin remains entrapped in the hydrogel post-crosslinking. FTIR results show an increase in secondary beta-sheet structures within hydrogels, with increasing concentrations of curcumin. Furthermore, we show that curcumin-silk di-tyrosine hydrogels are toxic to U2OS osteosarcoma cells, and most cancer cells are dead within short time scales of 4 h post-encapsulation. Full article

Interleukin-12 (IL-12), a crucial biomarker for immune and inflammatory responses, plays a pivotal role in diagnosing and managing diverse pathological conditions. Although colorimetric enzyme-linked immunosorbent assays (CELISAs) have been extensively employed to detect IL-12 in biological samples, their sensitivity is inherently limited by the catalytic efficiency of enzyme labels, presenting substantial challenges in achieving ultrasensitive detection and enabling pre-symptomatic diagnosis of diseases. In this study, we address this limitation by developing a novel peroxidase nanozyme, featuring ultrathin Pt skins consisting of only ~4 atomic layers, coated on Au nanoparticles (denoted as Au@Pt_4LNPs). These Au@Pt_4LNPs exhibit remarkable catalytic performance, achieving a ~1063-fold enhancement in peroxidase-like activity compared to horseradish peroxidase (HRP), while minimizing Pt consumption, thereby improving Pt utilization efficiency and reducing costs. This advancement facilitates the construction of an ultrasensitive CELISA capable of detecting IL-12 at femtomolar concentrations. Using Au@Pt_4LNPs as the signal labels, the developed CELISA demonstrates a quantitative detection range from 0.1 to 100 pg mL⁻¹, with a limit of detection (LOD) as low as 0.084 pg mL⁻¹ (1.1 fM), offering ~10 times greater sensitivity than the HRP-based CELISA. This study highlights the potential of Au@Pt_4LNP nanozymes as advanced signal labels, opening new avenues for next-generation ultrasensitive bioassays. Full article

Low-frequency electromagnetic fields, induced by alternating current (AC)-based equipment such as transformers, are known to influence the physicochemical properties and function of enzymes, including their catalytic activity. Herein, we have investigated how incubation near a 50 Hz AC autotransformer influences the physicochemical properties of horseradish peroxidase (HRP), by atomic force microscopy (AFM) and spectrophotometry. We found that a half-hour-long incubation of the enzyme above the coil of a loaded autotransformer promoted the adsorption of the monomeric form of HRP on mica, enhancing the number of adsorbed enzyme particles by two orders of magnitude in comparison with the control sample. Most interestingly, the incubation of HRP above the switched-off transformer, which was unplugged from the mains power supply, for the same period of time was also found to cause a disaggregation of the enzyme. Notably, an increase in the activity of HRP against ABTS was observed in both cases. We hope that the interesting effects reported will emphasize the importance of consideration of the influence of low-frequency electromagnetic fields on enzymes in the design of laboratory and industrial equipment intended for operation with enzyme systems. The effects revealed in our study indicate the importance of proper shielding of AC-based transformers in order to avoid the undesirable influence of low-frequency electromagnetic fields induced by these transformers on humans. Full article

Indium tin oxide (ITO) is a transparent conducting material with exceptional electrical and optical properties, widely used in biosensing and bioelectronics. Functionalization of ITO with linker molecules enables covalent attachment of biomolecules, allowing for dual-mode optical and electrochemical detection. Carbonic anhydrase IX (CA IX), a transmembrane enzyme overexpressed in hypoxic tumors, is a promising biomarker for cancer diagnostics due to its restricted expression in normal tissues. However, conventional detection methods are time-intensive and unsuitable for point-of-care applications. In this study, ITO surfaces were functionalized using silane-based chemistry to immobilize CA IX-specific antibodies, creating a novel biosensing platform. The biosensor utilized a secondary horseradish peroxidase (HRP)-conjugated antibody to catalyze the oxidation of luminol in the presence of hydrogen peroxide, producing a chemiluminescent and electrochemical signal. Characterization of the biosensor via a dual-mode optical and electrochemical approach revealed efficient antibody immobilization. Due to the high variation observed in the optical approach, limit of detection (LOD) experiments were conducted exclusively with electrochemistry, yielding an LOD of 266.4 ng/mL. These findings demonstrate the potential of ITO-based electrochemical biosensors for sensitive and selective CA IX detection, highlighting their applicability in cancer diagnostics and other biomedical fields. Full article