Search Results (151)

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

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

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

In recent years, hydrogel beads and in situ hydrogels have gained wide attention in various fields such as biomedicine. In this study, 3-(4-hydroxyphenyl) propionic acid (HP) was introduced into the side chain of poly(α,β-[N-(2-hydroxyethyl)-D,L-aspartamide]) (PHEA) to synthesize phenolic hydroxyl-functionalized poly(aspartamide) derivative PHEA-HP with enzyme-catalyzed cross-linking potential. First, the chemical structure of PHEA-HP was characterized by FT-IR, UV and ¹H NMR, and the results of in vitro cytotoxicity against L929 cell line and hemolysis experiment showed that PHEA-HP did not have toxicity to cells (viability > 90%) and had good blood compatibility. Then, rheological measurement confirmed the formation of PHEA-HP-based in situ hydrogel with a high storage modulus (G′) around 10⁴ Pa, and the vial-tilting method revealed that the gelation time of PHEA-HP aqueous solution could be tuned in the wide range of 5–260 s by varying the concentrations of hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP). Finally, hydrogel beads of different diameters containing methylene blue (for easy observation) were prepared using a coaxial needle and syringe pumps, and the effect of the flow rate of the outer phase on the diameters of the hydrogel beads was also investigated. Therefore, PHEA-HP may be a promising and safe poly(aspartamide) derivative that can be used to prepare in situ hydrogels and hydrogel beads for applications closely related to the human body. Full article

Prussian blue nanoparticles (PBNPs) have been identified as a promising candidate for biomimetic peroxidase (POD)-like activity, specifically due to the metal centres (Fe³⁺/Fe²⁺) of Prussian blue (PB), which have the potential to function as catalytically active centres. The decoration of PBNPs with desired functional polymers (such as amino- or carboxylate-based) primarily facilitates the subsequent linkage of biomolecules to the nanoparticles for their use in biosensor applications. Thus, the elucidation of the catalytic POD mimicry of these systems is of significant scientific interest but has not been investigated in depth yet. In this report, we studied a series of poly(ethyleneimine) (PEI)-mediated PBNPs (PB/PEI NPs) prepared using various synthesis protocols. The resulting range of particles with varying size (~19–92 nm) and shape combinations were characterised in order to gain insights into their physicochemical properties. The POD-like nanozyme activity of these nanoparticles was then investigated by utilising a 3,3′,5,5′-tetramethylbenzidine (TMB)/H₂O₂ system, with the catalytic performance of the natural enzyme horseradish peroxidase (HRP) serving as a point of comparison. It was shown that most PB/PEI NPs displayed higher catalytic activity than the PBNPs, with higher activity observed in particles of smaller size, higher Fe content, and higher Fe²⁺/Fe³⁺ ratio. Furthermore, the nanoparticles demonstrated enhanced chemical stability in the presence of acid, sodium azide, or high concentrations of H₂O₂ when compared to HRP, confirming the viability of PB/PEI NPs as a promising nanozymatic material. This study disseminates fundamental knowledge on PB/PEI NPs and their POD-like activities, which will facilitate the selection of an appropriate particle type for future biosensor applications. Full article

Hydrogels are widely used in tissue engineering due to their ability to form three-dimensional (3D) structures that support cellular functions and mimic the extracellular matrix (ECM). Despite their advantages, dextran-based hydrogels lack intrinsic biological activity, limiting their use in this field. Here, we present a strategy for developing bioactive hydrogels through sequential thiol–maleimide bio-functionalization and enzyme-catalyzed crosslinking. The hydrogel network is formed through the reaction of tyramine moieties in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), allowing for tunable gelation time and stiffness by adjusting H₂O₂ concentrations. Maleimide groups on the hydrogel backbone enable the coupling of thiol-containing bioactive molecules, such as arginylglycylaspartic acid (RGD) peptides, to enhance biological activity. We examined the effects of hydrogel stiffness and RGD concentration on human mesenchymal stem cells (hMSCs) during differentiation and found that hMSCs encapsulated within these hydrogels exhibited over 88% cell viability on day 1 across all conditions, with a slight reduction to 60–81% by day 14. Furthermore, the hydrogels facilitated adipogenic differentiation, as evidenced by positive Oil Red O staining. These findings demonstrate that DexTA–Mal hydrogels create a biocompatible environment that is conducive to cell viability and differentiation, offering a versatile platform for future tissue engineering applications. Full article

We revealed that the encapsulation of enzyme-immobilized silica particles in hollow-type spherical bacterial cellulose (HSBC) gels enables the use of the inside of HSBC gels as a reaction field. The encapsulation of horseradish peroxidase (HRP)-immobilized silica particles (Si-HRPs, particle size: 40–50 μm) within HSBC gels was performed by using a BC gelatinous membrane produced at the interface between Komagataeibacter xylinus suspension attached onto an alginate gel containing Si-HRPs and silicone oil. After the biosynthesis of the BC gelatinous membrane, formed from cellulose nanofiber networks, the alginate gel was removed via immersion in a phosphate-buffered solution. Si-HRP encapsulated HSBC gels were reproducibly produced using our method with a yield of over 90%. The pore size of the network structure of the BC gelatinous membrane was less than 1 μm, which is significantly smaller than the encapsulated Si-HRPs. Consequently, the encapsulated Si-HRPs could neither pass through the BC gelatinous membrane nor leak from the interior cavity of the HSBC gel. The activity of the encapsulated HRPs was detected using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H₂O₂ system, demonstrating that this method can encapsulate the enzyme without inactivation. Since HSBC gels are composed of a network structure of biocompatible cellulose nanofibers, immune cells cannot enter the hollow interior, thus, the enzyme-immobilized particles encapsulated inside the HSBC gel are protected from immune-cell attacks. The encapsulation technique demonstrated in this study is expected to facilitate the delivery of enzymes and catalysts that are not originally present in the in vivo environment. Full article

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (_oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity. Full article

A microfluidic immuno-biosensor detection system consisting of a microfluidic spectrum chip and a micro-spectrometer detection device is presented for the rapid point-of-care (POC) detection and quantification of high-sensitivity C-reactive protein (hs-CRP) in urine. The detection process utilizes a highly specific enzyme-linked immunosorbent assay (ELISA) method, in which capture antibodies and detection antibodies are pre-deposited on the substrate of the microchip and used to form an immune complex with the target antigen. Horseradish peroxidase (HRP) is added as a marker enzyme, followed by a colorimetric reaction using 3,3′,5,5′-tetramethylbenzidine (TMB). The absorbance values (a.u.) of the colorimetric reaction compounds are measured using a micro-spectrometer device and used to measure the corresponding hs-CRP concentration according to the pre-established calibration curve. It is shown that the hs-CRP concentration can be determined within 50 min. In addition, the system achieves recovery rates of 93.8–106.2% in blind water samples and 94.5–104.6% in artificial urine. The results showed that the CRP detection results of 41 urine samples from patients with chronic kidney disease (CKD) were highly consistent with the conventional homogeneous particle-enhanced turbidimetric immunoassay (PETIA) method’s detection results (R² = 0.9910). The experimental results showed its applicability in the detection of CRP in both urine and serum. Overall, the results indicate that the current microfluidic ELISA detection system provides an accurate and reliable method for monitoring the hs-CRP concentration in point-of-care applications. Full article

Glycerol is employed as a functional component of heat-transfer fluids, which are of use in both bioreactors and various biosensor devices. At the same time, flowing glycerol was reported to cause considerable triboelectric effects. Herein, by using atomic force microscopy (AFM), we have revealed the long-term effect of glycerol flow, stopped in a ground-shielded coiled heat exchanger, on horseradish peroxidase (HRP) adsorption on mica. Namely, the solution of HRP was incubated in the vicinity of the side of the cylindrical coil with stopped glycerol flow, and then HRP was adsorbed from this solution onto a mica substrate. This incubation has been found to markedly increase the content of aggregated enzyme on mica—as compared with the control enzyme sample. We explain the phenomenon observed by the influence of triboelectrically induced electromagnetic fields of non-trivial topology. The results reported should be further considered in the development of flow-based heat exchangers of biosensors and bioreactors intended for operation with enzymes. Full article

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H₂O₂ to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H₂O₂. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1–4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5–2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days. Full article

Removing organic pollutants from wastewater is crucial to prevent environmental contamination and protect human health. Immobilized enzymes are increasingly being explored for wastewater treatment due to their specific catalytic activities, reusability, and stability under various environmental conditions. Peroxidases, such as horseradish peroxidase (HRP) and myoglobin (Mb), are promising candidates for immobilized enzymes utilized in wastewater treatment due to their ability to facilitate the oxidation process of a wide range of organic molecules. However, the properties of the carrier and support materials greatly influence the stability and activity of immobilized HRP and Mb. In this research, we developed immobilized HRP and Mb using support material composed of sodium alginate and CaCl₂ as carriers and glutaraldehyde as a crosslinking agent. Following this, the efficacy of immobilized HRP and Mb in removing aniline, phenol, and p-nitrophenol was assessed. Both immobilized enzymes removed all three organic pollutants from an aqueous solution, but Mb was more effective than HRP. After being immobilized, both enzymes became more resilient to changes in temperature and pH. Both immobilized enzymes retained their ability to eliminate organic pollutants through eight treatment cycles. Our study uncovered novel immobilized enzyme microspheres and demonstrated their successful application in wastewater treatment, paving the way for future research. Full article