Encapsulated Horseradish Peroxidase Research Articles

DNA nanoflowers encapsulating horseradish peroxidase as a signal amplification tag for rapid diagnosis in cytology specimens

Rapid analysis of cytology samples obtained by minimally invasive sampling is essential in clinical diagnosis, yet existing diagnostic methods are often cumbersome and time-consuming. To rapidly detect the scanty number of cells in specimens, a multifunctional detection probe based on DNA nanoflower structure was prepared in this study, which possesses both signal output capability of fluorescence and oxidation reaction (colorimetric assay) for ultra-sensitive cancer cell detection. Firstly, DNA nanoflowers encapsulating horseradish peroxidase (HRP-DNA Nanoflowers, HDFs) were prepared by the rolling circle amplification (RCA) reaction and then hybridized with the aptamers labeled with fluorophores to construct the multivalent aptamer HDFs (Multi-Aptamer-HDFs) with fluorescence effect and efficient cancer cell targeting. The Multi-Aptamer-HDFs probe can be used to detect fresh clinical cytology specimens directly for fluorescence observation with sensitivity and specificity of 88.64%, and 84.85%, respectively. In addition, Multi-Aptamer-HDFs has peroxidase activity due to the encapsulation of HRP, which can catalyze the oxidation of substrates such as ABTS and DAB for color development. Therefore, we applied Multi-Aptamer-HDFs to one-step staining of clinical cytology cell blocks with a sensitivity of 88.41% and a specificity of 83.33%, which required fewer steps and reagents with a significantly shorter time required for immunocytochemical staining. The multifunctional probe, Multi-Aptamer-HDFs, prepared in this study is expected to be better applied in the rapid cellular analyses and one-step immunostaining, which is expected to provide a new means for rapid on-site assessment and on-site preliminary diagnosis.

A Near-Infrared-II Fluorescent Nanocatalyst for Enhanced CAR T Cell Therapy against Solid Tumor by Immune Reprogramming.

Chimeric antigen receptor (CAR) T cell therapy holds great promise in the treatment of hematological malignancies but performs poorly in solid tumors due to the tumor immunosuppressive microenvironment. Herein, a multifunctional nanocatalyst (APHA@CM) was prepared by encapsulating horseradish peroxidase (HRP)-loaded Au/polydopamine nanoparticles (Au/PDA NPs) and Ag2S quantum dots with CAR T cell membranes to improve the CAR T cell therapy in solid tumors. The APHA@CM has excellent multimodal imaging capability to precisely guide the scope and time window for nanocatalyst-induced tumor microenvironment regulation and CAR T cell therapy. The oxidase-like activity of Au NPs inhibited the glycolytic metabolism of tumor cells, reducing lactate efflux, reprogramming tumor immunosuppression, and ultimately increasing CAR T cell activation within the tumors. Additionally, the hypoxia environment of tumors could be relieved by HRP to enhance the Au/PDA NPs-induced synergistic sonodynamic/photothermal therapy (SDT/PTT), thereby promoting the immunogenic cell death of NALM 6 cells and enhancing CAR T cell-mediated immune microenvironment reprogramming. When this strategy was utilized to treat NALM 6 solid tumors, it not only completely eliminated tumors but also formed a long-term immune memory effect to inhibit tumor metastasis and recurrence. This work offers a strategy for CAR T cell therapy in solid tumor.

Pomegranate-Bionic Encapsulating Horseradish Peroxidase Using Dopamine Flexible Scaffold-Coated Multishell Porous ZIF-8 To Enhance Immunochromatographic Diagnosis.

Nanoparticle-natural enzyme complexes are receiving increasing attention as the promising signal reporters for colorimetric lateral flow immunoassay (LFIA). Nonetheless, it remains a challenge to develop the nanocomplexes with high loading efficiency, catalytic efficiency, and colorimetric signal brightness. Herein, inspired by pomegranate structure, we reported the synthesis of a colorimetric catalytic nanocomplex ((HRP@ZIF-8)3@PDA@HRP), using dopamine flexible scaffold-coated multishell porous zeolitic imidazolate framework-8 (ZIF-8) as a hierarchical scaffold to encapsulate horseradish peroxidase (HRP), and described its potential to promote an ultrasensitive colorimetric LFIA of cardiac troponin I (cTnI). (HRP@ZIF-8)3@PDA@HRP exhibited ultrahigh HRP loading efficiency and catalytic activity due to the epitaxial shell-by-shell overgrowth of porous ZIF-8 scaffold, which provided more cavities for enzyme immobilization and a diffusion path for the catalytic substrate. Furthermore, the polydopamine (PDA) layer on the (HRP@ZIF-8)3 surface both enhanced the colorimetric signal brightness and acted as a flexible scaffold to immobilize HRP, further increasing the amount of enzyme. Following integration with LFIA, the developed platform achieved an ultrasensitive colorimetric test strip assay for cTnI with pre- and postcatalytic naked-eye detection sensitivities of 0.5 ng mL-1 and 0.01 ng mL-1, respectively, which were 4/2- and 200/100-fold higher than gold nanoparticles (AuNPs)/PDA-based LFIA and comparable to chemiluminescence immunoassay. Further, the quantitative testing results of the developed colorimetric LFIA on 57 clinical serum samples agreed well with the clinical data. This work provides ideas for the design of natural enzymes-based colorimetric catalytic nanocomplex to encourage applications for the development of ultrasensitive LFIA for early diseases diagnosis.

Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λex = 488 nm, λem = 520 nm). To generate easily detectable UV–vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) to generate an optical signal (λabs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL−1 and 0.904 pg mL−1) and a wide linear range (0.001–100 ng mL−1). For detection of RBD in human saliva, the recovery was 93.0–100% for the fluorescence assay and 87.2–107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.Graphical

Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was defined as 4.3 CFU mL−1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.

Core-satellite nanoreactors based on cationic photosensitizer modified hollow CuS nanocage for ROS diffusion enhanced phototherapy of hypoxic tumor

Photodynamic therapy (PDT) efficiency is directly affected by the reactive oxygen species (ROS) generated by photosensitizers. However, ROSs' ultrashort life span and limited diffusion distance restrict the PDT efficiency. Therefore, it is important to control the delivery strategy of photosensitizers for PDT treatment. Herein, the core-satellite nanoreactors were fabricated with oxygen generation and ROS diffusion properties. The hollow CuS encapsulating horseradish peroxidase (HRP) was combined with the cationic photosensitizers (PEI-Ce6). The unique photosensitizers delivery strategy makes the nanoreactors achieve ROS diffusion-enhanced PDT effect. First, HRP in “core” (HRP@CuS) can decompose hydrogen peroxide (H2O2) to O2, increasing O2 levels on the surface of the nanoreactor. Second, the Ce6 molecules covalent-linked with PEI are uniformly dispersed on the surface of CuS as a “satellite”, avoiding Ce6 aggregation and causing more Ce6 molecules be activated to produce more 1O2. Due to the Ce6 was on the surface of the CuS nanocages, the generated ROS may ensure a larger diffusion range. Meanwhile, the inherently CuS nanocages exhibit photothermal and photoacoustic (PA) effect. The photothermal effect further enhances the ROS diffusion. Under the guidance of PA imaging, nanoreactors exhibit highly efficient hypoxic tumor ablation via photodynamic and photothermal effect. Overall, the core-satellite nanoreactors provide an effective strategy for tumor therapy, further promoting the research of photosensitizers delivery.

In-vitro and in-vivo efficacy of hollow gold nanoparticles encapsulating horseradish peroxidase: Oxidative stress-mediated tumor cell killing

This article reports the protocol for the synthesis of hollow gold nanoparticles using harmless template-material calcium phosphate nanoparticles for encapsulating enzyme to overcome the problem associated with antibody-directed enzyme pro-drug therapy (ADEPT) or chemotherapy against cancer. Horseradish peroxidase (HRP) could be effectively loaded inside the cavity of hollow gold nanoparticles. The high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) analysis confirmed monodispersed formulation demonstrating spherical morphology with an average diameter of 50 nm. Aqueous dispersion of these nanoparticles was highly stable under cold conditions. HRP loaded in the cavity of hollow gold nanoparticles showed more stability in response to change in temperature as compared to unloaded/free enzyme and followed Michaelis-Menten kinetics. From the in-vitro MTT assay, it is concluded that the prodrug - Indole acetic acid (IAA) is non-toxic, however, after oxidative decarboxylation by HRP, it became active/toxic. The cell death mechanism by IAA/entrapped-HRP combination confirmed apoptosis as demonstrated by DNA fragmentation and AO/PI staining. For assessing in-vivo efficacy, the hollow gold nanoparticles with HRP was topically administered into the xenographted tumor in mice. The benign substrate IAA was given either orally or by intra-peritoneal injection. It reached the tumor tissue through systemic circulation to get oxidized by HRP. This created severe and lethal oxidative stress to the cancer cells resulting in regression of tumor. The uniqueness and novelty of our work lies in the fact that we have used benign template to generate/create hollow formulation loaded with HRP, which could enhance the treatment efficacy by multi-fold as compared to the free HRP or solid formulation as more active sites are available due to internal mobility of the enzyme for interaction with the prodrug. From the survival percent graph, we concluded that all mice died after ∼32 days in case of free HRP/IAA combination but in case of HGNP-HRP/IAA combination, 100% mice were alive after ∼30 days and ∼50% of tumor bearing mice remained alive even after 60 days. In conclusion, both in vitro and in vivo experiments revealed that the combination of IAA/HRP entrapped in hollow gold nanoparticles could be efficiently and effectively used for cancer therapy.

Gastrointestinal Tract Stabilized Protein Delivery Using Disulfide Thermostable Exoshell System.

Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.

Amorphous metal-organic frameworks on PtCu hydrogels: Enzyme immobilization platform with boosted activity and stability for sensitive biosensing

Cell-free enzymatic catalysis (CFEC) is emerging biotechnology that simulates biological transformations without living cells. However, the high cost of separation and preparation of the enzyme has hindered the practical application of the CFEC. Enzyme immobilization technologies using solid supports to stabilize enzymes have been regarded as an efficient strategy to address this issue. Nevertheless, the activity and stability of the immobilized enzymes are still crucial challenges for working in vitro. Herein, an enzyme immobilization platform is developed by using PtCu hydrogels coated with amorphous metallic-organic frameworks (MOFs) as multifunctional carriers to encapsulate horseradish peroxidase (HRP). Specifically, PtCu hydrogels acting as a "reservoir of metal ions" can interact with the immobilized enzyme and facilitate electron transfer, leading to the boosted enzyme catalytic performances. Furthermore, amorphous MOFs on the surface of PtCu hydrogels serve as an "armor" to protect the internal enzymes from various perturbation environments. The resultant enzyme immobilization platform (PtCu@HRP@ZIF-8) not only shows an approximately 2.4-fold enhanced activity compared with free enzyme but also exhibits improved stability against harsh conditions. The PtCu@HRP@ZIF-8-based biosensor is constructed for sensitive sensing of organophosphorus pesticides (OPs). The proposed biosensor exhibits a favorable linear relationship with the concentration of paraoxon-ethyl from 6 to 800 ng/mL, with a low detection limit of 1.8 ng/mL. This work reveals the promising potential of our proposed enzyme immobilization platform in practical applications.

Removal of Persistent Sulfamethoxazole and Carbamazepine from Water by Horseradish Peroxidase Encapsulated into Poly(Vinyl Chloride) Electrospun Fibers.

Enzymatic conversion of pharmaceutically active ingredients (API), using immobilized enzymes should be considered as a promising industrial tool due to improved reusability and stability of the biocatalysts at harsh process conditions. Therefore, in this study horseradish peroxidase was immobilized into sodium alginate capsules and then trapped into poly(vinyl chloride) electrospun fibers to provide additional enzyme stabilization and protection against the negative effect of harsh process conditions. Due to encapsulation immobilization, 100% of immobilization yield was achieved leading to loading of 25 μg of enzyme in 1 mg of the support. Immobilized in such a way, enzyme showed over 80% activity retention. Further, only slight changes in kinetic parameters of free (Km = 1.54 mM) and immobilized horseradish peroxidase (Km = 1.83 mM) were noticed, indicating retention of high catalytic properties and high substrate affinity by encapsulated biocatalyst. Encapsulated horseradish peroxidase was tested in biodegradation of two frequently occurring in wastewater API, sulfamethoxazole (antibiotic) and carbamazepine (anticonvulsant). Over 80% of both pharmaceutics was removed by immobilized enzyme after 24 h of the process from the solution at a concentration of 1 mg/L, under optimal conditions, which were found to be pH 7, temperature 25 °C and 2 mM of H2O2. However, even from 10 mg/L solutions, it was possible to remove over 40% of both pharmaceuticals. Finally, the reusability and storage stability study of immobilized horseradish peroxidase showed retention of over 60% of initial activity after 20 days of storage at 4 °C and after 10 repeated catalytic cycles, indicating great practical application potential. By contrast, the free enzyme showed less than 20% of its initial activity after 20 days of storage and exhibited no recycling potential.

Development of site-specific antibody-conjugated immunoliposomes for sensitive detection of disease biomarkers.

Liposome-based immunoassay (LIA) is an attractive protocol for amplifying the detection signals because of the excellent ability of liposomes to encapsulate signal marker compounds. The antigen-binding activity of the conjugated antibodies on the liposomal surface is crucial for the specificity and sensitivity of LIA. We present here a general platform to ensure that antibodies can conjugate onto the surface of liposomes in a site-specific and oriented manner. A His-handle-modified antibody with Fc region-specific and covalent conjugation was first fabricated using a photoactivatable ZBpa-His tag that was engineered using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Based on the high affinity between the His tag and divalent metal ions, the novel His-modified antibody was oriented onto the surface of nickel ion-modified liposomes encapsulating horseradish peroxidase. With the prostate-specific antigen as a model, the detection efficiency of the new immunoliposomes was evaluated by chemiluminescence immunoassay. The immunoliposomes exhibited a limit of detection of 0.2 pg mL-1, which was a six time improvement compared with that of the chemical-coupled antibody-liposome conjugates. Thus, the proposed immunoliposomes are expected to hold potential applications for the sensitive detection of various biomarkers in complicated serum samples.

Removal of Diclofenac, Paracetamol, and Carbamazepine from Model Aqueous Solutions by Magnetic Sol-Gel Encapsulated Horseradish Peroxidase and Lignin Peroxidase Composites.

Sustainable and green synthesis of nanocomposites for degradation of pharmaceuticals was developed via immobilization and stabilization of the biological strong oxidizing agents, peroxidase enzymes, on a solid support. Sol–gel encapsulated enzyme composites were characterized using electron microscopy (TEM, SEM), atomic force microscopy, FTIR spectroscopy, and thermogravimetric analysis. Horseradish peroxidase (HRP) and lignin peroxidase (LiP) were adsorbed onto magnetite nanoparticles and sol–gel encapsulated in a surface silica layer. Encapsulation enhanced the stability of the biocatalysts over time and thermal stability. The biocatalysts showed appreciable selectivity in oxidation of the organic drinking water pollutants diclofenac, carbamazepine, and paracetamol with improved activity being pharmaceutical specific for each enzyme. In particular, sol–gel encapsulated LiP- and HRP-based nanocomposites were active over 20 consecutive cycles for 20 days at 55 °C (24 h/cycle). The stability of the sol–gel encapsulated catalysts in acidic medium was also improved compared to native enzymes. Carbamazepine and diclofenac were degraded to 68% and 64% by sol–gel LiP composites respectively at pH 5 under elevated temperature. Total destruction of carbamazepine and diclofenac was achieved at pH 3 (55 °C) within 3 days, in the case of both immobilized HRP and LiP. Using NMR spectroscopy, characterization of the drug decomposition products, and decomposition pathways by the peroxidase enzymes suggested.

Nanocapsulation of horseradish peroxidase (HRP) enhances enzymatic performance in removing phenolic compounds

As ubiquitous environmental pollutants, phenolic compounds are requested to be efficiently removed from wastewater. Enzymes, such as Horseradish peroxidase (HRP), have been demonstrated with great potential in removing phenolic compounds. Different from the general immobilization technology, the encapsulation of individual enzymes within nanogel has been employed in this work. Here we show that, the encapsulated HRP could remarkably enhance enzymatic performance, including thermostability, catalytic efficiency, environmental tolerance and, most importantly, the biodegradation of phenolic compounds. For instance, the removal efficiencies of phenol and BPA increased by 7-fold and 3.5-fold, respectively. On the other hand, the diverted removal efficiencies were obtained for a series of phenolic compounds. Based on molecular modelling, the biodegradabilities of phenolic compounds were rationalized according to their redox potentials and binding affinities with enzymes. In summary, our work indicates that the nanocapsulation of enzyme should be a promising strategy in removing different types of phenolic compounds from wastewater.

Construction of a novel electrochemical biosensor based on a mesoporous silica/oriented graphene oxide planar electrode for detecting hydrogen peroxide.

A constant magnetic field (CMF) was used to arrange the orientation of graphene oxide (GO) which was modified on a self-made screen-printed electrode. We evaluated the efficiency of this method for potential analytical application towards the sensing of hydrogen peroxide (H2O2). Mesoporous silica (MS)-encapsulated horseradish peroxidase (HRP) was immobilized on the electrode with vertically arranged GO to construct an H2O2 sensor (denoted as CMF/GO/HRP@MS). The linear range of the response of the CMF/GO/HRP@MS sensor to H2O2 was 0.1-235 μM, and the detection limit was as low as 0.01 μM. The results demonstrated that the vertical arrangement of GO resulting from the CMF on the electrode surface could increase the electron transfer rate. The excellent selectivity and anti-interference ability of this sensor to H2O2 in physiological samples may be attributed to the synergistic effect of mesoporous silica, GO and constant magnetic field.

Multifunctional Peroxidase-Encapsulated Nanoliposomes: Bioetching-Induced Photoelectrometric and Colorimetric Immunoassay for Broad-Spectrum Detection of Ochratoxins.

In this study, a versatile dual-modal readout immunoassay platform was achieved for sensitive and broad-spectrum detection of ochratoxins based on the photocurrent response of flexible CdS/ZnO nanorod arrays/reduced graphene oxide and the localized surface plasmon resonance (LSPR) peak shift of Au nanobipyramids (Au NBPs). By using nanoliposomes as the vehicle to carry the secondary antibody and encapsulate horseradish peroxidase (HRP), the photocurrent change and the peak shift can be greatly amplified. The reaction mechanism was investigated in detail, indicating that HRP can trigger enzymatic bioetching in the presence of H2O2. In the photoelectrochemical detection, the oxidized HRP can etch CdS on the photoelectrode, resulting in the photocurrent change, while in the colorimetric detection, HRP can oxidize H2O2 to produce hydroxyl radicals that can etch Au NBPs to form multiple color changes and LSPR shifts. Compared with the common single-modal immunoassay for ochratoxins, such dual-modal immunoassay is more precise and reliable, owing to the completely independent signal conversion and transmission mechanism. Therefore, we hope that this accurate, simple, and visualized strategy may create a new avenue and provide innovative inspiration for food analysis.

Adsorption of cholesterol oxidase and entrapment of horseradish peroxidase in metal-organic frameworks for the colorimetric biosensing of cholesterol

The co-immobilization of two enzymes onto single support commonly exhibits low efficiency due to the competition against limited sites. Water-stable metal–organic frameworks (MOFs) [i.e., PCN-333(Al)] with a high surface area and ultra-large cavities were employed to efficiently adsorb cholesterol oxidase (ChOx) and encapsulate horseradish peroxidase (HRP), respectively. The prepared PCN-333/ChOx&HRP was characterized through SEM, XRD, confocal microscopy, N2 adsorption isotherms, and thermal gravity analysis (TGA). The high surface area and high concentration of mesoporous cages resulted in the high loadings of both ChOx and HRP. The absorbed ChOx and the encapsulated HRP presented excellent activities without additional chemical modification. The immobilized enzymes were stable against protease digestion, organic solvents, temperature changes, and pH variation. Thus, a colorimetric biosensor for cholesterol detection was fabricated depending on cascade catalytic reactions of the immobilized bi-enzymes. An extended linear range from 0.0 to 40.0 μM with a low detection limit of 0.6 μM was obtained using the biosensor. The co-immobilization of the enzymes onto the surface and into the mesopores of MOFs provided a new and excellent platform for the development of highly stable and sensitive colorimetric biosensors.

The Search for Ways to Improve the Catalytic Activity of Encapsulated Horseradish Peroxidase

Abstract—We consider a previously proposed method for encapsulation of enzymes, which employs the layer-by-layer adsorption of oppositely charged polyelectrolytes onto composite spherulites (calcium carbonate/protein) followed by dissolution of the calcium carbonate support. The goal of this work is to choose conditions for encapsulation of the enzyme horseradish peroxidase by the method so that the catalytic activity of the encapsulated enzyme would be comparable with that of the soluble one. The steps of the fabrication of polyelectrolyte microcapsules with the studied enzyme have been tested. A protocol for obtaining composite spherulites of the desired size ranging from 2 to 10 µm has been developed. It is shown that the catalytic activity of horseradish peroxidase encapsulated in a microcapsule with a positively charged inner surface of the microcapsule envelope (the inner layer modified by polycation polyallylamine hydrochloride) is significantly higher than that of the enzyme encapsulated in a microcapsule with a negatively charged inner layer (modified by polyanion sodium polystyrene sulfonate). At the support dissolution step, ethylene glycol bis-(β-aminoethyl) tetraacetic acid (EGTA), a decalcifying agent used to dissolve CaCO3 from horseradish peroxidase microcapsules, is significantly less detrimental to enzymes than ethylene diamine tetraacetic acid. The catalytic activity of horseradish peroxidase encapsulated in polyallylamine hydrochloride/sodium polystyrene sulfonate/polyallylamine hydrochloride microcapsules (with positively charged inner surface of the microcapsule envelope) is 60–70% of the activity of the soluble enzyme (in experiments where the calcium-carbonate support of the composite spherulites is dissolved with EGTA).

DNA flower-encapsulated horseradish peroxidase with enhanced biocatalytic activity synthesized by an isothermal one-pot method based on rolling circle amplification.

DNA nanotechnology has been developed to construct a variety of functional two- and three-dimensional structures for versatile applications. Rolling circle amplification (RCA) has become prominent in the assembly of DNA-inorganic composites with hierarchical structures and attractive properties. Here, we demonstrate a one-pot method to directly encapsulate horseradish peroxidase (HRP) in DNA flowers (DFs) during RCA. The growing DNA strands and Mg2PPi crystals lead to the construction of porous DFs, which provide sufficient interaction sites for spontaneously incorporating HRP molecules into DFs with high loading capacity and good stability. Furthermore, in comparison with free HRP, the DNA flower-encapsulated HRP (termed HRP-DFs) demonstrate enhanced enzymatic activity, which can efficiently biocatalyze the H2O2-mediated etching of gold nanorods (AuNRs) to generate distinct color changes since the longitudinal localized surface plasmon resonance (LSPR) frequency of AuNRs is highly sensitive to the changes in the AuNR aspect ratio. Through rationally incorporating the complementary thrombin aptamer sequence into the circular template, the synthesized HRP-DF composites are readily used as amplified labels for visual and colorimetric detection of thrombin with ultrahigh sensitivity and excellent selectivity. Therefore, our proposed strategy for direct encapsulation of enzyme molecules into DNA structures shows considerable potential applications in biosensing, biocatalysis, and point-of-care diagnostics.

Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination.

As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1–40 μM in sulfide detection with a high sensitivity of 1720 μA mM−1 cm−2 and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported.

Fabrication of Lignosulfonate Vesicular Reverse Micelles to Immobilize Horseradish Peroxidase

Sodium lignosulfonate reverse micelles (SLRMs) with vesicular structure were prepared by self-assembling in ethanol–water media and applied to encapsulate horseradish peroxidase (HRP). Results showed that sodium lignosulfonate (SL) could not form SLRMs until the ethanol content reached 63% when its initial concentration was 7.5 g L–1. Owing to strong electrostatic repulsion, solid spherical SLRMs gradually swelled to stable vesicular structures with an average size of 240 nm. The shell of the SLRM thickened when NaCl was added to screen the electrostatic interaction. HRP can be effectively encapsulated while retaining its activity in the hydrophilic core of a SLRM. When hydrogen peroxide was added to initiate the catalytic activity of HRP, SL molecules would be polymerized and the structure of SLRMs would be fixed. Furthermore, HRP immobilized in polymerized SLRMs showed high activity at a more acidic pH of 4 and at a lower optimal temperature decrease of 35 °C compared to free HRP. SLRM allows enzymes su...