Peroxidase Substrate Research Articles

Dual responsive magnetic Au@Ni nanostructures loaded reduced graphene oxide sheets for colorimetric detection and photocatalytic degradation of toxic phenolic compounds.

We report the colorimetric detection and photocatalytic degradation of toxic phenolic compounds using Au@Ni loaded reduced graphene oxide (rGO) nanostructures. Core-shell nanoparticles of Au and Ni are successfully designed on rGO with size <8 nm by a solvothermal route which demonstrate excellent enzyme mimic behaviour towards the oxidation of 3,3',5,5' tetramethylbenzidine (TMB), a peroxidase substrate and towards colorimetric detection of phenols with detection limit as low as 1.68 μM, wide detection range of 1-300 μM and admirable selectivity. Additionally, the Au@Ni/rGO nanocomposite exhibits excellent photo responsive behaviour towards degradation of phenol, 2-chlorophenol (2-CP) and 2-nitrophenol (2-NP) under natural sunlight irradiation with more than 87% degradation. The superiority of the bimetallic nanocomposite is established by comparing its activity to its monometallic counterparts. The sustainability of the nanocomposite is assessed through the reusability in the photocatalytic reaction upto six consecutive cycles without significant loss in activity. This is the first study where nanomaterials are used for both detection and degradation of environmental pollutants with positive and encouraging results.

An efficient thin-walled Pd/polypyrrole hybrid nanotube biocatalyst for sensitive detection of ascorbic acid

Controllable fabrication of novel and uniform noble metal nanoparticles on a specific support with a superior catalytic or electrocatalytic performance is of significantly importance for practical applications. In this report, we demonstrated an effective way to fabricate uniform thin-walled Pd/polypyrrole (PPy) hollow nanotubes. The prepared Pd/PPy hybrid nanotubes exhibited an excellent peroxidase-like activity to oxidize a typical peroxidase substrate such as 3,3′,5,5′-tetramethylbenzidine in comparison with traditional Pd/C and Pd black catalysts. The outstanding catalytic activity of the Pd/PPy hybrid nanotubes for peroxidase mimicking could be resulting from their unique hollow characteristic and an interfacial effect between PPy and Pd components. Based on the favorable catalytic property of the Pd/PPy hybrid nanotubes, a convenient and rapid colorimetric way to sensitively determine ascorbic acid has been presented. The detection limit was around 0.062 μM and an excellent selectivity was also achieved. The developed detection system in this study could be extended to the fields of bioscience and biotechnology with promising prospects.

Green Synthesis of Fluorescent Carbon Quantum Dots from Azadirachta indica Leaves and Their Peroxidase-Mimetic Activity for the Detection of H2O2 and Ascorbic Acid in Common Fresh Fruits.

In the present study, an ecofriendly and zero-cost approach has been demonstrated for the preparation of carbon quantum dots by one-pot hydrothermal treatment of leaf extracts of neem (Azadirachta indica). The as-synthesized neem carbon quantum dots (N-CQDs) exhibited high fluorescent quantum yields (QYs) up to 27.2%. Moreover, N-CQDs also act with a peroxidase-like-mimetic activity toward the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in association with hydrogen peroxide (H2O2). Further, the kinetics of peroxidase-like catalytic activity follows the Michaelis-Menten and ping-pong pathway. In addition, the H2O2 sensitive TMB oxidation motivated the colorimetric detection of H2O2 which showed linearity from 0.1 to 0.5 mmol/L with a detection limit (LOD) of 0.035 mmol/L. Furthermore, the blue colors of oxidized TMB (ox-TMB) were selectively reduced in native TMB with ascorbic acid (AA) without any interference of other reducing agents. The linear range of AA detection was lying between 5 and 40 μM with a LOD up to 1.773 μM. The practicability assay of the proposed sensing system toward the detection of AA was also investigated in real sample analysis such as common fruits which showed good sensitivity to the presence of AA. Therefore, this convenient, ecofriendly, and cost-effective peroxidase-based sensing system opens a new platform for analysis of AA in real samples and in complex biological systems.

A Novel Method for the Quantification of White Wine Mannoproteins by a Competitive Indirect Enzyme-Linked Lectin Sorbent Assay (CI-ELLSA).

Mannoproteins (MPs) are cell wall proteoglycans released in wine by yeast during fermentation and ageing on lees, a procedure used for the production of several wines to enrich them in these components with consequences from both a technological and sensory point of view. Given the significance that wine MPs have for wine quality, winemakers would welcome a simple and accurate method for their quantification, as this would allow them to have a better control of this aspect at different winemaking stages. This study develops and validates a novel, simple and accurate method for MPs quantification in white wines based on a competitive indirect enzyme-linked lectin sorbent assay (CI-ELLSA), using the highly mannosylated yeast invertase as the standard. The method utilizes the lectin concanavalin A (ConA) as the immobilized ligand for MPs, and peroxidase, an enzyme rich in mannose, as the competitor for ConA. After addition of the peroxidase substrate, the intensity of the signal produced by the activity of this enzyme (absorbance at 450 nm) is inversely proportional to the amount of mannosylated proteins in the sample. Results have been validated on several wine styles including still, sparkling and sweet wines.

A novel biomimetic nanoenzyme based on ferrocene derivative polymer NPs coated with polydopamine

In this paper, ferrocene derivative polymer nanoparticles (FcP NPs) with uniform size and good photostability was synthesized using 1,1′-ferrocene dicarboxylic acid as precursor and methanol as solvent. FcP NPs-PDA was further obtained by coating of polydopamine (PDA) on FcP NPs in tris-HCl (pH=8.5) buffer solution at room temperature in the presence of dopamine (DA). The structure and morphology of FcP NPs and FcP NPs-PDA were characterized by transmission electron microscope (TEM), ultraviolet-visible spectroscopy (UV–Vis), and infrared radiation (FT-IR). The as-prepared FcP NPs-PDA showed better peroxidase-like activity than FcP NPs, which could catalyze the chromogenic reaction of peroxidase substrate TMB, OPD and ABTS. Based on the high peroxidase-like property of FcP NPs-PDA, a sensitive and convenient means to detect H2O2 has been proposed with TMB as the substrate, which displays wide linear range of 10–600 μM and 600 μM-4 mM with low detection limit of 5 μΜ. Compared with other Fe-containing NPs, such as magnetic materials and noble metal@Fe bimetallic NPs, the preparation approach for FcP NPs-PDA is simple, time and energy saving and environment friendly. This mild and simple synthesis route of FcP NPs-PDA will provide new ideas for the preparation of non-noble metal-based peroxidase-like nanomaterials and widen the applications in the fields of catalytic and analytical chemistry.

Ni-hemin metal\u2013organic framework with highly efficient peroxidase catalytic activity: toward colorimetric cancer cell detection and targeted therapeutics

BackgroundGiven the great benefits of artificial enzymes, a simple approach is proposed via assembling of Ni2+ with hemin for synthesis of Ni-hemin metal–organic-frameworks (Ni-hemin MOFs) mimic enzyme. The formation of the Ni-hemin MOFs was verified by scanning electron microscopy, Transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Energy-dispersive X-ray spectroscopy and UV–vis absorption spectroscopy. This novel nanocomposite exhibited surprising peroxidase like activity monitored by catalytic oxidation of a typical peroxidase substrate, 3,3,5,5′-tetramethylbenzidine, in the presence of H2O2. By using folic acid conjugated MOF nanocomposite as a recognition element, we develop a colorimetric assay for the direct detection of cancer cells.ResultsThe proposed sensor presented high sensitivity and selectivity for the detection of human breast cancer cells (MCF-7) and Human Caucasian gastric adenocarcinoma. By measuring UV–vis absorbance response, a wide detection range from 50 to 105 cells/mL with a detection limit as low as 10 cells/mLwas reached for MCF-7 cells. We further discuss therapeutics efficiency of Ni-hemin MOFs in the presence of H2O2 and ascorbic acid. Peroxidase-mimic Ni-hemin MOFs as reactive oxygen species which could damage MCF-7 cancer cells, however for normal cells (human embryonic kidney HEK 293 cells) killing effect was negligible.ConclusionsBased on these behaviors, the developed method offers a fast, easy and cheap assay for the interest in future diagnostic and treatment application.

Biosynthesized Quantum Dot Size Cu Nanocatalyst: Peroxidase Mimetic and Aqueous Phase Conversion of Fructose

AbstractEnvironmentally benign, stable quantum dot size spherical Cu particles with an average size of ∼ 4.5 nm, as measured via transmission electron microscope, were coherently tailored exploiting renewable ethno‐pharmacological Oxalis corniculata plant extract as both reducing plus capping agent. The reliable green aqueous synthesis approach completely excluded the usage of inert atmosphere and harmful chemicals including organic solvents. The ensuing cost effective Cu nanoparticles exhibited excellent intrinsic peroxidase like activity and superior nanozyme behaviour compare to earlier reports with Km value of 0.01426 M and rmax equal to 0.1399 Mmin−1 toward the oxidation of peroxidase substrate H2O2 in presence of o‐phenylenediamine, a paradigm reaction. Significantly, the as‐prepared Cu particles have been proven to be a novel catalyst for feasible selective aqueous phase conversion of fructose to levulinic acid with a maximum yield of ∼ 22.38% at 90°C in the absenteeism of prominent side product(s), auxiliaries and high temperature reaction condition. Lewis acid property and large surface area of the generated nanoscale particles attributed to their excellent catalytic potential introducing a new avenue in the continuous demand for better sensing, biomedical applications and cleaner energy production.

Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.

Beyond the Roles in Biomimetic Chemistry: An Insight into the Intrinsic Catalytic Activity of an Enzyme for Tumor-Selective Phototheranostics.

Protein-assisted biomimetic synthesis has been an emerging offshoot of nanofabrication in recent years owing to its features of green chemistry, facile process, and ease of multi-integration. As a result, many proteins have been used for biomimetic synthesis of varying kinds of nanostructures. Although the efforts on exploring new proteins and investigating their roles in biomimetic chemistry are increasing, the most essential intrinsic properties of proteins are largely neglected. Herein we report a frequently used enzyme (horseradish peroxidase, HRP) to demonstrate the possibility of enzymatic activity retaining after accomplishing the roles in biomimetic synthesis of ultrasmall gadolinium (Gd) nanodots and stowing its substrate 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), denoted as Gd@HRPABTS. It was found that ca. 70% of the enzymatic activity of HRP was preserved. The associated changes of protein structure with chemical treatments were studied by spectroscopic analysis. Leveraging on the highly retained catalytic activity, Gd@HRPABTS exerts strong catalytic oxidation of peroxidase substrate ABTS into photoactive counterparts in the presence of intrinsic H2O2 inside the tumor, therefore enabling tumor-selective catalytic photoacoustic (PA) imaging and photothermal therapy (PTT). In addition, the MR moiety of Gd@HRPABTS provides guidance for PTT and further diagrams that Gd@HRPABTS is clearable from the body via kidneys. Preliminary toxicity studies show no observed adverse effects by administration of them. This study demonstrates beyond the well-known roles in biomimetic chemistry that HRP can also preserve its enzymatic activity for tumor catalytic theranostics.

Dual Responsive Enzyme Mimicking of Ternary Polyaniline–MnO2–Pd Nanowires and Its Application in Colorimetric Biosensing

In recent years, a significant challenge in material science has been to fabricate novel functional nanomaterials to mimic the function of natural enzymatic systems. Herein, ternary polyaniline (PANi)–MnO2–Pd nanowires (NWs) are synthesized through a two-step redox reaction process. The prepared PANi–MnO2–Pd NWs were explored to exhibit dual responsive enzyme-like efficiencies, e.g., peroxidase and oxidase-like activities. With the assistance of H2O2, the obtained ternary PANi–MnO2–Pd NWs were able to oxidize the typical peroxidase substrate of 3,3′,5,5′-tetramethylbenzidine (TMB) to yield the oxidized TMB with a blue color, exhibiting a much better peroxidase-like activity than the synthesized PANi–MnO2 NWs alone and Pd nanoparticles owing to the interfacial electron transfer effect among the components. In addition, in the absence of H2O2, the prepared PANi–MnO2–Pd NWs could also oxidize TMB to display an excellent oxidase-like activity. Based on the dual responsive enzyme-like characteristic of the obt...

Sensitive biosensing of organophosphate pesticides using enzyme mimics of magnetic ZIF-8.

Development of a sensitive detection method for the reliable screening of widely used organophosphorus (OP) toxins is a crucial request to control their side-effects. Herein, a novel fluorometric assay based on the acetylcholinesterase (AChE) inhibited enzymatic activity and the new peroxidase-like Fe3O4 nanoparticles@ZIF-8 composite (Fe3O4 NPs@ZIF-8) was developed for the determination of OPs. Magnetic Fe3O4 NPs were encapsulated into ZIF-8 and the high mimetic activity of produced composite was assessed on the oxidation of substrates. This observation was applied to the rapid detection of diazinon as a model OP compound. The sensing tool contains AChE and choline oxidase (CHO) enzymes, peroxidase colorimetric or fluorometric substrate, and Fe3O4 NPs@ZIF-8 as the catalyst. In the presence of mimic Fe3O4 NPs@ZIF-8, the generated H2O2 from the enzymatic reactions of acetylcholine is decomposed to hydroxyl radicals. The radicals oxidize the peroxidase substrates to generate a detectable signal. However, due to the inhibition effect of OPs on the enzymatic activity of AChE, lower H2O2 amounts are produced in the presence of diazinon. Using the fluorometric detection system, the generated signal is decreased proportionally by increasing diazinon concentration in the range of 0.5-500 nM. The limit of detection was obtained 0.2 nM. Consequently, the usage of high performance peroxidase-mimic Fe3O4 NPs@ZIF-8 provided a sensitive bio-assay with a potential to be applied as screening tool for toxic OP compounds. The developed assay was successfully applied for the determination of diazinon in water and fruit juices.

Magnetite Fe3O4 Has no Intrinsic Peroxidase Activity, and Is Probably not Involved in Alzheimer's Oxidative Stress

AbstractDespite stated in some highly cited articles, magnetite is devoided of peroxidase activity. In fact, this very stable mixed valence FeIIO⋅FeIII2O3 complex is not catalytically competent to oxidize standard peroxidase substrates, especially at the biologically relevant pH value of 7.4. In addition, magnetite whose deleterious redox activity has been suspected in Alzheimer's disease brain damages, does not significantly interact with amyloid peptide Aβ in vitro, and is not able to induce, either in the presence or absence of Aβ, the reductive activation of dioxygen, the first step of an oxidative stress. In fact, this highly insoluble mineral iron derivative is probably not involved in the oxidative damage of brain neurons of patients with AD.

Magnetite Fe3 O4 Has no Intrinsic Peroxidase Activity, and Is Probably not Involved in Alzheimer's Oxidative Stress.

Despite stated in some highly cited articles, magnetite is devoided of peroxidase activity. In fact, this very stable mixed valence FeII O⋅FeIII2 O3 complex is not catalytically competent to oxidize standard peroxidase substrates, especially at the biologically relevant pH value of 7.4. In addition, magnetite whose deleterious redox activity has been suspected in Alzheimer's disease brain damages, does not significantly interact with amyloid peptide Aβ in vitro, and is not able to induce, either in the presence or absence of Aβ, the reductive activation of dioxygen, the first step of an oxidative stress. In fact, this highly insoluble mineral iron derivative is probably not involved in the oxidative damage of brain neurons of patients with AD.

Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control.

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.

Hybrid Hydrogels Based on insitu Interpenetrating Networks Graphene Oxide (GO) and Au Nanoparticles, and Its Application as Peroxidase Mimetics for Glucose Detection

AbstractGraphene oxide (GO) and Au Nanoparticles (NPs) hybrid hydrogels were prepared through one‐step hydrothermal method. The microstructure of GO/AuNPs hydrogels determined by SEM demonstrated that the hydrogels displayed three‐dimensional (3D) network structure. The freeze‐dried GO/AuNPs hydrogels (xerogels), i. e., GO/Au nanocomposites, could efficiently catalyze the oxidation reaction of peroxidase substrate, 3,3′,5,5′‐tetramethylbenzidine (TMB), in the presence of H2O2 to produce a blue color reaction. Compared with the natural enzyme, horse radish peroxidase (HRP), GO/Au nanocomposites exhibited higher peroxidase‐like activity. The reason might be that the porous 3D network structure of GO/Au nanocomposite enhanced the reaction activity. The catalytic reaction mechanism of GO/Au nanocomposite was followed the Michaelis‐Menten behavior. The combination of GO/Au nanocomposite with glucose oxidase (GOx) performed a selective colorimetric assay for glucose detection with a detection limit of 0.4728 μmol⋅L−1, which can be used as a promising candidate biosensor for glucose detection.

Determination of thiocyanate in exhaled breath condensate

Thiocyanate is a heme peroxidase substrate that scavenges oxidants produced during inflammation and regulates host defense. In cystic fibrosis (CF) patients, increased airway thiocyanate levels are associated with improved lung function. Research on airway thiocyanate is limited, however, because convenient non-invasive airway sampling methods, such as exhaled breath condensate (EBC), yield low concentrations that are difficult to detect with available assays. In the present study, we developed a method for the determination of thiocyanate in dilute samples using isotope dilution headspace gas chromatography-coupled high-resolution, accurate-mass mass spectrometry (GC-HRMS). The method reliably quantified as little as 4 pmol thiocyanate in EBC and could detect even lower amounts. We successfully measured thiocyanate in EBC from seven healthy donors, with a mean ± SD of 27 ± 16 nM and a median inter-assay coefficient of variation of 10.4% over six months. The method was applied to other biological fluids (plasma from the same visit as EBC donation; bronchoalveolar lavage fluid [BALF] from infants with CF; and healthy adult mouse BALF), giving reliable quantification of samples ranging from 10 nM to 100 µM. Thiocyanate concentrations in fluids besides EBC were (from lowest to highest): 0.73 ± 0.39 µM in BALF of healthy adult mice (n = 6); 1.4 ± 1.4 µM in BALF from infants with CF (n = 24); 46 ± 22 µM in the plasma of adult volunteers (n = 7). These results demonstrate the utility of this new method for clinical determination of thiocyanate in EBC and other biological fluids.

A 3-dimensional C/CeO2 hollow nanostructure framework as a peroxidase mimetic, and its application to the colorimetric determination of hydrogen peroxide.

Various3-dimensional C/CeO2 hollow nanostructure frameworks (3D C/CeO2 HNFs) were synthesized by using a polymer blowing process that is accelerated by adding a certain amount of cerium nitrate. Polyvinylpyrrolidone was used as the polymer. The resulting HNFs were characterized by scanning electron microscopy, energy dispersive spectrometry, X-ray diffraction, and X-ray photoelectron spectroscopy. The HNFs possess a large specific surface area, and the CeO2 nanocrystals consist of a single phase. The HNFs display intrinsic peroxidase-like activity and can catalyze the oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine in the presence of H2O2 to produce a blue product. The method was applied to the quantification of H2O2 with a 5.2 nM detection limit. The analytical range is from 10nM to 1μM. Graphical abstract Schematic of the preparation of a3-dimensional C/CeO2 hollow nanostructure framework by a polyvinylpyrrolidone-blowing process accelerated by Ce(NO3)3. They were applied to H2O2 detection by catalyzing the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) to produce blue-color reaction.

Graphene oxide-gold nanozyme for highly sensitive electrochemical detection of hydrogen peroxide

We fabricated a nafion/graphene oxide-gold nanoparticle (GO-AuNP) hybrid modified indium tin oxide (ITO) electrode and proposed an electrochemical method to detect hydrogen peroxide (H2O2) using 3,3,5,5,-tetramethylbenzidine (TMB) as a redox mediator. The GO-AuNP hybrids were employed as nanozymes, which function as peroxidase mimics and show highly effective catalytic activity. Based on the high catalytic activity, enzyme mimics were entrapped on the ITO electrode to construct an electrochemical H2O2 sensor by coating nafion polymer. During the catalytic reaction, the peroxidase substrate TMB was oxidized to form the TMB oxidation product, which not only produces a blue color detected by absorbance change, but also generates an electrochemical current. As a result, both spectrophotometric and electrochemical methods were used to determine H2O2 concentration. The spectrophotometric detection displayed a linearity for H2O2 concentration from 10 μM to 5 mM (r2 = 0.989), with an estimated detection limit of 2 μM. In the electrochemical detection, the TMB peak current had a good linear relationship with H2O2 concentration from 10 nM to 10 mM, with an estimated detection limit of 1.9 nM, which was much lower than that of the spectrophotometric method result.

Enzyme structure and function protection from gastrointestinal degradation using enteric coatings

Bovine Serum Albumin (BSA) and Horseradish Peroxidase (HRP) have been encapsulated within microparticulated matrices composed of Eudragit RS100 by the water-in-oil-in-water double emulsion solvent evaporation method. Good encapsulation efficiencies were achieved for BSA and HRP, 88.4 and 95.8%, respectively. The stability of the loaded proteins was confirmed by using circular dichroism and fluorescence. The gastroresistance of the protein-loaded microparticles was evaluated under simulated gastric conditions demonstrating the preservation of the structural integrity of the proteins loaded inside the particles. The enzymatic activity of HRP after being released from the enteric microparticles was evaluated by using the peroxidase substrate, revealing that the released enzyme preserved its 100% function. The high drug loadings achieved, reduced cytotoxicity and efficient gastric protection point out towards the potential use of those carriers as oral delivery vectors of therapeutic proteins offering a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption.

One-step three-dimensional printing of enzyme/substrate–incorporated devices for glucose testing

To substantially simplify the fabrication of analytical devices for rapid screening tests, in this study we employed multi-material fused deposition modeling-type three-dimensional printing (3DP) and two functionalized thermoplastic filaments—acrylonitrile butadiene styrene (ABS) incorporating peroxidase-mimicking iron oxide (Fe3O4) nanoparticles and polyvinyl alcohol (PVA) infiltrated with the chromogenic substrate o-phenylenediamine (OPD)—for the one-step manufacture of enzyme/substrate–incorporated multi-well plates. Upon contact with samples, these fabricated devices (i) released the chromogenic substrate OPD into the solution, (ii) efficiently catalyzed the oxidation of OPD mediated by the peroxidase substrate H2O2, (iii) enabled assays of those substances availably oxidized by their specific oxidases to generate H2O2, and (iv) facilitated colorimetric observation by the naked eye or through absorbance measurements after loading into a microplate reader. With glucose oxidase immobilized in each well, samples appropriately diluted could be directly loaded for derivatizing and analyzing glucose without adding any other reagents. After assay optimization, the limits of detection reached as low as 2.8 μM for H2O2 and 5.0 μM for glucose; the method's applicability was illustrated in terms of determining glucose concentrations in urine, serum, and plasma samples. These 3D-printed peroxidase mimic/chromogenic substrate–incorporated multi-well plates appear to be highly suitable for rapid and high-throughput screening of glucose in clinical samples. We demonstrate that adequate functionalization of raw materials for 3DP can contribute to the development of novel multifunctional devices with many potential practical applications.