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.

Highly-sensitive and simple fluorescent aptasensor for 17 b-estradiol detection coupled with HCR-HRP structure.

As an important kind of environmental endocrine disruptors, 17 ß -Estradiol (E2) plays a major role in affecting the growth of human including sexual characters, pregnancy system, etc. In the modern society, with the threat of abuse in breeding, it is imperative to design sensitive methods for detecting low concentration of E2 in environment. In this work, we constructed a highly sensitive and simple fluorescent aptasenor for detecting E2 via amplification of hybridization chain reaction (HCR) and horseradish peroxidase (HRP). Through the competitions between complementary strand (cmDNA) and E2 to E2 aptamer modified on magnetic beads, the unbound cmDNA would be collected and captured by polystyrene microspheres to induce HCR which brought abundant biotin sites. Subsequently, benefit from the excellent catalytic performance of streptavidin-horseradish peroxidase (SA-HRP), the highly sensitive fluorescence signals could be obtained in low concentration of E2. Under the optimal conditions, the prospered method for E2 detection was shown a good liner range from 1 to 100 pg/mL, with the lower detecting limit of 0.2 pg/mL compared with previous work. In addition, the recovery rates tested in the real samples of milk and water were 99.20%-108.06% and 91.07%-106.13%. In all, the assay may provide a perspective way for highly sensitive detection for various contaminants in the real samples.

Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Versatile electrochemical biosensor based on bi-enzyme cascade biocatalysis spatially regulated by DNA architecture.

The regulation of biocatalytic cascades in microenvironments for high performance and extended applications is still challenging. Herein, we develop a rolling circle amplification (RCA)-based one-pot method to prepare the micron-sized DNA flowers (DFs), which achieve the co-encapsulation and spatial regulation of bi-enzyme molecules, glucose oxidase (GOx) and horseradish peroxidase (HRP). In this system, GOx and HRP are integrated into the DFs simultaneously during RCA with the bridging of magnesium between enzyme residues and phosphate backbones on DFs. The cascade of GOx/HRP is regulated with the formation of highly ordered and hydrogen-bonded water environment in the cavity of DFs, resulting in an enhanced cascade catalytic efficiency compared with that in homogeneous solution. Moreover, the high density of DNA scaffold ensures the encapsulation of GOx/HRP with high efficiency. Accordingly, a glucose electrochemical biosensor with amplified signal response is fabricated using the as-prepared GOx/HRP DFs as biosensing interface, realizing sensitive detection of glucose. Further, through designing the complementary sequence of aptamer into the programmable circular template of RCA, the bi-enzyme co-encapsulated DFs are versatilely applied to sensitive and selective detection of cancerous exosomes and thrombin in "signal-on" and "signal-off" modes, respectively, which are further applied to the analysis of complex biological samples successfully. Overall, the encapsulation of multi-enzyme with DFs proposes a promising strategy to regulate the microenvironment of biocatalytic cascades, which hold great potential in biotechnology, bioanalysis and disease diagnosis.

In situ oxidation of canola meal sinapic acid by horseradish peroxidase (type II) and tyrosinase.

The enzymatic oxidation of sinapic acid catalyzed by horseradish peroxidase (HRP) or tyrosinase was investigated using model systems, which contained the pure compound or canola meal. Spectrophotometric scanning of pure sinapic acid solution in the presence of HRP (0.2 U) or tyrosinase (40.3 U) showed continuous decreases in absorbance at 304 nm over a period of 90 and 60 min, respectively. HPLC analyses of enzymatic end products, obtained by the catalysis with HRP or tyrosinase, indicated the presence of two main compounds (1 and 2). After alkaline hydrolysis of canola meal, sinapic acid that was released from sinapine was also converted to compounds 1 and 2 by HRP or tyrosinase. Enzyme reaction kinetics results indicate that the catalytic efficiency (CE = 0.538), reaction velocity (Vmax  = 5.67 ∆A/h), and Michaelis-Menten constant (Km  = 926.64 µM) of HRP are significantly higher than those of tyrosinase (CE = 0.041, Vmax  = 0.41 ∆A/h, Km  = 173.03 µM) at 50-250 µM pure sinapic acid concentrations. PRACTICAL APPLICATIONS: Canola meal contains a large amount of sinapine, which is the choline ester of sinapic acid, a strong antioxidant compound. However, the oxidation or decarboxylation products of sinapic acid could add value by increasing the level of electron-dense carboxylic and carbonyl compounds. In this study, enzymatic treatment of alkaline-hydrolyzed canola meal with horseradish peroxidase (HRP) and tyrosinase was investigated and shown to be suitable for converting sinapic acid into oxidized compounds. Therefore, the enzymatic treatment is a potential application for value-added processing of canola meal.

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP.

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications.

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.

Polymeric Caffeic Acid Is a Safer Mucosal Adjuvant That Augments Antigen-Specific Mucosal and Systemic Immune Responses in Mice.

Infections remain a major threat to human lives. To overcome the threat caused by pathogens, mucosal vaccines are considered a promising strategy. However, no inactivated and/or subunit mucosal vaccine has been approved for human use, largely because of the lack of a safe and effective mucosal adjuvant. Here, we show that enzymatically synthesized polymeric caffeic acid (pCA) can act as a potent mucosal adjuvant in mice. Intranasal administration of ovalbumin (OVA) in combination with pCA resulted in the induction of OVA-specific mucosal IgA and serum IgG, especially IgG1. Importantly, pCA was synthesized from caffeic acid and horseradish peroxidase from coffee beans and horseradish, respectively, which are commonly consumed. Therefore, pCA is believed to be a highly safe material. In fact, administration of pCA did not show distinct toxicity in mice. These data indicate that pCA has merit for use as a mucosal adjuvant for nasal vaccine formulations.

Efficient preparation of surface imprinted magnetic nanoparticles using poly (2-anilinoethanol) as imprinting coating for the selective recognition of glycoprotein.

In view of the significance of glycoprotein biomarkers for early clinical diagnostics and treatments of diseases, it is essential to develop efficient and selective enrichment approaches for glycoproteins. Molecularly imprinted polymers (MIPs) have found important applications for separation and enrichment of glycoproteins. In this study, we use boronate affinity-based controllable oriented surface imprinting to prepare glycoprotein-imprinted magnetic nanoparticles. A glycoprotein was first immobilized onto the surface of boronic acid functionalized magnetic nanoparticles by boronate affinity. Subsequently, self-polymerization of 2-anilinoethanol was carried out to form thin imprinting coating on the magnetic nanoparticles surface with appropriate thickness. After removing the template with an acidic solution containing sodium dodecyl sulfate, 3D cavities complementary to the template were efficiently formed in the imprinting layer. The imprinting coating was highly hydrophilic and presented limited residual boronic acid, thus non-specific binding was avoided. Using horseradish peroxidase as a model target, the effects of imprinting conditions on the properties and performance of the prepared MIPs were investigated. The obtained MIPs exhibited several highly favorable features, including excellent specificity, high binding strength and low binding pH. The MIPs were successfully applied to the analysis of transferrin (TRF) in human serum.

A Graphene Quantum Dots-Enzyme Hybrid System for the Fluorescence Assay of Alkaline Phosphatase Activity and Inhibitor Screening.

A graphene quantum dots (GQDs) and horse radish peroxidase (HRP) hybrid system was designed for the sensing of alkaline phosphatase (ALP) activity and inhibitor screening. We found that the photoluminescence (PL) intensity of GQDs could be quenched efficiently in the presence of phenol, H2O2 and HRP. Moreover, ALP could hydrolyze disodium phenyl phosphate (DPP) to produce phenol, and also could result in the photoluminescence quenching of GQDs. The decrease in the PL intensity was linear to the activity of ALP in the concentration range of 0.02 - 0.8 U/L, with a detection limit of 0.008 U/L. The proposed GQDs/HRP hybrid system was successfully applied to ALP determination in human serum samples. The inhibition study was further analyzed, and Na3VO4 (as an ALP inhibitor) showed a clear inhibition effect. The results suggest that the GQDs/HRP hybrid system has good potential applications for the assay of ALP activity and inhibitors screening in related biochemical fields.

A novel multi-purpose enzymatic system and procedures for the rapid fluorescent determination of flavonoids in herbal pharmaceuticals and plant materials.

The paper presents a novel multi-purpose enzymatic system and procedures for fluorescent determination of several flavonoids in herbal pharmaceuticals and plant materials after their enzyme-catalyzed oxidation by hydrogen peroxide and further derivatization with meso-1,2-diphenylethylenediamine. This system may be used for rapid (15-30min/20 samples) simultaneous screening of samples containing a certain flavonoid in a standard microplate, or as a HPLC detection system for analyzing plant extracts with uncertain composition. In the first case, this indicator system provides sensitive and reproducible microplate determination of quercetin, epicatechin, caffeic acid, and taxifolin in the ranges 0.1-5, 1-10, 0.1-10, 0.5-5µM, respectively. In the second case, quercetin, epicatechin, and caffeic acid can be determined in the ranges 0.05-0.75, 0.05-0.75, and 0.01-0.75µg/ml (0.16-2.5, 0.17-2.6, 0.06-4.2µM), respectively. We have demonstrated the application of the system for the analysis of 3 pharmaceuticals and 3 types of plants.

2,4-Dichlorophenol Enzymatic Removal and Its Kinetic Study Using Horseradish Peroxidase Crosslinked to Nano Spray-Dried Poly(Lactic-Co-Glycolic Acid) Fine Particles.

Horseradish peroxidase (HRP) catalyzes the oxidation of aromatic compounds by hydrogen peroxide via insoluble polymer formation, which can be precipitated from the wastewater. For HRP immobilization, poly(lactic-co-glycolic acid) (PLGA) fine carrier supports were produced by using the Nano Spray Dryer B-90. Immobilized HRP was used to remove the persistent 2,4-dichlorophenol from model wastewater. Both extracted (9-16 U/g) and purified HRP (11-25 U/g) retained their activity to a high extent after crosslinking to the PLGA particles. The immobilized enzyme activity was substantially higher in both the acidic and the alkaline pH regions compared with the free enzyme. Optimally, 98% of the 2,4-dichlorophenol could be eliminated using immobilized HRP due to catalytic removal and partly to adsorption on the carrier supports. Immobilized enzyme kinetics for 2,4-dichlorophenol elimination was studied for the first time, and it could be concluded that competitive product inhibition took place.

Protection of enzymes from photodegradation by entrapment within alumina.

Most enzymes are highly sensitive to UV-light in all of its ranges and their activity can irreversibly drop even after a short time of exposure. Here we report a solution of this problem by using sol-gel matrices as effective protectors against this route of enzyme inactivation and denaturation. The concept presented here utilizes several modes of action: First, the entrapment within the rigid ceramic sol-gel matrix, inhibits denaturation motions, and the hydration shell around the entrapped protein provides extra protection. Second, the matrix itself - alumina in this report - absorbs UV light. And third, sol-gel materials have been shown to be quite universal in their ability to entrap small molecules, and so co-entrapment with well documented sun-screening molecules (2-hydroxybenzophenone, 2,2'-dihydroxybenzophenone, and 2,2'-dihydroxy-4-methoxybenzophenone) is an additional key protective tool. Three different enzymes as models were chosen for the experiments: carbonic anhydrase, acid phosphatase and horseradish peroxidase. All showed greatly enhanced UV (regions UV-A, UV-B, and UV-C) stabilization after entrapment within the doped sol-gel alumina matrices.

Encapsulated Hydrogels by E-beam Lithography and Their Use in Enzyme Cascade Reactions.

Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 µm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/ AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions.

Coil-like Enzymatic Biohybrid Structures Fabricated by Rational Design: Controlling Size and Enzyme Activity over Sequential Nanoparticle Bioconjugation and Filtration Steps.

Well-defined enzymatic biohybrid structures (BHS) composed of avidin, biotinylated poly(propyleneimine) glycodendrimers, and biotinylated horseradish peroxidase were fabricated by a sequential polyassociation reaction to adopt directed enzyme prodrug therapy to protein-glycopolymer BHS for potential biomedical applications. To tailor and gain fundamental insight into pivotal properties such as size and molar mass of these BHS, the dependence on the fabrication sequence was probed and thoroughly investigated by several complementary methods (e.g., UV/vis, DLS, cryoTEM, AF4-LS). Subsequent purification by hollow fiber filtration allowed us to obtain highly pure and well-defined BHS. Overall, by rational design and control of preparation parameters, e.g., fabrication sequence, ligand-receptor stoichiometry, and degree of biotinylation, well-defined BHS with stable and even strongly enhanced enzymatic activities can be achieved. Open coil-like structures of BHS with few branches are available by the sequential bioconjugation approach between synthetic and biological macromolecules possessing similar size dimensions.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

Enzymatic amplification detection of peanut allergen Ara h1 using a stem-loop DNA biosensor modified with a chitosan-mutiwalled carbon nanotube nanocomposite and spongy gold film.

In this paper, a highly sensitive biosensor was constructed for peanut allergen Ara h1 detection. The biosensor was constructed by coating a glassy carbon electrode with a chitosan-mutiwalled carbon nanotube nanocomposite and then adding a spongy gold film via electro-deposition to increase the effective area. The probe switched from an "on" to an "off" state in the presence of target DNA, which detached biotin from the electrode surface. This also detached streptavidin-horseradish peroxidase (HRP-SA), which was bound to the electrode via specific interaction with biotin. The HRP-SA catalyzed chemical oxidation of hydroquinone by H2O2 to form benzoquinone, and when it was detached, electrochemical reduction of the signal of benzoquinone could be used to monitor DNA hybridization via chronoamperometry. Under optimum conditions, a wide dynamic detection range (3.91 × 10(-17)-1.25 × 10(-15) mol L(-1)) and a low detection limit (1.3 × 10(-17) mol L(-1)) were achieved for the complementary sequence. Furthermore, the DNA biosensor exhibited an excellent ability to discriminate between a complementary target and a one-base mismatch or non-complementary sequence. The sensor was successfully applied to Ara h1 analysis in peanuts.

Colorimetric assay for T4 polynucleotide kinase activity based on the horseradish peroxidase-mimicking DNAzyme combined with λ exonuclease cleavage.

T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5'-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked 'HRPzyme' sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL(-1) and a wide detection range from 0.06 to 100 U mL(-1). Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs.

Quantification and evaluation of kinetic bio-catalytic pathway of horseradish peroxidase in an electron mediated reaction system and its applications in plant extracts.

The intermolecular coupling of 2,5-dimethoxyaniline (DMA) as mediated electron transfer reaction in presence of H(2)O(2) and peroxidase in acetate buffer of pH 4.2 resulting green colored product having maximum absorption at λ(max)=740 nm was investigated by spectrophotometer. Under optimum conditions, linearity range for the quantification of H(2)O(2) was 2.0-288.0 µM and for peroxidase were 0.59-9.46 and 0.443-9.46 nM by kinetic and fixed-time method, respectively. The catalytic efficiency and catalytic power were K(eff)(D)=2.354 × 10(5)M(-1)min(-1) and K(pow)(D)=4.59 × 10(-4)min(-1), respectively. From the plot of d(1/D(o)) vs d(1/V(o)) and d(1/H(o)) vs d(1/V(o)), Michaelis-Menten constants for DMA and H(2)O(2)were found that K(m)(D)=1,458 µM and [Formula: see text] =301 µM. Applicability of the method was tested for peroxidase activity in some plant extracts and compared with guaiacol/peroxidase system. Regarding superiority of the method, it is suggested that DMA/peroxidase system can be a better hydrogen donor for HRP assay than guaiacol system as evident from kinetic data.

Nutritional restriction of omega-3 fatty acids alters topographical fine tuning and leads to a delay in the critical period in the rodent visual system.

The development and maturation of sensory systems depends on the correct pattern of connections which occurs during a critical period when axonal elimination and synaptic plasticity are involved in the formation of topographical maps. Among the mechanisms involved in synaptic stabilization, essential fatty acids (EFAs), available only through diet, appear as precursors of signaling molecules involved in modulation of gene expression and neurotransmitter release. Omega-3 fatty acids, such as docosahexaenoic acid (DHA), are considered EFAs and are accumulated in the brain during fetal period and neonatal development. In this study, we demonstrated the effect of omega-3/DHA nutritional restriction in the long-term stabilization of connections in the visual system. Female rats were fed 5 weeks before mating with either a control (soy oil) or a restricted (coconut oil) diet. Litters were fed until postnatal day 13 (PND13), PND28 or PND42 with the same diets when they received an intraocular injection of HRP. Another group received a single retinal lesion at the temporal periphery at PND21. Omega-3 restriction induced an increase in the optical density in the superficial layers of the SC, as a result of axonal sprouting outside the main terminal zones. This effect was observed throughout the SGS, including the ventral and intermediate sub-layers at PND13 and also at PND28 and PND42. The quantification of optical densities strongly suggests a delay in axonal elimination in the omega3(-) groups. The supplementation with fish oil (DHA) was able to completely reverse the abnormal expansion of the retinocollicular projection. The same pattern of expanded terminal fields was also observed in the ipsilateral retinogeniculate pathway. The critical period window was studied in lesion experiments in either control or omega-3/DHA restricted groups. DHA restriction induced an increased sprouting of intact, ipsilateral axons at the deafferented region of the superior colliculus compared to the control group, revealing an abnormal extension of the critical period. Finally, in omega-3 restricted group we observed in the collicular visual layers normal levels of GAP-43 with decreased levels of its phosphorylated form, p-GAP-43, consistent with a reduction in synaptic stabilization. The data indicate, therefore, that chronic dietary restriction of omega-3 results in a reduction in DHA levels which delays axonal elimination and critical period closure, interfering with the maintenance of terminal fields in the visual system.