Horseradish Peroxidase Molecules Research Articles

Revisiting the catalytic activity of single horseradish peroxidase clusters through electrochemical collision technique: Effect of electrolyte and substrate

Horseradish peroxidase (HRP) is a versatile biosensing label and signal reporter owing to its broad-spectrum catalytic ability. In present work, we characterized HRP's catalytic performance with various substrates using electrochemical collision technique and analyzed the associated electron transfer processes. Different electrolyte solutions greatly affected enzyme dispersibility and zeta potential, thereby impacting HRP collision dynamics in single H2O2 substrate system. The maximum turnover number (kcat) for single HRP molecules was calculated to be 3.611 ± 0.149 × 103 s−1 in 0.85 % NaCl and 2.967 ± 0.286 × 103 s−1 in 0.1 M PBS solution, reflecting differences in cluster size induced by the electrolyte conditions. More severe agglomeration of HRP molecules was observed in double-substrate systems, where the hydrophilic mediator (K4Fe(CN)6) and lipophilic mediator (ABTS) served as electron donors and signal reporters. The calculated kcat value of single HRP molecules in ABTS-H2O2 was 7.6 times higher than that in K4Fe(CN)6–H2O2. This difference is attributed to mediators' solubility, lipophilicity, and HRP's affinity for different substrates, with HRP demonstrated stronger affinity for ABTS-H2O2 substrates, which realized more efficient electron transfer and compensated for the low diffusion coefficient of ABTS. This work provides a comprehensive analysis of the effects of electrolytes and substrates on HRP collision and catalytic behavior, offering valuable insights for the advanced design of HRP-based biosensors and diagnostic platforms.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Droplet-free digital immunoassay based on electrochemiluminescence

Here, we report a droplet-free digital immunoassay for goat immunoglobulin G (IgG) based on electrochemiluminescence (ECL) for simplified digital immunoassay. Digital enzyme-linked immunosorbent assay (ELISA) enabled ultrasensitive detection using digital counting immuno-reaction-induced femtoliter-sized droplets. However, the complicated preparation process of femtoliter-sized droplet assay limits the simplicity of digital ELISA. In this study, droplet-free digital ELISA using ECL was developed with digital counting ECL-induced beads instead of droplets along with a labeling reaction of immuno-reacted beads with ECL luminophore of [Ru(bpy)3]2+ in bulk solution. After antigen–antibody reaction on the beads using poly horseradish peroxidase (HRP) as a label, HRP reaction with [Ru(bpy)3]2+-conjugated tyramide was induced to modify the bead with [Ru(bpy)3]2+. The number of beads emitting ECL was counted on an indium tin oxide (ITO) electrode using tri-n-propylamine as a co-reactant. From result of poly HRP assay, our digital assay system required two poly HRP molecules on the bead to confirm ECL. Finally, a digital immunoassay was performed. The limit of detection for goat IgG was 0.7 pg/mL (4.7 fmol/L). Our simplified digital ELISA was less sensitive than conventional digital ELISA by 10-100-fold, however, we successfully performed digital ELISA based on ECL without complicated procedures for droplet array.

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate.

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4°C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20-30µm as well as below 30nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.

Photoelectrochemical immunoassay for thyroglobulin on nanogold-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation

Methods derived from photoelectrochemical (PEC) have been constructed for immunoassays, but most involve the split-type immunoreaction modes, and thus easily cause unpredictable intermediate precision. Herein, we innovatively designed an integrated PEC immunosensing platform for the quantitative monitoring of thyroglobulin (TG) on the gold nanoparticles (AuNPs)-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation (EBCP). This sensing system could simultaneously implement the immunoreaction and photocurrent measurement. Anti-TG capture antibodies were modified onto AuNPs-decorated BiVO4 photoelectrode. A sandwich-type immunoreaction was carried out in the presence of target TG using horseradish peroxidase (HRP)-conjugated anti-TG detection antibody. The carried HRP molecules catalyzed 4-chloro-1-naphthol (4-CN) to generate an insoluble benzo-4-chlorohexadienone product on the photoanode in the presence of peroxide hydrogen, thereby decreasing the photocurrent. Under optimal conditions, the PEC immunosensors gave good photocurrent responses toward target TG within the dynamic range of 0.01–10 ng mL−1 at a detection limit of 7.6 pg mL−1. Good repeatability and precision, high specificity and acceptable storage stability were acquired during the measurement. No significant differences were encountered for screening 15 human serum specimens between the developed PEC immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) method for the detection of target TG. Significantly, PEC immunosensing system offers promise for simple and cost-effective analysis of disease-related biomarkers.

Activity of AC electrokinetically immobilized horseradish peroxidase.

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500nm diameter and 20nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60fg HRP by observing the enzymatic turnover of Amplex Red and H2 O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.

Thermosonication inactivation of horseradish peroxidase with different frequency modes: Effect on activity, structure, morphology and mechanisms

This study aimed to investigate the effect of thermosonicaion (TS) with different frequency modes on the horseradish peroxidase (HRP) inactivation from the aspects of ultrasound field and enzyme structural changes. The relationships between the activity and structural modification were also evaluated. A thermal-only treatment was performed to estimate the action of ultrasound in TS treatment. Compared to mono-frequency, especially the 33 kHz, TS with dual- and tri-frequency modes showed an intensive inactivation impact on HRP. Exogenous hydroxyl radical test showed the hydroxyl radical played a vital role in HRP inactivation. Changes in hematin group, tertiary and secondary structures were also confirmed, and the inactivation process followed an all-or-none model. Morphological images indicated the aggregation and deformation of HRP molecules during TS treatment, and roughness values were changed accordingly. Pearson correlation analysis implied that the structural destruction on the spatial conformation of HRP was strongly related to its activity.

Effect of high hydrostatic pressure on activity, thermal stability and structure of horseradish peroxidase

The mechanism of the high hydrostatic pressure (HHP) effect on horseradish peroxidase (HRP) is still unclear. The activity, thermal stability and structural changes of HRP after HHP treatments were studied in this work. Compared with the untreated sample, the enzyme activity reduces by 36% after 800 MPa processing. The results indicated that the conformation of the enzyme active center changes under pressure. Furthermore, HHP also changes the conformation of disulfide bonds and some secondary structures in HRP. These structural and conformational changes induce decreased activity. In addition, differential thermal scanning (DSC) results showed that the thermal denaturation temperature decreased from 103.74 °C to 85.78 °C after pressure treatment, suggesting HRP molecules formed large aggregates after pressure treatment. In this study, the interaction mechanism between pressure and enzyme was studied as well, and the results can provide some guidance for the application of HHP technology in fruit and vegetable products processing.

Microwave-assisted rapid conjugation of horseradish peroxidase-dextran aldehyde with Schiff base reaction and decolorization of Reactive Blue 19.

Microwave irradiation has become a routine technique in homogeneous and effective heating in organic synthesis. However, its application in enzyme-containing reactions is limited since it can cause denaturation of the enzyme. In this study, we have briefly investigated the effect of microwave heating on the conjugation reaction of horseradish peroxidase (HRP) with aldehyde derivative of dextran (D-CHO). The reaction was irradiated by microwave at 50 °C for 5 min. The conjugate was confirmed via GPC, in which the conjugates of HRP and D-CHO coexist with free unbound HRP molecules. Activity studies of HRP revealed that there is a small decrease in conjugate activity relative to the free enzyme after a short bioconjugation reaction with microwave irradiation. In decolorization studies of the textile dye Reactive Blue 19 (RB19), 99% of RB19 was decolorized through the free enzyme at 35 °C while the decolorization of the dye was 96% at 25-35 °C by the conjugate, which is a critical result showing clearly that the HRP conjugated via D-CHO is not denatured and still active after microwave-assisted reaction. This phenomenon is due to the multiple point conjugation of D-CHO on the surface of HRP and locking the 3D structure which may prevent changes in the secondary or tertiary structure of the enzyme. The results reveal that microwave irradiation can be used in production of covalently modified enzymes.

Peptide-functionalized metal-organic framework nanocomposite for ultrasensitive detection of secreted protein acidic and rich in cysteine with practical application

In this work, we have prepared peptide-functionalized metal-organic frameworks (MOFs) as signal-amplifying tags for the detection of secreted protein acidic and rich in cysteine (SPARC). Furthermore, enzyme-MOF nanocomposites are fabricated via a coprecipitation strategy between horse radish peroxidase (HRP) and ZIF-90, where ZIF-90 is used as a protective support for HRP immobilization. Meanwhile, the peptide sequence has been designed as SPARC-binding peptide, which imparts biorecognition functionality to HRP@ZIF-90 for performing a colorimetric sensor. Therefore, during the test, HRP molecules can be quickly released from nanocomposites by acidic condition to catalyze chromogenic reaction, enabling the ultrasensitive detection of SPARC with a low detection limit of 30 fg/mL. Moreover, the content of SPARC in colon cancer tissues with different degrees of differentiation can be determined with this sensor, demonstrating that the expression of SPARC is closely related to the occurrence, invasion and metastasis of human colon cancer. These results may show the potential applications of this biosensor in SPARC fundamental research as well as clinical diagnosis in the future.

Signal amplified enzyme-linked immunosorbent assay with gold nanoparticles for sensitive detection of trace furaltadone metabolite

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for detection of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). However, conventional ELISA bears drawback of unsatisfactory sensitivity. Hence, development of high-performance ELISA is of great significance for food safety. We report a signal amplified ELISA strategy for sensitive detection of AMOZ by incorporating gold nanoparticles (AuNPs) into conventional ELISA. In this system, AMOZ competes with bovine serum albumin-AMOZ (BSA-AMOZ) to bind to limited amount of monoclonal antibody; and a large amount of reporter antibody and horseradish peroxidase molecules are loaded onto AuNPs to amplify colorimetric signals. Under the optimized conditions, the signal amplified ELISA achieves a limit of detection of 10−2 ng/mL, which is 500-fold improvement than traditional ELISA in sensitivity. Moreover, pork liver samples spiked with different amount of AMOZ are successfully measured, suggesting this signal amplified ELISA can be used as a general signal enhancement strategy for determination of low-molecular weight contaminants in various foods.

Scanning Electrochemical Potential Microscopy Imaging Horseradish Peroxidase on HOPG

Electrochemical scanning tunneling microscopy (EC-STM) has been successfully applied to study the electron-transfer (ET) reactions of biomolecules at solid-liquid interfaces.1–4 However, there are still limitations, including requirement of sample conductivity and potential damage to the biomolecules from strong tunneling current, to be overcome.A different type of probe microscopy, scanning electrochemical potential microscopy (SECPM), can be used as a promising alternative. SECPM shares the similar hardware setup with EC-STM, except for the current amplifier being substituted by a potentiometer to measure the local electrochemical potential as feedback signal. This feature enables SECPM to work on less conductive biomolecular samples. It has been shown that SECPM is capable of imaging horseradish peroxidase (HRP) at single molecular level under in situ conditions, with image resolution comparable or even better than EC-STM (Figure 1). 5 The electrochemical double layer (EDL) existing at the solid-liquid interface in an electrochemical cell can be investigated by SECPM using tip-distance curves. Preliminary results will be shown.In this work, we use SECPM to image HRP molecules adsorbed on HOPG under in situ conditions, with varying electrochemical potentials applied to the substrate. HRP redox reactions were controlled by varying the substrate potential, and the corresponding change of the potential distribution in the EDL covering the adsorbed HRP surface was recorded by SECPM. The experimental results could offer a novel perspective to understand how the ET reactions influences the EDL, and then help the further research on biosensors and EDL capacitors.This work is supported in part by the North-East Centre for Energy Materials (EP/R021503/1) funded by EPSRC.

Boosting the biocatalytic precipitation with enzyme-loaded liposomes: Toward a general platform for amplified photoelectrochemical immunoassay

Liposome-assisted photoelectrochemical (PEC) bioanalysis represents one of the latest frontiers in the arena of PEC bioanalysis. This work reports a general enzyme-amplified liposomal PEC bioanalysis protocol via the use of enzyme-loaded liposomes to boost the biocatalytic precipitation (BCP) effect. In the representative system, the horseradish peroxidase (HRP)-loaded liposome (HRPLL) and the Au nanoclusters (NCs)/Au nanoparticles (NPs)/TiO2 nanotubes (NTs) framework (AATF) were used as liposomal label and photoelectrode, respectively. In the detection, the sandwich immunocomplex reaction was accomplished in a 96-well plate to confine the HRPLL label, which was then lysed to release the HRP molecules to initiate the BCP process. Due to the amplified formation of HRP-induced BCP on the AATF scaffold, the photo-current response correlated closely with the immunorecognition process and the analyte could be detected very sensitively. This work features the first integration of enzyme-loaded liposomes and the BCP for sensitive PEC bioanalysis, which to our knowledge has not been reported. With the use of various other enzymes, this work could serve as a general basis for the PEC bioanalysis of numerous other target of interest.

Immobilization of horseradish peroxidase on polyglycerol-functionalized magnetic Fe3O4/nanodiamond nanocomposites and its application in phenol biodegradation

In this study, Fe3O4/nanodiamond nanocomposites (MND) were synthesized by polyglycerol-mediated covalent bonding. The horseradish peroxidase (HRP) was successfully immobilized on PG layer of MND by interaction between functional groups of MND and HRP, where the HRP molecules became tridimensionally connected outside the MND. The physicochemical properties of MND were analyzed by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy measurements, vibrating sample magnetometer, Fourier transform infrared spectroscopy and zeta potential. The optimal conditions for the immobilization of HRP by MND are 7 mg/L, 35 °C and 3 h, respectively. Then, the effects of temperature, pH and storage time on the activity of immobilized enzyme and free enzyme were studied. The results showed that the relative activities of immobilized enzymes were higher than free enzymes at different temperatures, pH and storage time. In addition, the reusability experiments of immobilized enzymes showed that after six cycles, the immobilized HRP retained a relative activity of 75%. Applied to the removal of phenol, the effects of phenol concentration, H2O2/phenol molar concentration, immobilized HRP concentration and temperature on phenol removal were investigated. The results displayed that the removal efficiency of phenol reached a maximum when the phenol concentration was 75 mg/L, the ratio of H2O2 to phenol was 1, the immobilized HRP concentration was 0.25 mg/L and the temperature was 30 °C. The above results indicate that the immobilized HRP exhibits high removal efficiency and has great potential for removing phenol.

Enzyme-loaded liposome with biocatalytic precipitation for potentiometric immunoassay of thyroid-stimulating hormone in thyroid carcinoma

A simple and feasible potentiometric immunosensing platform based on enzymatic biocatalytic precipitation technique was designed for the sensitive detection of thyroid-stimulating hormone (TSH; a typical kind of biomarkers for thyroid carcinoma), using horseradish peroxidase (HRP)-loaded liposome for the signal amplification. To construct such an assay system, a sandwich-type immunoreaction was readily carried out on monoclonal anti-TSH capture antibody-coated electrode by using polyclonal anti-TSH secondary antibody-conjugated HRP-loaded liposome. Accompanying the formation of sandwich-type immunocomplex, the carried liposome was lysed through the added Triton X-100 to release the entrapped HRP molecules, which catalyzed the oxidation of 4-chloro-1-naphthol to produce an insoluble and uncharged organic precipitation on the electrode surface, thereby causing the change of the local electrical potential. Two labeling protocols with and without the liposome were investigated for detection of target TSH, improved analytical features were achieved with HRP-entrapped liposome. Under optimal conditions, the potentiometric immunosensor had good responses for TSH detection within the linear range of 0.01–30 μIU/mL at a detection limit of 0.0067 μIU/mL. Good reproducibility, high specificity and long-time stability were acquired during the assay procedure. Importantly, a well-matched accuracy between the potentiometric immunosensor and commercial human TSH ELISA kit was gave for the analysis of human serum samples.

Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization.

The quantification of anammox bacteria is crucial to manipulation and management of anammox biosystems. In this study, we proposed a protocol specifically optimized for quantification of anammox bacteria abundance in anammox sludge samples using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and flow cytometry (FCM) in combination (Flow-CARD-FISH). We optimized the pretreatment procedures for FCM-compatibility, as well as the permeabilization, hybridization and staining protocols of the CARD-FISH. The developed method was compared with other methods for specific bacteria quantification (standard FISH, 16S rRNA sequencing and quantitative polymerase chain reaction). Anammox sludge samples could be disaggregated effectively by sonication (specific energy of 90 kJ·L-1 with MLVSS of 3-5 g·L-1) with the mixed ionic and nonionic dispersants Triton X-100 (5%) and sodium pyrophosphate (10 mM). Lysozyme treatment for permeabilizing bacterial cell walls and H2O2 incubation for completely quenching endogenous peroxidase of anammox sludges were essential to fluorescence enhancement and false positive signals control, respectively. Horseradish peroxidase molecules labeling at 20 °C for 12 h and the fluorescent tyramide labeling at 25 °C for 30 min with a fluorescent substrate concentration of 1:50 maintained the balance between increasing the signal and preventing nonspecific binding. Flow-CARD-FISH results showed that anammox bacteria absolute abundance in two different sludge samples were (2.31 ± 0.01) × 107 and (1.20 ± 0.06) × 107 cells·mL-1, respectively, with the relative abundances of 36.7 ± 4.1% and 26.5 ± 3.7%, respectively, comparable with those of qPCR and 16S rRNA sequencing analysis. The enhanced fluorescence signals induced by CARD-FISH combined with the high quantitative fluorescence sensitivity of FCM provide a rapid and sensitive method that yields accurate quantification results that will be valuable in future studies of microbial community determination.

SPM Imaging of DNA, Redox Proteins and Bacteria Spores Under in-Situ Conditions

Scanning probe microscopy (SPM) can be successfully used in biological investigations1. Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) are two main SPM techniques and they are capable of imaging biological objects at molecular level under in situ conditions. With this advantage, STM and AFM are applied to study not only the topographical structures of bio-samples in their natural environment, but also their electrochemical properties at liquid-solid interfaces. The electrochemical properties of biomolecules are usually studied by integral electrochemical methods, like cyclic voltammetry. The degree of surface coverage, the surface orientation of enzymes and the activity of a single enzyme remain a question of dispute, however, electrochemical SPM techniques can bring clarification. Electrochemical scanning tunnelling microscopy (EC-STM) and electrochemical atomic force microscopy (EC-AFM) provide visual topological information of biomolecules adsorbed on the electrode surface at single-molecular level. Some experimental issues, such as the requirement of the sample being conductive, potential damage to samples caused by the tunnelling current or tip-sample force, may limit their application. Scanning electrochemical potential microscopy (SECPM), a technique that measures the surface potential distribution at zero current, can be a promising alternative. SECPM shares most of the hardware setup with EC-STM except for the current amplifier being substituted by a potentiometer. It is easy to switch between SECPM and EC-STM to compare the topographical profile of the same area by mapping electron density and surface potential2. In our study, SPM techniques are used to study various biological objects, from nanometer-sized DNA and redox proteins to micrometer-sized bacteria spores. Since high-resolution images of DNA origami nanostructures can be achieved by using AFM3, we use both EC-AFM and SECPM techniques to investigate the morphology and potential-dependent adsorption behaviour of DNA scaffold and origamis4 on the modified HOPG surface. We found DNA scaffold can adsorb on HOPG in a certain potential range, and the adsorption structure changes with the applied electrochemical potential. Horseradish peroxidase (HRP), a redox enzyme, was studied using both, EC-STM and SECPM under in situ conditions5. HRP molecules were adsorbed on highly oriented pyrolytic graphite (HOPG) surfaces and imaged in phosphate buffered saline. We found that SECPM shows a better resolution than EC-STM in imaging this biomolecule. SECPM is also capable of measuring the surface charge distribution of the adsorbed biomolecules at the electrochemical interfaces. AFM in liquid was used to investigate the morphology and cellular process of Streptomycetes venezuelae both the wild type and the mutant6. To immobilize the bacteria on the surface for imaging, we use SiC patterned by photolithography with hole arrays (1.5×1.5×0.6 µm) to trap the bacteria and then measure under the physiological condition. The designed hole arrays trap the bacteria without causing any denaturation and due to the high electronic conductivity, they can serve as the working electrode under electrochemical conditions for EC-AFM and SECPM studies. EC-AFM was applied to image the morphology of the bacterial spore under different potential, and we found the morphology of the spore didn’t change in the wide potential window. (1) Choi, E.; Kim, A.; Son, H.; Pyo, S. G. J. Nanosci. Nanotechnol. 2014, 14 (1), 924. (2) Herpich, M.; Friedl, J.; Stimming, U. In Surface Science Tools for Nanomaterials Characterization; Springer Berlin Heidelberg: Berlin, Heidelberg, 2015; pp 1–67. (3) Bald, I.; Keller, A. Molecules 2014, 19 (9), 13803. (4) Kozyra, J.; Ceccarelli, A.; Torelli, E.; Lopiccolo, A.; Gu, J.-Y.; Fellermann, H.; Stimming, U.; Krasnogor, N. ACS Synth. Biol. 2017, 6 (7), 1140. (5) Baier, C.; Stimming, U. Angew. Chem. Int. Ed. Engl. 2009, 48 (30), 5542. (6) Santos-Beneit, F.; Gu, J.-Y.; Stimming, U.; Errington, J. Heliyon. Accepted

Dynamics of Single-Enzyme Activity in a Nanopore Confinement

Enzymatic reactions play a crucial role in controlling and performing a number of biological processes. The ability to directly probe catalysis of a single enzyme can potentially enable a deeper understanding of the enzymatic mechanisms that are not distinguishable in ensemble measurements. Herein we report the direct observation of the electrocatalytic process of a single enzyme molecule in nanopore confinement. Horseradish peroxidase (HRP), a heme-containing enzyme that is widely used in immunoassays and bioassay, was chosen as a model enzyme in our studies. We used nanopore-based techniques to electrochemically capture a single HRP molecule in its confined space, which enable long-term observation of only a single enzyme molecule. Then the bipolar electrochemical reactions catalyzed by HRP enzyme were occurring at the two poles of the polarized gold layer. The enzyme-catalyzed electrochemical reactions resulting in easily distinguished ionic current signals enable us to monitor the catalytic process of single enzyme molecules. Using this system, we investigated the single HRP enzyme catalysis in the confined spaces of nanopore and found the heterogeneous catalytic activity between individual enzymes. This methodology provides a new tool to investigate the single-molecule catalysis of redox enzymes, which will lead to a better understanding of the enzymatic mechanisms and biocatalytic processes.

Reactive bienzyme systems fabricated through immobilization of biotinylated glucose oxidase and peroxidase molecules onto neutralized avidin-conjugated liposomes

Immobilization of multiple enzymes onto solid surface is of significance for efficient consecutive reactions. In this work, the neutralized avidin (NA) molecules were covalently conjugated at pH 7.5 to the liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 20 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 15 mol% N-(succinimidyloxy-glutaryl)-l-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl (POPE-NHS). The apparent number of NA molecules conjugated per liposome, nNA was estimated on the basis of fluorescence quenching of biotin-4-fluorescein (B4F) in the presence of neutralized avidin-conjugated liposomes (NALs). The NALs possessed the nNA value of (4.2±1.8)×102 and the mean hydrodynamic diameter of about 150nm. The biotinylated glucose oxidase and horse radish peroxidase molecules (b-GO and b-HRP) were mixed with NALs at pH 7.5 under the condition that the total concentration of b-GO and b-HRP ([b-GO]=[b-HRP]) is equal to the apparent concentration of NA (500nM) in the NAL suspension. When the immobilization was performed at [Lipid]=0.35mM, a NAL possessed the enzyme activities corresponding to 1.4 free GO and 50 free HRP molecules. The b-GO and b-HRP-immobilized NALs could continuously catalyze the oxidation of glucose with molecular oxygen producing H2O2 which was consumed for the catalytic oxidation of 2,2′-Azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) (ABTS2–). The results obtained demonstrate that the reactive bienzyme system can be fabricated under mild conditions by using the NALs as colloidal scaffolds.

Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles.

A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski's base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Because of the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS2- (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS2- inside the vesicles does not occur. Therefore, using PPD and ABTS2- in separate assays allows distinguishing between vesicle-trapped HRP and HRP in the external bulk solution. In this way, the storage stability of HRP-containing POPC vesicles was investigated in terms of HRP leakage and activity of entrapped HRP. It was found that pH 7.0 suspensions of POPC vesicles (2.2 mM POPC) containing on average about 12 HRP molecules per vesicle are stable for at least 1 month without any significant HRP leakage, if stored at 4 °C. Such high stability is beneficial not only for bioanalytical applications but also for exploring the kinetic properties of vesicle-entrapped HRP through simple spectrophotometric absorption measurements with PPD as a sensitive and cheap substrate.