Graphene quantum dots on TiO2 nanotubes as a light-assisted peroxidase nanozyme.

Hybrid nanozyme graphene quantum dots (GQDs) deposited TiO2 nanotubes (NTs) on titanium foil (Ti/TiO2 NTs-GQDs) were manufactured by bestowing the hybrid with the advantageous porous morphology, surface valence states, high surface area, and copious active sites. The peroxidase-like activity was investigated through the catalytic oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, which can be visualized by the eyes. TiO2 NTs and GQDs comprising oxygen-containing functional groups can oxidize TMB in the presence of H2O2 by mimicking peroxidase enzymes. The peroxidase-mimicking activity of hybrid nanozyme was significantly escalated by introducing light illumination due to the photosensitive features of the hybrid material. The peroxidase-like activity of Ti/TiO2 NTs-GQDs enabled H2O2 determination over the linear range of 7 to 250 µM, with a LOD of 2.1 µM. The satisfying peroxidase activity is possibly due to the unimpeded access of H2O2 to the catalyst's active sites. The porous morphology provides the easy channeling of reactants and products. The periodic structure of the material also gave rise to acceptable reproducibility. Without material functionalization, the Ti/TiO2 NTs-GQDs can be a promising substitute for peroxidases for H2O2 detection.

Photosystem II as a chemiluminescence-induced photosensitizer for photoelectrochemical biofuel cell-type biosensing system.

Herein, photosystem II (PSII), extracted from spinach, is used for the first time as an efficient and green sensitizer for a photobioanode in a photoelectrochemical glucose biofuel cell (PBFC) setup. The concept is based on the formation of hemin-catalyzed luminol chemiluminescence (CL) after the enzymatic oxidation of glucose and the simultaneous production of hydrogen peroxide by glucose oxidase. The photosynthetic enzyme PSII, combined with an osmium polymer serving as mediator and photosensitizer, is immobilized and wired on microporous carbonaceous material (MC) for the chemiluminescence-induced oxidation of water to O2 at the photobioanode (GCE|MC|Os polymer|PSII). Also, bilirubin oxidase immobilized on multiwalled carbon nanotubes (MWCNTs) coated electrode (GCE|MWCNT|BOx) serves as a biocathode. The photoelectrochemical biofuel cell (PBFC) is applied to a biosensor model system to validate the appropriateness of such a bioanode operating in a self-powered mode. Os redox polymer attached to MCs provides abundant PSII immobilization and a reliable electron transfer pathway. The well-matching energy levels of photosensitive entities reduce recombination phenomena while MC enhances the charge collection. Substantial photocatalytic water oxidation was observed under CL due to the well-matched CL emission and PSII absorption. The electrode is rationally designed to gain the maximum luminol CL power for the photobioanode. The open circuit potential of PBFC linearly increased with the CL power intensity and, in turn, glucose concentrations in the range of 0-6 mmol L-1. The PBFC yielded an OCP of 0.531 V in 30 mmolL-1 glucose. The study may open a new horizon to the green and pioneering PEC biosensing realm.

Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode.

Herein, we report a membraneless glucose and air photoelectrochemical biofuel cell (PBFC) with a visible light assisted photobioanode. Flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH) was immobilized on the combined photobioanode for the visible light assisted glucose oxidation (GCE|MWCNT|g-C3N4|Ru-complex|FADGDH) with a quinone mediated electron transfer. Bilirubine oxidase (BOx) immobilized on MWCNT coated GCE (GCE|BOx) was used as the cathode with direct electron transfer (DET). An improvement of biocatalytic oxidation current was observed by 6.2% due in part to the light-driven electron-transfer. The large oxidation currents are probably owing to the good contacting of the immobilized enzymes with the electrode material and the utilization of light assisted process. Under the visible light, the photobioanode shows an anodic photocurrent of 1.95 µA cm2 at attractively low potentials viz. -0.4 vs Ag/AgCl. The lower-lying conduction band of g-C3N4 as compared to Ru-complexes decreases the rate of hole and electron recombination and enhances the charge transportation. The bioanode shows maximum current density for glucose oxidation up to 6.78 µA cm-2 at 0.2 V vs Ag/AgCl at pH:7. The performance of three promising Ru-complexes differing in chemical and redox properties were compared as electron mediators for FADGDH. Upon illumination, the PBFC delivered a maximum power density of 28.5 ± 0.10 µW cm-2 at a cell voltage of +0.4 V with an open circuit voltage of 0.64 V.

A self-powered photoelectrochemical biosensor for H2O2, and xanthine oxidase activity based on enhanced chemiluminescence resonance energy transfer through slow light effect in inverse opal TiO2.

TiO2 inverse opal photonic crystals (IOPCs) were fabricated by using polystyrene template. TiO2 IOPCs based photoelectrochemical (PEC) biosensor was fabricated for the precise and stable detection of Heme without external irradiation. Then, the sensitization of TiO2 IOPCs was fulfilled with CdS quantum dots (QDs) by SILAR method to form ITO-TiO2 IOPCs-CdS:Mn electrode, which in turn was used to construct a PEC biosensor. The uniform porous structure of IOPCs with a large surface area is conducive to the excellent electronic transmission and QDs deposition. Also, the energy level matching between the conduction bands of CdS QDs and TiO2 IOPCs widened the range of light absorption, allowing for electron injection from excited CdS QDs to TiO2 upon luminol chemiluminescence, which enhanced the photocurrent. Furthermore, when the red edge of the photonic stop band of TiO2 IOPCs overlapped with the band gap of TiO2, and chemiluminescence emission of luminol, a substantial photocurrent increment was observed due in part to the slow light effect. The biosensor possesses a large linear detection range of 0.063-4 mM with a LOD of 19 µM for H2O2. Also, xanthine oxidase activity was determined with a linear measurement range of 0.01-15 mU/mL. Our strategy opens a new horizon to IOPCs based and QDs sensitized PEC sensing, which could be more sensitive, convenient and inexpensive for clinical and biological analysis. As far as we know, the largest photocurrent generation by luminol chemiluminescence was observed thanks to the use of semiconducting hybrid IOPCs material even at 0 V.

Novel hydroxyapatite/graphene oxide/collagen bioactive composite coating on Ti16Nb alloys by electrodeposition.

A novel implant coating material containing graphene oxide (GO) and collagen (COL), and hydroxyapatite (HA) was fabricated with the aid of tannic acid by electrodeposition. The surface of Ti16Nb alloy was subjected to anodic oxidation, and then HA-GO coating was applied to Ti16Nb surface by cathodic method. Then, COL was deposited on the surface of the HA-GO coating by the biomimetic method. HA, HA-GO, HA-GO-COL coatings on the surface of the Ti16Nb alloy have increased the corrosion resistance by the formation of a barrier layer on the surface. For HA-GO-COL coating, the highest corrosion resistance is obtained due to the compactness and homogeneity of the coating structure. The contact angle of the bare Ti16Nb is approximately 65°, while the contact angle of the coated samples is close to 0°. Herein, the increased surface wettability is important for cell adhesion. The surface roughness of the uncoated Ti16Nb alloy was between 1 and 3 µm, while the surface roughness of the coated surfaces was measured between 20 and 110 µm. The contact between the bone and the implant has been improved. Graphene oxide-containing coatings have improved the antibacterial properties compared to the GO-free coating using S. aureus. The hardness and elastic modulus of the coatings were measured by the nanoindentation test, and the addition of GO and collagen to the HA coating resulted in an increase in strength. The addition of GO to the HA coating reduced the viability of 3 T3 fibroblast cells, whereas the addition of collagen to HA-GO coat increased the cell adhesion and viability.

A self-powered photoelectrochemical glucose biosensor based on supercapacitor Co3O4-CNT hybrid on TiO2.

In this study, a photoelectrochemical (PEC) biosensor was constructed by depositing supercapacitor carbon nanotubes (CNT) and Co3O4 onto the anatase TiO2 coated ITO electrodes. Herein, supercapacitor Co3O4 was employed as a semiconductor with a band gap of ~2.07 eV, and the supercapacitor behavior of the PEC system was improved by introducing CNT into the electrode material. Furthermore, a self-empowering glucose biosensor operating at 0 V was constructed for the first time. Also, upon the formation of p-n junction, Co3O4 was rendered electron accepting material, unlike its usual use in photocatalytic systems. Co3O4-CNT-anatase TiO2 semiconductor hybrid was used to reduce recombination of exited electrons, and increasing the visible light absorption. Prior to enzyme immobilization, CNT containing electrode material was modified with 1-pyrene boronic acid via π-π interactions. The enzyme immobilization was carried out through covalent esterification between the boronic acid moiety and the carbohydrate part of GOx. Enzyme immobilization way enabled the close contact between FAD and electrode material, and the electron donor FADH2 forming after the enzymatic reaction can give electrons to the photogenerated holes of Co3O4 through CNT along with H2O2 by enhancing the photocurrent. The obtained PEC biosensor demonstrated acceptable reproducibility and decent stability with a linear measurement range of 0-4 mM, a sensitivity of 0.3 µA mM-1 cm-2, and lower detection limit of 0.16 µM. Thus, a self-powered biosensor was constructed by combining the PEC, and supercapacitor behavior of Co3O4 for the first time, and the utilization of the present PEC material can be extended to the other analytes detection through photoelectrochemistry. The supercapacitor materials led to the high current at direct electron transfer potential range, and this phenomenon implies that the PEC electrode can also be used in biofuel cells to obtain high power.

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid-based nanofiber membrane.

In this study, polyacrylic acid-based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid-based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid-based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn2+, Ni2+, Cu2+, Zn2+, Mg2+, Ca2+ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis-Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis-Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE-based biosensors.

Efficient protein digestion using immobilized trypsin onto tannin modified Fe3O4 magnetic nanoparticles.

Fe3O4 magnetic nanoparticles (MNPs) were prepared via solvothermal method. A commercially available trypsin was covalently immobilized onto MNPs modified with tannin (T) via a novel binding process. The morphology, structure, surface and magnetic properties of the obtained nanostructures were characterized comprehensively. The Fe3O4 MNPs had a saturation magnetization value of 60.18emu/g at room temperature, while the tannin modified Fe3O4 MNPs, and the trypsin immobilized on tannin modified-Fe3O4 MNPs possessed a saturation magnetization value of 57.82emu/g and 55.16emu/g, respectively, which indicated the decent tannin coating and trypsin immobilization. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of widely used bovine serum albumin (BSA). The immobilized trypsin was investigated by conducting the tryptic digestion of BSA within 1min, 5min and 15min. Also, microwave-assisted digestion was carried out for 15s. The digested protein fragments were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and a satisfactory peptide numbers of 39, and a superior sequence coverage of 84% for 1min digestion were obtained. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the satisfactory digestion of BSA and egg white proteins by immobilized trypsin.

Covalent immobilization of trypsin onto modified magnetite nanoparticles and its application for casein digestion.

The immobilization method consists of the production magnetite nanoparticles (Fe3O4) by solvothermal treatment of FeCl3 and sodium acetate (NaAc) in the presence of ethylene glycol. Subsequently, the surface of magnetite nanoparticles was modified with a well-known polyphenol tannic acid. Trypsin was covalently immobilized on the tannic acid modified magnetite nanoparticles after exposing the modified nanoparticles to pH 9.4. Then, tryptic digestion of casein by free and immobilized trypsin was carried out for 13h and 1h, respectively. TGA curves, FTIR spectra, and magnetization curves demonstrated the decent amount of trypsin immobilization without compromising the enzyme activity. Digestion efficiency of casein was investigated using liquid chromatography-mass spectrometry (LC-MS/MS) technique. LC-MS chromatograms confirmed the efficient digestion of casein by immobilized trypsin compared to free trypsin owing to prevention of autohydrolysis. Also, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the satisfactory digestion of casein by immobilized trypsin.

Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk.

Trypsin (EC 3.4.21.4) was successfully immobilized on the surface of Fe3O4 magnetic nanoparticles that had been pre-treated with gallic acid (GA). Measurements of protein load by using Bradford assay and the trypsin-catalyzed hydrolysis of Nα-Benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) were made for the immobilized enzyme. By using magnetic nanoparticles, which provides easy separation and decent support material for enzyme immobilization with high surface area to volume ratio, and by employing biocompatible material gallic acid, immobilized enzyme system was synthesized along with improving trypsin activity and stability. Immobilized trypsin (TR) was more stable than the free one and demonstrated higher enzymatic activity at elevated temperatures (45-55°C) and in the alkaline pH region (6-10.5). Fe3O4 NPs-GA-TR retained 92% of its initial activity after 120days of storage at 4°C in sodium phosphate buffer (0.1M, pH 7.5), whereas the free trypsin maintained about 64% of its initial activity during the same storage period. In addition, activity of the immobilized trypsin was preserved 54.5% of its initial activity after eight times successive reuse. The Michaelis-Menten kinetic constant (Km) and maximum reaction velocity (Vmax) for free trypsin were 5.1mM and 23mM/min, respectively, whereas Km and Vmax values of immobilized trypsin were 7.88mM and 18.3mM/min, respectively. The performance of the immobilized trypsin was demonstrated by carrying out the hydrolysis of bovine serum albumin (BSA) within 1h, and the assay was performed by using liquid chromatography-mass spectrometry (LC-MS/MS) technique. The hydrolysis of bovine milk as a real food was investigated by immobilized trypsin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

A facile and effective immobilization of glucose oxidase on tannic acid modified CoFe2O4 magnetic nanoparticles.

This article presents a study of glucose oxidase (GOx) immobilization by employing tannic acid (TA) modified-CoFe2O4 (CFO) magnetic nanoparticles which demonstrates novel aspect for enzyme immobilization. By using the strong protein and tannic acid binding, GOx immobilization was carried out via physical adsorption in a simpler way compared with the other immobilization methods which require various chemicals and complicated procedures which is difficult, expensive, time-consuming, and destructive to the enzyme structure. CFO was synthesized by hydrothermal synthesis and modified with TA to immobilize GOx. The immobilized GOx demonstrated maximum catalytic activity at pH 6.5 and 45 °C. The samples were characterized by vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential, and fourier transform infrared spectroscopy (FTIR), all of which confirm the surface modification of CFO and GOx immobilization. Also, field emission scanning electron microscope (FESEM) and X-ray diffraction (XRD) were performed to demonstrate the surface morphology and chemical structure of samples. According to the Lineweaver-Burk plot, GOx possessed lower affinity to glucose after immobilization, and the Michelis-Menten constant (KM) of immobilized and free GOx were found to be 50.05 mM and 28.00 mM, respectively. The immobilized GOx showed excellent reusability, and even after 8 consecutive activity assay runs, the immobilized GOx maintained ca. 60% of its initial activity.