Microstructural modifications induced by the entrapped glucose oxidase in cross-linked polyacrylamide microgels used as glucose sensors (original) (raw)
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Kinetics of glucose oxidase immobilized in p(HEMA)-hydrogel microspheres in a packed-bed bioreactor
Journal of Molecular Catalysis B: Enzymatic, 2002
Glucose oxidase (GO x ) was immobilized via both physical entrapment and covalent linkage to crosslinked poly(hydroxyethyl methacrylate-co-dimethylaminoethyl methacrylate, (p(HEMA-DMEMA)) hydrogel microspheres (20-150 m in diameter) that were synthesized by inverse suspension polymerization. Loading capacities of 7-8 mg GO x per gram of hydrogel were achieved with physical entrapment, compared to <1.8 mg GO x per gram of gel with the covalent technique. The microspheres containing physically entrapped enzyme were packed into bioreactors and the kinetics of the immobilized enzyme investigated under various flow conditions. Flow rate dependence of K m(app) and C max , when extrapolated to near diffusion-free conditions, resulted in values of 13.2 mM and 2.7 × 10 −3 mol min −1 , respectively, for the immobilized enzyme. Studies of pH-dependence of K m(app) and C max suggest that the imidazolium and sulphydryl groups may be involved at the active site of the immobilized GO x . Studies of the temperature dependence of C and C max confirm lower activation energies for the oxidation of glucose at temperatures >35 • C, suggesting the influence of diffusional limitations within the hydrogel.
Analytica Chimica Acta, 2004
An optical glucose biosensor was fabricated by entrapping glucose oxidase (GOx) within the xerogel that was derived from tetraethylorthosilicate and hybridised with hydroxyethyl carboxymethyl cellulose polymer. The entrapped-GOx was mainly characterised with its long-lasting apparent biocatalytic activity as compared to that being entrapped in only sol-gel matrix. The biocatalytic activity of the entrapped-enzyme has extended its shelf lifetime up to 3 years. This long-term stability was closely correlated with the reduction in the shrinkage process of the hybrid gel being used. In conjunction with an optical oxygen transducer, the entrapped-GOx was assembled as an optical glucose biosensor comprised a sample flow system with which the dissolved oxygen in the sample could be precisely controlled and varied. The analytical working range was tuneable within 9.0 M-100 mM range depending on the dissolved oxygen concentration in the test solution. The time taken to reach a 95% steady signal was 6-9 min at flow rate of 1.0 mL min −1 . The glucose biosensor has been satisfactorily applied to the determination of glucose contents of urine samples.
Electroanalysis, 2018
Thermosensitive microgel particles based on the polymeric network synthesized from N-isopropylacrylamide, sodium acrylate and N,N'-bisacryloylcystine have been functionalized with aminoferrocene, a mediatior, and a model enzyme-glucose oxidase (GOx). The product was very stable. A thin coating of that material has been successfully attached to the glassy carbon electrode surface to form a sensing layer. A temperature increase to a value higher than the volume phase transition temperature led to a substantial increase in the amperometric signal. That behavior was well switchable. The amperometric signal was linearly dependent on glucose concentration in the physiological concentration range. It appeared that the synthesized and functionalized microgel was a kind of microreactor and might be very useful for the construction of the platforms that can enhance the activity of enzymes in biosensors.
Sensors and Actuators B: Chemical, 2015
The development of a sandwich-type biosensor for glucose quantification is presented. This work is focused on the optimization of the enzymatic matrix of the biosensor. The best performance was found for an enzymatic matrix composed by 30% w/w mucin, 70% w/w albumin, 1.35 U glucose oxidase (GOX) per sensor, and glutaraldehyde diluted to 3%. The crosslinking with glutaraldehyde transforms this mixture into a hydrogel that is entrapped between two membranes of polycarbonate. The selected sandwich-type biosensor showed very good response time, sensitivity, stability, and sensor-to-sensor reproducibility. According to the results presented in this manuscript, a biosensor prepared with very high amount of enzyme would not necessarily increase the analytical signal. Simulated curves are compared with experimental data to explain the dependence of sensitivity on the concentration of enzyme. In addition, this kind of comparison represents a quite simple way to estimate the value of v max ≈0.13 M s −1 from the amperometric response of a sensor prepared with 1.34 U of GOX. Considering that sandwich-type biosensors are commonly assembled as part of devices where the sample is diluted with buffer, the more than 3 orders of magnitude of linear behavior of this sensor would ensure the possibility for assessing any sample.
Journal of Molecular Catalysis B: Enzymatic, 2003
Poly(N-vinylimidazole), PVIm, gels were prepared by ␥-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption-desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.
Engineering in Life Sciences
The aim of this study was to formulate silica and alginate hydrogels for immobilization of β-glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate-polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β-glucosidase was loaded into glass-silicon-glass microreactors and catalysis of 4-nitrophenyl β-d-glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.
Development of Glucose Biosensor by Using Gelatin and Gelatin-Polyacrylamide Supporting Systems
Artificial Cells, Blood Substitutes, and Biotechnology, 2006
In this work an amperometric glucose biosensor based on surface immobilization method was developed. Glutaraldehyde was used as cross-linker to establish the immobilization of glucose oxidase onto gelatin (carrier=coating reagent). In order to increase the porosity of coating material, immobilization media was further treated by polyacrylamide. Although this treatment increased the performance of biosensor to a large extent with respect to current densities obtained, it negatively affected the long-term stability. Our biosensor showed linear response in the physiological range of blood glucose (0.05 to 6 mM), had an acceptable response time (60 seconds) and was stable for 17 repeated usages in 51 days. We obtained best results with pH values very close to physiological pH and our biosensor could work efficiently in the tested temperature range 15 to 65 C.
Application of sol gel technique for glucose oxidase immobilization in biosensor application
A sol gel based biosensor using 3-glycidoxypropyl dimethylethoxysilane, as a silane agent was developed as a novel method for biosensor enzyme immobilization. The key materials applied were tetra methyl orthosilicate (TMOS), and the cross link agent, 3-Glycidoxypropyl dimethylethoxysilane, (GDP). Three compositions with different amount of GDP and glucose oxidase were experimented with the amount of TMOS was kept constant. The first coating consisted of 2mg of glucose oxidase and 20μl of GDP. The second trial involved higher GDP composition which is 100μl. The final trial applied higher content of glucose oxidase which is 12 mg with 20μl of GDP. The result showed that sensors with high composition of crosslink agent coating was capable to exhibit reliable glucose detection. The crosslink agent insufficiency in the first composition failed to provide good attachment for the enzyme on the electrode. Thus, the test was halted after few readings due to the inability of the sensor to detect glucose increment. For the high amount glucose oxidase composition, failure happened due to the deficiency of GDP to retain the enzyme thus contributed to the glucose oxidase leaching. We conclude that adequate amount of crosslink agent is vital for a sol gel based biosensor to function successfully.