Immobilization of lactate oxidase in a poly(vinyl alcohol) matrix on platinized graphite electrodes by chemical cross-linking with isocyanate

doi:10.1016/0039-9140(91)80007-M

Talanta

Volume 38, Issue 1, January 1991, Pages 37-47

https://doi.org/10.1016/0039-9140(91)80007-M Get rights and content

Abstract

A new method for development of an electrochemical sensor based on lactate oxidase is dedbed. Platinized spectroscopic-grade graphite electrodes were modified by chemically cross-linking l-lactate oxidase from Pediococcus species into a poly(vinyl alcohol) network through reaction with a tri-isocyanate. The immobilized enzyme exhibits high activity and long-term stability. The sensor provides a linear response to l-lactate over a concentration range of 2 × 10⁻⁵−4 × 10⁻³M and a sensitivity of 1.71 μA.1. mmole⁻¹. The response time of the sensor is 10–45 sec and the detection limit is 10μM. Stable response to the substrate was obtained over a period of 3 months. The new sensor was also used for the analysis of some dairy products without any special pretreatment.

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However, LOD-sensor's performance was better when mixing with redox conjugates in pH 6.5. The reported optimum pH range for the free and immobilized LOD is 6.0–7.0 (Hajizadeh et al., 1991; Duncan et al., 1989), when the intrinsic pH of the redox matrix is sufficiently mild to permit retention of enzymatic activity and stability, the enzymatic reaction should retain persistence (Stoisser et al., 2016). Hence, we have inferred that LOD gradually diminished its stability and activity when mixing with redox conjugates in pH below 6.5 so that the sensor failed after several hours testing.
We reported a proof-of-concept study of developing an electrochemical biosensor for prolonged continuous monitoring free flap failure caused by vascular occlusion after reconstructive surgery. Ferrocene (Fc)-containing Chitosan-cografted-Branched Polyethylenimine redox conjugates (CHIT-Fc-co-BPEI-Fc) were used as pH-tuneable matrix to attach the target enzymes (glucose oxidase ≡ GOD and lactate oxidase ≡ LOD, respectively) to build up the corresponding GOD-sensor and LOD-sensor. The sensitivity of GOD-/LOD-sensor was found to be 2.89(±0.06)μA/mM and 2.95(±0.19)μA/mM with a limit of detection (LOD) of 0.047 mM and 0.172 mM, respectively. The sensor performance was further pre-clinically validated via rabbit study, which confirmed that the designed biosensors could electrochemically detect the changes of metabolite levels caused by flap failure within minutes. This sensor protocol is able to be fabricated as embedded biosensor for prolong continuous detection for clinical application/research.
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Non-toxicity, biocompatibility alongside remarkable chemical and thermal stability, has made PVA a viable choice in biosensor applications [6]. Most of the researchers have studied different methods (e.g. cross-linking with aromatic tri-isocyanates, UV or gamma irradiation, etc.) to immobilize enzymes in PVA matrices and have led to applicable biosensors [7–10]. One of the crucial factors in the rate of enzyme absorption by polymer surfaces is the chemical features of the surface, i.e. functional groups on the surface.
Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO₂, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.
Biosensors based on electrochemical lactate detection: A comprehensive review
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They can be easily being prepared in laboratory. Amucin/albumin hydrogel matrix/Nafion Polymer Pt electrode [70], Chitosan/MWCNT/ferrocyanide/gold electrode [80], Chitosan/poly-vinyl imidazole Os (PVI-Os)/CNT/gold electrode [81], poly-vinyl alcohol (PVA) matrix on platinized graphite electrodes [82], polyethyleneimine (PEI) and poly(carbamoyl)sulfonate (PCS) hydrogel/screen-printed graphite/nafion/Pt electrode [83], sol–gel film derived from MTEOS/Os-polymer /GCE [84], hydrogel based osmium complexe [Os(byp)2ClPyCH2NH Poly(allylamine)] redox polymer/GCE [85], Laponite-chitosan hydrogel/GCE [86], Nafion/cobalt phthalocyanine poly-vinyl alcohol/screen-printed carbon electrode [60]. Can be prepared easily; provide a favorable microenvironment for the enzyme molecules to enhance storage stability, variety of supports can be synthesized due to the possibility chemical modifications.
Lactate detection plays a significant role in healthcare, food industries and is specially necessitated in conditions like hemorrhage, respiratory failure, hepatic disease, sepsis and tissue hypoxia. Conventional methods for lactate determination are not accurate and fast so this accelerated the need of sensitive biosensors for high-throughput screening of lactate in different samples. This review focuses on applications and developments of various electrochemical biosensors based on lactate detection as lactate being essential metabolite in anaerobic metabolic pathway. A comparative study to summarize the L-lactate biosensors on the basis of different analytical properties in terms of fabrication, sensitivity, detection limit, linearity, response time and storage stability has been done. It also addresses the merits and demerits of current enzyme based lactate biosensors. Lactate biosensors are of two main types – lactate oxidase (LOD) and lactate dehydrogenase (LDH) based. Different supports tried for manufacturing lactate biosensors include membranes, polymeric matrices-conducting or non-conducting, transparent gel matrix, hydrogel supports, screen printed electrodes and nanoparticles. All the examples in these support categories have been aptly discussed. Finally this review encompasses the conclusion and future emerging prospects of lactate sensors.
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In the first instance, we utilized Nafion as a permselective layer that presumably repels AA due to electrostatic interactions. Nafion has been widely used for interference rejection in biosensors [42,43], even in the detection of lactate [46,47]. Fig. 6 shows the results for the measurement of l-lactate levels over a range present in the tear fluid without and with 50 μM AA in solution.
The tear film is a valuable diagnostic fluid for a continuous, minimally invasive surveillance of health conditions in a patient. We developed an electronic enzymatic l-lactate sensor on a polymer substrate molded into contact lens shape for potential in situ monitoring of l-lactate levels in tear fluid. The platinum sensing structures were functionalized by cross-linkage of lactate oxidase with glutaraldehyde and bovine serum albumin, and coated with medical grade polyurethane. Different approaches for the suppression of interfering species present in the tear film were evaluated. The sensors show a quick response time of 35 s, an average sensitivity of ∼53 μA mM⁻¹ cm⁻² within their linear range, and sufficient resolution within the physiological range of lactate concentrations. A dual sensor design was found appropriate for the suppression of interfering signals. The sensors are functional at temperatures comparable to those at the surface of the eye, and their response is stable for up to 24 h.
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2012, Analytica Chimica Acta
Citation Excerpt :
However, the determination of l-lactate in real samples by the direct amperometric measurement of H2O2 can be affected by interfering species that are oxidized at the high potential required for H2O2 detection. In order to minimize the contribution of interfering substances to the biosensor response, the use of a permselective membrane [13–18] and electrocatalytic mediators has been reported [16–18,22–25]. Regarding the former, it has been reported that a Nafion layer has high anti-interfering ability and can eliminate interference from electroactive species [13–15].
A novel amperometric biosensor for the measurement of l-lactate has been developed. The device comprises a screen-printed carbon electrode containing cobalt phthalocyanine (CoPC-SPCE), coated with lactate oxidase (LOD) that is immobilized in mesoporous silica (FSM8.0) using a polymer matrix of denatured polyvinyl alcohol; a Nafion layer on the electrode surface acts as a barrier to interferents. The sampling unit attached to the SPCE requires only a small sample volume of 100 μL for each measurement. The measurement of l-lactate is based on the signal produced by hydrogen peroxide, the product of the enzymatic reaction. The behavior of the biosensor, LOD-FSM8.0/Naf/CoPC-SPCE, was examined in terms of pH, applied potential, sensitivity and operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.4 and an applied potential of +450 mV. The determination range and the response time for l-lactate were 18.3 μM to 1.5 mM and approximately 90 s, respectively. In addition, the sensor exhibited high selectivity for l-lactate and was quite stable in storage, showing no noticeable change in its initial response after being stored for over 9 months. These results indicate that our method provides a simple, cost-effective, high-performance biosensor for l-lactate.
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We report on the formation of colloidal biocomposites resulting from the electrostatic self-assembly of negatively charged lactate oxidase (LOD) and oppositely charged nanoscaled cobalt phthalocyanine (NanoCoPc) colloid under enzyme-friendly conditions. The biocomposites were further used to fabricate lactate biosensors due to their excellent film-forming ability and strong adsorbability on the surface of glassy carbon electrode. The electrochemical assays of such films revealed a large capacity of NanoCoPc for the retention of LOD. Here, NanoCoPc colloid was not only used as carriers for immobilization of LOD, but also displayed intrinsic electrocatalytic activity for the oxidation of H₂O₂, a product of enzymatic reaction. A chitosan film containing MnO₂ nanoparticles was further electrodeposited as an external layer, which could effectively eliminate the interference from ascorbic acid. Under optimal conditions, the biosensor showed a wide linear response to lactate in the range of 0.020–4.0 mM, with high sensitivity (3.98 μA cm⁻² mM⁻¹), as well as good reproducibility and long-term stability. The biosensor has been used for the determination of lactate in real samples with an acceptable accuracy.

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Immobilization of lactate oxidase in a poly(vinyl alcohol) matrix on platinized graphite electrodes by chemical cross-linking with isocyanate

Abstract

Biosensors

Biosensors

Biosensors

Anal. Chim. Acta

J. Chromatog.

Anal. Chim. Acta

Anal. Chim. Acta

Biosensors

Anal. Chim. Acta

J. Biol. Chem.

Anal. Chim. Acta

J. Theor. Biol.

Lancet

J. Electrochem. Soc.

Clin. Chem. News

Clin. Chem.

Biotechnol. Bioeng.

Clin. Chem.

Biotechnol. Bioeng.