脱氢酶生物传感器研究关键技术与进展

自然界中依赖烟酰胺类辅酶(NAD+或NADP+)的脱氢酶是氧化还原酶中最重要的一类,基于此类酶的生物传感器应用前景广阔,近年来发展迅速。构建这类传感器需要两项关键技术,即氧化型辅酶在电极表面的再生和辅酶固定化。本文介绍了辅酶电化学再生的主要方法、辅酶固定化的常见手段,以及相关的研究进展。     

The slow-down of the CALB immobilization rate permits to control the inter and intra molecular modification produced by glutaraldehyde

Lipase B from Candida antarctica (CALB) has been immobilized on octyl-agarose in two ways: rapidly, in 5 mM sodium phosphate (85% immobilization yield after 30 min), or slowly, in the presence of 30% (v/v) ethanol (40% immobilization yield after 30 min). Both biocatalysts were treated with glutaraldehyde in order to obtain different modification degrees on their amino groups (25, 50 and 100% modification). SDS-PAGE and detergent desorption experiments showed that, when the immobilization was performed in absence of ethanol, very large aggregates were formed by intermolecular crosslinking, while when 30% ethanol was added during immobilization, almost 90% of the enzyme remained as a monomer. The stability of both derivatives improved upon modification, both in thermal inactivation experiments (at pHs 5, 7 and 9) or in the presence of 50% (v/v) dimethylsulfoxide, achieving stabilization values ranging between 5 and 20 depending on the inactivation conditions. The stability increased proportionally with the modification degree, and was also higher when intermolecular bonds were performed (by a 2–4 factor). Moreover, the activity/pH profile was completely altered after enzyme modification, and, under certain conditions, the activity of the modified biocatalysts doubled that of the non-modified immobilized CALB. Results show that the addition of ethanol permits to have a distance between enzyme molecules that did not allow intermolecular crosslinking, and this has permitted to distinguish between the effects of intramolecular glutaraldehyde modifications and intermolecular glutaraldehyde crosslinking. The simple and controlled treatment of CALB-octyl with glutaraldehyde has proved to be an effective way to obtain a biocatalyst with improved activity and stability under different conditions.     

Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

Sensitive detection of an anthrax biomarker using a glassy carbon electrode with a consecutively immobilized layer of polyaniline/carbon nanotube/peptide

Sensitivity of Anthrax protective antigen (PA) detection has been improved by directly immobilizing a PA-specific peptide onto a multi-wall carbon nanotube (MWCNT). The MWCNT was covalently immobilized onto a polyaniline (PANI) electrode, which was prepared via electropolymerization of the aniline monomer onto a glassy carbon electrode (GCE). Then, the PA-specific peptide was covalently immobilized to the MWCNT layer for measurement. When comparing this technique to that of PA immobilization on an insulting self assembled organic layer, the advantages of the MWCNT are clear. The MWCNT sensor resulted in enhanced electron transfer across the sensing layer. The resulting limit of detection (LOD) was 0.4 pM, a 13-fold improvement over that of our previous self-assembled organic layer was used for immobilization of the same peptide. Neither positive nor negative interferences were observed when a sample containing both 100 pM PA and bovine serum albumin (BSA) was measured, indicating good selectivity of the proposed sensor.     

Conversion of cellobiose and xylose to ethanol by immobilized growing cells of Clostridium saccharolyticum on charcoal support

Summary An immobilization technique has been developed for the conversion of both cellobiose and xylose to ethanol, which may be considered as one stage of a process for the conversion of cellulosic biomass to ethanol. Relatively inexpensive charcoal was used as a support material, with 23 mg dry weight of Clostridium saccharolyticum cells per g dry weight of support. Tests were run for 170 h at 0.15 1/h dilution rate. From a 3% (w/v) sugar mixture, 0.7% (w/v) ethanol was obtained with over 97% cellobiose and 62% xylose utilization.     

An all-solid-state amperometric ethanol sensor

An all-solid-state pellet electrode for ethanol determination has been developed and compared with the membrane-layered sensor. It is found that the new pellet electrodes have reliable responses (current vs. concentration) to ethanol from 0.1 to 10 m with response times of about 2 min. The current response decreases about 10% h⁻¹ during continuous operation, compared with a drop of 50% h⁻¹ with normal membrane electrodes. Fresh electrodes can be stored in a freezer for 2 weeks without apparent activity loss. The measurement procedure is convenient and it is probable that reproducible, disposable electrodes can be made at low cost. This format is general and can easily be extended to many other systems with β-NAD⁺/NADH as coenzyme.     

Designing enzymatic kyotorphin synthesis in organic media with low water content

Design of enzymatic kyotorphin synthesis in low water media has been carried out as a function of enzyme nature, the immobilization support material and the reaction medium, by using N-benzoyl-L-tyrosine ethyl ester and L-argininamide as substrates. Native and chemically-glycated alpha-chymotrypsin deposited on supports with different degrees of aquaphilicity (celite, polypropylene PP, and polyamide PA6) were used as catalysts. Binary organic solvent systems of ethanol and different water-immiscible organic cosolvents (ethylacetate, tert-butanol, chloroform, toluene, n-hexane, and n-octane) were studied as reaction media at constant water content (3% v/v). The greater the water binding affinity of the support the lower the synthetic activity of deposited enzymes: the activity of the celite derivative was 4x greater than the polyamide derivative. The enzyme glycation process hardly modified the catalytic ability of the celite derivative, but resulted in a moderate increase in operational stability. The presence of hydrophobic organic cosolvents in the water/ethanol reaction medium significantly increased enzyme activity, whereas the selectivity of the reaction remained high. Hexane was shown to be the best cosolvent, the synthetic activity of the celite derivative in hexane-ethanol (77 : 20%, v/v) being 130x greater than that in 97% (v/v) ethanol.     

Ethanol production from sucrose by immobilizedZymomonas mobilis cells in polyurethane foam

Summary The possibility of using polyurethane foam as a support for the immobilization ofZymomonas mobilis cells to carry out sucrose conversion to ethanol was investigated. Sucrose hydrolysis efficiencies of 90% and higher, volumetric reactor productivity of 20 gL^–1h^–1 and final ethanol concentration of 6.3% (v/v) at a dilution rate of 0.4 h^–1 show the good performance of polyurethane foams for whole cell immobilization.     

Regeneration of NAD(+) cofactor by photosensitized electron transfer in an immobilized alcohol dehydrogenase system

The irradiation with visible light of a photosensitizer dye like methylene blue was used to regenerate by electron transfer the oxidized form of a pyridine nucleotide coenzyme (NAD(+)). The process has been studied on a common enzymatic reaction: ethanol oxidation by alcohol-NAD(+) oxidoreductase immobilized on polyacrylamide gel or porous glass balls. In the experimental conditions used, the initial NAD(+) recycling rates were 2.33 x 10(4) cycles/h (polyacrylamide) and 3 x 10(4) cycles/h (glass balls). A total number of 49.5 x 10(4) cycles was obtained for 13 runs of 2 h. The enzyme immobilization strongly increased its stability: after 28 days at 20 degrees C, the residual activity was 25% of the initial value.     

Needle-type lactate biosensor

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.     

Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half-life period of the reactor activity after one-shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes-which is of more general use.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Amperometric glucose biosensors based on Prussian Blue- and polyaniline-glucose oxidase modified electrodes.

The properties of glucose sensors fabricated by immobilization of glucose oxidase in a layer of electrochemically deposited polyaniline were investigated. Selective amperometric glucose sensors were prepared by immobilization of glucose oxidase on a Prussian Blue-modified platinum electrode in a layer of polyaniline during a one-step electropolymerization procedure from phosphate buffer. The influence of ascorbic acid and acetaminophen was completely eliminated due to impermeability of polyaniline to these substances.     

Miniaturized multi-enzyme biosensors integrated with pH sensors on flexible polymer carriers for in vivo applications

An electrochemical glucose sensor has been integrated, together with a pH sensor, on a flexible polyimide substrate for in vivo applications. The glucose sensor is based on the measurement of H₂O₂ produced by the membrane-entrapped enzyme glucose oxidase (GOD). To minimize electrochemical interference, an electrode configuration was designed to perform differential measurements. The solid-state pH sensor employs a PVC-based neutral carrier membrane. The enzymes GOD and catalase were immobilized into two layers of photolithographically patterned hydrogels. The intended use of this device is the short-term monitoring of glucose and pH in intensive care units and operating theatres, especially for neurosurgical applications. The developed immobilization technique can also be used to create integrated multi-sensor chips for clinical analysers. The glucose and pH sensor exhibited excellent performance during tests in buffer solutions, serum and whole blood.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.     

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5''-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.     

The effect of soluble alginate and calcium on β-galactosidase activity produced by the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus imb3

Since it has previously been demonstrated that ethanol production by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 is more efficient in calcium alginate-based immobilization systems during growth on lactose-containing media, it was decided to examine the separate effects of soluble alginate and free calcium on the β-galactosidase activity produced by that organism. It was found that the presence of Ca²⁺ significantly increased the thermal stability of the activity at 45?°C, although the pH?and temperature optima remained the same in the presence and absence of that cation. It was also found that the presence of 2% (w/v) sodium alginate (soluble) had a very limited positive effect on the thermal stability of the enzyme at 45?°C, although it was found that activity was very significantly stimulated at that temperature. The activity was found to have an enhanced thermal stability at 30?°C in the presence of sodium alginate. The presence of sodium alginate in assay mixtures had no significant effect on the Km of the activity for the substrate o-nitrophenyl-β-D-galactoside. The results observed in the presence of either free calcium or soluble alginate may at least partially explain enhanced ethanol production by this microorganism in alginate-based immobilization systems.