基于酶电极的乳酸检测生物传感器

乳酸(C₃H₆O₃),又名2-羟基丙酸、丙醇酸,属于羟基酸的一种。乳酸在食品工业、临床医学、生物技术等行业具有极其重要的意义,因此如何高通量检测不同样品中的乳酸成为目前业界研究的重点。传统乳酸检测方法操作繁琐、费时费力或需要昂贵的检测设备,乳酸生物传感器可以克服这些限制,不需要样品制备,能够快速、简便、可靠地定量测定食品或血浆中的乳酸,具有广阔的应用前景。乳酸酶电极生物传感器主要有两种类型——基于L-乳酸氧化酶(L-LOD)和L-乳酸脱氢酶(L-LDH)的乳酸生物传感器。本文综述了L-LOD和L-LDH结构特征、来源及催化机理,讨论了改善基于酶电极的乳酸传感器性能的3种策略(电极材料改造策略、酶固定化策略、酶分子工程改造策略),还根据用于制造乳酸生物传感器的不同载体包括膜、透明凝胶基质、水凝胶载体、纳米颗粒等对乳酸生物传感器进行了归类分析,最后本文将目前商品化应用的酶电极乳酸生物传感器特点进行了对比总结讨论,阐述了乳酸生物传感器的未来应用方向,并对未来发展前景进行了展望。     

Development of a screen-printed amperometric biosensor for the determination of L-lactate dehydrogenase level.

We attempted to develop a screen-printed biosensor for the amperometric determination of L-lactate dehydrogenase (LDH) level on the basis of NAD(+)/NADH-dependent dehydrogenase reaction. The printing ink for the working electrode consisted of L-lactate, NAD(+), composite polymer of hydroxyethyl cellulose with ethylene glycol, 3,4-dihydroxybenzaldehyde (3,4-DHB) as an electron transferring mediator, and graphite as the conducting material. The 3,4-DHB was electropolymerized on the carboneous working electrode by potential cycling between -200 and +300 mV vs. Ag/AgCl reference electrode. Through the electrocatalytic reaction with immobilized 3,4-DHB, the NADH generated by the LDH reaction could be efficiently oxidized at lower potential than the unmodified carbon electrode. The analytical performance of the electrode was characterized in terms of linear sensing range and detection limit for LDH. The response from the developed biosensor was linear up to 500 U/l of LDH, and the detection limit of 50 U/l was observed at the signal-to-noise ratio of 3.     

A novel L-lactate-selective biosensor based on flavocytochrome b2 from methylotrophic yeast Hansenula polymorpha

A novel amperometric biosensor highly selective to L-lactate has been developed using L-lactate-cytochrome c oxidoreductase (flavocytochrome b2) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface. Moreover, the possibility of direct electron transfer from the reduced form of FCb2 to carbon electrodes has been evaluated. The bioanalytical properties of FCb2-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for L-lactate determination.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase.

Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.     

Citric acid cycle biomimic on a carbon electrode

The citric acid cycle is one of the main metabolic pathways living cells utilize to completely oxidize biofuels to carbon dioxide and water. The overall goal of this research is to mimic the citric acid cycle at the carbon surface of an electrode in order to achieve complete oxidation of ethanol at a bioanode to increase biofuel cell energy density. In order to mimic this process, dehydrogenase enzymes (known to be the electron or energy producing enzymes of the citric acid cycle) are immobilized in cascades at an electrode surface along with non-energy producing enzymes necessary for the cycle to progress. Six enzymatic schemes were investigated each containing an additional dehydrogenase enzyme involved in the complete oxidation of ethanol. An increase in current density is observed along with an increase in power density with each additional dehydrogenase immobilized on an electrode, reflecting increased electron production at the bioanode with deeper oxidation of the ethanol biofuel. By mimicking the complete citric acid cycle on a carbon electrode, power density was increased 8.71-fold compared to a single enzyme (alcohol dehydrogenase)-based ethanol/air biofuel cell.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles‐modified pencil graphite electrode

A novel amperometric lactate biosensor was developed based on immobilization of lactate dehydrogenase onto graphene oxide nanoparticles‐decorated pencil graphite electrode. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and cyclic voltammetry at different stages of its construction. The biosensor showed optimum response within 5 s at pH 7.3 (0.1 M sodium phosphate buffer) and 35°C, when operated at 0.7 V. The biosensor exhibited excellent sensitivity (detection limit as low as 0.1 μM), fast response time (5 s), and wider linear range (5–50 mM). Analytical recovery of added lactic acid in serum was between 95.81–97.87% and within‐batch and between‐batch coefficients of variation were 5.04 and 5.40%, respectively. There was a good correlation between serum lactate values obtained by standard colorimetric method and the present biosensor (r = 0.99). The biosensor measured lactate levels in sera of apparently healthy subjects and persons suffering from lactate acidosis and other biological materials (milk, curd, yogurt, beer, white wine, and red wine). The enzyme electrode lost 25% of its initial activity after 60 days of its regular uses, when stored dry at 4°C.     

A protein biosensor for lactate

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.     

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme–electrode contact junction electrode

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5–40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

     

Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase

A subzero temperature operating biosensor was constructed using immobilized quinoprotein glucose dehydrogenase (PQQGDH), glassy carbon electrode, soluble electron mediator (ferrocene monocarboxylic acid), and an organic solvent, ethylene glycol, as an antifreezing reagent. Using this biosensor, glucose concentration can be determined even at -7 degrees C. At this temperature, the response was 20% of that obtained at 20 degrees C. This is the first study describing a subzero temperature operating biosensor. (c) 1993 John Wiley & Sons, Inc.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.     

New redox mediator-modified polysulfone composite films for the development of dehydrogenase-based biosensors

This work presents polysulfone membranes as new materials for the development of compact dehydrogenase-based biosensors. Composite films were prepared by mixing polysulfone with graphite and were deposited on epoxy-graphite composite electrodes. Redox mediators were successfully immobilized in the composite film leading to highly reproducible biosensors, without leakage of the immobilized species. This results in a more reliable analytical system as, at the same time, problems of electrode fouling related to the detection of the coenzyme nicotinamide adenine dinucleotide (NADH) on which is based the amperometric detection of dehydrogenase-based biosensors are avoided. Scanning electron microscopy was used to study the morphological characteristics of the surface and the cross-section of the polysulfone-graphite composite films. Several procedures to immobilize enzymes in these membranes were demonstrated. Glutamate dehydrogenase (GlDH) was immobilized as an example of dehydrogenase enzyme, in this case for the development of an ammonium biosensor. High sensitivity, good selectivity, wide linear ranges and short response times were obtained for the optimized sensors and biosensors. Their good performance combined with the simplicity of the construction method, make the polysulfone-graphite composite films attractive matrices for the development of new enzyme-based biosensors, especially those based on dehydrogenase enzymes.     

Capillary-driven multiparametric microfluidic chips for one-step immunoassays

A new zinc oxide nanoparticles/chitosan/carboxylated multiwall carbonnanotube/polyaniline (ZnO-NPs/CHIT/c-MWCNT/PANI) composite film has been synthesized on platinum (Pt) electrode using electrochemical techniques. Three enzymes, creatinine amidohydrolase (CA), creatine amidinohydrolase (CI) and sarcosine oxidase (SO) were immobilized on ZnO-NPs/CHIT/c-MWCNT/PANI/Pt electrode to construct the creatinine biosensor. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The enzyme electrode detects creatinine level as low as 0.5 μM at a signal to noise ratio of 3 within 10s at pH 7.5 and 30°C. The fabricated creatinine biosensor showed linear working range of 10-650 μM creatinine with a sensitivity of 0.030 μA μM(-1)cm(-2). The biosensor shows only 15% loss of its initial response over a period of 120 days when stored at 4°C. The fabricated biosensor was successfully employed for determination of creatinine in human blood serum.     

Formation and dissimilation of oxalacetate and pyruvate Pseudomonas citronellolis grown on noncarbohydrate substrates.

Metabolism of lactate as a carbon source by Pseudomonas citronellolis occurred via a nicotinamide adenine dinucleotide (NAD)-independent L-lactate dehydrogenase, which was present in cells grown on DL-lactate but was not present in cells grown on acetate, aspartate, citrate, glucose, glutamate, or malate. The cells also possessed a constitutive, NAD-independent malate dehydrogenase instead of the conventional NAD-dependent malate dehydrogenase instead of the conventional NAD-dependent enzyme in the tricarboxylic acid cycle. Both enzymes were particulate and used dichlorophenolindo-phenol or oxygen as an electron acceptor. In acetate-grown cells, the activity of pyruvate dehydrogenase and NAD phosphate-linked malate enzyme decreased, cells grown on glucose or lactate. This was consistent with the need to maintain a supply of oxalacetate for metabolism of acetate via the tricarboxylic acid cycle. Changes in enzyme activities suggest that gluconeogenesis from noncarbohydrate carbon sources occurs via the malate enzyme (when oxalacetate decarboxylase is inhibited) or a combination of the NAD-independent malate dehydrogenase and oxalacetate decarboxylase.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Characterization of sand as a support for immobilized enzymes.

The potential of sand as a support for immobilized enzymes was investigated by preparing alkylamine sand and devising methods to measure the total number of amine groups present and the fraction available for immobilization of enzymes. Alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1.) and lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) were immobilized on alkylamine sand, and the stability of the immobilized protein and dehydrogenase activity was measured. Urease (urea amidohydrolase, EC 3.5.1.5) was also immobilized on sand to test the applicability of these methods to larger scale immobilizations. Results suggest that sand shows promise as a support for immobilized enzymes.     

一种新的检测黄曲霉毒素B1的酶生物传感器的制作

本文报道了一种新的检测黄曲霉毒素B1的生物传感器,该传感器以开管的多壁纳米碳管固定化黄曲霉毒素氧化还原酶制作传感电极检测黄曲霉毒素B1,其线性范围达到0.16μM-3.2μM,当把特异性的黄曲霉毒素B1抗体与黄曲霉毒素氧化还原酶通过多壁纳米碳管共固定化制作修饰电极,传感器的检测限提高到16nM,灵敏度提高了10倍。用这种方法制作黄曲霉毒素酶生物传感器,使黄曲霉毒素酶生物传感器向实用化迈进了一步。     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide