壳聚糖-二茂铁复合物的制备及其在电化学传感器上的应用

壳聚糖-二茂铁复合物(CHIT-Fc)由二茂铁羧酸的羧基和脱乙酰壳多糖的羟基缩合合成,并通过红外光谱检测.合成得到的壳聚糖-二茂铁复合物通过物理吸附作为固定胆固醇氧化酶(CHOx)的基体.同时,使用Nafion稀释液可以消除诸如抗坏血酸和尿酸的影响.最佳测试条件下,采用示差脉冲(DPV)研究胆固醇生物传感器的响应,在4.0×10-6mol/L～1.0×10-4 mol/L范围内,氧化峰电流与胆固醇浓度呈现良好的线性关系,线性方程为Ipa=0.0223c-0.0875(Ipa:μA,c:μmol/L,R=0.9982),检测限为5.0×10-7mol/L(S/N=3).     

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

RE-SELEX is the first homogenous method for in vitro evolution of structure-switching DNA aptamers.     

The application of boron-doped diamond electrodes in amperometric biosensors

Boron-doped diamond (BDD) electrodes outperform conventional electrodes in terms of high stability, chemical inertness, wide potential window and low background current. Combining the superior properties of BDD electrodes with the merits of biosensors, such as specificity, sensitivity, and fast response, amperometric biosensors based on BDD electrodes have attracted the interests of many researchers. In this review, the latest advances of BDD electrodes with different surfaces including hydrogen-terminated, oxygen-terminated, metal nanoparticles-modified, amine-terminated, and carboxyl-terminated thin films, and microelectrodes, for the construction of various biosensors or the direct detection of biomolecules were demonstrated. The future trends of BDD electrodes in biosensing were also discussed.     

Graphene electrochemistry: fabricating amperometric biosensors

The electrochemical sensing of hydrogen peroxide is of substantial interest to the operation of oxidase-based amperometric biosensors. We explore the fabrication of a novel and highly sensitive electro-analytical biosensor using well characterised commercially available graphene and compare and contrast responses using Nafion -graphene and -graphite modified electrodes. Interestingly we observe that graphite exhibits a superior electrochemical response due to its enhanced percentage of edge plane sites when compared to graphene. However, when Nafion, routinely used in amperometric biosensors, is introduced onto graphene and graphite modified electrodes, re-orientation occurs in both cases which is beneficial in the former and detrimental in the latter; insights into this contrasting behaviour are consequently presented providing acuity into sensor design and development where graphene is utilised in biosensors.     

Enzyme-based amperometric galactose biosensors: a review

This review (with 35 references) summarizes the various strategies used in biosensors for galactose, and their analytical performance. A brief comparison of the enzyme immobilization methods employed and the analytical performance characteristics of a range of galactose biosensors are first summarized in tabular form and then described in detail. Selected examples have been included to demonstrate the various applications of these biosensors to real samples. Following an introduction into the field that covers the significance of sensing galactose in various fields, the review covers biosensors based on the use of galactose oxidase, with a discussion of methods for their immobilization (via cross-linking, adsorption, covalent bonding and entrapment). This is followed by a short section on biosensors based on the use of galactose dehydrogenase. The conclusion section summarizes the state of the art and addresses current challenges.

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Graphical abstract Fabrication of a disposable screen-printed (a) electrochemical galactose biosensor (b) for real sample analysis and a dummy biosensor (c) for compensating the effect of interferences

     

Three approaches to the development of selective bilayer amperometric biosensors for glucose by in situ electropolymerization

Three different glucose oxidase biosensors for the enzymatic determination of glucose, based on bilayer polymer coatings consisting of polypyrrole (PPy) and poly(o-phenylenediamine), were developed. The electrode substrates are Pt metal, carbon paste and an organic conducting salt (tetrathiafulvalene-tetracyanoquinodimethane), which introduces appreciable variability in the electrode construction process. The three sensors were compared with one another and with bare and PPy monolayer-coated sensors in terms of performance in the determination of glucose in a synthetic serum sample. These configurations provide improved selectivity against the interferences of electroactive species such as ascorbic acid and uric acid, frequently present in biological samples, and the differences between them can be taken advantage of in application to different kinds of samples.     

Uncertainty estimation of ethanol concentration in simulated breath using enzyme-based biosensors

Exhaled breath (EB) contains volatile and nonvolatile compounds that are correlated with physiological processes in the body, and these breath biomarkers hold enormous diagnostic potential when they are adequately measured and monitored. Thus, the development of instrumentation, including enzyme-based biosensors, for breath monitoring applications has been expanding rapidly. In this paper, the process of estimating the overall combined uncertainty in predicting ethanol concentration, u(C _v)_pred, using a calibrated alcohol oxidase-based amperometric biosensor is presented. Components that contributed to u(C _v)_pred were the standard uncertainties associated with simulation of a breath sample with trace ethanol concentration, sampling temperature, biosensor instrumentation, and regression analysis. In both EB and exhaled breath condensate (EBC) sensing, the largest contributor to overall uncertainty was the random effects captured by the regression model at 38.2 % and 39.8 %, respectively, for EB and EBC. This was followed by biosensor instrumentation (34.5 %) and simulation (25.3 %) in EB sensing. The trend was reversed in EBC sensing with EB simulation having a larger contribution (33.8 %) than biosensor instrumentation (25.5 %) owing to a better repeatability of amperometric measurements with aqueous samples. The remaining 2.0 % and 0.9 % were due to breath sampling temperatures in EB and EBC sensing, respectively. This study provides a framework for how to incorporate uncertainty estimation in both breath monitoring and is applicable to biosensing of other breath biomarkers.     

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

Compact amperometric algal biosensors for the evaluation of water toxicity

Unicellular microalga Chlorella vulgaris was entrapped in an alginate gel or a polyion complex membrane immobilized directly on the surface of a transparent indium tin oxide electrode. Photosynthetically generated oxygen of the immobilized algae was monitored amperometically. Responses of the algal biosensor to four toxic compounds, 6-chloro-N-ethyl-N-isopropyl-1,3,5-triazine-2,4-diamine (atrazine), 3-(3,4-dichlorophenyl)-1,1-diethylurea (DCMU), toluene and benzene, were evaluated as inhibition ratios of the reduction current. The concentrations that give 50% inhibition of the oxygen reduction current (IC^′₅₀) for atrazine, DCMU, toluene and benzene were 2.0, 0.05, 1550 and 3000 μmol dm⁻³, respectively. There was a good correlation between these data and those of the conventional standard growth test. In comparison with the conventional algal biosensors based on the Clark-type oxygen electrode, the present sensor is much smaller and less expensive, and its assay time is much shorter (≤200 s).     

Electrocatalytic reduction of hydrogen peroxide on modified graphite electrodes: application to the development of glucose biosensors

The electrocatalytic activities of a series of compact graphites modified with microquantities of platinum metals (Pd or Pt+Pd) towards the electrochemical reduction of hydrogen peroxide were characterised. Operational parameters such as the optimal working potential, the influence of temperature and the resulting electrode characteristics were examined. The benefits of using graphite modified with Pt+Pd (mixture ratio 30%:70%) as the basic transducer in a glucose biosensor with improved sensitivity were demonstrated. It was proven that, under the working conditions chosen, the selected electrode (whether bare or covered with an enzyme layer) did not respond to any glutathione, uric acid or ascorbic acid (which all normally occur in biological fluids) present.     

Simultaneous determination of creatine and creatinine using amperometric biosensors

In order to determine creatine and creatinine amperometric biosensors were proposed. A bienzymatic biosensor based on creatinase (CI) and sarcosine oxidase (SO) was used for the assay of creatine and a trienzymatic biosensor based on CI, SO and creatininase (CA) for the assay of creatinine. The linear concentration ranges are of pmol l⁻¹ to nmol l⁻¹ magnitude order, with very low limits of detection. The biosensors proved high reliability for determination of creatine and creatinine as raw material, and in the pharmaceutical formulation.     

Affinity and enzyme-based biosensors: recent advances and emerging applications in cell analysis and point-of-care testing

The applications of biosensors range from environmental testing and biowarfare agent detection to clinical testing and cell analysis. In recent years, biosensors have become increasingly prevalent in clinical testing and point-of-care testing. This is driven in part by the desire to decrease the cost of health care, to shift some of the analytical tests from centralized facilities to "frontline" physicians and nurses, and to obtain more precise information more quickly about the health status of a patient. This article gives an overview of recent advances in the field of biosensors, focusing on biosensors based on enzymes, aptamers, antibodies, and phages. In addition, this article attempts to describe efforts to apply these biosensors to clinical testing and cell analysis.     

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2+/-0.72 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-2)M(-1)). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination.     

Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

Developmental aspects of amperometric ATP biosensors based on entrapped enzymes

A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5′-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 µm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5?±?0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/µM.     

Mechanisms controlling the sensitivity of amperometric biosensors in flow injection analysis systems

This paper numerically investigates the sensitivity of an amperometric biosensor acting in the flow injection mode when the biosensor contacts an analyte for a short time. The analytical system is modelled by non-stationary reaction-diffusion equations containing a non-linear term related to the Michaelis-Menten kinetics of an enzymatic reaction. The mathematical model involves three regions: the enzyme layer where enzymatic reaction as well as the mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region. The biosensor operation is analysed with a special emphasis to the conditions at which the biosensor sensitivity can be increased and the calibration curve can be prolonged by changing the injection duration, the permeability of the external diffusion layer, the thickness of the enzyme layer and the catalytic activity of the enzyme. The apparent Michaelis constant is used as a main characteristic of the sensitivity and the calibration curve of the biosensor. The numerical simulation was carried out using the finite difference technique.     

Solubilization of carbon nanotubes by Nafion toward the preparation of amperometric biosensors

The ability to solubilize single-wall and multiwall carbon nanotubes (CNT) in the presence of the perfluorinated polymer Nafion is described. Such use of Nafion as a solubilizing agent for CNT overcomes a major obstacle for creating CNT-based biosensing devices. Their association with Nafion does not impair the electrocatalytic properties of CNT. The resulting CNT/Nafion modified glassy-carbon electrodes exhibit a strong and stable electrocatalytic response toward hydrogen peroxide. The marked acceleration of the hydrogen peroxide redox process is very attractive for the operation of oxidase-based amperometric biosensors, as illustrated for the highly selective low-potential (-0.05 V vs Ag/AgCl) biosensing of glucose. These findings open the door for using CNT in a wide range of chemical sensors and nanoscale electronic devices.     

Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids

Screen-printed three-electrode amperometric sensors incorporating L- and/or D-amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D-amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume.     

Self-encapsulation of oxidases as a basic approach to tune the upper detection limit of amperometric biosensors

This study describes a new, basic procedure for the tuning of some analytical parameters of enzymatic biosensors that are based on hydrogen peroxide-producing oxido-reductases. An amperometric biosensor based on glucose oxidase (GOx) (EC 1.1.3.4) from Penicillum vitale, immobilized on a carbon rod electrode by cross-linking with glutaraldehyde, was exploited as a model system for demonstration of the approach described here. Such an important analytical parameter as the upper detection limit was dramatically changed by the formation of a polypyrrole conducting polymer layer by the GOx-induced polymerization of polypyrrole (Ppy). An increase in the upper detection limits for differently modified electrodes was estimated by calculation of the apparent Michaelis-Menten constant [K(M(app))]. A significant increase in the long-term stability of the GOx-based electrode modified by Ppy (GOx/Ppy) was detected compared with that of an unmodified one. Further application of this approach, based on the self-encapsulation of glucose oxidase and other oxidases, is predicted for such biosensors where extension of the detection rate as well as K(M(app)) are required.