Self-containing reactant biochips for the electrochemiluminescent determination of glucose, lactate and choline

A multi-parametric biochip for glucose, lactate and choline has been developed based on luminol/hydrogen peroxide electrochemiluminescence (ECL). The sensing layers developed were composed of enzyme-bound beads co-entrapped in a photopolymer with luminol-charged beads and spotted at the surface of a glassy carbon electrode (GCE). Lactate oxidase, choline oxidase and luminol were immobilised via electrostatic interactions with DEAE (diethylaminoethyl)-Sepharose whereas glucose oxidase was immobilised, after modification with histidine, by chelation on nickel-charged IDA (imidodiacetic acid)-Sepharose. The luminol immobilisation enabled the achievement of micro-biosensors, present at the surface of the same GCE and free of lateral contamination between each spot by the other’s reaction product. After optimisation of the applied potential, the integration time and the luminol charge in the sensing layer, the electrochemiluminescent H₂O₂ sensor exhibited a detection limit of 2 μM and a working range from 2 μM to 0.5 mM.

The multi-biosensor enabled the concomitant detection of glucose, lactate and choline in the ranges 20 μM–2 mM, 2 μM–0.2 mM and 2 μM–0.2 mM, respectively. Glucose and lactate measurements in complex matrix such as human serum, were in good agreement with the reference methods, without internal calibration of the sample, demonstrating the absence of matrix interference with the present analytical system.     

Amperometric biosensor for catechol using electrochemical template process

A novel amperometric biosensor for the determination of catechol was developed accordingly to the electrochemical template procedure. The optimum fabricating conditions of the biosensor were studied. The resulting biosensor with the limit of less than 0.05 μM can be used for detection of catechol in the linear range of 2.5-140 μM. The maximum response current (I_max) and the Michaelis-Menten constant (k′_m) are 3.08 μA and 77.52 μM, respectively. The activation energy (E_a) of the polyphenol oxidase (PPO) catalytic reaction is 25.56 kJ mol⁻¹ in the B-R buffer. The stability of the PANI-CA biosensor fabricated with the electrochemical template process (retains 86% of the original activity after four months) is much higher than that fabricated with one-step and two-step processes (retains 75% of the original activity after four months). The effects of potential and pH on the response current of the biosensor are also described.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

基于空心微针阵列的连续监测葡萄糖传感器

研制了一种基于空心微针阵列并可微痛刺入皮下实时连续监测人体血糖浓度的葡萄糖传感器.微针阵列由等间距的三根微针组成,分别作为工作电极、辅助电极和参比电极,其中工作电极与辅助电极采用表面溅射了Ti/Pt的空心不锈钢微针来制作,采用戊二醛-牛血清白蛋白交联法将葡萄糖氧化酶固定并仅置于作为工作电极的针尖通孔处;参比电极采用Ag/AgCl实心微针制作.测试结果表明:葡萄糖浓度在0～21 mmol·L-1范围内传感器输出电流为线性,灵敏度为0.575 μA/(mmol·L-1),响应时间为16s,且具有很好的抗干扰性.     

Gold nanoparticles-coated eggshell membrane with immobilized glucose oxidase for fabrication of glucose biosensor

This work reports the fabrication and application of a glucose biosensor based on the catalytic effect of gold nanoparticles (AuNPs) on enzymatic reaction for blood glucose determination. AuNPs were initially in situ synthesized on the surface of an eggshell membrane (ESM) which was subsequently immobilized with glucose oxidase (GO_x) to produce a GO_x-AuNPs/ESM. The GO_x-AuNPs/ESM was positioned on the surface of an oxygen electrode to form a GO_x-AuNPs/ESM glucose biosensor. The effects of pH, concentration of phosphate buffer solution and amount of GO_x on the response of the GO_x-AuNPs/ESM glucose biosensor were studied in detail. AuNPs on GO_x/ESM can improve the calibration sensitivity (30% higher than GO_x/ESM without AuNPs), stability (87.3% of its initial response to glucose after 10-week storage) and shortens the response time (<30 s) of the glucose biosensor. The linear working range for the GO_x-AuNPs/ESM glucose biosensor is 8.33 μM to 0.966 mM glucose with a detection limit of 3.50 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose in human blood serum samples and the results compared well to a standard spectrophotometric method commonly used in hospitals. Our work demonstrates that the developed GO_x-AuNPs/ESM glucose biosensor has potential in biomedical analysis.     

Evaluation of amperometric glucose biosensors based on glucose oxidase encapsulated within enzymatically synthesized polyaniline and polypyrrole

In this article potential and suitability of enzymatically synthesized conducting polymers polyaniline (PANI) and polypyrrole (PPY) for fabrication of enzymatic amperometric glucose biosensors were evaluated. The polymerisation of these polymers was induced by catalytic activity of glucose oxidase (GOx) from Penicillium vitale cross-linked by glutaraldehyde (GA) on the graphite rod electrode (GOx-electrode) surface. The main precursors for initiation of polymerisation reactions were hydrogen peroxide as an initiator of polymerisation reaction and β-d-gluconic acid as a medium, which reduced the pH towards acidic one is the most suitable for the formation of PANI and PPY. During the polymerisation reactions the immobilized GOx was self-encapsulated within formed PANI or PPY layers in order to form GOx/PANI- and GOx/PPY-modified electrodes (GOx/PANI-electrode and GOx/PPY-electrode, respectively). Kinetic properties of GOx, which is acting as a biocatalyst in GOx/PANI- and GOx/PPY-electrodes, were studied and results were compared with GOx-electrode. The results show that in both GOx/PANI- and GOx/PPY-electrodes self-encapsulated GOx exhibited different parameters of catalysed reaction kinetics due to increasing diffusion limitations if compared with that of the GOx-electrode and it allowed the detection of glucose in a wider concentration interval. Moreover, both GOx/PANI- and GOx/PPY-electrodes exhibited good operational stability and reproducibility of analytical signal. The electrochemical characteristics of formed PANI and PPY in the GOx/PANI- and GOx/PPY-electrodes were also determined. In addition, the influence of temperature, pH and common interfering compounds on the steady-state current response of modified electrodes were investigated and discussed.     

A new glucose sensor based on encapsulated glucose oxidase within organically modified sol–gel glass

A non-mediated glucose biosensor is reported based on encapsulated glucose oxidase (GOD) within the composite sol–gel glass, which is prepared using optimum concentrations of 3-aminopropyltriethoxy silane, 2-(3, 4-epoxycyclohexyl)-ethyltrimethoxy silane, GOD dissolved in double distilled water and HCl. A white, smooth film of sol–gel glass with controlled thickness is also prepared at the surface of a Pt disk electrode without GOD to study the electrochemistry of ferrocene monocarboxylic acid at the surface of the modified electrode. The electrochemistry of ferrocene monocarboxylic acid at composite sol–gel glass electrode with varying thickness is reported. The GOD-immobilized film over the Pt disk surface shows a yellow colour. The new sol–gel glass in the absence and the presence of GOD is characterized by scanning electron microscopy (SEM). The enzyme-immobilized film of different thickness is made using varying concentrations of soluble sol–gel components applied to the well of the Pt disk electrode. The enzyme is cross-lined with the 3-aminopropyltriethoxysilane, one of the composite component of sol–gel glass using glyoxal at 4°C for 4 h. The response of non-mediated enzyme sensor is studied based on cyclic voltammetry and amperometric measurements. A typical amperometric response of the enzyme sensor having varying thickness of the modified sol–gel glass film is reported. The variation of the response time as a function of the film thickness is reported. The stability of cross-linked GOD to sol–gel glass is found to be more than a month without loss of enzymatic activity when the enzyme sensor is stored at 4°C.     

电化学式葡萄糖传感器阳极的研究

制备了一种石墨粉含量高的混合物导电材料,研究了该材料的导电性能及水中贮存稳定性,并将其作为电化学式葡萄糖传感器的阳极,以此电极作为固定化酶载体得到的葡萄糖氧化酶电极对葡萄糖的响应时间为２０ ～３０ ｓ、灵敏度为６０ ｎＡ／（ｍｍｏｌ·Ｌ－１） 、线性响应范围为０ ．０２ ～７ ．０ ｍ ｍｏｌ／Ｌ,在水溶液中储存２０ 天,传感器的响应值基本保持不变。     

基于应用于生物传感器的新型有机-无机杂化材料

制备了一种应用于生物传感器的新型有机-无机杂化材料,这种杂化材料由二氧化硅溶胶和聚乙烯醇接枝巯基乙酸组成。此杂化材料克服了普通溶胶凝胶容易脆裂的特点,杂化材料中的巯基可以与金原子形成牢固的配位键从而使杂化材料很好地附着在金电极表面。对杂化材料进行了红外光谱、石英电子微天平、原子力显微镜分析。为了检验杂化材料的应用效果,以此杂化材料作为包埋材料在金电极上制备了葡萄糖氧化酶电极。研究了此酶电极的响应速度和稳定性,结果表明此酶电极具有较快的响应速度(响应时间为2.2s)、较好的稳定性(响应电流在70d后衰减9.6%)。     

用于血糖无创检测的葡萄糖传感器研究

研制一种新型葡萄糖传感器,初步实现用于反离子电渗透技术提取皮下组织液的葡萄糖的浓度测量。用铁氰化钾作为电子媒介体,固定在聚环氧乙烷凝胶里的葡萄糖氧化酶与溶液中的葡萄糖催化氧化生成葡萄糖酸和亚铁氰化钾,通过检测该反应产生的氧化还原电流的大小来计算葡萄糖溶液的浓度。新型葡萄糖传感器的检测浓度范围2～22 mmol/L内线性度较好,传感器的一致性测试表明:同一传感器多次测量的偏差不超过2%,反应时间较短,接近于1 s。