基于碳纳米管和铁氰酸镍纳米颗粒协同作用的葡萄糖生物传感器

将制备的铁氰酸镍纳米颗粒(NiNP)与多壁碳纳米管(CNT)混合, 分散于壳聚糖溶液中, 形成一种新的纳米复合成分(NiNP-CNT-CHIT), 将其修饰在玻碳电极表面. 新复合膜体现了NiNP和CNT之间的协同作用, 由于CNT的良好的传递电子性能, 促使NiNP催化氧化还原能力有了较大的提高. 此NiNP-CNT-CHIT复合膜修饰的玻碳电极在较低电位下对过氧化氢具有良好的电催化性能, 与NiNP-CHIT膜比较, 测定H₂O₂的灵敏度增大了50倍. 通过戊二醛在电极表面固定葡萄糖氧化酶制备了一种新的葡萄糖传感器. 该传感器在－0.2 V下对葡萄糖的线性范围为0.05～10 mmol/L, 检测下限为10 μmol/L.     

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

Multiwalled Carbon Nanotubes‐Chitosan Modified Single‐Use Biosensors for Electrochemical Monitoring of Drug‐DNA Interactions

A multiwalled carbon nanotubes (CNT)‐chitosan (CHIT) modified pencil graphite electrode (CNT‐CHIT/PGE) was developed for the first time herein for electrochemical monitoring of the interaction of an anticancer drug, mitomycin C (MC) and DNA. The characterization of unmodified PGE, CHIT/PGE, CNT/PGE and CHIT‐CNT/PGE were performed by scanning electron microscopy and cyclic voltammetry techniques. The oxidation signals of MC and guanine were measured before and after interaction at the surface of CNT‐CHIT/PGEs using differential pulse voltammetry. Electrochemical impedance spectroscopy technique was also successfully utilized for monitoring of the interaction process at the surface of CNT‐CHIT/PGEs in different interaction times.     

Measurement of binding kinetics between PI3-K and phosphorylated IGF-1R using a surface plasmon resonance biosensor

A high-performance amperometric glucose biosensor was developed, based on immobilization of glucose oxidase (GOx) on a copper (Cu) nanoparticles/chitosan (CHIT)/carbon nanotube (CNT)-modified glassy carbon (GC) electrode. The Cu and CNT had a synergistic electrocatalytic effect toward the reduction of hydrogen peroxide in the matrix of biopolymer CHIT. The Cu/CHIT/CNT modified GC electrode could amplify the reduction current of hydrogen peroxide greatly. Besides, the Cu/CHIT/CNT modified GC electrode reduces hydrogen peroxide at a much lower applied potential and inhibit the responses of interferents. With GOx as an enzyme model, a new glucose biosensor was fabricated. The sensitivity of the sensor is due not only to the large microscopic area but also to the high efficiency of transformation of H₂O₂ generated by enzymatic reaction to current signal. The biosensor exhibited excellent sensitivity (the detection limit is down to 0.02 mM), fast response time (less than 4 sec), wide linear range (from 0.05 to 12 mM), and perfect selectivity. Correspondence: Wanzhi Wei, State key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China     

Direct electrochemistry of glucose oxidase immobilized on NdPO4 nanoparticles/chitosan composite film on glassy carbon electrodes and its biosensing application

The direct electrochemistry of glucose oxidase (GOx) immobilized on a composite matrix based on chitosan (CHIT) and NdPO(4) nanoparticles (NPs) underlying on glassy carbon electrode (GCE) was achieved. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. In deaerated buffer solutions, the cyclic voltammetry of the composite films of GOx/NdPO(4) NPs/CHIT showed a pair of well-behaved redox peaks that are assigned to the redox reaction of GOx, confirming the effective immobilization of GOx on the composite film. The electron transfer rate constant was estimated to be 5.0 s(-1). The linear dynamic range for the detection of glucose was 0.15-10 mM with a correlation coefficient of 0.999 and the detection limit was estimated at about 0.08 mM (S/N=3). The calculated apparent Michaelis-Menten constant was 2.5 mM, which suggested a high affinity of the enzyme-substrate. The immobilized GOx in the NdPO(4) NPs/CHIT composite film retained its bioactivity. Furthermore, the method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Direct electrochemistry of glucose oxidase immobilized on porous carbon nanofiber/room temperature ionic liquid/chitosan composite film and its biosensing application

The direct electron transfer of glucose oxidase (GOD) immobilized on a composite matrix based on porous carbon nanofibers (PCNFs), room-temperature ionic liquid (RTIL), and chitosan (CHIT) underlying on a glassy carbon electrode was achieved. The combination of the PCNFs, RTIL, and CHIT provided a suitable microenvironment for GOD to transfer electron directly. In deaerated buffer solutions (pH 7.0), the cyclic voltammetry of the GOD/PCNFs/RTIL/CHIT composite films showed a pair of well-defined redox peaks with the formal potential of −0.45 V (vs. SCE). The synergistic effort of the PCNFs, RTIL, and CHIT also promoted the stability of GOD in the composite film and retained its bioactivity.     

Amperometric biosensing of glutamate using carbon nanotube based electrode

Amperometric biosensing of glutamate using nanobiocomposite derived from multiwall carbon nanotube (CNT), biopolymer chitosan (CHIT), redox mediator meldola’s blue (MDB) and glutamate dehydrogenase (GlDH) is described. The CNT composite electrode shows a reversible voltammetric response for the redox reaction of MDB at −0.15 V; the composite electrode efficiently mediates the oxidation of NADH at −0.07 V, which is 630 mV less positive than that on an unmodified glassy carbon (GC) electrode. The CNTs in the composite electrode facilitates the mediated electron transfer for the oxidation of NADH. The CNT composite electrode is highly sensitive (5.9 ± 1.52 nA/μM) towards NADH and it could detect as low as 0.5 μM of NADH in neutral pH. The CNT composite electrode is highly stable and does not undergo deactivation by the oxidation products. The electrode does not suffer from the interference due to other anionic electroactive compounds such as ascorbate (AA) and urate (UA). Separate voltammetric peaks have been observed for NADH, AA and UA, allowing the individual or simultaneous determination of these bioanalytes. The glutamate biosensor was developed by combining the electrocatalytic activity of the composite film and GlDH. The enzymatically generated NADH was electrocatalytically detected using the biocomposite electrode. Glutamate has been successfully detected at −0.1 V without any interference. The biosensor is highly sensitive, stable and shows linear response. The sensitivity and the limit of detection of the biosensor was 0.71 ± 0.08 nA/μM and 2 μM, respectively.     

新型CNT/nano-TiO_2复合膜电极的制备及其异相电催化性能

采用溶胶-凝胶法制备了碳纳米管/纳米TiO2(CNT/nano-TiO2)复合溶胶,通过提拉法将复合溶胶涂覆在Ti基体上制得CNT/nano-TiO2复合膜修饰电极(C电极),其电化学性能经循环伏安、计时库仑、交流阻抗谱(EIS)等方法研究.研究结果表明,CNT可阻碍nano-TiO2粒子团聚.在循环伏安图中,C电极的氧化还原峰电流比nano-TiO2膜修饰电极(P 电极)的高出两倍多.通过对草酸溶液的异相电催化反应进一步证明C电极比P电极具有更高的电催化活性,而且对双氧水也有很强的异相电催化还原能力.     

固载于壳聚糖-纳米金复合膜修饰玻碳电极上的葡萄糖氧化酶的直接电化学研究及其生物传感应用

通过将葡萄糖氧化酶固载于壳聚糖-纳米金复合膜内所构置的传感器,实现了葡萄糖氧化酶的直接电化学,并采用循环伏安法与电化学阻抗法对修饰电极进行了表征。研究表明：在除氧缓冲溶液中,葡萄糖氧化酶-壳聚糖-纳米金复合膜修饰电极表现出一对良好的氧化还原峰,这对峰归因于葡萄糖氧化酶的氧化还原,证明葡萄糖氧化酶被成功固载于复合膜内。电子传递速率常数为15.6 s-1,说明葡萄糖氧化酶的电活性中心与电极之间的电子传递很快。将壳聚糖与纳米金相结合还提高了葡萄糖氧化酶在复合膜内的稳定性并保持其生物活性,并可以用于葡萄糖检测。计算得到其表观米氏常数为10.1 mmol·L-1。而且,该生物传感器可以用于血样中葡萄糖含量的测定。     

Coulometric determination of NAD+ and NADH in normal and cancer cells using LDH, RVC and a polymer mediator

An electrochemical method for the measurement of NAD(+) and NADH in normal and cancer tissues using flow injection analysis (FIA) is reported. Reticulated vitreous carbon (RVC) electrodes with entrapped l-lactate dehydrogenase (LDH) and a new redox polymer containing covalently bound toluidine blue O (TBO) were employed for this purpose. Both NAD(+) and NADH were estimated coulometrically based on their reaction with LDH. The latter was immobilized on controlled pore glass (CPG) by cross-linking with glutaraldehyde and packed within the RVC. The concentrations of NAD(+) and NADH in the tissues, estimated using different electron mediators such as ferricyanide (FCN), meldola blue (MB) and TBO have also been compared. The effects of flow rate, pH, applied potential (versus Ag/AgCl reference) and adsorption of the mediators have also been investigated. Based on the measurements of NAD(+) and NADH in normal and cancer tissues it has been concluded that the NADH concentration is lower, while the NAD(+) concentration is higher in cancer tissues. Amongst the electron mediators TBO was found to be a more stable mediator for such measurements.     

碳纳米管的快速功能化及电催化

将耐尔兰(Nile Blue, NB)分子修饰到碳纳米管(CNT)表面形成NB-CNT纳米复合体, 谱学结果表明, NB不仅能快速、高效地修饰到CNT表面, 而且还能有效地改善CNT在水溶液中的分散性能. 将NB-CNT修饰到玻碳(GC)电极表面制备了NB-CNT/GC电极, 循环伏安结果显示, 其伏安曲线上不仅表现出一对良好的、几乎对称的NB单体的氧化还原峰, 式量电位E⁰'几乎不随扫速而变化[其平均值为(－0.422±0.002) V (vs. SCE, 0.1 mol/L PBS, pH 7.0)]; 而且还显示出NB聚合体分子的氧化还原峰, E⁰'为－0.191 V (100 mV/s时). 进一步的实验结果表明, NB和CNT对NADH(即还原型β-烟酰胺腺嘌呤二核苷酸, 又称还原型辅酶I)的电化学氧化具有协同催化作用, 能使其氧化过电位降低多于560 mV; NB-CNT/GC电极还能较好地响应脱氢酶催化底物氧化过程中体系内NADH浓度的变化. 本文对碳纳米管功能化方法具有简单快速、电极制作容易以及催化效率高等优点, NB-CNT/GC电极有望在制作脱氢酶传感器方面得到应用.     

Disposable electrochemical biosensor with multiwalled carbon nanotubes-chitosan composite layer for the detection of deep DNA damage.

A novel electrochemical DNA-based biosensor for the detection of deep DNA damage was designed employing the bionanocomposite layer of multiwalled carbon nanotubes (MWNT) in chitosan (CHIT) deposited on a screen printed carbon electrode (SPCE). The biocomponent represented by double-stranded (ds) herring sperm DNA was immobilized on this composite using layer-by-layer coverage to form a robust film. Individual and complex electrode modifiers are characterized by a differential pulse voltammetry (DPV) with the DNA redox marker [Co(phen)(3)](3+), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with [Fe(CN)(6)](3-) as a redox probe in a phosphate buffer solution (PBS). A good correlation between the CV and EIS parameters has been found, thus confirming a strong effect of MWNT on the enhancement of the electroconductivity of the electrode surface and that of CHIT on the MWNT distribution at the electrode surface. Differences between the CV and EIS signals of the electrodes without and with DNA are used to detect deep damage to DNA, advantageously using simple working procedures in the same experiment.     

Electrochemical biosensors based on redox carbon nanotubes prepared by noncovalent functionalization with 1,10-phenanthroline-5,6-dione

To improve the electrocatalytic activities of carbon nanotubes (CNT) towards the oxidation of nicotinamide adenine dinucleotide (NADH), we derive them with a redox mediator, 1,10-phenanthroline-5,6-dione (PD), by the noncovalent functionalization method. The redox carbon nanotubes (PD/CNT/GC) show excellent electrocatalytic activities towards the oxidation of NADH (catalytic reaction rate constant, k(h) = 7.26 × 10(3) M(-1) s(-1)), so the determination of NADH can be achieved with a high sensitivity of 8.77 μA mM(-1) under the potential of 0.0 V with minimal interference. We also develop an amperometric ethanol biosensor by integration of alcohol dehydrogenase (ADH) within the redox carbon nanotubes (PD/CNT/GC). The ethanol biosensor exhibits a wide linear range up to 7 mM with a lower detection limit of 0.30 mM as well as a high sensitivity of 10.85 nA mM(-1).     

Stabilization role of a phenothiazine derivative on the electrocatalytic oxidation of hydrogen via Aquifex aeolicus hydrogenase at graphite membrane electrodes

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage.     

Effect of Dosimetric and Physiological Factors on the Lethal Photosensitization of Porphyromonas gingivalis in vitro

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui-dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (³H-TBO). In the presence of TBO, a light dose-dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.     

Electrochemical Study of Chitosan‐SiO2‐MWNT Composite Electrodes for the Fabrication of Cholesterol Biosensors

We report a novel composite electrode made of chitosan‐SiO₂‐multiwall carbon nanotube (CHIT‐SiO₂‐MWNT) composite coated on the indium‐tin oxide (ITO) glass substrate. Cholesterol oxidase (ChOx) was covalently immobilized on the CHIT‐SiO₂‐MWNT/ITO electrode that resulted in a ChOx/CHIT‐SiO₂‐MWNT/ITO cholesterolactive bioelectrode. The CHIT‐SiO₂‐MWNT/ITO and ChOx/CHIT‐SiO₂‐MWNT/ITO electrodes were characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of various parameters was investigated, including the applied potential, pH of the medium, and the concentration of the enzyme on the performance of the biosensor. The cholesterol bioelectrode exhibited a sensitivity of 3.4 nA/ mgdL^?1 with a response time of five seconds. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode retained its original response after being stored for six months. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode showed a linear current response to the cholesterol concentration in the range of 50–650 mg/dL.     

Rapid functionalization of carbon nanotube and its electrocatalysis

It was reported that carbon nanotube (CNT) was functionalized with the electroactive Nile blue (NB), which is a phenoxazine dye, by a method of adsorption to form a NB-CNT nanocomposite. The NB-CNT nanocomposite was characterized by several spectroscopic techniques, for example, Ultraviolet-visible spectroscopy (UV-VIS), Fourier transform infrared (FTIR), Raman spectroscopy and scanning electron microscopy (SEM) etc., and the results showed that NB could rapidly and effectively be adsorbed on the surface of CNT with a high stability without changing the native structure of NB and the structure properties of CNT. Moreover, it was shown that the dispersion ability of CNT in aqueous solution had a significantly improvement after CNT functionalized with NB even at a level of high concentration, for example, 5 mg of NB-CNT per 1 mL of H₂O. The NB-CNT/ glasssy carbon (GC) electrode was fabricated by modifying NB-CNT nanocomposite on the GC electrode surface and its electrochemical properties were investigated by cyclic voltammetry. The cyclic voltammetric results indicate that CNT can improve the electrochemical behavior of NB and greatly enhance its redox peak currents. While the NB-CNT/GC electrode exhibited a pair of well-defined and nearly symmetrical redox peaks with the formal potential of (−0.422±0.002) V (versus SCE, 0.1 mol/L PBS, pH 7.0), which was almost independent on the scan rates, for electrochemical reaction of NB monomer; and the redox peak potential of NB polymer located at about −0.191 V. The experimental results also demonstrated that NB and CNT could synergistically catalyze the electrochemically oxidation of NADH (β-nicotinamide adenine dinucleotide, reduced form) and NB-CNT exhibited a high performance with lowing the overpotential of more than 560 mV. The NB-CNT/GC electrode could effectively sense the concentration of NADH, which was produced during the process of oxidation of substrate (e.g. ethanol) catalyzed by dehydrogenase (e.g. alcohol dehydrogenase). The presented method for functionalization of CNT had several advantages, such as rapid and facile CNT functionalization, easy electrode fabrication and high electrocatalytic activity, etc., and could be used for fabrication electrochemical biosensor on the basis of dehydrogenase. __________ Translated from Acta Chimica Sinica, 2007, 65(1): 1–9 [译自: 化学学报]     

Chitosan based hydrogel microspheres as drug carriers

Chitosan/tripolyphosphate (CHIT/TPP) and chitosan/tripolyphosphate/chondroitin sulfate (CHIT/TPP/CHS) core-shell type microspheres were prepared by polyelectrolyte complexation in order to develop a biocompatible matrix for drug delivery. The continual method using a multi-loop reactor under sterile conditions was applied for microsphere preparation. All the types of microspheres produced were spherical in shape and had a porous structure. The mechanical resistance of the microspheres increased in the presence of CHS as the second polyanion, which toughened the microsphere shell structure. For a drug release application, the process of microsphere preparation was modified by dissolving ofloxacin (OFL), the fluoroquinolone antibiotic, in CHIT solution before complex formation. This study shows the difference in OFL release comparing the microspheres CHIT/TPP and CHIT/TPP/CHS and implies the potential to control this process.     

壳聚糖-纳米金-葡萄糖氧化酶生物传感器的制备及其应用于葡萄糖的测定

采用了一种简便快捷的电沉积方法制备了壳聚糖-纳米金复合膜并应用于葡萄糖生物传感器的构建.氯金酸和壳聚糖的混合液在玻碳电极表面电化学还原为金纳米粒子,再将葡萄糖氧化酶通过戊二醛交联的方式固定在纳米金复合膜修饰的玻碳电极表面,制成一种新型的葡萄糖氧化酶生物传感器.该传感器对葡萄糖的响应十分快速,在5 S内即达到平衡.测定葡萄糖的线性范围为20μmol·L-1～5 mmol·L-1,检出限(3S/N)为12μmol·L-1.     

Chitosan‐Glutamate Oxidase Gels: Synthesis,Characterization, and Glutamate Determination

The biopolymer chitosan (CHIT) was chemically modified with glutaric dialdehyde (GDI) and used for the covalent immobilization of enzyme glutamate oxidase (GmOx). The relationships between the loaded, retained, and active units of GmOx in the CHIT‐GDI‐GmOx gels were determined by electrochemical assays. The latter indicated that on average ca. 95% of the GmOx was retained in the CHIT‐GDI matrix that was loaded with 0.10–3.0 units of the enzyme. The maximum activity of the GmOx immobilized in the gels corresponded to ca. 5% of the activity of the free enzyme. Platinum electrodes coated with CHIT‐GDI‐GmOx gels (films) were used as amperometric biosensors for glutamate. Such biosensors displayed good operational and long‐term stability (at least 11 h and 100 days, respectively) in conjunction with low detection limit of 0.10 μM glutamate (S/N=3), linear range up to 0.5 mM (R²=0.991), sensitivity of 100 mA M^?1 cm^?2, and short response time (t_90%=2 s). This demonstrated an efficient signal transduction in the Pt/CHIT‐GDI‐GmOx+glutamate system. The CHIT‐GDI‐GmOx gels constitute a new biosensing element for the development of glutamate biosensors.