Use of biologically designed gold nanowire for biosensor application

A highly sensitive tyrosinase (TYR)-based amperometric biosensor is prepared using biologically designed gold nanowires (AuNWs) for pesticide detection. The AuNWs were synthesized by dodecapeptide Midas-11 and were modified with the formation of self-assembled monolayer (SAM), followed by covalent binding with TYR. The prepared TYR-AuNWs-SPCE (screen printed carbon electrode) was compared with bare, AuNWs-, modified-AuNWs-SPCE by the measurement of cyclic voltammetry. The quantitative relationship between the inhibition percentage and the pesticide concentration at the TYR-AuNWs-SPCE was obtained by measuring the current response in various concentrations of pesticides. The reasonable detection range of parathion was determined to be 0.1 ppt through 10 ppb (R ² =0.990) with 0.087 ppt of detection limits. The higher sensitivity and wider detection range of the TYR-based biosensor was achieved by the use of biologically synthesized AuNWs.     

酶的固定化及在生物传感器的应用

简要介绍了传统的酶固定化方法,对这一领域的新进展进行了综述.对酶生物传感器在食品分析、环境监测、医学和军事上的应用作了介绍,并对其发展前景进行了展望.     

Electrochemical glucose biosensor by electrostatic binding of PQQ-glucose dehydrogenase onto self-assembled monolayers on gold

Efficient binding of enzymes onto the electrode surface has been prerequisite for the construction of sensitive biosensors and biochips. Here, a simple and robust construction of electrochemical glucose biosensor based on pyrroloquinoline quinone-glucose dehydrogenase was demonstrated. The glucose biosensor was fabricated by binding the enzyme onto the anionic self-assembled monolayers on gold electrode via electrostatic interactions. The resulting glucose biosensor gave rise to twofold higher detection sensitivity than that by covalent conjugation under the same condition. Surface plasmon resonance and atomic force microscopy analyses revealed that electrostatic binding of the enzyme leads to much higher surface density of the enzyme. This approach will find wide applications to the development of robust enzyme-based biosensors and biochips.     

A sensitive biosensor for cyanide detection using modified glassy carbon electrode with reduced graphene oxide and immobilization of horseradish peroxidase

Cyanide is a highly poisonous and hazardous substance which may release into the environment from natural sources or industrial effluent; therefore, cyanide detection is a fundamental step to prevent environmental pollution and secure health and safety. In this study, we prepared a sensitive amperometric inhibition biosensor for cyanide detection by immobilization of horseradish peroxidase (HRP) enzyme and reduced graphene oxide (rGO) on the surface of glassy carbon electrode (GCE). To do so, we performed the amperometric measurement by modified GCE to test its efficiency in detecting cyanide. The optimum conditions of pH equal to 7.5, −100 mV applied potential, 0.7 μM mediator concentration, and 0.5 mM substrate concentration were found. Then, experiments were performed at different boundary conditions in a range of 0.1 to 10 μM cyanide concentration at optimal conditions and a low detection limit of 0.01 μM was obtained. Also, the possible mechanism of inhibition was analyzed based on the Michalis–Menten equation and non-competitive inhibition was observed. Due to high sensitivity, low detection limit, and low cost, this biosensor is proposed as a useful method for cyanide determination in real samples.     

l-Lactic acid biosensor based on multi-layered graphene

Pristine graphene platelets and graphene oxide were used as electrode modifiers, aiming the investigation of their electrochemical efficacy towards β-nicotinamide adenine dinucleotide (NADH). The electrochemical detection of NADH is one of the most studied areas of bioelectroanalysis because of the ubiquity of NAD(P)H-based enzymatic reactions in nature. Commercially available graphene and laboratory prepared graphene oxide were used to modify glassy carbon electrodes and the behaviour of such modified electrodes against potassium ferricyanide (III) and NADH was reported. Relying on the graphene-modified transducer, l-lactic dehydrogenase (l-LDH) was successfully immobilised in a 1 % Nafion^® membrane. The developed biosensor, working at +250 mV versus Ag/AgCl reference electrode, was used to assess l-lactic acid in four different types of yogurts, revealing an l-lactic acid concentration ranging between 0.3 and 0.6 %.     

Fabricating a new immobilization matrix based on a conjugated polymer and application as a glucose biosensor

A new monomer named 4-(dihexylamino)9,12-di(thiophen-2-yl)-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (ThBN) is synthesized and used as a potential glucose biosensor after electropolymerization of the ThBN on the graphite pencil electrode. The amount of glucose is determined according to the decrease in the amount of oxygen by using cyclic voltammetry technique. Herein, conjugated polymer of ThBN is used as a immobilization matrix. The synthesized PThBN is found to be effective enzymatic biosensor having wide linear glucose determination range between 2.975 × 10⁻³ and 2.087 mM with a limit of detection of 0.0304 mM and a sensitivity of 0.1326 μA/mM cm². This potential enzymatic biosensor has been also tested in commercial samples and found to be useful to detect the glucose concentration.     

Synthesis of nickel nanosheet/graphene composites for biosensor applications

The growth of graphene-based nanostructures using chemical vapor deposition (CVD) is a promising approach for novel applications. CVD processes typically use high-quality metal films as catalyst substrates, and require accurate control over the experimental conditions. Here, we report the direct synthesis of Ni nanosheet/graphene composites using a DC arc plasma jet CVD method, using Ni(NO₃)₂ as a catalyst precursor. The composites consisted of graphene nanosheets, graphene nanoribbons, and core–shell Ni/graphene nanosheets. In this process, no catalyst substrate was required, and the very high-quality graphene grew at the {1 1 1} plane of the Ni. It was demonstrated that the Ni nanosheet/graphene composites could be used as sensitive films for l-alanine sensing. The strong electrocatalytic properties resulted from the synergetic effects of the graphene, which enhanced the electron transfer, and the high catalytic activity of the Ni.     

DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

In this paper, single-stranded DNA (ss-DNA) is demonstrated to functionalize graphene (GR) and to further guide the growth of PtAu bimetallic nanoparticles (PtAuNPs) on GR with high densities and dispersion. The obtained nanocomposites (PtAuNPs/ss-DNA/GR) were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectrometer (EDS), and electrochemical techniques. Then, an enzyme nanoassembly was prepared by self-assembling glucose oxidase (GOD) on PtAuNP/ss-DNA/GR nanocomposites (GOD/PtAuNPs/ss-DNA/GR). The nanocomposites provided a suitable microenvironment for GOD to retain its biological activity. The direct and reversible electron transfer process between the active site of GOD and the modified electrode was realized without any extra electron mediator. Thus, the prepared GOD/PtAuNP/ss-DNA/GR electrode was proposed as a biosensor for the quantification of glucose. The effects of pH, applied potential, and temperature on the performance of the biosensor were discussed in detail and were optimized. Under optimal conditions, the biosensor showed a linearity with glucose concentration in the range of 1.0 to 1,800 μM with a detection limit of 0.3 μM (S/N = 3). The results demonstrate that the developed approach provides a promising strategy to improve the sensitivity and enzyme activity of electrochemical biosensors.     

Covalent immobilization of glucose oxidase to poly(O‐amino benzoic acid) for application to glucose biosensor

A biosensor for glucose utilizing glucose oxidase (GOX) covalently coupled to poly(o‐amino benzoic acid) (PAB; a carboxy‐group‐functionalized polyaniline) is described. Amperometric response measurements conducted via unmediated and mediated (with ferrocene carboxylic acid and tetrathiafulvalene) reoxidation of GOX show that glucose can be detected over a wide range of concentrations. An enzyme‐conducting polymer‐mediator model provides for better charge transport in a biosensor. The optimal response, obtained at pH 5.5 and 300 K, lies in the 1–40 mM range. A kinetic plot yields the value of the apparent Michaelis–Menten constant, K_m^app. The operational stability of the PAB‐based glucose biosensor was experimentally determined to be about 6 days. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 662–667, 2000     

A novel electrochemical DNA biosensor based on graphene and polyaniline nanowires

A novel DNA biosensor based on oxidized graphene and polyaniline nanowires (PANIws) modified glassy carbon electrode was developed. The resulting graphene/PANIw layers exhibited good DPV current response for the complementary DNA sequences. The good electron transfer activity might be attributed to the effect of graphene and PANIw. Graphene and PANIw nanolayers film with highly conductive and biocompatible nanostructure were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immobilization of the probe DNA on the surface of electrode was largely improved due to the unique synergetic effect of graphene and PANIw. Under optimum conditions, the biosensor exhibited a fast amperometric response, high sensitivity and good storage stability for monitoring DNA. The current response of the sensor increases linearly with the concentration of target from 2.12 × 10⁻⁶ to 2.12 × 10⁻¹² mol l⁻¹ with a relative coefficient of 0.9938. The detection limit (3σ) is 3.25 × 10⁻¹³ mol l⁻¹. The results indicate that this modified electrode has potential application in sensitive and selective DNA detection.     

肌醇六磷酸钙膜固定辣根过氧化酶制备过氧化氢生物传感器

利用肌醇六磷酸钙(Ca-IP6)的磷酸酯键及Ca2+对HRP具有静电吸引和螯合作用,将其自组装于玻碳电极(GCE)表面,制备Nafion/HRP/Ca-IP6/GCE生物传感器。用紫外-可见吸收光谱、电化学阻抗光谱和循环伏安法对Nafion/HRP/Ca-IP6/GCE膜进行了表征。实验结果表明,吸附了HRP仍然能够保持原有的生物活性,实现了辣根过氧化物酶在电极表面的直接电子转移。该修饰电极对过氧化氢有很好的电催化活性,线性范围为2.67×10-7~1.067×10-6mol/L,最低检测限为1.3×10-7mol/L(信噪比S/N=3),米氏常数为0.259 mmol/L。该生物传感器拥有较低的检测限,较高的电催化效率和较好的稳定性及重现性。     

An electrochemical immunosensor for ochratoxin A based on immobilization of antibodies on diazonium-functionalized gold electrode

A novel electrochemical immunosensor for sensitive detection of ochratoxin A (OTA) was reported. An electrochemical and chemical reaction protocol was elaborated to modify the gold electrode. A screen-printed gold electrode (SPGE) was modified with a layer of 4-nitrophenyl, assembled from 4-nitrophenyl diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were electrochemically reduced to amines followed by activation with glutaraldehyde to give a stable intermediate derivative that covalently binds antibodies against OTA during the second step, thereby tailoring an immunosensor for ochratoxin A. The utility of the electrochemical immunosensor for a competitive immunoassay was demonstrated. A competition between OTA and fixed concentration of a horseradish peroxidase-labeled OTA (OTA-HRP) for the immobilized antibodies was realized. The activity of the bound OTA-HRP was electrochemically measured by chronoamperometry (CA) using 3,3′,5,5′-tetramethylbenzidine (TMB) as substrate. The immunosensor obtained using this novel approach enabled a detection limit of 12 ng mL⁻¹ and a dynamic range up to 60 ng mL⁻¹ of OTA. Precision, accuracy and stability studies were satisfactory for the developed immunosensor.     

Synthesis of few-layer graphene over gold nanoclusters supported on MgO

We present the successful design and synthesis of few-layer graphenes over an Au/MgO catalytic system by chemical vapor deposition with methane as the carbon source. The resulting structures were composed of a few graphitic layers, and the highest synthesis yield was found to be 62% when the methane flow rate was 120 mL/min. The dimension of the in-plane crystallites was found to increase with the reaction time. A simple purification step was used to remove the MgO support that resulted in graphene sheets decorated with Au nanoparticles. Such an approach makes it possible to synthesize high purity Au–graphene nanostructured composites by using a single reaction step and without the need for additional functionalization or attachment.     

Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode

A simple and sensitive DNA impedance sensor was prepared for the detection of chronic lymphocytic leukemia. The DNA electrochemical biosensor is worked based on the electrochemical impedance spectroscopic (EIS) detection of the sequence-specific DNA related to chronic lymphocytic leukemia. The ssDNA probe was immobilized on the surface of the gold nanoparticles. Compared to the bare gold electrode, the gold nanoparticles-modified electrode could improve the density of the probe DNA attachment and hence the sensitivity of the DNA sensor greatly. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were performed in a solution containing 1.0 mmol L⁻¹ K₃[Fe(CN)₆]/K₄[Fe(CN)₆] and 50 mmol L⁻¹ phosphate buffer saline pH 6.87 plus 50 mmol L⁻¹ KCl. In the CV studied, the potential was cycled from 0.0 to +0.65 V with a scan rate of 50 mV s⁻¹. Using EIS, the difference of the electron transfer resistance (ΔR_et) was linear with the logarithm of the complementary oligonucleotides sequence concentrations in the range of 7.0 × 10⁻¹²–2.0 × 10⁻⁷ mol L⁻¹, with a detection limit of 1.0 × 10⁻¹² mol L⁻¹. In addition, the DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles.     

氧化石墨烯和过渡金属碳/氮化合物固定化酶

二维纳米材料具有高机械强度和比表面积、大量表面官能团、良好的亲水性及生物相容性,是固定化酶的良好载体。本文选取经典的氧化石墨烯（GO）以及新型的过渡金属碳/氮化合物（MXenes）,分别介绍了它们的制备方法和结构、物理和化学性质,综述了它们在固定化酶领域的应用研究,并进行了比较。文中指出：GO由石墨烯经化学氧化再剥离制得,MXenes由其前体经刻蚀制得,不同的氧化或刻蚀方法制得的材料在组成、结构、性能等方面存在差异。GO表面的可反应官能团更多,包括羟基、羧基和环氧基,故在固定化酶领域应用广泛。MXenes固定化酶则主要利用表面的羟基反应或负电荷吸附,目前主要用于制备生物传感器。最后指出这两种材料还存在制备效率低、纳米片易聚集、循环利用性差等问题。今后的发展方向是要开发更为简单和安全的材料制备方法,探索更为有效的插层和剥离手段以及改善固定化酶的回收策略,进一步推进二维纳米材料在固定化酶领域的应用。     

Amperometric study of hydrogen peroxide biosensor with butadiene rubber as immobilization matrix

A carbon paste electrode bound by butadiene rubber has been newly constructed and its electrochemical properties have been investigated to test the practicability of the enzyme electrode. The binder of carbon powder was butadiene rubber dissolved in toluene and ground cabbage tissue was embedded in the matrix as an enzyme source. The electrode, which showed a mechanical robustness after volatilization of solvent, displayed good catalytic power (detection limit = 2.5 × 10^?5 M, S/N = 2) and electrochemically irreversible characteristics. Its symmetry factor and the exchange current density of the electrode used were 0.23 and 1.71 × 10^?3 A cm^?2, respectively.     

Assembled gold nanoparticles on nitrogen-doped graphene for ultrasensitive electrochemical detection of matrix metalloproteinase-2

A new electrochemical immunosensor was developed for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), which is one of the key biomarkers in blood. In our approach, an effective assembly of well-defined gold nanoparticles on nitrogen-doped graphene sheets was demonstrated. The composite facilitated robust immobilization of antibodies, promoted electron transfer and exhibited excellent electrochemical activity, which are suitable for biosensing. The design of the immunosensor also involved a polydopamine functionalized graphene oxide hybrid conjugated to horseradish peroxidase-secondary antibodies by covalent bonds as a multi-labeled and biocompatible probe to increase the electrochemical response. This novel signal amplification strategy with a sandwich-type immunoreaction significantly enhanced the sensitivity of detection of biomarkers. The proposed immunosensor displayed excellent analytical performance in the detection of MMP-2 ranging from 0.0005 to 50 ng mL⁻¹, with a detection limit of 0.11 pg mL⁻¹. Furthermore, it not only exhibited good stability with adequate reproducibility and accuracy, but also demonstrated efficiency in the detection of MMP-2 in real samples.