基于铂/碳球的乙酰胆碱酯酶传感器在农药乐果检测中的应用研究

基于农药乐果对乙酰胆碱酯酶的抑制作用,构建生物传感器,实现了农药乐果的快速、高灵敏检测。合成了纳米材料铂/碳球(Pt/Cs),利用其比表面积大、导电性好的优势,构建乙酰胆碱酯酶(ACh E)传感器。铂/碳球修饰电极比裸电极的阻抗更低,峰电流增加了147.06%,说明该材料能很好地保持酶的催化活性。在最优实验条件下,用ACh E传感器检测农药乐果,在1.0×10~(-9)~1.0×10~(-6)g/L范围,乐果浓度的负对数与抑制率呈良好的线性关系,其检出限为7.3×10~(-12)g/L(按抑制率为10%计算)。对纺织品样品进行加标回收实验,测得回收率为86.2%~101.7%。     

Amine-terminated organosilica nanosphere functionalized prussian blue for the electrochemical detection of glucose

A new glucose biosensor had been developed by immobilizing positively charged gold nanoparticles (PGNs) on organosilica nanosphere functionalized prussian blue (OSiFPB)-modified gold electrode. The OSiFPB compound could not only effectively prevent the leakage of the PB mediator during measurements, but also easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore, with the negatively charged surface of OSiFPB, this film could be used as an interface to adsorb the PGNs, which provided a congenial microenvironment for adsorbing biomolecules and decreased the electron-transfer impedance. So, with glucose oxidase as a model biomolecular, the proposed sensor showed rapid and highly sensitive amperometric response to glucose and this immobilization approach effectively improved the stability of the electron-transfer mediator. This work would be promising for construction of biosensor and bioelectronic devices.     

Electrocatalytic activity of horseradish peroxidase/chitosan/carbon microsphere microbiocomposites to hydrogen peroxide

Colloidal carbon microspheres (CMS) are dispersed in chitosan (CHIT) solution to form an organic-inorganic hybrid with excellent micro-environment for the immobilization of biomolecules. A novel amperometric biosensor for the determination of hydrogen peroxide (H(2)O(2)) has been constructed by entrapping horseradish peroxidase (HRP) in as-synthesized CMS/CHIT hybrid. The modification of glassy carbon electrode is made by a simple solution-evaporation method. The electrochemical properties of the biosensor are characterized in electrochemical methods. The proposed biosensor shows high sensitive determination and fast response to H(2)O(2) at -0.15 V. The constructed HRP/CHIT/CMS/GC electrode also exhibits a fine linear correlation with H(2)O(2) concentration. The calculated value of the apparent Michaelis-Menten constant, 2.33 mM, suggests that the HRP in CMS/CHIT hybrid keeps its native bioactivity and has high affinity for H(2)O(2).     

空壳纳米金修饰的新型DNA传感器

利用新型材料金纳米空球, 通过层层修饰的技术, 分别将壳聚糖、空壳纳米金、L-半胱氨酸、细胞色素c以及ssDNA探针修饰到玻碳电极表面, 制备了一种新型的DNA生物传感器. 以紫外及透射电子显微镜(TEM)表征了空壳纳米金, 以循环伏安法、阻抗谱图等电化学方法研究了传感器的特性, 通过原子力显微镜方法观察了该DNA生物传感器不同层之间的形态差异. 结果表明, 该修饰电极所吸附的ssDNA探针为1.672×10^―10 mol•cm^－2. 在指示剂柔红霉素的帮助下, DNA探针可与互补的DNA进行杂交, 借此以微分脉冲伏安法测定DNA.     

基于拟糖蛋白的电化学阻抗生物传感器用于糖与蛋白质相互作用的研究

以D-甘露糖和伴刀豆球蛋白(ConA)的相互作用为研究对象,利用美拉德反应将D-甘露糖共价结合到负载蛋白牛血清蛋白(BSA)的表面形成拟糖蛋白,然后将拟糖蛋白固定到玻碳电极表面,以拟糖蛋白表面的D-甘露糖为分子识别物质,构建了检测伴刀豆球蛋白(ConA)的电化学阻抗传感器。拟糖蛋白制备过程简单,D-甘露糖负载量大,在空间中提供多个结合位点,因此能与ConA形成多价复合物,提高了传感器的灵敏度。该传感器的响应值与ConA浓度的对数在5.0×10-11~5.0×10-9mol/L之间呈良好的线性关系,检出限为1.7×10-11mol/L,D-甘露糖和ConA之间的结合常数为2.6×106L/mol。该方法简单,可适用于不同糖和蛋白质相互作用的研究,为构建高灵敏度的电化学阻抗传感器提供了新思路。     

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

纳米金颗粒增强信号的电化学生物传感器用于谷胱甘肽和半胱氨酸的检测

构建了一种高灵敏检测谷胱甘肽(GSH)和半胱氨酸(Cys)的新型电化学生物传感器. 先将富含T碱基的DNA1和DNA2探针分别修饰在金电极和纳米金颗粒(AuNPs)上, 再加入Hg²⁺, 通过形成T-Hg²⁺-T结构使AuNPs结合到金电极表面. 当加入GSH(或Cys)后, GSH(或Cys)可以竞争结合T-Hg²⁺-T结构中的Hg²⁺, 使AuNPs离开电极表面. 由于AuNPs上修饰的DNA探针能够静电吸附大量电活性物质六氨合钌(RuHex), 因此该过程可引起计时电量信号的显著变化, 据此实现了GSH(或Cys)的高灵敏检测. 该传感器的检出限达10 pmol/L, 比荧光法或比色法降低了2~3个数量级. 实验结果表明, 该传感器具有较好的选择性.     

Electrochemical Fabrication of Prussian Blue Nanocube‐decorated Electroreduced Graphene Oxide for Amperometric Sensing of NADH

In this study, Prussian blue (PB) film on the electroreduced graphene oxide (ERGO)‐modified Au electrode surface (ERGO/PB) is easily prepared by means of cyclic voltammetric technique in the mixture of K₃Fe(CN)₆ and FeCl₃. Its electrochemical behaviors for NADH biosensor are studied. The structural and morphological characters of modified electrode material are analyzed with using of XPS, XRD, Raman, EDS, and SEM techniques. ERGO/PB hybrid nanocomposite for NADH biosensor is exhibited to the higher catalytic effect (linear range from 1.0 to 100 μM, detection limit of 0.23 μM at S/N=3) compared to naked Au, ERGO‐modified Au, and PB‐modified Au electrodes. In addition to, ERGO/PB electrode was used to voltammetric and amperometric detection of H₂O₂. ERGO/PB electrodes also showed the same behavior as the NADH sensor. This ERGO/PB‐modified electrode supplied a simple, new, and low‐cost route for amperometric sensing of both NADH and H₂O₂.     

Quantitative Analysis of a Promising Cancer Biomarker,Calretinin, by a Biosensing System Based on Simple and Effective Immobilization Process

Calretinin (CAL) is calcium binding protein, and its levels in blood and cerebrospinal fluids are increased, since its expression is increased various cancer types. A novel biosensor system fabricated by immobilization of a specific antibody to CAL, anti‐Calretinin (anti‐CAL), onto a gold electrode surface via an effective covalent binding method using mercaptohexanol, epichlorohydrin, and ethanolamine was reported for the sensitive, selective, and accurate analysis of CAL. The proposed biosensor showed a linear calibration range between 1 ng/mL and 5 ng/mL. LOD and LOQ values were determined as 0.11 ng/mL and 0.38 ng/mL, respectively. The standard deviation related to the reproducibility of the new biosensor system was calculated as 3.95 %. Lastly, in order to state the applicability of the biosensor to early diagnosis of CAL in practice, artificial serum samples spiked with CAL have been analyzed by the proposed biosensor.     

Detection of Wild-Type Hypoxanthine Guanine Phosphoribosyl Transferase of Lymphocytes in Gamma-Irradiated Mice with Surface Plasmon Resonance

A quick and sensitive detection of the wild-type hypoxanthine guanine phosphoribosyl transferase (HPRT), which is known as a biomarker for radiation exposure, was developed. The conventional HPRT measurement technique is to detect the mutant HPRT, which is time-consuming and has low sensitivity. In this study, the wild-type HPRT was detected as a gamma radiation biomarker using a surface plasmon resonance (SPR) biosensor with 6-thioguanine (6-TG) as a probe and the anti-HPRT antibody as a signal amplification factor. First, we used this system to measure the wild-type HPRT dissolved in PBS. Six-TG immobilized on the surface can specifically detect the wild-type HPRT, and the anti-HPRT antibody enhances about 10 times the primary signal produced by the binding of the wild-type HPRT with 6-TG. A linear relationship (r = 0.991) was obtained between the concentration of the wild-type HPRT and the enhanced signal. The low detection limit (LDL) is 2.1 ng/mL. The regeneration using glycine-HCl was also investigated. Six-TG immobilized on the surface can be used in 9 injection-regeneration cycles. The relative standard deviation (RSD) of baseline changes after regenerations is 2.8%. The applicability of the method to real samples has been demonstrated with comparative analyses of lymphocyte extracts in gamma irradiated and unirradiated mice. The irradiated sample displays a significantly lower level of the wild-type HPRT compared to that in the unirradiated sample. The single sample detection only needs about 20 min. Thus, the SPR biosensor could potentially serve as an attractive technique for rapid and sensitive detection of the wild-type HPRT, a gamma radiation biomarker.     

Determination of radon using solid state nuclear tracks wireless sensing method

A highly sensitive and selective electrochemiluminescent (ECL) biosensor for the determination of adenosine was developed. Single DNA (capture DNA) was immobilized on the gold electrode through Au-thiol interaction at first. Another DNA modified with tris(2,2'-bipyridyl) ruthenium(II)-doped silica nanoparticles (Ru-SNPs) that contained adenosine aptamer was then modified on the electrode surface through hybridizing with the capture DNA. In the presence of adenosine, adenosine-aptamer complex is produced rather than aptamer-DNA duplex, resulting with the dissociation of Ru-SNPs-labeled aptamer from the electrode surface and the decrease in the ECL intensity. The decrease of ECL intensity has a direct relationship with the logarithm of adenosine concentration in the range of 1.0×10(-10) to 5.0×10(-6)molL(-1). The detection limit of the proposed method is 3.0×10(-11)molL(-1). The existence of guanosine, cytidine and uridine has little interference with adenosine detection, demonstrating that the developed biosensor owns a high selectivity to adenosine. In addition, the developed biosensor also demonstrates very good reusability, as after being reused for 30 times, its ECL signal still keeps 91% of its original state.     

TPD修饰电化学生物传感器测定DNA片段序列

合成了一种新的吸附偶联试剂-硫酚乙酸琥珀酰亚胺酯(TPD),将TPD吸附在金的表面,利用其琥珀酰亚胺基与客体氨基的反应,将单链DNA修饰在金电极上作为探针,以电化学方法检测能与之互补DNA样品的碱基序列和含量。杂交后的DNA采用自行合成的双[1-二茂铁按甲酰丙基-四氢化吡嗪-4-丙基氨甲酰吡啶]并吩嗪(FCZ)作指示剂进行检测,该化合物能够嵌入DNA双螺旋结构的碱基对中,显示出高度电化学活性。该传感的电流信号与DNA深度在2×10^-8～1×10^-7mol/L范围有线性关系,检测限为8×10^-10mol/L。本方法可用于检测爱滋病和乙肝病毒DNA特征序列的研究。     

Facile and controllable preparation of glucose biosensor based on Prussian blue nanoparticles hybrid composites

A glucose biosensor based on polyvinylpyrrolidone (PVP) protected Prussian blue nanoparticles (PBNPs)-polyaniline/multi-walled carbon nanotubes hybrid composites was fabricated by electrochemical method. A novel route for PBNPs preparation was applied in the fabrication with the help of PVP, and from scanning electron microscope images, Prussian blue particles on the electrode were found nanoscaled. The biosensor exhibits fast current response (<6 s) and a linearity in the range from 6.7x10(-6) to 1.9x10(-3) M with a high sensitivity of 6.28 microA mM(-1) and a detection limit of 6x10(-7) M (S/N=3) for the detection of glucose. The apparent activation energy of enzyme-catalyzed reaction and the apparent Michaelis-Menten constant are 23.9 kJ mol(-1) and 1.9 mM respectively, which suggests a high affinity of the enzyme-substrate. This easy and controllable construction method of glucose biosensor combines the characteristics of the components of the hybrid composites, which favors the fast and sensitive detection of glucose with improved analytical capabilities. In addition, the biosensor was examined in human serum samples for glucose determination with a recovery between 95.0 and 104.5%.     

检测痕量Hg~(2+)的DNA电化学生物传感器

通过自组装方法将修饰有二茂铁基团的富T序列DNA分子(DNA-Fc)固定在金电极表面,得到了一种基于DNA修饰电极的电化学汞离子(Hg2+)传感器.当溶液中有Hg2+存在时,Hg2+可与修饰电极上DNA的T碱基发生较强的特异结合,形成T-Hg2+-T发卡结构,使DNA分子构象发生改变,其末端具有电化学活性的二茂铁基团远离电极表面,电化学响应随之发生变化.示差脉冲伏安法(DPV)结果显示:DNA末端二茂铁基团的还原峰在0.26V(vs饱和甘汞电极(SCE))附近,峰电流随溶液中Hg2+浓度的增加而降低;Hg2+浓度范围在0.1nmol·L-1-1μmol·L-1时,电流相对变化率与Hg2+浓度的对数呈现良好的线性关系.该修饰电极对Hg2+的检测限为0.1nmol·L-1,可作为痕量Hg2+检测的电化学生物传感器.干扰实验也表明,该传感器对Hg2+具有良好的特异性与灵敏度.     

Electropolymerized network of polyamidoamine dendron-coated gold nanoparticles as novel nanostructured electrode surface for biosensor construction

Polyfuntionalized gold nanoparticles were prepared by using 2-mercaptoethanesulfonic acid, p-aminothiophenol and cysteamine core polyamidoamine G-4 dendron as capping ligands. The nanoparticles were electropolymerized on a Au electrode surface through the formation of a bisaniline-cross-linked network. The enzyme tyrosinase was further crosslinked on this nanostructured matrix. The enzyme electrode, poised at -100 mV, was used for the amperometric quantification of cathecol. The biosensor showed a linear response from 50 nM to 10 μM cathecol, with a low detection limit of 20 nM and a sensitivity of 1.94 A M(-1) cm(2). The electrode retained 96% and 67% of its initial activity after 16 and 30 days of storage at 4 °C under dry conditions.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H2O2)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10-15~1.0×10-10 mol/L,检出限(S/N=3)为4.4×10-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

A Label-Free Amperometric Immunoassay for Thrombomodulin Using Graphene/Silver-Silver Oxide Nanoparticles as a Immobilization Matrix

A simple, sensitive, and label-free electrochemical immunosensor has been developed for the measurement of serum thrombomodulin (TM), an endothelial glycoprotein which is associated with the progression and metastasis of tumors. At first, the graphene nanosheets, which were dispersed in Nafion solution, were used to coat the bare gold electrode. Then, silver-silver oxide nanoparticles (Ag-Ag₂O NPs) were immobilized on the graphene-modified electrode by a one-step electrochemical deposition method. Lastly, a thrombomodulin antibody (anti-TM) was attached via amido-Ag affinity. This strategy combines graphene/Ag-Ag₂O NPs hybrid materials as an immobilization matrix and Ag-Ag₂O NPs also as an electrochemical signal indication reagent. The main advantage of this strategy has two important aspects. One is the high stability and unique electronic properties of the graphene nanostructure. The other is the use of Ag-Ag₂O NPs as the immobilization matrix and redox probes, thus avoiding the laborious labeling protein operation. Using this strategy, the concentration of TM in the range of 0.1 to 20 ng/mL was detected, with a detection limit of 31.5 pg/mL (at 3σ). The proposed methodology demonstrates that the nanocomposite film composed of graphene and Ag-Ag₂O NPs is a potential for biosensor applications.     

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.     

Electrochemical Behavior of Glucose Oxidase Immobilized on Pd‐Nanoparticles Decorated Ionic Liquid Derived Fibrillated Mesoporous Carbon

Pd nanoparticles with an average diameter of 5 nm were decorated on the surface of ionic liquid derived fibrillated mesoporous carbon (IFMC) to prepare a novel nano‐hybrid material (Pd@IFMC). Thereafter, glucose oxidase was immobilized on Pd@IFMC modified glassy carbon electrode to fabricate an enzymatic glucose biosensor. A pair of well‐defined redox peaks was recorded for direct electron transfer of the immobilized glucose oxidase at the formal potential of ? 0.418 V with a peak to peak separation of 25 mV. Electron transfer rate constant of was calculated to be 14.6 s^?1. The response of fabricated biosensor was linear towards glucose concentration.     

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)₆]^3−/4−. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.