电化学阻抗谱在生物传感器研究中的应用进展

电化学阻抗谱(EIS)是近年快速发展起来的一种电化学分析方法。它具有良好的界面表征作用,特别适合于分析电极表面生物敏感膜的制备、生物学反应的动力学机制,已逐渐成为生物传感器研究的有效辅助方法。简要概述了EIS技术的原理,及其在各种生物传感器中的应用进展。     

细菌内毒素电化学生物传感器的构建及性能表征

细菌内毒素为外源性致热源,内毒素的检测在生物制品生产过程中至关重要。构建了一种用于检测细菌内毒素的电化学核酸适体生物传感器,以氨基修饰的内毒素核酸适体EAQ2为配体,通过3-巯基丙酸(MPA)中间连接物,固定修饰在金电极表面,并通过循环伏安(CV)和电化学交流阻抗谱(EIS)两种方式共同表征了生物传感器的构建过程。结果发现MPA组装时间在6 h时能在金电极表面形成稳定的自组装单分子层。构建的生物传感器检测限达0.001 EU/ml,低于目前报道的其他内毒素检测方法的检测限,在0.001~0.1 EU/ml内毒素浓度范围内具有较好的线性关系,相关系数R~2=0.9878,在实际生物样品的检测上具有一定的应用前景。     

生物电化学及其在环境监测中的应用

简要概述了生物电化学的研究领域,包括生物体系和生物界面模拟、生物分子的电化学、生物电催化、光合作用模拟和活组织电化学;总结了生物电化学传感器、生物芯片和生物电化学反应器在环境监测中的应用现状,并提出了其发展趋势,即不断向商品化方向发展,实现环境污染物的在线检测;利用基因技术,创造出检测能力更强的生物传感器和生物芯片;与其他精密分析仪器相结合,向多功能、集成化、智能化、微型化方向发展。     

生物电阻抗特征参数测量数据采集系统的研究

为了实现生物电阻抗参数成像,我们建立了一个快速、精确的生物电阻抗特性参数测量的数据采集系统,采取多频率组合激励并扫基频的方法,在多个激励频率不同测量阻抗信息,同时采用了一种新的高精度解调方法;正交序列数字解调,并对阻抗模型参数的估算方法进行了改进。结果表明,系统可以在1．6kHz-380kHz的频带范围内以小于100Hz的跨度编程选择工作频率,最多时可同时在四种频率下测量阻抗信息,系统的噪声水平在－80dB左右。在每周期采样64点的情况下,解调算法可将信噪比提高5．6倍以上。     

基于生物阻抗谱的生物细胞活性免标记检测方法

目的 基于生物阻抗谱（bioelectrical impedance spectroscopy,BIS）技术,提出一种免标记的生物细胞活性实时检测方法。该方法依据不同浓度、生理、病理状态下细胞组织的电学特性差异来判断细胞是否具有活性,以协助医师在临床手术中快速精准定位患者烫伤组织并实现有效切除。方法 使用具有活性的斑马鱼胚胎干细胞来模拟人体烫伤组织,采用生物阻抗谱技术来鉴别细胞组织的生理状态。结果 在不同状态下,细胞的阻抗幅值变化有显著的差异,可以从中发现同等浓度下活性细胞的阻抗幅值比死亡细胞平均高出17.25%,活性细胞发生弛豫频率的时间也比死亡细胞早25%。结论 实验数据表明,生物阻抗谱法能有效区分胚胎干细胞的两类生理状态;从聚类区域中可以看出,BIS检测法具有明显的细胞活性及浓度区分能力,理论上能够快速地协助医师完成对患者烫伤组织检测。     

葡萄糖氧化酶在枯草芽孢杆菌芽孢表面的展示及其酶电极制备

葡萄糖生物传感器是目前最为常见的电化学生物传感器,绝大多数葡萄糖生物传感器采用在电极表面修饰葡萄糖氧化酶的方法来制备,但是,在电极的固定化过程中需要酶的纯化,使得成本增加,已成为固定化酶电极开发领域的瓶颈。文中主要以芽孢衣壳蛋白CotX为锚定蛋白将葡萄糖氧化酶(Glucose oxidase,GOD)展示到枯草芽孢杆菌芽孢表面,通过Western blotting分析、免疫荧光分析以及酶活检测均证明GOD在芽孢表面有效表达,发酵获得重组芽孢(Spore-GOD)。再采用滴涂法和电沉积法制备了氧化石墨烯/普鲁士蓝沉积膜修饰玻碳电极,将Spore-GOD固定在修饰电极表面,最后滴加一层Nafion溶液,制成了电化学生物传感器,用于葡萄糖的灵敏测定。葡萄糖在该酶电极传感器上的循环伏安图表明,该反应在0.42V处出现明显的氧化峰,并且氧化还原峰电流与葡萄糖浓度在0.1–7.0mmol/L之间具有良好的线性关系,校正曲线方程为:I=1.3047C_glucose+3.639(R²=0.9929),其检测限为7.5μmol/L(S/N=3)。此修饰电极具有...     

皮肤肿瘤早期筛查的快速无创电阻抗检测方法

目的 随着环境问题和臭氧层空洞化的加剧,皮肤肿瘤的患病率也大幅增加,但是皮肤肿瘤前期隐蔽性高、症状不明显,导致大部分病例都是在中晚期发现的。因此,本文基于生物阻抗谱(bioimpedance spectroscopy,BIS)技术,提出一种皮肤肿瘤早期筛查的快速无创电阻抗检测方法。方法 首先,建立四层皮肤模型,采用数值分析方法研究角质层对BIS测量的阻碍作用。其次,使用去除角质层的皮肤模型研究混有不同半径和浸润深度的皮肤肿瘤组织电学特性。最后,使用凝胶处理后的猪皮组织实验验证肿瘤浸润深度(h)的影响。结果 角质层仿真结果表明,去除角质层的皮肤对激励信号的响应更明显。皮肤肿瘤模型仿真表明,当肿瘤半径(R_tumor)及h>1.5 mm时能够区分肿瘤组织与正常组织。同时根据实验结果中正常组织与肿瘤组织虚部弛豫阻抗(Z_imag-relax)定义了组织病变度(ε_worse,为肿瘤组织虚部阻抗相对于正常组织虚部阻抗变化的百分比),并绘制了肿瘤组织浸润深度(Depth)与Z_imag-relax的拟合曲线。当...     

适配子纳米生物传感器在青霉素检测中的应用

通过脉冲腐蚀法对硅片进行多孔硅的制备,利用玻片通过对共价法、离子吸附法和APTES修饰的戊二醛交联法3种固定适配子方法的对比,以确定较好的固定青霉素适配子的方法。将适配子固定在多孔硅上后,利用交流阻抗法对加入青霉素前后传感器阻抗值进行测定、对比,构建等效电路并进行阻抗拟合。对多孔硅传感器的Nyqu ist谱图进行分析以确定多孔硅表面成功固定了青霉素适配子,从而证明构建纳米生物传感器成功。传感器的线性检测范围为0.05~0.2 mg/L,检测限为0.05 mg/L。     

压电体声波阻抗生物传感器技术在血培养中的应用评价

评价全自动压电体声波阻抗生物传感器血液培养系统的应用价值,并与Bactec9120血培养系统相比较。将2005年7~12月临床标本407例分别同时接种于2种不同的血培养系统中进行培养,对其阳性检出率、阳性检出时间、假阳性率及相关因素进行综合性评估。407份标本全自动压电体声波阻抗生物传感器血液培养系统检测出阳性标本58株,阳性率为14.3%,平均检出时间为(11.58±7.62)h;Bactec9120血培养系统培养阳性菌株61株,阳性率为15.0%,平均检出时间为(18.71±12.89)h。经统计学处理,两种检测方法阳性率无显著性差异(χ2=0.089,P>0.5),但检出时间有显著性差异(t=3.64,P<0.001)。全自动压电体声波阻抗生物传感器血液培养系统快速、简单、准确、便宜,可在各医院推广使用。     

基于径向基函数神经网络的肺部加权频差电阻抗成像方法

目的 对肺通气过程进行床旁实时连续图像监控，是机械通气患者和临床医生的迫切需求。肺部电阻抗成像（EIT）可反映呼吸引起的胸腔电特性变化分布，在肺通气监测方面具有天然的优势。本文目的在于建立基于径向基函数神经网络（RBFNN）的肺部加权频差电阻抗成像（wfd-EIT）方法，实现对肺通气的高空间分辨率成像。方法 利用肺部wfd-EIT成像方法实时描绘胸腔电导率分布状况，再通过RBFNN将目标区域可视化并精准识别其边界信息。首先通过数值分析模拟，在各个激励频率利用COMSOL与MATLAB软件建立2 028个仿真样本，分为训练样本集和测试样本集，验证所提出成像方法的可行性和有效性。其次，为了验证仿真结果，建立肺部物理模型，选用具有低电导特性的生物组织模拟肺部通气区域，对其进行成像实验，并采用图像相关系数（ICC）和肺区域比（LRR）定量数据衡量成像方法的准确性。结果 wfd-EIT方法可以在任意时刻进行图像重建，并能够准确反映出目标区域的电特性分布；利用基于RBFNN的算法能够增强目标区域的成像精度，ICC可达0.94以上，更好地凸显其边界轮廓信息。结论 通过wfd-EIT成像方法，利用多频阻抗谱同步测量实现目标区域的快速可视化，并结合RBFNN网络逼近任意非线性函数的优点，实现对目标区域电特性变化的精准识别，为下一步进行临床肺通气的EIT图像监测奠定了理论和技术基础。     

Factors affecting electrical impedance of internodal stem sections

A sample holder was designed and built to facilitate measuring the magnitude and phase angle of the electrical impedance of internodal stem sections from Cornus stolonifera Michx. A nonpolarizing, electrically conducting manganese dioxidecarbon paste used between the stem sample and the electrodes of the sample holder allowed measurement of impedance at frequencies from 50 hertz to 500 kilohertz without electrode polarization or electrical interference. The impedance magnitude was linearly dependent on the sample length, but this dependence was minimized by computing a normalized impedance magnitude. The normalized impedance magnitude (Z_nf) was calculated using the impedance magnitude (Z) at any specified frequency (f) and the impedance magnitude at 500 kilohertz (Z_{500 khz}) in the following formula: Z_nf = (Z - Z_{500 khz})/Z_{500 khz}. The normalized impedance magnitude was sensitive to injury produced by boiling and peeling the sample. Electrical impedance measurements on the bark and wood separately demonstrated that they have different electrical properties.     

体外循环血栓非侵入在线电阻抗成像方法

目的 在体外循环系统中,血栓的在线检测和可视化具有重要意义。本文提出了基于电阻抗成像（EIT）的体外循环血栓非侵入在线检测方法。方法 首先通过联合仿真研究了传感器尺寸对成像效果的影响。其次,根据仿真结果设计了直径为20 mm的16铜质电极EIT传感器,搭建了循环流动实验平台,并设计了静态和循环流动实验。使用尺寸为3～6 mm的猪血块代替血栓,将血块置于新鲜猪血样本中,采用Tikhonov正则化算法进行成像。将3 mm和5 mm的血块分别置于循环系统中,重建血块在传感器截面的大小和位置图像,并与高速相机拍摄结果进行对比。结果 仿真结果显示当目标物与传感器面积比（AR）不小于0.01时,传感器直径为20 mm和30 mm对应的图像相关系数（IC）均大于0.06,成像效果较好。静态成像结果显示,相对尺寸覆盖率误差（RCR）小于等于0.1。循环流动实验显示,血块经过传感器时,检测到归一化后的相对电导率变化值分别为80和200,结果显示该方法能够检测到循环系统中的血块。结论 该方法具有实时性和非侵入的优点,有望应用于体外血栓的检测。     

Sexual communication in the termite Prorhinotermes simplex (Isoptera, Rhinotermitidae) mediated by a pheromone from female tergal glands

We studied the post-flight behavior and sex attraction in imagoes of the termite Prorhinotermes simplex (Rhinotermitidae, Prorhinotermitinae). Pairing is mediated by the secretion from tergal glands, exposed by females in a calling posture and highly attractive to males. Analysis of extracts of these glands by means of gas chromatography with electroantennographic detection indicated a chromatographic area corresponding to an intense physiological response of males. The retention characteristics of this area proved to be identical with those of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. Electroantennographic and behavioral assays revealed that units of picograms of the compound represent a stimulus qualitatively and quantitatively equivalent to one female tergal gland. Thus, we hypothesize that (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol is a major component of the female sex pheromone in P. simplex.     

Synthesis of (2RS,8R,10R)-YM-193221 and an Improved Approach to Tyroscherin,Bioactive Natural Compounds from Pseudallescheria sp.

Short-step syntheses of (2RS,8R,10R)-YM-193221 (1) and tyroscherin (2), which are biologically active compounds isolated from Pseudallescheria sp., were accomplished in six and eight steps from L-tyrosine. The relative stereochemistry of natural YM-193221 was determined to be 8R ^*,10R ^*.     

Enantiotopically Selective Oxidation of 1,5-Diols with Gluconobacters Preparation of (S)-Mevalonolactone

(±)-(2Z,4E)-α-Ionylideneacetic acid (2) was enantioselectively oxidized to (?)-(l′S)-(2Z,4E)-4′-hydroxy-α-ionylideneacetic acid (3), (+)-(1′R)-(2Z,4E)-4′-oxo-α-ionylideneacetic acid (4) and (+)-abscisic acid (ABA) (1) by Cercospora cruenta IFO 6164, which can produce (+)-ABA and (+)-4′-oxo-α-acid 4. This metabolism was confirmed by the incorporation of radioactivity from (±)-(2-¹⁴C)-(2Z,4E)-α-acid 2 into three metabolites. (?)-4′-Hydroxy-α-acid 3 was a diastereoisomeric mixture consisting of major 1′,4′-trance-4′-hydroxy-α-acid 3a and minor 1′,4′-cis-4′-hydroxy-α-acid 3b. These structures, 3a and 3b, were confirmed by ¹³C-NMR and ¹H-NMR analysis. Also, the enantioselectivity of the microbial oxidation was reexamined by using optically pure α-acid (+)-2 and (?)-2, as the substrates.     

Real time detection and quantification of inclusion bodies expressed in Escherichia coli by impedance measurements

Escherichia coli expressing human growth hormone as inclusion bodies was cultured in a fermenter. Real time, non-invasive detection and quantification of inclusion body protein expressed in E. coli was performed by impedance measurements at 50 MHz and 180 MHz. At 50 MHz rotation of dipoles of the protein and their proton fluctuation, i.e., -dispersion of protein aggregates formed inside the cell as a result of expressed protein, results in an additional decrease in impedance. At 180 MHz the impedance remained at a plateau. In a high cell density E. coli culture, after induction with IPTG, when the cell mass remained unchanged, an increase in the magnitude of -dispersion was observed at 50 MHz. This was due to the formation and subsequent increase in the concentration of r-human growth hormone which aggregate as inclusion bodies. The estimation of inclusion bodies by taking the ratio of impedance at 180 MHz and at 50 Mhz matched with the amount of protein estimated after extraction and purification (coefficient of correlation was 0.92). This is the first report of real time detection and monitoring of recombinant protein expressed as inclusion bodies by impedance measurements.     

E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。     

Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalis L.)

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV–vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5′), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5′Z,9′Z)-lycopene (3), (5′Z)-γ-carotene (4), and (5′Z,9′Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5′Z)-lycopene (2) has been reported only as a synthesized compound.     

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging for Monitoring the Instantly Therapeutic Efficacy of Radiofrequency Ablation in Rabbit VX2 Tumors without Evident Links between Conventional Perfusion Weighted Images

ObjectiveTo investigate the intravoxel incoherent motion diffusion weighted imaging (IVIM-DWI) as a potential valuable marker to monitor the therapy responses of VX2 to radiofrequency ablation (RF Ablation).MethodsThe institutional animal care and use committee approved this study. In 10 VX2 tumor–bearing rabbits, IVIM-DWI examinations were performed with a 3.0T imaging unit by using 16 b values from 0 to 800 sec/mm². The true diffusion coefficient (D), pseudodiffusion coefficient (D^*) and perfusion fraction (f) of tumors were compared between before and instantly after RF Ablation treatment. The differences of D, D^* and f and conventional perfusion parameters (from perfusion CT and dynamic enhanced magnetic resonance imaging, DCE-MRI) in the coagulation necrosis area, residual unablated area, untreated area, and normal control had been calculated by compared t- test. The correlation between f or D^* with perfusion weighted CT including blood flow, BF (milliliter per 100 mL/min), blood volume, BV (milliliter per 100 mL/min), and capillary permeability–surface area, PMB (as a fraction) or from DCE-MRI: transfer constant (K_trans), extra-vascular extra-cellular volume fraction (V_e) and reflux constant (K_ep) values had been analyzed by region-of-interest (ROI) methods to calculate Pearson’s correlation coefficients.ResultsIn the ablated necrosis areas, f and D^* significantly decreased and D significantly increased, compared with residual unblazed areas or untreated control groups and normal control groups (P < 0.001). The IVIM-DWI derived f parameters showed significant increases in the residual unablated tumor area. There was no significant correlations between f or D^* and conventional perfusion parameters.ConclusionsThe IVIM-DW derived f, D and D^* parameters have the potential to indicate therapy response immediately after RF Ablation treatment, while no significant correlations with classical tumor perfusion metrics were derived from DCE-MRI and perfusion-CT measurements.