成骨细胞介电特性检测的方法研究

目的:以小鼠成骨细胞为材料,建立一套完整的用于成骨细胞的介电特性检测方法。方法:本研究首先是设计制作不同形状和尺寸的细胞介电谱检测装置(测量池),其次进行测量池的生物相容性评价,包括浸提液毒性实验,HE染色观察细胞形态。最后将细胞培养在测量池中,连入阻抗分析仪在20Hz~10MHz检测并联电导Gp和电容Cp并绘制介电谱图。结果:生物相容性检测表明测量池对细胞无毒性,可使用。此外,介电特性检测结果表明随着频率的增大,细胞的电容减小,电导略微增大后减小;随着培养时间的增长,电容值增大而电导值减小;经中强磁场处理后,细胞的电容和电导值都有下降。结论:该方法能够为其他贴壁细胞的介电特性检测提供研究基础,并对于在此基础上展开的贴壁细胞介电特性解析提供参考。     

金属离子及CTAB对酵母细胞介电行为的影响——相互作用的模型化解析

利用介电谱方法详细研究了Cu2＋、Zn2＋、Cd2＋、Pb2＋、Al3＋、Ni2＋等金属离子以及阳离子表面活性剂CTAB：十六烷基三甲基溴化铵）对典型的真核细胞——酵母细胞介电性质的影响。在时间变化和浓度变化的情况下,对上述体系在40HZ～110MHz宽频范围进行了介电测量,Cole-Cole拟合确定了介电参数,定性讨论了不同试剂的时间和浓度的各种作用效果。通过无作用的对照细胞和有离子作用的作用组细胞10小时内的介电谱图比较发现,Cu2＋、Pb2＋及CTAB对酵母细胞介电行为的影响是以孵化时间依赖的方式发生;对有时间作用的三者选用不同的浓度进行作用,结果发现Cu2＋及CTAB对酵母细胞的作用同样是以浓度依赖的方式进行,而不同浓度的Pb”的作用效果接近。进一步,根据酵母细胞的结构特点采用双壳介电模型,理论计算了相参数,并结合细胞生理学知识对细胞受金属离子或特殊试剂作用后的相参数变化原因给予了解释;给出了金属离子,特别是Cu2＋以及CTAB与酵母细胞作用的可能机制。此外,模拟了实验条件下细胞悬浮液中各组成相参数对介电谱的依存关系,给出了一些有益的暗示：介电增量主要受细胞膜介电常数和细胞体积分数影响;特征频率尼与液泡膜介电常数以及细胞质的电导率等物理参数有关;而液泡内电导率支配高频的弛豫行为。这些模拟将对酵母细胞介电实时监测技术的实现提供基础参考。     

间断性低氧对大鼠腓肠肌介电性能的影响

目的：在40Hz～110MHz频率范围观察间断性低氧暴露4周大鼠离体腓肠肌细胞介电性能的改变。方法：采用低压氧舱建立模拟低氧模型,雄性SD大鼠随机分为间断低氧组和正常对照组。利用Agilent 4294A阻抗分析仪测量了离体大鼠腓肠肌的交流阻抗,通过频域介电谱、Cole—Cole图、介电损耗因子频谱、电导率虚部频谱和介电损耗角正切频谱的数据分析,观察间断性低氧暴露对大鼠离体腓肠肌细胞介电性能的影响。结果：间断性低氧暴露4周大鼠腓肠肌的介电常数（εL,εh）降低,介电增量△ε减小,绝缘性降低;低频电导率κL升高,高频电导率κh降低,电导率增量△κ降低;特征频率（f1,f2）增加;介电损失峰值ε”peak、电导率虚部峰值κ”peak和损耗角正切峰值婶谳均降低。结论：间断性低氧暴露致骨骼肌细胞介电性能降低,但其特征频率增加。     

100 Hz ~ 100 MHz蛙骨骼肌介电谱的椭圆壳理论解析

在100Hz～100MHz频率范围内,应用浓厚系介电椭圆壳理论分析了蛙骨骼肌介电谱,提出了蛙骨骼肌细胞的椭圆壳模型参数。讨论了骨骼肌介电谱的高频段平行方向与垂直方向的电导率不相等的理论问题。明确了决定骨骼肌高频率段(10^6～10^8Hz)介电数据的物质基础主要是骨骼肌细胞内部的肌原纤维,其次是胞浆内的微小颗粒(线粒体、肌质网)。     

脉冲磁场对神经元动作电位发放的影响

磁场作用于生物体产生的生物效应被广泛研究。本文将脉冲磁场作用于神经元细胞,试图观察Na+通道的变化及其引起的动作电位的变化。选择频率15 Hz、强度1 mT的脉冲磁场刺激昆明小鼠大脑皮质神经元,随后进行全细胞膜片钳实验。结果显示,脉冲磁场延缓了Na+通道电流的激活,促进Na+通道电流的失活。基于经典的细胞三层介电模型,模拟了在脉冲磁场下细胞膜电位分布的极化图,结果显示诱发的膜电位大小与脉冲磁场的频率和强度有关。基于Hodgkin-Huxley(H-H)模型,仿真了脉冲磁场感应的电流作用于离子通道所产生的动作电位曲线,与不加刺激时候的曲线相比较,当外加电流在-1.32~0μA时,动作电位的产生频率减小,幅值降低;当外加电流大于0μA时,动作电位的产生频率增大,幅值变化不明显;当外加电流小于-1.32μA时,动作电位的上升时间快速变短,峰值剧烈降低,无法形成完整的动作电位,即无动作电位。以上结果提示,磁场刺激可通过调节Na+通道影响神经元动作电位的发放频率和幅值。     

50例兔在体和离体器官组织射频介电性能的测量与分析

根据同轴传输线反射原理,建立起计算机控制的生物组织介电测量系统;在６０—３０００ＭＨｚ频率范围对５０例兔器官组织进行了有效的在体和离体介电测量。测量结果的统计分析表明,不同兔器官组织之间介电参数的最大差异可达３０·４％;在体和离休测量得出的介电常数之间未见显著性差异,但电导率有频率依赖性的显著差异,因此将人体的离体介电参数延用到实际活体场合时,必须慎重。     

多层生物介质传感器的特性研究（英文）

目的 多层生物介质的生物传感器被广泛应用于各大领域,其检测特性对于传感器优劣的评估尤为重要。本文目的在于量化表征多层生物介质的电学特征。方法 基于生物电阻抗谱技术来探究多层生物介质的电化学阻抗谱特性,并结合保角映射的方法来量化表征多层生物介质,阐明其对阻抗的影响规律,继而为生物传感器的研制与开发提供理论基础。有效提取各生物介质层修饰后电阻抗参数(Z^*),从而量化表征多层生物介质层的电阻抗谱特性。结果 对多层模型进行了理论计算并构建了相关试验测试系统,研究结果表明,随着生物介质层的逐步修饰,检测区域电阻抗参数(Z^*)在f=0.1～50 MHz下持续上升,理论计算结果趋势与试验结果趋势较好吻合,论证了此理论计算方法的正确性。结论 本文证实了可根据生物电阻抗谱和保角映射方法量化表征多层生物介质的电阻抗谱特性,对生物传感器的研制与开发有一定的实用价值。     

骨骼肌细胞频响特性的数值分析

在100 Hz-100MHz频率范围内,利用非线性数值计算和曲线拟合分析,验证了蛙离体骨骼肌细胞的频率响应特性满足Cole-Cole公式(误差<3．45％),通过频域介电谱、 Cole-Cole图、介电损耗因子和介电损耗角正切频率谱的曲线拟合分析,确定了蛙骨骼肌细胞的 Cole-Cole参数:高频段相对介电常数εh=78,第一相对介电增量εt=113000,第二相对介电增量ε2=45000,第一特征频率fc1=9 KHz。第二特征频率fc2=158KHz,第一相位角β1=0．881,第二相位角β2=0．984,低频段电导率κ1=0．55mS／cm,A=35,m=1．08．     

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。     

近红外与荧光光谱法检测血糖试验研究

采用荧光光谱法检测血糖。结果表明,检测血清的最佳激发波长为340 nm,血清中葡萄糖的发射峰位置为470 nm。随着血清中葡萄糖浓度的增加,荧光光谱图中荧光峰处荧光强度,面积积分强度整体呈上升趋势,荧光峰的半峰全宽整体呈现下降趋势。利用近红外光谱法检测血糖,血清可分辨的光谱区域位于5 600~6 060 cm-1范围内,不同血糖浓度的吸收峰均位于5 768 cm-1处。随着血糖浓度的增大,其吸收峰处的吸光度逐渐增大,面积积分强度也呈上升趋势。分析二者的优缺点,荧光分析法受外界因素的干扰更小,精确度更高,光谱参数与血糖浓度之间的规律更明显,在有创检测方面具有更明显的优势,而近红外光谱法检测血糖则在无创血糖检测方面具有更大的潜力。     

Dielectric approach to the investigation of erythrocyte aggregation: I. Experimental basis of the method

A method based on dielectric properties of dispersed systems was developed to investigate red blood cell (RBC) aggregation in blood and RBC suspensions. Measurements of capacitance and resistance were made in a rectangular channel at low (0.2 MHz) and high (14 MHz) frequencies relative to the mid-point of the beta-dispersion range. Compared to capacitance, minimal post-shearing changes of resistance were observed; capacitance changes at 0.2 MHz were two orders of magnitude larger than those at 14 MHz and hence subsequent measurements were carried out at the lower frequency. It is shown that post-shearing changes in the capacitance are affected by the recovery of RBC shape and relaxation processes at the electrode-suspension interface. However, the dominant factor contributing to time-dependent changes in the capacitance is the dynamic process of RBC aggregation. It is experimentally shown that the time record of the capacitance at 0.2 MHz quantitatively reflects the aggregation process in RBC-plasma suspensions with hematocrit up to 0.56 (v/v) and in suspensions of RBCs in artificial aggregating media. It is concluded that a dielectric approach to the study of RBC aggregation in whole blood offers great potential for basic studies and for diagnostic use.     

Changes in surface capacitance and conductance parallel to phospholipid membranes associated with phase transition: effects of halothane

The effects of phase transition on the surface capacitance and conductance parallel to dipalmitoyl- (DPPC) and dimyristoyl-phosphatidylcholine (DMPC) membranes were studied by impedance dispersion. The phospholipid aggregates were embedded into pores of a polycarbonate filter and the impedance dispersions were measured at a frequency range from 30 Hz to 1.0 MHz. When the frequency was below 120 kHz, the capacitance showed a peak at the pretransition temperature and a steep rise at the main-transition temperature. In this system, the observed capacitance consists of frequency-dependent and -independent parts. The frequency-dependent part is a surface phenomenon and arises from the lateral motion of counterions at the membrane/water interface. The frequency-independent part represents mainly the properties of the bulk lipid phase. Addition of halothane decreased the total capacitance of the DPPC aggregates at the low frequency range to 1/2 to 1/8 of the control depending upon the temperature. The surface component was solely responsible for this capacitance decrease, because the non-surface component was slightly increased instead. The data suggest that halothane inhibited the lateral ionic flow parallel to the interface.     

Impedance spectroscopy flow cytometry: on-chip label-free cell differentiation.

BACKGROUND: The microfabricated impedance spectroscopy flow cytometer used in this study permits rapid dielectric characterization of a cell population with a simple microfluidic channel. Impedance measurements over a wide frequency range provide information on cell size, membrane capacitance, and cytoplasm conductivity as a function of frequency. The amplitude, opacity, and phase information can be used for discrimination between different cell populations without the use of cell markers. METHODS: Polystyrene beads, red blood cells (RBCs), ghosts, and RBCs fixed in glutaraldehyde were passed through a microfabricated flow cytometer and measured individually by using two simultaneously applied discrete frequencies. The cells were characterized at 1,000 per minute in the frequency range of 350 kHz to 20 MHz. RESULTS: Cell size was easily measured with submicron accuracy. Polystyrene beads and RBCs were differentiated using opacity. RBCs and ghosts were differentiated using phase information, whereas RBCs and fixed RBCs were differentiated using opacity. RBCs fixed using increasing concentrations of glutaraldehyde showed increasing opacity. This increased opacity was linked to decreased cytoplasm conductivity and decreased membrane capacitance, both resulting from protein cross-linking. CONCLUSIONS: This work presents label-free differentiation of cells in an on-chip flow cytometer based on impedance spectroscopy, which will be a powerful tool for cell characterization.     

Dielectric relaxation in lecithin/cholesterol/water-mixtures

The dielectric spectrum of aqueous solutions of dimyristoyl-l-3-phosphatidylcholine and dipalmitoyl-l-3-phosphatidylcholine with admixed cholesterol has been determined by means of a pulse reflection method which was used to measure the complex permittivity of the solutions as a function of frequency between 100 kHz and 50 MHz. Measurements have been performed at various concentrations of cholesterol in dependence of temperature around the crystal-line/liquid-crystalline phase transition temperature of the solutions.The measured dielectric spectra are treated in terms of a Debye-function. The dielectric relaxation strength and the relaxation time decrease distinctly with increasing cholesterol concentration. In addition, the data are treated on the basis of a theoretical solution model in order to allow for conclusions concerning the lecithin head group motion in the lipid bilayer surface. One important result is that increasing cholesterol concentration affects the interaction of the lecithin head groups and increases their mobility. These effects already occur at small concentrations of cholesterol.     

PGE(2) activation of apical membrane Cl(-) channels in A6 epithelia: impedance analysis.

Measurements of transepithelial electrical impedance of continuously short-circuited A6 epithelia were made at audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E(2) (PGE(2)) modulates Cl(-) transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral membrane resistances were determined by nonlinear curve-fitting of the impedance vectors at relatively low frequencies (<50 Hz) to equations (P?unescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838--851) where depressed Nyquist impedance semicircles were characteristic of the membrane impedances under control Na(+)-transporting and amiloride-inhibited conditions. In all tissues (control, amiloride-blocked, and amiloride-blocked and furosemide-pretreated), PGE(2) caused relatively small (< approximately 3 microA/cm(2)) and rapid (<60 s) maximal increase of chloride current due to activation of a rather large increase of apical membrane conductance that preceded significant activation of Na(+) transport through amiloride-sensitive epithelial Na(+) channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion whose relaxation frequency was near 150 Hz. Analysis of the time-dependent changes of the complex frequency-dependent equivalent capacitance of the cells at frequencies >1.5 kHz revealed that the mean 9.8% increase of capacitance caused by PGE(2) was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical membrane Na(+) transport.     

Microwave dielectric relaxation in muscle. A second look.

The dielectric permittivity and conductivity of muscle fibers from the giant barnacle, Balanus nubilus, have been measured at 1, 25, and 37 degrees C, between 10 MHz and 17 GHz. The dominant microwave dielectric relaxation process in these fibers is due to dipolar relaxation of the tissue water, which shows a characteristic relaxation frequency equal to that of pure water, ranging from 9 GHz (1 degree C) to 25 GHz (37 degree C). The total permittivity decrease, epsilon 0 -- epsilon infinity, due to this process accounts for approximately 95% of the water content of the tissue; thus, the major fraction of tissue water is dielectrically identical to the pure fluid on a picosecond time scale. A second dielectric process contributes significantly to the tissue dielectric properties between 0.1 and 1--5 GHz, and arises in part form Maxwell-Wagner effects due to the electrolyte content of the tissue, and in part from dielectric relaxation of the tissue proteins themselves.     

Electrical Properties of Phospholipid Vesicles

The capacitance of the membrane of phospholipid vesicles and the electrical properties of the vesicle interior have been determined. To this end the electrical properties of phospholipid vesicles have been investigated over a frequency range extending from 1 kHz to 100 MHz. The dielectric behavior is characterized by two dispersions, one placed between 1 kHz and 1 MHz and the other between 1 and 100 MHz. The relaxational behavior at low frequencies is explained by counterion movement tangential to the vesicle surface and a reasonable value for the fixed charge of the vesicles is calculated from the dispersion magnitude. The relaxation at high frequencies is of the Maxwell-Wagner type and appears caused by the phospholipid bilayer bounding the interior phase of the vesicles. It is consistent with the existence of a closed bilayer with a capacitance of about 2 μF/cm² and an internal phase similar to the vesicle suspending medium. There is no indication of other than normally structured water inside the small vesicles.     

Electrical Impedance Analysis in Plant Tissues11

Electrical impedance was measured at a range of AC frequenciesof 100 Hz to 1 MHz in potato tubers and carrot roots. Detailsof a method for analysing the data in relation to electricalmodels, using complex non-linear least squares (CNLS), are described.The measured data were analysed in relation to four previously-describedelectrical models for plant tissues. The results showed thatplant tissue conforms well to a double-shell model which includescomponents (resistors and capacitors) representing the vacuole,as well as cytoplasm, plasma membrane, and extracellular space.The method permitted the calculation of specific membrane capacitance,which was found to approximate l0µF cm^–2. Key words: Electrical impedance, complex non-linear least squares (CNLS), electrical modelling, membrane capacitance     

Modeling error and stability of endothelial cytoskeletal membrane parameters based on modeling transendothelial impedance as resistor and capacitor in series

Transendothelial impedance across an endothelial monolayer grown on a microelectrode has previously been modeled as a repeating pattern of disks in which the electrical circuit consists of a resistor and capacitor in series. Although this numerical model breaks down barrier function into measurements of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance, such solution parameters can be inaccurate without understanding model stability and error. In this study, we have evaluated modeling stability and error by using a ² evaluation and Levenberg-Marquardt nonlinear least-squares (LM-NLS) method of the real and/or imaginary data in which the experimental measurement is compared with the calculated measurement derived by the model. Modeling stability and error were dependent on current frequency and the type of experimental data modeled. Solution parameters of cell-matrix adhesion were most susceptible to modeling instability. Furthermore, the LM-NLS method displayed frequency-dependent instability of the solution parameters, regardless of whether the real or imaginary data were analyzed. However, the LM-NLS method identified stable and reproducible solution parameters between all types of experimental data when a defined frequency spectrum of the entire data set was selected on the basis of a criterion of minimizing error. The frequency bandwidth that produced stable solution parameters varied greatly among different data types. Thus a numerical model based on characterizing transendothelial impedance as a resistor and capacitor in series and as a repeating pattern of disks is not sufficient to characterize the entire frequency spectrum of experimental transendothelial impedance. cell-cell adhesion; cell-matrix adhesion; cell membrane capacitance; mathematical computation     

Impedance spectroscopy of solutions at physiological glucose concentrations

Impedance spectroscopy has been proposed as possible approach for non-invasive glycaemia monitoring. However, few quantitative data are reported about impedance variations related to glucose concentration variations, especially below the MHz band. Furthermore, it is not clear whether glucose directly affects the impedance parameters or only indirectly by inducing biochemical phenomena. We investigated the impedance variations in glucose-water, glucose-sodium chloride, and glucose-blood samples, for increasing glucose values (up to 300 mg/dl). In all the frequency range (0.1-10(7) Hz) glucose-water samples showed impedance modulus increases for increasing glucose values (up to 135%). In blood and sodium chloride samples the impedance modulus showed only slight variations (2% and 1.4%), but again in wide frequency ranges. Therefore: i) glucose directly affects the impedance parameters of solutions; ii) effects are more relevant at frequencies below the MHz band; iii) the influence on the impedance is decreased in high conductivity solutions, but still clearly present.