面向血液葡萄糖测量的生物组织阻抗模型

本文推导了细胞膜电容与血糖浓度之间的关系,并将这种关系应用到Pauly和Schwan提出的弛豫模型中,再将弛豫模型应用于多重Cole-Cole介电模型,最后通过介电模型得出阻抗模型,建立起人体血糖浓度与组织介电谱,阻抗谱之间的联系。通过分析发现,细胞膜电容随着血糖浓度增加而增大,且当血糖浓度趋于无穷大时,细胞膜电容等于血糖浓度为0时细胞膜电容的1.53倍。对介电谱的仿真得出,当外加信号频率小于1 MHz时,介电常数实部随着葡萄糖浓度改变而发生明显变化。阻抗谱的研究结果表明,随着葡萄糖浓度的升高,阻抗的实部减小,阻抗虚部模增大,且分别在频率为1.48 MHz和0.55 MHz时变化得最快。本文建立的模型为血糖无创检测提供了一种新的方法和手段。     

Studies of Electric Capacitance of Membranes: I. A Model Membrane Composed of a Filter Paper and a Lipid Analogue

A hydrophobic filter paper of a given pore size containing a synthetic lipid, i.e. dioleyl phosphate, was interposed between aqueous electrolyte solutions having the same chemical composition and temperature. The electric capacitance and conductance of the membrane immersed in various concentrations of KCl were measured in the frequency range from 20 to 3 × 10⁶ cycle/sec. The observed capacitance and conductance were found to be strongly dependent on the applied frequency. A theory is proposed to account for this dispersion of impedance observed in the present membrane-electrolyte system. The dispersion is attributed to the formation of bilayer membranes of the lipid inside the filter paper. The effects of the salt concentration, the adsorbed quantity of the lipid, and the pore size of the filter paper on the capacitance and conductance of the membrane are discussed in terms of the distribution function of bilayers formed within the filter paper.     

Alamethicin adsorption to a planar lipid bilayer.

The effect of alamethicin and its derivatives on the voltage-dependent capacitance of phosphatidylethanolamine (squalane) membranes was measured using two different methods: lock-in detection and voltage pulse. Alamethicin and its derivatives modulate the voltage-dependent capacitance at voltages lower than the voltage at which alamethicin-induced conductance is detected. The magnitude and sign of this alamethicin-induced capacitance change depends on the aqueous alamethicin concentration and the kind of alamethicin used. Our experimental data can be interpreted as a potential-dependent pseudocapacitance associated with adsorbed alamethicin. Pseudocapacitance is expressed as a function of alamethicin charge, its concentration in the bathing solution and the applied electric field. The theory describes the dependence of the capacitance on applied voltage and alamethicin concentration. When alamethicin is neutral the theory predicts no change of the voltage-dependent capacitance with either sign of applied voltage. Experimental data are consistent with the model in which alamethicin molecules interact with each other while being adsorbed to the membrane surface. The energy of this interaction depends on the alamethicin concentration.     

Alternating current studies of charge carrier transport in lipid bilayers. Pentachlorophenol in lecithin-cholesterol membranes.

Surface and interior electrical properties of lecithin-cholesterol bilayer membranes treated with the uncoupler pentachlorophenol have been determined on the basis of a.c. measurements over a wide range of frequencies (0.02 to 1000 kHZ). The method used depends on accurately determining the resistance of the aqueous solution in series with each individual membrane by extrapolating admittance data into infinite frequency. Loss tangent vs. frequency curves are corrected by subtracting out a loss contribution which is present in untreated membranes and is due, presumably, to dielectric relaxation. The results, which are useful below 100 kHZ, can be fitted to loss tangent curves computed for a three-element equivalent circuit consisting of frequency independent conductance-capacitance pairs, arranged in series to represent surface and interior properties of membranes. Interior conductances agree with net conductances obtained from d.c. measurements. The pH and concentration dependence of surface conductance is consistent with a scheme of transport in which a fixed number of surface binding sites are filled preferentially with neutral pentachlorophenol molecules, which in turn dissociate to supply protons to the aqueous phase. Surface capacitances range from 15 to 90 times that of interior capacitance and show a systematic increase with pentachlorophenol concentration at high pH, and a decrease with concentration at low pH.     

Effect of ion concentration,solution and membrane permittivity on electric energy storage and capacitance

Bio-membranes as capacitors store electric energy, but their permittivity is low whereas the permittivity of surrounding solution is high. To evaluate the effective capacitance of the membrane/solution system and determine the electric energy stored within the membrane and in the solution, we estimated their electric variables using Poisson-Nernst-Planck simulations. We calculated membrane and solution capacitances from stored electric energy. The effective capacitance was calculated by fitting a six-capacitance model to charges (fixed and ion) and associated potentials, because it cannot be considered as a result of membrane and solution capacitance in series. The electric energy stored within the membrane (typically much smaller than that in the solution), depends on the membrane permittivity, but also on the external electric field, surface charge density, water permittivity and ion concentration. The effect on capacitances is more specific. Solution capacitance rises with greater solution permittivity or ion concentration, but the membrane capacitance (much smaller than solution capacitance) is only influenced by its permittivity. Interestingly, the effective capacitance is independent of membrane or solution permittivity, but rises as the ion concentration increases and surface charge becomes positive. Experimental estimates of membrane capacitance are thus not necessarily a reliable index of its surface area.     

Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

Membrane capacity of squid giant axon during hyper- and depolarizations

Summary The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarized and depolarized membranes. The duration of DC pulses is 20–40 msec and is long enough to permit the admittance measurements between 1 and 50 kHz. The amplitudes of DC pulses were varied between 0 and 40mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.     

On the frequency response of prestin charge movement in membrane patches

Outer hair cell (OHC) nonlinear membrane capacitance derives from voltage-dependent sensor charge movements within the membrane protein prestin (SLC26a5) that drive OHC electromotility. The ability of the protein to influence hearing depends on its reaction to membrane receptor potentials across auditory frequency. Estimates of prestin’s frequency response have been evaluated by several groups out to tens of kHz in voltage-clamped macro-patches of OHC membrane. The response is a power function of frequency that is down 40 dB at 77 kHz. Despite these observations, concerns remain that the macro-patch approach is flawed due to mechanical constraints of pipette solution column load or patch size itself. In the absence of these influences, prestin’s frequency response is posited by some to be ultrasonic in nature. Here we evaluate the influence of these putative confounding factors on prestin’s frequency response. We show that neither pipette column height nor negative or positive pipette pressure substantially influence total sensor charge frequency response. Additionally, patch surface area has negligible influence. We conclude that the speed of voltage-driven conformational changes in prestin within the plasma membrane is accurately assessed with the macro-patch technique, permitting investigations of membrane characteristics that can substantially alter prestin’s performance bandwidth. We illustrate significant alterations in bandwidth by perturbation of membrane fluidity and chloride anion concentration. Finally, we speculate that OHC membrane characteristics may differ along the tonotopic axis of the cochlea to tune nonlinear membrane capacitance frequency cutoffs.     

Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na⁺ currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. The frequency dependent capacitance increment, ΔC, was of sigmoidal shape and decreased at high frequencies. The midpoint frequency, f ₀, depended on the ionic strength of the buffer. At 150 mm NaCl f ₀ was about 200 Hz and decreased to 12 Hz at high ionic strength (1 M). At low frequencies (f≪f ₀) the capacitance increment became frequency independent. It was, however, dependent on Na⁺ concentration and on the membrane potential which was generated by the charge transferred. A simple model is presented to analyze the experimental data quantitatively as a function of two parameters, the capacitance of the adsorbed membrane fragments, C _P, and the potential of maximum capacitance increment, ψ ₀. Below 5 mm Na⁺ a negative capacitance change was detected which may be assigned to electrogenic Na⁺ binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes. Received: 3 November 1997 / Revised version: 19 February 1998 / Accepted: 21 February 1998     

Fluctuation of motor charge in the lateral membrane of the cochlear outer hair cell

Functioning of the membrane motor of the outer hair cell is tightly associated with transfer of charge across the membrane. To obtain further insights into the motor mechanism, we examined kinetics of charge transfer across the membrane in two different modes. One is to monitor charge transfer induced by changes in the membrane potential as an excess membrane capacitance. The other is to measure spontaneous flip-flops of charges across the membrane under voltage-clamp conditions as current noise. The noise spectrum of current was inverse Lorentzian, and the capacitance was Lorentzian, as theoretically expected. The characteristic frequency of the capacitance was approximately 10 kHz, and that for current noise was approximately 30 kHz. The difference in the characteristic frequencies seems to reflect the difference in the modes of mechanical movement associated with the two physical quantities.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics.

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Photocurrents generated by bacteriorhodopsin on planar bilayer membranes

When purple-membrane fragments from Halobacterium halobium are added to one aqueous phase of a positively-charged black lipid membrane, the membrane becomes photoelectrically active. Under normal conditions the steady-state photo-current is extremely low, but increases considerably when the lipid bilayer is doped with proton-permeable gramicidin channels or with a lipophilic acid-base system. These findings indicate that the purple-membrane sheets are bound to the surface of the bilayer, forming a sandwich-like structure. The time-behaviour of the photocurrent may be interpreted on the basis of a simple equivalent circuit which contains the conductance and capacitance of the purple membrane in series with the conductance and capacitance of the lipid bilayer. From the dependence of the photocurrent on the polarization of the exciting light the average angle between the transition moment of the retinal chromophore and the plane of the bilayer was calculated to be about 28 degrees. Furthermore, it was shown that chromophore-free apomembrane binds to the lipid bilayer and that its photoelectrical activity can be restored in situ by adding all-trans-retinal to the aqueous phase.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Studies of electric capacitance of membranes

Summary The electric capacitance and conductance of a model membrane composed of a hydrophobic filter paper and a synthetic lipid analogue, i.e., dioleylphosphate, immersed in an electrolyte solution were observed with various frequencies ranging from 20 to 3×10⁶ Hz. With successive increase of salt concentration in the external solution, the capacitance and conductance of the membrane increased discontinuously at a certain critical value of the external salt concentration. This variation of the capacitance and conductance of the membrane with the salt concentration was found to be reversible, and the critical value of salt concentration was independent of the adsorbed quantity of the lipid, and of the pore size of the filter paper as far as the adsorbed quantity of the dioleylphosphate was large.A theoretical analysis based on the membrane model for the filter paper-phospholipid system proposed in Part I of this series revealed that the dioleylphosphate impregnated in the filter paper changed its conformation from oil droplets or globular micelles to a number of bilayer membranes when the salt concentration reached the critical value for a given pair of electrolyte species and the membrane. The conformational change of the lipid analogue in the filter paper is discussed in connection with the ability of formation and stability of a black bilayer membrane of the dioleylphosphate.     

Effect of the anesthetics benzyl alcohol and chloroform on bilayers made from monolayers.

The neutral anesthetics chloroform and benzyl alcohol, at concentrations that block the nerve impulse, greatly modify the transport parameters of positive and negative ions in lipid bilayers made from monolayers. Both chloroform and benzyl alcohol increase the membrane permeability to these ions and increase the translocation rate for tetraphenylborate. It was found that both anesthetics increase the membrane permeability to positive ions more markedly than to negative ions. It was also found that the membrane capacitance increases lineary with the concentration of benzyl alcohol. At 51 mM benzyl alcohol, the increase in capacitance is approximately 6%. Chloroform also increases the membrane capacitance; the increase in capacitance was found to be 6% at 18 mM chloroform. An analysis of the changes in the transport parameters of the lipophilic ions, together with the changes in membrane capacitance, suggests that benzyl alcohol and chloroform modify the dipole potential and dielectric constant of the membrane. Benzyl alcohol may also increase the "fluidity" of the lipid bilayer membranes. At 36 mM benzyl alcohol, the membrane permeability to acetamide increases by 38%.     

Frequency domain analysis of membrane capacitance of cultured cells (HeLa and myeloma) using the micropipette technique.

The membrane capacitance and conductance of cultured cells (HeLa and mouse myeloma) are investigated using the micropipette method. Mean values of the membrane capacities were found to be 1.9 microF/cm2 for HeLa cells and 1.0 microF/cm2 for myeloma cells. These values are in agreement with those obtained using the suspension method. Whereas the suspension method is unable to provide the information on membrane conductance, the micropipette method is able to measure even an extremely small membrane conductance if leakage current is negligibly small. The membrane conductances were found, using this technique, to be approximately 90-100 microS/cm2 for both HeLa and myeloma cells. One of the purposes of this study is to establish the frequency profile of membrane capacitance. It was found, however, that membrane capacitances of these cells are independent of frequency between 1 Hz and 1 KHz within the resolution of this technique.     

The molecular organisation of bimolecular lipid membranes. The effect of benzyl alcohol on the structure

The separate effects of benzyl alcohol on the hydrocarbon and polar-head region capacitances and conductances of phosphatidylcholine bimolecular lipid membranes were obtained from measurements of the very low frequency impedance dispersion. It was found that the conductance of the hydrocarbon region (and, to a lesser extent, the polar-head region) increased progressively with increasing concentrations of benzyl alcohol in the external solution. The polar-head capacitance did not show a systematic dependence on the concentration of benzyl alcohol.At low concentrations of benzyl alcohol (7.5 μM) the capacitance of the hydrocarbon region was not significantly affected by the alcohol. At high concentrations (? 7.5 mM) of benzyl alcohol, however, the capacitance of this region was reduced by ≈25%. This is interpreted in terms of an increase in the thickness of this region caused by a straightening of the otherwise kinked, folded (across neighbouring molecules) and perhaps even partially interdigitated hydrocarbon tails of the phosphatidylcholine molecules. This effect of benzyl alcohol is probably closely related also to the apparent increase in the fluidity of the membrane. The effect of benzyl alcohol on the thickness of the hydrocarbon region of the membrane provides a ready insight into its mode of action as a local anaesthetic.     

Impedance spectroscopy of bilayer lipid membranes self-assembled on agar support - interaction with HDL

The electrical properties of the supported lipid bilayer membrane (s-BLM) of egg phosphatidylcholine (PC) self-assembled on agar surface were examined. To characterize the insulating properties of s-BLMs, electrochemical impedance spectroscopy (EIS) was used. The analysis of impedance spectra in terms of an equivalent circuit of agar/electrolyte and agar/s-BLM/electrolyte in the frequency range of 0.1 Hz-10 kHz was performed. The high-density lipoproteins (HDL)/s-BLM interaction in the concentration range from 20 microg/ml to 80 microg/ml of HDL was investigated. It is evident that treatment of s-BLM with HDL resulted in an increase of the lipid film resistance and a decrease of membrane capacitance.     

大鼠嗜铬细胞分泌的膜电容和微碳纤电极检测

在原代培养的大鼠肾上腺嗜铬细胞上,综合运用细胞内钙测定法和全细胞膜片钳法,以检测膜电容变化为手段测定单一肾上腺嗜铬细胞的胞吐过程。－７０ｍＶ到＋２０ｍＶ去极化引起的钙电流和细胞膜电容的变化以及吹加６０ｍｍｏｌ／ＬＫＣｌ时,细胞内游离钙离子浓度［Ｃａ２＋］ｉ和细胞膜电容变化的同时检测,表明了Ｃａ２＋对细胞胞吐的控制作用。而用微碳纤电极则能检测到吹加６０ｍｍｏｌ／ＬＫＣｌ导致嗜铬细胞胞吐时儿茶酚胺的量子化释放。细胞膜电容检测和微碳纤电极检测从不同侧面动态的反映了细胞胞吐过程与Ｃａ２＋的相关性