Interfacial behavior of immortalized hypothalamic mouse neurons detected by acoustic wave propagation

The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 μM), colchicine (1 μM), cytochalasin B (10 μM) and cytochalasin D (2 μM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.     

电化学石英晶体阻抗系统研究金电极上Fe(CN)_6~(3-)/Fe(CN)_6~(4-)电化学过程

电化学过程的石英晶体阻抗分析法已用于现场获取电活性聚合物粘弹性等信息［１,２］．本文联用ＨＰ４３９５Ａ阻抗／网络／频谱分析仪和ＥＧ＆ＧＭ２８３恒电位仪开发出电化学石英晶体阻抗系统（ＥｌｅｃｔｒｏｃｈｅｍｉｃａｌＱｕａｒｔｚＣｒｙｓｔａｌＩｍｐｅｄａｎ...     

Surface immobilisation and properties of smooth muscle cells monitored by on-line acoustic wave detector

The attachment of rat aortic smooth muscle cells to various surfaces has been monitored by a thickness shear mode acoustic wave device incorporated into an on-line configuration. Using the total injection analysis method, laminin and fibronectin were adsorbed to the device surface, to be followed by introduction of cells into the system. The results of these experiments in terms of frequency and motional resistance measurements were also compared with those for cell attachment to the bare gold electrode of the sensor. The responses of the surface-bound cells to the introduction of various ions, depolarisation events and damage subsequent to exposure to hydrogen peroxide were also observed. Morphological changes in the cells, as confirmed by scanning electron microscopy, are correlated with results of the acoustic wave measurements.     

A comparative study on the viscoelasticity and morphology of polyaniline films galvanostatically grown on bare and 4-aminothiophenol-modified gold electrodes using an electrochemical quartz crystal impedance system and SEM.

The equivalent-circuit parameters of the 9-MHz piezoelectric quartz crystal (PQC) resonance were measured in situ during the galvanostatic polymerization of aniline on 4-aminothiophenol(4-ATP)-modified and bare Au electrodes for ca. 2000 s, respectively. Two polymerization media, 0.100 mol L-1 aniline in 1.0 mol L-1 H2SO4 and in 2.0 mol L-1 HClO4 aqueous solutions, and two values of the current density, 12 and 36 microA cm-2, were used. At identical levels of the resonant frequency shifts in the solutions, obviously greater increases in the motional resistance (R1) were found after aniline polymerization on bare Au electrodes, though the absolute values of delta f0/delta R1 were all large; also, the resonant frequency shifts in air (delta f0g) were considerably smaller for PANI films grown on bare Au electrodes. It is thus concluded that, under identical polymerization conditions, (1) the PANI film grown on a bare gold electrode is rougher, less compact, and can entrap solution more notably; (2) the deposition efficiency of PANI is higher on a 4-ATP-modified Au electrode, owing to a significantly greater observed "dry" frequency shift, and thus a greater "net" mass value of the polyaniline backbone. SEM observations have confirmed that PANI films on 4-ATP-modified Au electrodes were smoother and more compact than those grown on bare Au ones under identical polymerization conditions. In addition, a technique of simultaneous measurements of the electroacoustic admittance of the PQC resonance and the electrochemical impedance was used to monitor the adsorption of 4-ATP onto a PQC gold electrode.     

High sensitive piezoelectric quartz crystal liquid density sensor

<正>We report for the first time a cleavage phenomenon in the resonant peak of a piezoelectric quartz crystal(PQC) in liquid phase.In the presence of a strong longitudinal wave effect,an additional resonant peak appears in the conductance-frequency curve.With gradually increasing liquid density,the additional peak moves from low to high frequency region then disappears.The frequency of the additional resonant peak is sensitive to the change in liquid density.The frequency shift of the additional peak is linear with the liquid density in a given range.For a 5 MHz PQC with a reflection distance of 16 mm for longitudinal wave,the sensitivity to liquid density is 2.61×10~6 Hz g~(-1) cm~3.The overlap between the primary resonant peak and the additional resonant peak causes a decrease in the intensity of the former and an increase in the intensity of the latter.In a combined impedance analysis method,the changes in surface mass loading,density and viscosity of the liquid were monitored simultaneously by a PQC sensor.     

Electrode modification and the response of the acoustic shear wave device operating in liquids

The effect of electrode polarity, geometry, and stray capacitance on the performance of the thickness-shear mode acoustic wave sensor operating in electrolytes and solutions of biomolecules has been studied. In contrast to the well-known mass-based response of the device operating in the gas phase, the response in a liquid is governed by several factors including acoustoelectric and fringing field effects, which are known to be active at the edges of the electrodes. In order to investigate and utilize these effects, we modified the electrode geometry to increase the edge length, which, in turn, raises the sensitivity of the device. These changes which constituted either complete coverage of the back of the device with electrode material, or the removal of disks and lines from the electrode surface, resulted in a two to three times enhancement of sensor response. Such modifications that extend device sensitivity beyond the electrode area to the quartz region of the sensing structure also provide a better surface for the immobilization of various probes. We verified the enhancing ability of the modified electrodes for the case of adsorption of the protein avidin and neutravidin, followed by their affinity reactions with biotinylated biomolecules. It was found that the active electrode in contact with electrolyte exhibits a sensitivity of about twice that of the grounded electrode. The existence of stray capacitance around the cell was confirmed by shielding the cell assembly with a bath of concentrated KCl solution. This shielding effect was measured to be about 25-60 Hz in series resonant frequency and -1000 Hz in parallel resonant frequency.     

Different experimental results for the influence of immersion angle on the resonant frequency of a quartz crystal microbalance in a liquid phase: with a comment

This paper presents different experimental results of the influence of an immersion angle (θ, the angle between the surface of a quartz crystal resonator and the horizon) on the resonant frequency of a quartz crystal microbalance (QCM) sensor exposed one side of its sensing surfaces to liquid. The experimental results show that the immersion angle is an added factor that may influence the frequency of the QCM sensor. This type of influence is caused by variation of the reflection conditions of the longitudinal wave between the QCM sensor and the walls of the detection cell. The frequency shifts, measured by varying θ, are related to the QCM sensor used. When a QCM sensor with a weak longitudinal wave is used, its resonant frequency is nearly independent of θ. But, if a QCM sensor with a strong longitudinal wave is employed, the immersion angle is a potential error source for the measurements performed on the QCM sensor. When the reflection conditions of the longitudinal wave are reduced, the influence of θ on the resonant frequency of the QCM sensor is negligible. The slope of the plot of frequency shifts (ΔF) versus (ρη)^1/2, the square root of the product of solution density (ρ) and viscosity (η), may be influenced by θ in a single experiment for the QCM sensor with a strong longitudinal wave in low viscous liquids, which can however, be effectively weakened by using the averaged values of reduplicated experiments. In solutions with a large (ρη)^1/2 region (0-55 wt% sucrose solution as an example, with ρ value from 1.00 to 1.26 g cm⁻³ and η value from 0.01 to 0.22 g cm⁻¹ s⁻¹, respectively), the slope of the plot of ΔF versus (ρη)^1/2 is independent of θ even for the QCM sensor with a strong longitudinal wave in a single experiment. The influence of θ on the resonant frequency of the QCM sensor should be taken into consideration in its applications in liquid phase.     

High-pressure Raman study of vibrational relaxation in N2O

The vibrational widths of the ν₁ and ν₃ Raman bands of N₂O were determined at pressures ranging from 8 bar to 2 kbar and temperatures varied from 25 to 150°C. The different dephasing theories including motional narrowing collisional models and resonant vibrational energy transfer theory were tested. A comparison of the theoretical predictions with the experimental data indicates the resonance VV transfer represents the dominant broadening mechanism. The observed frequency shifts between isotropic and anisotropic components of the bands were interpreted in terms of dipole-dipole interactions in dense N₂O.     

A study of adsorption behavior of human serum albumin and ovalbumin on hydroxyapatite/chitosan composite

In situ adsorption of human serum albumin (HSA) and ovalbumin (OVA) was real-time monitored by piezoelectric quartz crystal impedance (PQCI) technique to fully understand the initial cellular response on hydroxyapatite/chitosan (HAP/CS) composite. The PQCI parameters, such as resonant frequency (f), static capacitance (C_s), and motional resistance (R_m) were measured for investigating the kinetic adsorption behaviors of both proteins. The change in frequency shifts (Δf) depends on the amount of the adsorbed protein, and the change in motional resistance (ΔR_m) results from the microporosity variation of HAP/CS coating. The results show that the amount of the absorbed HSA is much greater than that of OVA on HAP/CS coating because of the unique construction of HSA as well as a flexible protein. Furthermore, Δf and ΔR_m data were fitted according to the kinetic exponential decay equations. It can be seen that there is only one adsorption process for OVA, but the absorption process for HSA is followed by a rearrangement process, and the former process is faster than the rearrangement process. Subsequently, the composite binding with proteins were demonstrated by the Fourier transform infrared (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).     

QCM/HCC as a platform for detecting the binding of warfarin to an immobilized film of human serum albumin

Quartz crystal microbalance/heat conduction calorimetry (QCM/HCC) is a new measurement technology that has been used to monitor simultaneously the mass and motional resistance of a thin film in conjunction with the heat flow produced by a chemical change in the film initiated by reaction with a gas. In this work we examine the applicability of the QCM/HCC in detecting chemical changes at the solution/thin film interface. Human serum albumin (HSA) was bound to the gold electrode of a 5 MHz AT-cut quartz resonator using three types of linkers and then exposed to buffered solutions of the anticoagulant drug warfarin. Changes in resonator frequency and motional resistance as well as changes in heat flow produced by warfarin binding to HSA were monitored as a function of the warfarin concentration. Differences in frequency and motional resistance changes depend upon the linker and vary both in magnitude and sign, whereas the integrated heat signal is proportional to the concentration of warfarin and independent of the linker chemistry. Quartz crystal microbalance/heat conduction calorimetry can thus be a useful tool for studying protein-ligand interactions at the solution-surface interface, even though the quartz resonator does not behave as a microbalance.     

pH-dependent behavior of surface-immobilized artificial leucine zipper proteins

The coiled-coil protein motif occurs in over 200 proteins and has generated interest for a range of applications requiring surface immobilization of the constituent peptides. This paper describes an investigation of the environment-responsive behavior of a monolayer of surface-immobilized artificial proteins, which are known to assemble to form coiled-coil structures in bulk solution. An extended version of the quartz crystal microbalance (QCM-D) and surface plasmon resonance (SPR) are independently employed to characterize the adsorption of the proteins to a gold surface. The data suggest that the molecules arrange in a closely packed layer orientated perpendicular to the surface. QCM-D measurements are also employed to measure pH-induced changes in the resonant frequency (f) and the energy dissipation factor (D) of a gold-coated quartz crystal functionalized with the formed monolayer. Exposure of the protein monolayer to a pH 4.5 solution results in a shift of 43 Hz in f and a shift of -0.7 x 10(-6) in D as compared to pH 7.4. In contrast, increasing the pH to 11.2, results in f and D shifts of -17 Hz and 0.6 x 10(-6), respectively. The magnitude of the observed shifts suggests that the proteins form a rigid layer at low pH that can be hydrated to a fluid layer as the pH is increased. These observations correlate with spectroscopic changes that indicate a reduction in the helical content of the protein in bulk solutions of high pH.     

H2S gas sensor based on integrated resonant dual-microcantilevers with high sensitivity and identification capability

Detection of trace-level hydrogen sulfide (H₂S) gas is of great importance whether in industrial production or disease diagnosis. This research presents a novel H₂S gas sensor based on integrated resonant dual-microcantilevers which can identify and detect trace-level H₂S in real-time. The sensor consists of two integrated resonant microcantilever sensors with different functions. One cantilever sensor can identify H₂S by outputting positive frequency shift signals, while the other cantilever sensor will detect H₂S as a normally used cantilever sensor with negative frequency shifts. Combined the two cantilever sensors, the proposed gas sensor can distinguish H₂S from a variety of common gases, and the detection limit to H₂S of the sensor is as sensitive as below 1 ppb.     

The study of surface properties of an IgE-sensitive aptasensor using an acoustic method

We applied the acoustic transverse shear mode (TSM) method for study of the surface properties of a DNA aptasensor that specifically binds human immunoglobulin E (IgE). The biotinylated 45-mer DNA aptamers were immobilized on the surface of a self-assembled layer composed of a mixture of polyamidoamine dendrimers of the fourth generation with 1-hexadecanetiol covered by neutravidin. Using the TSM method, we studied the kinetics of changes of the series resonant frequency, f _s, and the motional resistance, R _m, of a quartz crystal transducer, used as a support for formation of the sensing layer. We have shown that attachment of the biotinylated DNA aptamers onto the surface covered by neutravidin results in a decrease of f _s, but in an increase of R _m. Similar changes of f _s and R _m were observed following addition of IgE. This suggests the contribution of friction forces to the crystal oscillation, which was taken into account in the calculation of the mass changes at the sensor surface following binding processes.     

Multi-modal biochip for simultaneous, real-time measurement of adhesion and electrical activity of neurons in culture

Recent evidence suggests that integrin-mediated adhesion of neurons has immediate functional implications for learning and memory. In addition, adhesion of neurons to artificial substrates often determines the effectiveness and life of implants in the brain and peripheral nervous system. In this study, we present a novel biochip capable of simultaneous, quantitative, real-time monitoring of integrin-mediated adhesion and electrophysiology of primary neurons in vitro. The proposed technology combines acoustic micro-resonators capable of tracking changes in mechanics of the adhering neuronal layer, and microelectrode arrays for recording extracellular unit activity. Our results showed in four different experimental paradigms that the acoustic sensor response to adhering cells is correlated to integrin-mediated adhesion and that the micro-sensor is capable of monitoring the dynamics of neuronal adhesion over a period of 9 days. Finally, using our unique dual measurement platform, we performed simultaneous, real-time measurement of integrin-mediated adhesion and single cell electrophysiology in a neuronal culture. The sensitivities of the micro-resonators were 4-5 orders of magnitude greater than the sensitivity of the macro-scale resonators in response to adhering neurons. This multi-functional sensor platform offers insight into the interplay between integrin-mediated adhesion and neural function on a temporal resolution beyond any currently available experimental method and can therefore potentially lead to novel discoveries on the interactions between neuronal adhesion and function.     

Measuring cell adhesion on RGD-modified, self-assembled PEG monolayers using the quartz crystal microbalance technique

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.     

Acoustic coupling of transverse waves as a mechanism for the label-free detection of protein-small molecule interactions

An on-line acoustic transverse wave device has been used to study the binding interactions of human serum albumin with the small molecule drug, warfarin. Four linking systems for the covalent attachment of the protein to the surface of the gold electrode of the sensor were employed, namely thioctic acid, cysteamine, an N-hydroxysuccinimide ester and 11-mercaptoundecanoic acid. All the attachment protocols involve the ability of thiols to form gold-sulfur bonds at the metal surface. The functional group present at the distal end of each thiol was chemically activated in order to facilitate covalent attachment of the protein. On-line sensor measurements of acoustic parameters show that the binding of warfarin to the protein can be detected, and depending on the linking monolayer used three of four possible combinations of changes in series resonance frequency and motional resistance are observed. Calculations of possible mass and thickness viscoelastic effects demonstrate that these conventional notions are invalid in terms of an explanation of the acoustic signals observed for the warfarin-protein interaction. The responses are ascribed to acoustic coupling phenomena.     

基于微电极阵列的睡眠剥夺大鼠海马区神经电活动检测

Sleep deprivation (SD) is the partial or complete loss of sleep and has long been used as a tool in sleep research to interfere with normal sleep cycles in rodents and humans. The progressively-accumulating sleep pressure induced by sleep deprivation can lead to a variety of physiological changes and even death. Compared to traditional detection methods, in vivo detection of neuronal activity using micro-electromechanical system (MEMS) technology following sleep deprivation can help fully elucidate the effects of sleep deprivation at the cellular level. Herein, a computer-controlled rotary roller was used to completely deprive rats of sleep for 14 days and 16-channel microelectrode arrays (MEAs) were fabricated and implanted into the rat hippocampus to measure neural spikes and local field potentials (LFPs) in real-time. The hippocampus is involved in learning and memory and has been the focus of intensive research aimed at understanding the function of sleep. This study was performed to measure the changes in neuronal activity in the rat hippocampus induced by sleep deprivation as well as their overall impact on the brain. After sleep deprivation, both the pyramidal- and inter-neurons showed a higher amplitude and more intense firing patterns. The fast-firing pattern of the neurons after sleep deprivation indicated elevated excitability in the prolonged awake state. In addition, the LFP of the sleep deprived rats fluctuated more frequently. The power of the LFPs in the low-frequency band (0–50 Hz) was calculated, showing increased power of the delta, theta, alpha, and beta bands after sleep deprivation, especially for the delta band (0.1–4 Hz). Generally, LFPs are generated by all types of neural activity in the neural circuit, and the changes in the low frequency band power suggested decreased arousal and increased sleep pressure induced by sleep deprivation, which could further impair brain function. This study was mainly aimed at measuring electrophysiological changes induced by sleep deprivation in the rat brain. Typically, neuronal activity changes were accompanied by the alternation of specific neurotransmitters in the brain. In the future, it will be essential to focus on measuring the concurrent change of electrophysiological and neurochemical signals to better examine the impact of sleep deprivation on brain function.     

Influence of molecular adsorption on the dielectric properties of a single wall nanotube: a model sensor

Recent measurements of the resonance frequency of a copper disk covered with carbon nanotube bundles have shown characteristic resonance shifts during exposure with various gas molecules. The shifts were interpreted as the change of the dielectric permittivity of the system forming the sensor due to the electric properties of the adsorbed molecules. Starting from a simplified sensor model formed by one single wall nanotube, we develop a self-consistent approach to describe the variation of the linear dielectric susceptibility of the tube at the atomic scale when molecules are adsorbed at its external surface. The sensitivity of this model sensor is tested as a function of the apolar or polar nature of the admolecules, their adsorption geometry, their concentration, and the characteristics of the tube (length, diameter,...). The comparison with data on dielectric constant changes vs adsorption, coming from measurements of the resonance frequency shifts, displays striking agreement for most of the molecular species considered.     

A Novel QCM-based Biosensor for Detection of Microorganisms Producing Hydrogen Sulfide

Abstract

A novel piezoelectric quartz crystal microbalance (QCM) device with gas permeable membrane is proposed for the detection of microorganisms producing hydrogen sulfide (H₂S). The detection theory is based on the adsorption of hydrogen sulfide onto the silver electrode of the piezoelectric crystal sensor, which causes a dramatic decrease in the resonant frequency of QCM. A 100 Hz frequency shift is chosen as the criteria value to judge the presence of microorganisms producing H₂S. Factors affecting detection were investigated. Desiccant is of great practical importance in sensor response. This new biosensor can be a potential candidate for detecting bacteria which produce hydrogen sulfide.     

Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers

Lead Zirconate Titanate (PZT)-embedded microcantilevers were fabricated with dimensions of 30 × 90 × 3 μm(3) (width × length × thickness). A thicker PZT layer improved the actuation and enabled long-term data acquisition in common aqueous buffers with a frequency resolution of 20 Hz. A quantitative assay was conducted in the range of 1-20 μM and the resonant frequency was found to increase with the concentration of target DNAs and the probe DNAs were almost saturated at 20 μM. Back-filling with ethyleneglycol-modified alkanethiol was shown to facilitate the hybridization efficiency and stabilize the surface reaction, resulting in a signal enhancement of 40%. We report for the first time how secondary structures in oligonucleotide monolayer change the surface property of a dynamic mode microcantilever and subsequently affect its oscillating behavior. Using fabricated microcantilevers, the real time changes in resonant frequency upon hybridization were measured by utilizing different probe and target sets. The results revealed that the microcantilevers experienced a resonant frequency upshift during the hybridization with complementary DNAs if a dimer structure was present between DNA probes. A resonant frequency downshift was observed for DNA probes that did not contain any complex secondary structures. In addition, the results demonstrate the potential of using these microcantilevers to extract structural information of oligonucleotides.