耗散型石英晶体微天平在生物医用高分子材料中的应用

石英晶体微天平(QCM)是一种基于石英晶体压电效应的分析检测技术,可实时在线提供石英晶体表面吸附层质量、厚度、粘弹性等信息,由此获得表面分子相互作用关系。 耗散型石英晶体微天平(QCM-D)因其独特的对粘弹性的解析,使其在高分子材料中的应用迅速发展,尤其是生物医用高分子材料领域,已用来评价生物医用高分子材料的表界面相互作用,力学和生物相容性等。 本文简单介绍了耗散型石英晶体微天平的基本原理及理论模型,重点综述了近几年QCM-D在高分子链构象、蛋白质吸附、生物大分子相互作用、药物释放以及水凝胶中的应用,并且展望了QCM-D的未来发展趋势。     

石英晶体微天平生物传感体系构建及其分析应用研究进展

石英晶体微天平(Quartz Crystal Microbalance,QCM)技术是免标记和实时在线表征及研究分子间相互作用的重要工具。近年来在分子生物学、疾病诊断、药物分析、环境监测等研究领域引起广泛关注。本文评述了2011年以来QCM生物传感体系构建及其在分析应用中的研究进展,并对QCM技术在生物传感领域的未来研究动向进行了展望。     

石英晶体微天平的新进展

石英晶体微天平（quartz crystal microbalance,QCM）是一种对界面变化敏感的仪器,它已经在物理、化学、生物学、药物学、临床医学、环境科学等学科的界面问题研究中得到了一定的应用.然而,QCM在液相下的应用和推广一直受限于QCM数据定量解释的困难.为此,科研工作者发展了多种高级的QCM,比如带阻抗分析功能的QCM（impedance QCM,i-QCM）或带能量耗散监测功能的QCM（QCM with dissipation,QCM-D）,同时还发展了许多相应的理论模型.但是,对于多数生物、化学工作者来说,这些理论过于复杂.这极大地限制了QCM的推广和潜力发挥.本文以我们小组在QCM方面的研究工作为线索,对已报道的分析方法、模型和方程按5类应用条件进行了整理,给出了明确的界定标准：它们是：1,固-气界面;2,牛顿流体;3,固-液界面的薄膜;4,固-液界面厚膜;5,固-液界面超厚膜.对于每一类情况,我们将用通俗易懂的语言描述如何对QCM数据进行简化却又保证研究精度需要的定量分析.对于液态环境下的QCM数据的分析,我们着重介绍了＂固化水层＂模型,该模型允许QCM在一定的条件下成为一把＂分子尺＂,工作范围从几个纳米到数百纳米.该分子尺在多个创新界面问题研究中得到很好的应用.最后,我们从理论上分析了QCM作为生物传感器的先天缺陷--因基于面均质量检测的原理,QCM技术对溶液中蛋白的检测下限仅在1μg mL-1数量级.进一步,我们探索了QCM的发展方向和潜在应用领域,希望籍此能进一步推广QCM在各个学科界面问题中的研究应用.     

石英晶体微天平传感器的膜厚敏感性

近几年,石英晶体微天平(QCM)作为一种简易、灵敏的实时监测手段,广泛地应用于研究生物活性分子之间的相互作用,其中包括蛋白质吸附动力学、抗原/抗体相互作用、DNA杂交、适配体-蛋白相互作用等领域.但是QCM作为商业化的生物传感器一直发展不顺利.主要因为QCM在液态环境中的非理想行为导致了对QCM数据分析困难.我们利用阻抗分析法对QCM的数据进行分析,分离出质量和粘弹性两种因素引起的频率改变,解决粘弹性的困扰问题.同时,我们提出了"固化水层"模型,合理地处理了溶剂的影响.在该模型的基础上发展了一种基于QCM的分子尺技术,该技术能够简便、有效地测量出固定在固-液界面的生物大分子的纳米尺寸.我们的主要工作是将"固化水层"模型拓展到三维结构的高分子基质中.我们联合QCM和表面等离子共振(SPR)技术研究基于高分子基质的蛋白质的固定,抗原/抗体识别的过程,进一步采用"固化水层"模型解释高分子的溶胀行为、羧基活化、抗体固定、抗原、抗体识别等过程.理论分析表明,结合生物分子势必排出相同体积的溶剂,由于溶剂的密度接近于蛋白质溶液的密度.从而导致"固化水层"质量增加不明显.实验上也证实了石英晶体微天平的响应主要取决于"固化水层"的厚度变化(T2-T1),而并非固定的生物分子的质量.我们利用QCM实时监测在高分子基质中IgG的固定以及IgG与anti-IgG识别的过程,并将石英晶体微天平监测的频率变化与相应的厚度变化直接关联.这一方法的建立在一定的应用范围内简化了QCM的定量分析模型,有望实现QCM作为传感器在界面物理与化学等相关领域研究中的应用.     

石英晶体微天平在高分子研究中的应用

石英晶体微天平(QCM)作为一种强有力的表征工具已被广泛应用于高分子研究之中.本文中,作者介绍了QCM的发展简史、基本原理以及实验样品制备方法 .在此基础上,介绍了如何基于带有耗散测量功能的石英晶体微天平(QCM-D)及相关联用技术研究界面接枝高分子构象行为、高分子的离子效应以及高分子海洋防污材料,展示了QCM-D技术在高分子研究中的广阔应用前景. QCM-D可同时检测界面高分子薄膜的质量变化和刚性变化,从而反映其结构变化.与光谱型椭偏仪联用后,还可同步获取界面高分子薄膜的厚度变化等信息,可以有效解决相关高分子研究中的问题.希望本文能够对如何利用QCM-D技术开展高分子研究起到一定的启示作用,使这一表征技术能够为高分子研究解决更多问题.     

基于石英晶体微天平的信号放大技术在肿瘤标志物检测中的应用

石英晶体微天平(quartz crystal microbalance, QCM)是一种质量敏感型传感器,近年来被广泛应用于多个领域的分析检测.为了进一步提高检测灵敏度,开发了多种QCM信号放大方法.本文综合介绍了各种QCM信号放大方法,主要聚焦于肿瘤标志物检测中的应用,涉及对核酸序列、蛋白以及肿瘤细胞的检测. QCM的信号放大方法主要是基于质量放大的原理,主要放大技术包括:生物分子偶联、纳米颗粒偶联、生物催化产生不溶物沉淀、金属还原沉积、DNA复制/杂交、晶体原位生长.质量放大子的设计和使用大大增强了QCM的检测能力,提高了其检测灵敏度,拓宽了QCM的应用范围.     

“固化水层”模型拓展石英晶体微天平的应用

石英晶体微天平(QCM)在液态环境下的应用一直由于数据分析复杂而受到限制。最近,我们提出了"固化水层"模型(SLL)以简化QCM数据分析过程。本文进一步提供了SLL模型的有关数据。根据SLL模型,1 Hz QCM频率信号对应于0.18 nm SLL层厚度的变化,而大多数商业的QCM均可以达到该精度。因此,根据SLL模型设计的检测方案将具有很高的灵敏度。QCM作为生物传感器的应用也得以拓展,可以相信SLL模型将大大增强QCM的分析能力。     

石英晶体微天平在手性识别中的应用

石英晶体微天平(QCM)是一类重要的质量型检测器,因具有灵敏度高、分析速度快、检测成本低等优点而具有极好的应用前景,现已广泛应用于环境监测、药物分析、食品质量控制等诸多领域。手性工程的崛起对简单、快速、在线的手性检测技术提出了挑战,QCM手性传感器就是其中一个重要的发展分支。该文简要介绍了QCM的典型实验装置和基本传感原理,详细综述了近年来QCM在手性识别领域的研究进展,包括以环糊精衍生物、分子印迹聚合物、氨基酸衍生物等为手性主体的QCM在手性识别中的应用,并对其今后的发展进行了展望。     

环介导扩增与石英晶体微天平快速检测核酸

建立环介导恒温扩增(LAMP)-石英晶体微天平(QCM)原位快速检测核酸的方法。将环介导恒温核酸扩增(LAMP)技术与石英晶体微天平(QCM)技术相结合,采用巯基化试剂分子组装方法,将LAMP反应体系中的4个引物之一固定于QCM电极上,在安装所述电极的QCM检测池中配置LAMP反应体系并进行环介导恒温核酸扩增,用QCM仪器在线原位检测频率变化,判断LAMP反应是否发生,进而判断体系中是否存在目标核酸特异基因。该方法检测核酸特异性强、灵敏度高,并且操作简便,有望发展成为快速筛查检测核酸的有效手段。     

不同种类敏感膜修饰的QCM气体传感器研究现状

石英晶体微天平(quartz crystal microbalance,QCM)是一种对质量变化敏感的器件,具有灵敏度高、成本低廉、操作简单、可实时在线检测等优点,在气体传感领域受到了广泛关注。敏感材料是石英晶体微天平气体传感器的关键组成部分,本文综述了不同敏感材料包括有机聚合物、超分子化合物、离子液体和分子液体以及近年来备受关注的纳米材料修饰的QCM对特定气体传感检测的研究现状,详细介绍了纳米材料为敏感膜的QCM气体传感器对不同气体传感检测的研究现状及相关敏感机理。最后,在国内外研究现状的基础上,展望了敏感材料的发展前景。QCM作为一种成本低廉、操作方便、测量精度高的气体传感检测器件,将会有更加广阔的应用前景。     

Surface immobilization of bio-functionalized cubosomes: sensing of proteins by quartz crystal microbalance

A strategy for tethering lipid liquid crystalline submicrometer particles (cubosomes) to a gold surface for the detection of proteins is reported. Time-resolved quartz crystal microbalance (QCM-D) was used to monitor the cubosome-protein interaction in real time. To achieve specific binding, cubosomes were prepared from the nonionic surfactant phytantriol, block-copolymer, Pluronic F-127, and a secondary biotinylated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], which enabled attachment of the particles to a neutravidin (NAv)-alkanethiol monolayer at the gold surface of the QCM sensor chip. A second set of cubosomes was further functionalized with addition of the glycolipid (G(M1)) to facilitate a specific binding uptake of the protein, cholera toxin B subunit (CT(B)), from solution. QCM-D confirmed the specificity of the cubosome-NAv binding. The analysis of titration experiments, also performed with QCM, suggests that an optimal concentration of cubosomes is required for the efficient packing of the particles at the surface: high cubosome concentrations lead to chaotic cubosome binding onto the surface, sterically inhibiting surface attachment, or require significant reorganization to permit uniform cubosome coverage. The methodology enabled the straightforward preparation of a complex nanostructured edifice, which was then used to specifically capture analyte proteins (cholera toxin B subunit or free NAv) from solution, supporting the potential for development of this approach as a biosensing platform.     

Immunodetection by quartz crystal microbalance

Biodetection is one of the most important challenges for the twenty-first century: many fields are concerned, mainly environmental and medical. The quartz crystal microbalance (QCM) may offer great possibilities for this purpose: a direct response signal, which characterizes the binding event between a sensitive layer, immobilized onto the surface transducer, and the analyte to be detected, can be obtained. However, for the detection of small biomolecules such as antigens, it is quite difficult to obtain an observable signal that corresponds directly to the binding event. In general, this is owing to the lack of mass sensitivity of the commonly used QCM, with 5- to 10-MHz quartz crystals. For improving this mass sensitivity, a 27-MHz quartz resonator was developed and incorporated in a flow-through microcell. Two biospecies, IgG rabbit and peroxidase enzyme, were studied with this ultra-sensitive QCM in terms of specificity, detection limit, and calibration curve.     

Influence of salt and rinsing protocol on the structure of PAH/PSS polyelectrolyte multilayers

A quartz crystal microbalance (QCM) and dual polarization interferometry (DPI) have been utilized to study how the structure of poly(allylamine hydrochloride) (PAH)/poly(styrene sulfonate) (PSS) multilayers is affected by the rinsing method (i.e., the termination of polyelectrolyte adsorption). The effect of the type of counterions used in the deposition solution was also investigated, and the polyelectrolyte multilayers were formed in a 0.5 M electrolyte solution (NaCl and KBr). From the measurements, it was observed that thicker layers were obtained when using KBr in the deposition solution than when using NaCl. Three different rinsing protocols have been studied: (i) the same electrolyte solution as used during multilayer formation, (ii) pure water, and (iii) first a salt solution (0.5 M) and then pure water. When the multilayer with PAH as the outermost layer was exposed to pure water, an interesting phenomenon was discovered: a large change in the energy dissipation was measured with the QCM. This could be attributed to the swelling of the layer, and from both QCM and DPI it is obvious that only the outermost PAH layer swells (to a thickness of 25-30 nm) because of a decrease in ionic strength and hence an increase in intra- and interchain repulsion, whereas the underlying layers retain a very rigid and compact structure with a low water content. Interestingly, the outermost PAH layer seems to obtain very similar thicknesses in water independent of the electrolyte used for the multilayer buildup. Another interesting aspect was that the measured thickness with the DPI evaluated by a single-layer model did not correlate with the estimated thickness from the model calculations performed on the QCM-D data. Thus, we applied a two-layer model to evaluate the DPI data and the results were in excellent agreement with the QCM-D results. To our knowledge, this evaluation of DPI data has not been done previously.     

Study of film structure and adsorption kinetics of polyelectrolyte multilayer films: effect of pH and polymer concentration

The alternate adsorption of polycation poly(allylamine hydrochloride)(PAH) and the sodium salt of the polymeric dye poly(1-[ p-(3'-carboxy-4'-hydroxyphenylazo)benzenesulfonamido]-1,2-ethandiyl)(PCBS) on quartz crystals coated with silica was studied to understand the structural properties and adsorption kinetics of these films using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D), absorbance, and ellipsometry measurements. In-situ deposition of the polycation PAH on QCM crystals was monitored, followed by rinsing with water and then deposition of the polyanion PCBS. The effects of polymer concentration and pH on film structure, composition and adsorption kinetics were probed. The polymers were adsorbed at neutral pH conditions and at elevated pH conditions where PAH was essentially uncharged to obtain much thicker films. The change in the resonant frequency, Deltaf, of the QCM-D showed a linear decrease with the number of bilayers, a finding consistent with absorbance and ellipsometric thickness measurements which showed linear growth of film thickness. By using the Delta f ratios of PCBS to PAH, the molar ratios of repeat units of PCBS to PAH in the bilayer films as determined by QCM-D were approximately 1:1 at polyelectrolyte concentrations 5-10 mM repeat unit, indicating complete dissociation of the ionic groups. The frequency and dissipation data from the QCM-D experiments were analyzed with the Voigt model to estimate the thickness of the hydrated films which were then compared with thicknesses of dry films measured by ellipsometry. This led to estimates of the water content of the films to be approximately 45 wt %. In addition to the QCM-D, some films were also characterized by a QCM which measures only the first harmonic without dissipation monitoring. For the deposition conditions studied, the deposited mass values measured by the QCM's first harmonic were similar to the results obtained using higher harmonics from QCM-D, indicating that the self-assembled polyelectrolyte films were rigid.     

The change in thickness of the solidified liquid layer rather than the immobilized mass determines the frequency response of a quartz crystal microbalance

The "solidified liquid layer" model has been examined using a quartz crystal microbalance(QCM) with a polymeric matrix.The model is shown to give a reasonable explanation for the following experimental observations:(i) The opposite response of the QCM and surface plasmon resonance(SPR) for the activation process;(ii) the marked difference in the responses for IgG/anti-IgG interaction between QCM and SPR.Theoretical analysis and experimental results indicated that QCM is sensitive to the thickness change of the "solidified liquid layer" but not the mass of captured biomolecules(i.e.,the immobilized mass),implying caution must be taken in interpreting QCM results.     

一种新的压电免疫传感器中生物分子固定化方法的研究

生物分子固定化或传感界面设计技术是研制压电免疫传感器的关键之一。本文 结合自组装单分子膜（SAMs）和聚电解质静电吸附组装技术，提出了一种新的压电 免疫传感器中生物分子固定化方法，研制成一种检测补体C_3的压电免疫传感器。 先在石英晶振的金电极表面组装一层胱胺SAMs，再在膜上组装带相反电荷的聚苯磺 酸钠（PSS）单层膜，通过静电吸附作用固定抗体（抗原），实现对相应抗原（抗 体）的检测。利用扫描电镜技术，从形态上考察了晶振组装胱氨SAMs与PSS及固定 补体C_3抗体后的表面形貌。研究了抗体的固定化条件，探讨了传感器采用这种固 定化方法的响应与再生性能，并与戊二醛键合固定法进行比较。结果表明，这种固 定化方法不仅对蛋白质类生物分子的固定化具有普适性，而且对所固定的生物分子 的活性影响小，传感器的响应的频移值大，灵敏度高，选择性和再生性能均较好。     

Highly sensitive biomolecule detection on a quartz crystal microbalance using gold nanoparticles as signal amplification probes.

We report here a novel strategy for the high-sensitive detection of target biomolecules with very low concentrations on a quartz crystal microbalance (QCM) device using gold nanoparticles as signal enhancement probes. By employing a streptavidin-biotin interaction as a model system, we could prepare biotin-conjugated gold nanoparticles maintaining good dispersion and long-term stability by controlling the biotin density on the surface of gold nanoparticles that have been investigated by UV-vis spectra and AFM images. These results showed that 10 microM N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) was the critical concentration to prevent the nonspecific aggregation of gold nanoparticles in this system. For sensing streptavidin target molecules by QCM, biotinylated BSA was absorbed on the Au surface of the QCM electrode and subsequent coupling of the target streptavidin to the biotin in the sensing interface followed. Amplification of the sensing process was performed by the interaction of the target streptavidin on the sensing surface with gold nanoparticles modified with 10 microM biotin-HPDP. The biotinylated gold nanoparticles were used as signal amplification probes to improve the detection limit, which was 50 ng/ml, of the streptavidin detection system without signal enhancement, and the calibration curve determined for the net frequency changes showed good linearity over a wide range from 1 ng/ml to 10 microg/ml for the quantitative streptavidin target molecule analysis. In addition, the measured dissipation changes suggested that the layer of biotin-BSA adsorbed on the Au electrode and the streptavidin layer assembled on the biotin-BSA surface were highly compact and rigid. On the other hand, the structure formed by the biotinylated gold nanoparticles on the streptavidin layer was flexible and dissipative, being elongated outward from the sensing surface.     

Hydrogels from a water-soluble zwitterionic polythiophene: dynamics under pH change and biomolecular interactions observed using quartz crystal microbalance with dissipation monitoring

The water-soluble zwitterionic polythiophene, poly(3-((S)-5-amino-5-carboxyl-3-oxapentyl)-2,5-thiophene) hydrochloride (POWT), is a conjugated polyelectrolyte (CPE) with properties well suited for biochip applications. CPEs readily form hydrogels when exposed to water-based buffer solutions or biomolecule solutions. In this work, we used in situ quartz crystal microbalance with dissipation (QCM-D) monitoring to collect information on the interaction between POWT films exposed to buffers with different pH and POWT/DNA chains. Our data show that POWT swells significantly when exposed to low-pH buffers, such as pH 4 acetate, this is seen as an increase in thickness and decrease in viscosity obtained via a Voight-based modeling of combined f and D QCM-D measurements. The magnitude of thickness and viscosity change upon changing from a pH 10 carbonate buffer to pH 4 acetate is 100% increase in thickness and 50% decrease in viscosity. The response of the hydrogel under pH change is well correlated with fluorescence data from POWT films on glass. The state of the hydrogel is important during interaction with biomolecules; illustrated by the observation that a swollen CPE hydrogel adsorbs a higher amount of DNA than a compacted one. In agreement with previous results, the QCM-D data confirmed that the POWT/DNA hydrogel sense complementary DNA specifically and with negligible binding of noncomplementary DNA. These results are important for efficient constructions of biochips in water environments using this class of materials.