【作者】 肖锡林；

【导师】 姚守拙； 蔡青云；

【作者基本信息】 湖南大学 ， 分析化学， 2010， 博士

【摘要】 本论文开展了细胞传感新方法和纳米材料生物相容性的研究。纳米生物材料是纳米材料和生物材料交叉而成的一个全新领域。纳米尺度的特殊生物效应使得纳米生物材料具有广泛的应用前景。细胞体外分析对细胞生理学行为的研究以及药物的体外筛选都具有重要意义。纳米生物材料细胞相容性界面的构建对促进细胞传感器的研制、组织工程的发展和组织工程材料的临床应用都起着重要的作用。磁弹性传感器是近几年迅速发展起来的无线无源传感器,在活体、在体及无损检测等领域具有广泛的应用前景,特别适用于需无菌操作(如细胞检测)的科学实验中。另外,细胞的新陈代谢、呼吸作用、光合作用、信息传递、物质的跨膜运输等活动都涉及到细胞荷电粒子或电活性粒子的定向有序传递、传导或转移,即涉及到细胞物质有序、专一、特异的氧化和还原。电化学方法具有简单、快速、方便等优点。因此,可用电化学手段研究细胞的生理行为或评估药物与细胞的相互作用等。基于上述描述,本论文主要进行了以下几方面的研究工作：1.乳腺癌细胞(MCF-7)无线磁弹性传感器制备及应用。本工作应用无线传感器原位监测了MCF-7细胞的正常生长及抗癌药物作用下的生长过程,评价了5-氟尿嘧啶和顺铂对MCF-7细胞生长的抑制效果。无线传感器是由非晶态金属铁合金磁条带切割而成的矩形磁振片,表面涂一层聚氨酯膜保护磁振片不被氧化腐蚀,提供一个细胞相容性好的细胞生长附着面。传感器在一定频率范围内的激发磁场中振动,振动产生的电磁流可以通过感应线圈远距离遥感检测,而传感器与检测系统无需任何物理连接,这一特性有利于实现生物实验过程的无菌操作,特别是细胞培养过程。MCF-7细胞贴附在传感器表面生长,可引起传感器共振振辐减小,并且共振振幅的减少与细胞的初始接种浓度成线性关系,线性范围为5×104-1×106 cells mL-1,检出限为1.2×104 cells mL-1。本工作中通过增大激发共振振辐来评价细胞贴壁的生长能力,并用磁传感器评价了5-氟尿嘧啶和顺铂这两种抗癌药物对MCF-7细胞生长的抑制作用。当MCF-7细胞生长到20 h时,5-氟尿嘧啶和顺铂的半数致死浓度(LC50)分别为19.9和13.1μM。2. Escherichia coli O157:H7 (E. coli O157:H7)无线磁弹性传感器制备及应用。以预聚合了聚氨酯膜的磁性膜片为敏感元件,制备了无线磁弹性微生物传感器；研究了微生物生长导致培养基性质(如黏度、密度等)改变对传感器共振频率的影响,考察了细菌在传感器表面吸附对传感器响应的贡献；研究了庆大霉素注射液对E. coli 0157:H7生长的抑制作用,依据庆大霉素注射液对细菌的抑制作用可以进行抗生素的药效评测。E. coli O157:H7的生长和繁殖导致溶液黏度减小,从而引起磁弹性传感器共振频率增大；细菌生长过程中在传感器表面有一定吸附,从而引起传感器共振频率减小,溶液性质变化和细菌吸附程度共同影响共振频率的变化。该传感器可测定的E.coli O157:H7浓度为2×102-3×106 cellsmL-1,检测限为102 cells mL-1。基于此,建立了可用于细菌早期诊断和快速分析的无线磁弹性传感检测新方法。3.支原体无线磁弹性传感器制备及应用。支原体是最小、最简单、能够自我复制的无细胞壁致病微生物。本文应用无线磁弹性传感器实时监测了生殖支原体的正常生长及抗生素抑制生长过程,评价了四环素和左氧氟沙星对生殖支原体的抑制效果。无线传感器是由28μm厚的带状非晶态铁磁合金2826MB(Fe40Ni38Mo4B18)切割而成的矩形磁振片,表面涂一层聚氨基甲酸酯膜保护磁振片不被氧化腐蚀,为生殖支原体提供了一个生长附着面。传感器在一定频率范围内的激发磁场中纵向振动,振动产生的磁通量可以通过感应线圈远距离遥感检测,而传感器与检测系统无需任何物理连接,这一特性有利于实现生殖支原体培养过程的无菌操作。生殖支原体黏附在传感器表面生长使传感器共振频率下降,共振频率下降值与生殖支原体的接种浓度存在线性关系,通过细菌生长抑制实验评价了两种抗生素的抑制效果。由实验得到,当细菌生长时间为120 h时,四环素的平均抑制浓度(MIC50)为1.5μg mL-1,左氧氟沙星的MIC50为0.5μg mL-1。4.大肠杆菌碳纳米管方波伏安传感器制备及应用。采用循环伏安和方波伏安技术研究了多壁碳纳米管(MWCNTs)修饰玻碳电极上大肠杆菌细胞的电化学行为。与裸玻碳电极相比,修饰电极对细胞悬浮液中电化学活性物质的氧化有催化作用。在细胞生长过程中,各个时期的细胞活性与方波伏安分析信号趋势一致。利用该技术评估了抗生素庆大霉素对大肠杆菌细胞的活性抑制作用,同时用形态学技术进行了验证。5.TiO2纳米管(TiO2 NTs)阵列的细胞相容性及应用。在本工作中,应用纯Ti片阳极氧化技术制得管径大约为100 nm,且易于被酒精分散或从Ti片基底上分离的良好的TiO2 NTs。为了探索此种纳米结构的细胞相容性及组织工程应用的可能,通过使用荧光显微镜,研究和比较了不同的癌细胞以及正常细胞在此种材料表面生长的情况。实验结果表明此种新的生物材料能够维持细胞的生长,并且不需要被覆细胞外基质(Extracellular matrix, ECM)。相继研究了纳米管中顺铂药物的释放动力学及对成骨肉瘤细胞(human osteosarcoma, MG-63)贴壁生长的影响。表面积为0.75 cm×1 cm的二氧化钛基底上实验载药量分别为75μg、150μg和225μg顺铂。实验结果表明,此种TiO2 NTs易于载抗癌药物,并且载药TiO2 NTs能够有效地减少MG-63细胞在纳米管表面的贴附。这些研究发现表明,TiO2 NTs是一种良好的潜在载药材料,并且有希望在移植工程(尤其是肿瘤切除病例)中发挥巨大的作用。更多还原

【Abstract】 This dissertation is mainly devoted to study the biocompatibility of nanobiomaterials and the new sensor method for cell detection. Nanobiomaterial is a new field which combines nanomaterials and biological materials. Nanobiomaterials have many potential applications due to their special size effect on bio-systems. In vitro assays using cultured cells are of great significance for studying many aspects of cell biology and drug screening. The construction of cellular compatible (cytocompatible) interface of nanomaterials plays a key role for the fabrication of cell-based biosensors, and could undoubtedly promote the development of tissue engineering. Magnetoelastic sensors are wireless passive sensors and developed rapidly in recent years. The wireless nature of the magnetoelastic sensor makes it a powerful candidate for in situ and in vivo analysis, particularly appropriate for aseptic operations (such as cell detection) experiment. Moreover, the metabolism, respiration, photosynthesis, message communication and substance transportation between membranes of living cells are all related to electron or electroactive species transfering in order and specific manner. Electrochemical technique is very simple, rapid and cheap. Therefore, it can be easily used to study many aspects of cell biology and drug screening. Based on the above description, this dissertation is devoted to the wireless magnetoelastic/electrochemical cell sensors and the cytocompatibility of TiO2 nanotube arrays. The details of work are summarized as follows:1. The fabrication of wireless magnetoelastic human breast cancer cells (MCF-7) sensor and its application. A wireless sensing device was developed for the in-situ monitoring of the growth of MCF-7 cells and evaluation of the cytotoxicity of the anticancer drugs fluorouracil and cisplatin. The sensor is fabricated by coating a magnetoelastic ribbon-like sensor with a layer of polyurethane that protects the iron-rich sensor from oxidation and provides a cell-compatible surface. In response to a time-varying magnetic field, the magnetoelastic sensor longitudinally vibrates, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic biological operation, especially in cell culture as illustrated in this work. The adhesion of cells on the sensor surface results in a decrease of the resonance amplitude, which is proportional to the cell concentration. A linear response was obtained in cell concentrations of 5×104-1×106 cells mL-1, with a detection limit of 1.2×104 cells mL-1. The adhesion strength of cells on the sensor is qualitatively evaluated by increasing the amplitude of the magnetic excitation field. And the cytotoxicity of the anticancer drugs fluorouracil and cisplatin is evaluated by the magnetoelastic biosensor. The cytostatic curve is related with the quantity of cytostatic drug. The LC50 (Lethal Concentration) for cells incubated in the presence of drugs for 20 h is calculated to be 19.9μM for fluorouracil and 13.1μM for cisplatin.2. The fabrication of wireless magnetoelastic E. coli O157:H7 sensor and its application. The microorganism sensor is fabricated by coating a magnetoelastic-ribbon with a polyurethane protecting film. Bacteria consume nutrients from the culture medium and produce small molecules during the growing process, causing a corresponding change in viscosity of culture medium. The resonant frequency of a magnetoelastic sensor changes due to the properties changes of a liquid culture medium and the adhesion of bacteria to the sensor surface. The effect of gentamycin sulfate injection (GSI) on proliferation of the bacteria was investigated, which can be used to evaluate the medicament effect of antibiotic. The growth and reproduction of E. coli O157:H7 decreases the solution viscosity, and in turn the resonance frequency of the magnetoelastic sensor increases, the bacteria adhesion reversely results in the decrease of the resonance frequency, change of solution properties and bacteria adhesion affect the resonance frequency together. Using the described sensor we are able to directly quantify E. coli O157:H7 concentrations over the range of 2×102-3×106 cells mL-1, with a detection limit of 102 cells mL-1. Thus, a wireless magnetoleastic microorganism sensor is developed for the early diagnosis and rapid detection of bacteria.3. The fabrication of wireless magnetoelastic Mycoplasma genitalium (Mg) sensor and its application. Mg is the smallest and simplest self-replicating bacteria lacking of cell wall and is a human pathogen causing various diseases. This paper describes the real-time, long-term and in-situ monitoring of the growth of Mg and evaluation of the effect of the antibiotics tetracycline and levofloxacin on the growth using a wireless magnetoelastic sensor. The sensor is fabricated by coating a magnetoelastic strip with a polyurethane protecting film, which protects the iron-rich sensor from oxidation and provides an Mg-compatible surface. In response to a time-varying magnetic field, the sensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic operation. The adhesion of Mg on the sensor surface results in a decrease in the resonance frequency, which is proportional to the concentration of Mg. The shift of the resonance frequency-time curves shows that under routine culture condition the growth curve of Mg is composed of three phases which are lag, logarithmic and stationary phase, respectively. In the presence of the antibiotics, the lag phase in the growth inhibition curves is prolonged obviously and the stationary phase is substituted by a decline phase. The growth inhibition of Mg is related to the concentration of the antibiotics. The MIC50 (Minimal Inhibitory Concentration required to inhibit the growth of 50% of organisms) of Mg incubated in the presence of the antibiotics for 120 h is calculated to be 1.5 and 0.5μg mL-1 for tetracycline and levofloxacin respectively.4. The fabrication of square-wave voltammetry (SWV) carbon nanotubes sensor for E. coli and its application. The electrochemical behavior of E. coli cell suspension on multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode was studied by using cyclic voltammetry (CV) and SWV. Compared with bare glassy carbon electrode, the MWCNTs-modified electrode shows electrocatalytic property to the oxidation of electroactive species in the cell suspension. The baseline-corrected SWV signal is found to be related to the viability of cells and the technique was used for monitoring the growth of E. coli cells. The effect of antibiotics drug GSI on the growth of E. coli cells was also investigated by SWV and microscopy methods.5. The cytocompatibility of TiO2 nanotube arrays and its application. We have developed well-controlled TiO2 nanotubes (NTs) array by anodic oxidation of a pure titanium sheet in an electrolyte solution containing sodium fluoride. To explore the cytocompatibility of this novel nanostructured surface, the growth of different cancer and normal cells on the TiO2 NTs substrate was investigated by fluorescence microscopy, with the aim to evaluate NTs films as substrates for cell-based and tissue-based applications. Our results strongly suggest that this new biomaterial supports normal growth and adhesion of cells with no need for coating with ECM protein. We have loaded these TiO2 nanotube substrates (0.75 cm x 1 cm) with 75μg, 150μg and 225μg of cisplatin. The cisplatin release kinetics from these nanotubes and its effect on MG-63 cell adhesion was investigated. Our results indicate that we can effectively fill the nanotubes with the antineoplastic drug and the drug eluting nanotubes significantly reduce MG-63 adhesion on the surface. These findings suggest that TiO2 nanotube is a potential candidate for a delivery vehicle of therapeutic agents in implantable drug delivery applications.更多还原