Studies On Biosensors For Cells And Biocompatibility Of Nanomaterials

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.