Analytical Application Of Liposome And Single Wall Carbon Nanotubes In The Bioassay And Diagnosis In Clinic

Cholera toxin (CT) is a protein enterotoxin secreted by the bacterium Vibrio cholerae that can cause an epidemic disease leading to rapid dehydration, acidosis, and even death in several hours without appropriate treatment. Most cholera cases are reported in many underdeveloped countries, and it is estimated that cholera causes approximately 120,000 deaths annually. As a typical biomarker of Vibrio cholerae, CT has become a very important target in biological detection, and there has been increasing interest in the development of rapid and sensitive methods for the determination of CT. In addition, prostate cancer (PCa) has become a most widespread and stubborn diseases and a major cause of death in the old age male population nowadays. It admits of no delay for the sensitive diagnosis and efficient treatment of PCa. Porstate-specific antigen (PSA), a 33-kD single-chain glycoprotein with chymotrypsin-like protease activity, has been used as the most validated marker for the detection of PCa in screening, diagnosis and monitor of disease recurrence after surgical prostatectomy.Use of chemo/biosensing techniques to detect the meaningful bio-markers for the accurate and rapid diagnosis of clinical diseases as well as for the field screening and mass monitoring of the epidemic diseases has been a novel, attractive and hot topic in the current medical studies. In the past few years, nanomaterials have been widely researched and used. Applying nanomaterials for the fabrication of biosensors will greatly improve the performance of the resulting biosensor.liposomes are formed spontaneously when phospholipids are dispersed into water. Liposomes can simply be portrayed as spherical vesicles consisting of one or more phospholipid bilayers surrounding an aqueous cavity. Molecules such as fluorophores, enzymes or drugs, present in the aqueous phase during the preparation, can be encapsulated in the liposome. Liposomes have been widely used as analytical tools in immunochemistry and can be implemented with hardly any modifications. Since colloidal Au has a very large surface area and good bio-compatibility, the use of colloidal particles as versatile and efficient templates for the immobilization of biomolecules has been recognized since the early 1980s. As a new kind of carbon materials, carbon nanotubes (CNTs) have attracted considerable interest because of their unique structural, mechanic, electronic, magnetic and optical characteristics. Biomolecule-functionalized carbon nanotubes have rapidly attracted substantial research interests for its application in the biosensor and electrochemistry detection.This dissertation focuses on developing a series of nanoparticules-based biosensors for the diagnosis of some clinical serious diseases including prostate cancer, Vibrio cholera andβ-thalassemia diseases etc. by probing their specific markers (sometimes as the model test reagents). The detailed materials are shown as follows:(1) A piezoelectric immunoagglutination assay has been proposed for rapid detection of human immunoglobulin G (hIgG) using antibody-modified liposoms (in Chapter 2). liposomes are employed as replacements for the traditional latex to be labeled with goat anti-human IgG antibody, the specific agglutination event in the presence of corresponding antigen was monitored by the sensing probe modified with bovine serum albumin (BSA). It is found that the frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05～6μg mL?1 with a detection limit of 50 ng mL?1, without necessity for immobilization of immunoactive entities and purification of samples. Results of evaluating practical specimens show that the analytical ability of the developed piezoelectric technique is comparable to that of the ELISA method. (2) A novel reusable piezoelectric biosensor has been developed for the detection of cholera toxin (CT) based on analyte-specific surface agglutination of ganglioside GM1-functionalized liposomes on supported lipid membrane incorporated with ganglioside GM1 (in Chapter 3). Compared with traditional piezoelectric biosensor, the proposed technique combines the detection of both the gravimetric and the viscoelastic effects, thereby enabling vast sensitive enhancement in protein determination. Besides the use of stabilized response for the quantification of CT, the initial response rate, defined by the slope of the frequency change curve at the initial stage, could also be utilized for the detection of CT, which might be of particular significance in rapid assay practices. This method had an analytical interval of 0.1～5μg mL-1, with a detection limit of 25 ng mL-1. (3) An ultrasensitive chemiluminescence biosensor was developed for the detection of CT based on a supported lipid membrane as sensing surface and the HRP/GM1-functionalized liposome as detection probe (in Chapter 4). The supported lipid based biosensing surface could be renewed easily and rapidly. The application of enhanced chemiluminescence reaction in the detection of HRP-bearing liposome afforded a further signal amplification and background alleviation. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1 pg mL-1 to 1 ng mL-1 with readily achievable detection limit of 0.8 pg mL-1. (4) Another highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome (in Chapter 5). The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed lumino/peroxide/enhancer system. The chemiluminescence intensity was directly proportional to the concentration of PSA in sample solution in the range from 7.4 pgmL-1 to 74 ng mL-1 with a detection limit of 4 pg mL-1. In this part, the application of several kinds of functionalized liposome for the construction of biosensor have greatly improved the performance of the resulting biosensor.In this dissertation, the Au nanoparticles and the carbon nanotubes have also been used for the fabrication of biosensor and combined with electrochemical detection methods:(5) A highly sensitive electrochemical impedance immunosensor was developed by using a novel amplification procedure with the application of an Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with an Au-colloid labeled secondary antibody and an Au-colloid labeled antibody(in Chapter 6). Colloidal Au was used as a versatile and efficient template for the immobilization of antibody due to its relatively large surface and good biocompatibility. A novel amplification method was developed with the application of a Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with a Au-colloid labeled secondary antibody and a Au-colloid labeled antibody. The results obtained revealed that the sensitivity could be substantially improved via the amplification step and a detection limit as low as 4.1 ng L?1 could be reached for a model analyte of human immunoglobulin G (hIgG). (6) A novel approach for scanning of unknown gene mutations was developed based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated MB markers for electrochemical signal generation(in Chapter 7). The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. The adsorption of heteroduplex on the electrode surface could then be probed electrochemically by a certain redox indicator as methylene blue (MB) that was selectively intercalated in nucleic acid duplexes. The proposed approach has been successfully implemented for the identification of single-base mutation in ?28 site of theβ-thalassemia gene with a detection limit of 5.6×10-13M, providing a highly specific and cost-efficient approach for point mutation detection. (7) An electrochemical label-free detection technique was fabricated for the detection of nucleic acids based on the precipitation of SWNTs by DNA hybridization (in Chapter 8). The DNA hybridization between target DNA and the DNA probe coating on the nanotubes could actively remove DNA probe from the SWNTs surface. The precipitation of SWNTs onto the n-octadecyl mercaptan (C18H37SH) modified Au electrode substantially restores heterogeneous electron transfer between bare Au electrode and redox species in solution phase which was almost totally blocked by the SAM of C18H37SH, and as a result, the electrical signal of the electrode was correlated with the concentration of target DNA in the range from 6.4pM～50nM with a detection limit of 3.2pM.