Carbon Nanotube-based Electrochemical Cytosensor And Cell Apoptosis Study

Cancer is a class of diseases, which could seriously affect the health of human body. Early detection of cancer greatly increases the chances for successful treatment. Recently, electrochemical cytosensor has attracted increasing attention in the detection of cancer cells and related biomarkers, which offers distinctive advantages of high sensitivity and selectivity, rapid response, and low cost. Carbon nanotubes have shown attractive prospect in the biosensing field due to their unique one-dimensional, nano tubular structure, unusual thermal, electrical and mechanical properties. On the other hand, apoptosis is an important biological process that is associated with pathogeny and chemotherapy response for a variety of diseases. It is beneficial to develop highly sensitive and convenient detection approach for apoptosis diagnosis.Based on the electrochemical technology, this dissertation presents an exploration of the use of functionalized carbon nanotubes in cytosensing and apoptosis diagnosis. The main results were summarized as follows:1. Synthesis of Gelatin-Stabilized Gold Nanoparticles and Assembly of Carboxylic Single-walled Carbon Nanotubes/Au Composites for Cytosensing and Drug UptakeGelatin-Stabilized gold nanoparticles with hydrophilic and biocompatible were prepared with a simple and "green" route by reducing in situ tetrachloroauric acid in gelatin. The nanoparticles showed the excellent colloidal stability. UV-vis spectra, transmission electron microscopy, and atomic force microscopy revealed the formation of well-dispersed AuNPs with different sizes. By combining the biocompatibility of AuNPs and excellent conductivity of carboxylic single-walled carbon nanotubes, a novel nanocomposite was designed for the immobilization and cytosensing of HL-60cells at electrodes. The immobilized cells showed sensitive voltammetric response, good activity and increased electron-transfer resistance. It can be used as a highly sensitive impedance sensor for HL-60cells ranging from1×104to1×107cell mL-1with a limit of detection of5×103cell mL-1. Moreover, the nanocomposite could effectively facilitate the interaction of Adriamycin with HL-60cells and remarkably enhance the permeation and drug uptake of anticancer agents in the cancer cells, which could readily lead to the induction of the cell death of leukemia cells.2. Mussel-Inspired Preparation of Carbon Nanotube/Polydopamine/Folic Acid Nanocomposites for Targeted Cell DetectionThis paper reported a facile approach for surface modification of carboxylic multi-walled carbon nanotubes through self oxidative polymerization of dopamine. The obtained nanocomposite was further conjugated with folic acid by a carbodiimide coupling reaction. This nanoprobe incorporated both the excellent electronic property of carbon nanotube and the specific recognition ability of folic acid for folate receptor on tumor cell membrane. Scanning electron microscopy, transmission electron microscopy and contact angle analysis revealed that the novel nanoprobe was uniform and biocompatible. With the help of electrochemical impedance technology, an electrochemical label-free method was developed to detect folate receptor positive tumor cells by making use of the interaction between folic acid and its receptor over-expressed on tumor cell membrane. The proposed cytosensor showed an excellent analytical performance for the detection of folate receptor positive tumor cells, such as human cervical cancer cells and human promyelocytic leukemia cells. This strategy offers great promise to extend its application in studying the interaction between ligand and cell-surface receptor.3. Design and Implementation of Electrochemical Cytosensor for Evaluation of Cell Surface Carbohydrate and GlycoproteinA new strategy for assessing cell surface carbohydrates and P-glycoprotein (P-gp) expression status and quantifying the cell numbers with electrochemical immunoassay was designed. In order to construct the base of the cytosensor, a novel3-D architecture was initially fabricated by combining nitrogen-doped carbon nanotubes, thionine, and gold nanoparticles via a simple layer-by-layer method. The formed architecture provided an effective matrix for Concanavalin A (ConA) binding and made the immobilized ConA hold high stability and bioactivity. On the basis of the specific recognition of cell surface mannosyl groups to ConA, the ConA/3-D architecture interface showed a predominant capability for cell capture. By coupling with another signal amplification based on a enzymatic catalytic reaction of HRP toward the oxidation of thionine by the H2O2, which was induced by two-step immunoreactions, the proposed cytosensor showed an excellent analytical performance for the detection of HeLa cells ranging from8.0×102to2.0×107cells mL-1with a limit of detection of500cells mL-1. Moreover, with the use of pre-blocking procedures, the mannosyl groups and P-gp on single HeLa cell could be further detected to be (4×2)×1010molecules of mannose moieties and8.47×106molecules of P-gp. This strategy offers great promise for sensitive detection of cancer cells and cell surface receptors, thus may help improve cancer diagnosis and treatment.4. Dual Amplification Strategy for Sensitive Detection of Early Apoptotic Cells based on Nitrogen-doped Carbon Nanotubes and Quantum Dot-Labeled Silica NanosphereA novel lectin functionalized nanoprobe was designed by noncovalent assembly of ConA on CdTe quantum dot (QD)-labeled silica nanosphere with poly(allylamine hydrochloride) as a linker. This nanoprobe incorporated both the specific recognition ability of ConA for cell-surface mannosyl groups and the electrochemical signal amplification property of multi-labeled SiO2@QDs nanocomposites, and was further used as a label in cytosensor. In order to construct the base of the cytosensor, a novel3-D architecture was initially fabricated by combining nitrogen-doped carbon nanotubes and gold nanoparticles via a simple layer-by-layer method. The formed architecture provided an effective matrix for Annexin Ⅴ binding and made the immobilized Annexin V hold high stability and bioactivity, offering the possibility of sensitivity enhancement. On the basis of the specific recognition between Annexin V and phosphatidylserine on the early apoptotic cell membrane, the Annexin V/3-D architecture interface showed a predominant capability for early apoptotic cell capture. By coupling with SiO2@QDs-ConA nanoprobe and electrochemical stripping analysis, a novel electrochemical method for cytosensing was then developed for detection of early apoptotic cells. This facile method showed excellent sensitivity and selectivity, revealing great potential in cell apoptosis analysis.5. Electrochemical Sensing for Caspase3Activity and Inhibition Using Quantum Dot Functionalized Carbon Nanotube LabelsThis work constructed a novel electrochemical sensing platform for sensitive determination of caspase3activity and inhibition by combining the site-specific recognition and cleavage of the DEVD-peptide with quantum dots as signal amplification. A signaling probe composed of CdTe QDs, carbon nanotubes, and streptavidin was first prepared via the layer-by-layer assembly approach. Then, a smart, caspase-3-responsive, and biotinylated DEVD peptide was designed and immobilized on the gold electrode surface, which could be specifically recognized and cleaved by active caspase3in the apoptotic cell lysates. Using the QDs-based nanoprobes, an electrochemical biosensor was then developed for monitoring the caspase3activity and inhibition during cell apoptosis. This electrochemical strategy exhibited attractive advantages of ease of performance, high sensitivity and specificity. It presents a significant tool for efficient screening of the potential caspase3inhibitors and anticancer drugs, suggesting promising applications in cancer research.6. Ultrasensitive Detection of Human Interleukin6Using a Competitive Immunosensor Based on a Disposable Electrically Heated Screen-printed Carbon ElectrodeA facile one-step chemical reduction method was developed to prepare the polydopamine functionalized gold nanoparticle, followed by ultrasonically assembled on the surface of graphene nanosheets. The resulting hybrid material incorporated both the high-binding capability of graphene nanosheets, favorable biocompatibility of gold nanoparticles, and high protein affinity of polydopamine, which thus offered a promising template for biomolecule immobilization and biosensor fabrication. A novel competitive electrochemical immunoassay was then proposed by combining the RGO-AuNPs platform with quantum dots functionalized carbon nanotube labels for the sensitive detection of human interleukin6. Enhanced sensitivity was obtained by combining the advantages of high-binding capability of graphene nanocomposite with the QDs-based signal amplification. More importantly, an electrically heated screen-printed carbon electrode was induced in the detection procedure of the immunosensor, and further improved the sensitivity. The immunosensor exhibited a wide linear response to IL-6ranging from0.1to100pg mL-1with a detection limit of0.033pg mL-1. The proposed method showed good precision, acceptable stability and reproducibility, and could be used for the detection of IL-6in real samples, which possessed promising application in clinical research.