Electrochemical Detection And Cell Imaging Analysis Of Glycan Expression On Tumor Cell Surface

Cells are the elements of all living organisms, thus deep research into cellular structure and function is the key for the conquest of diseases. The glycan molecules on the membrane surface can dictate proteins and lipid functions through conjugation, mediate and control cellular adhesion behavior and signal transduction. Many of the growth characteristics of malignant tumor cells are manifested by structural and functional changes in the glycans. Thus, specific detection of cell surface glycans is important for unraveling the role of glycosylation change in cellular adhesion and migration, formation and metathesis of malignant tumors, and developing new methods for the treatment of cancer. Traditional research methods only offer the information of the average expression level of a cell population, thus, possibly enshroud the heterogeneity of single cells, which might be indicative of disease in the early stage, leading to the wrong result. The analysis of cellular composition and physiological behavior at the single-cell level is mostly desired for the accurate early warning of disease. In this dissertation, by integrating nanotechnology, surface science and chemical biology, in situ electrochemical detection of cell glycans has been realized. The effect of drugs on the cell surface glycan expression has been investigated. Furthermore, in situ electrochemical imaging method for monitoring cell membrane glycan and cell viability at the single-cell level has also been proposed by combining scanning electrochemical microscopy with the micromachining technique. This dissertation includes the following four parts:1. Noncovalent functionalization of carbon nanotubes with lectin for label-free dynamic monitoring of cell-surface glycan expressionA kind of concanavalin A functionalized multi-walled carbon nanotubes (ConA-MWCNT) was constructed by noncovalent assembly of ConA on carboxylated MWCNT with poly(diallyldimethylammonium) as a linker. The novel nanomaterial was characterized with scanning electron microscopy and atomic force microscopy. It incorporated both the specific recognition ability of lectin for cell-surface mannosyl groups and the unique electronic and mechanical properties of MWCNT. An electrochemical label-free method for cytosensing was proposed by constructing a ConA-MWCNT interface on a glassy carbon electrode, which showed a linear response to K562 cells ranging from 1×104 to 1×107 cells mL-1. The ConA-MWCNT interface could be further used for monitoring of dynamic variation of glycan expression on K562 cells in response to drug. A facile and high-throughput optical method for the analysis of dynamic glycan expression on living cells was also developed by constructing an array of ConA-MWCNTs spots on a glass slide. This method showed acceptable rapidity and low cost. The noncovalent functionalization of MWCNTs with lectins could be potentially applied in cell biological study based on cell-surface glycan expression.2. A simple electrochemical lectin-probe for in situ homogeneous cytosensing and facile evaluation of cell surface glycanThis work constructed a novel electrochemical lectin-probe, ferrocene-concanavalin A (Fc-ConA), for in situ monitoring of cell surface glycan by incorporating the specific recognition ability of lectin to glycan and favorable electrochemical property of ferrocenyl group. The covalent conjugation of ConA with ferrocenyl group was achieved by a carbodiimide coupling reaction and proved with UV-vis absorption spectroscopy and infrared spectroscopy. Cyclic voltammetric behavior of Fc-ConA at glassy carbon electrode demonstrated a reversible diffusion-controlled process. A facile homogeneous cytosensing strategy was then developed by using Fc-ConA probe for detection of K562 cells. The suspending cells specifically captured Fc-ConA via membrane mannosyl groups and decreased the concentration of free Fc-ConA, producing a response correlative with cell number and the content of cell surface glycan. A wide linear response to cells ranging from 1×104 to 1×107 cells mL-1 with a calculated detection limit of 3000 cells mL-1 was obtained. The lectin-probe could be conveniently used to in situ evaluate cell surface glycan. The average number of mannose moieties on single living K562 cell was detected to be 3.0×1010, while this value increased by 81% on drug-treated cells. These results agreed with those from flow cytometric detection. This strategy presented a promising platform for homogeneous sensitive cytosensing and facile monitoring of carbohydrate expression on living cells in response to drugs.3. Real-time monitoring of cell viability by its nanoscale height change with oxygen as endogenous indicatorThis work developed a novel method for real-time monitoring of nanoscale height change of single cell by scanning electrochemical microscopy (SECM) with oxygen as an endogenous indicator. The Pt nanodisk electrode was firstly prepared as a SECM probe by assembling Pt wire-inserted quartz capillary in a glass tube, which produced a taper tip with controllable radius down to 5 nm. The early apoptosis of adherent BGC-823 human gastric carcinoma (BGC) cell could sensitively be monitored by the decrease of oxygen reduction current during the constant-height scan around single BGC cell, which was characterized with AnnexinⅤ-FITC apoptosis detection kit coupled with a confocal fluorescent microscope. A step-approaching method was proposed to separate the negative feedback current from the total reduction current above single living cell for testing the morphological effect of cell. The height change of BGC cells exposed to 25℃PBS in 2 h was estimated to be in the range of 55 to 365 nm (n=8). The practicability of the designed strategy was demonstrated by treating the model cells with paclitaxel and real-time SECM monitoring. This work provided a powerful protocol to monitor the nanoscale height change of living cells in physiological environment for real-time cell viability evaluation.4. In situ electrochemical imaging of membrane glycan expression on micropatterned adherent single cellsA scanning electrochemical microscopic (SECM) method for in situ imaging of four types of membrane glycan motifs on single adherent cells was proposed using BGC-823 human gastric carcinoma (BGC) cells as the model. These adherent cells were firstly micropatterned in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the localized surface interaction, and the membrane glycans were then specifically recognized with corresponding lectins labeled with horseradish peroxidase (HRP). Based on the enzymatic oxidization of ferrocenylmethanol (FMA) by H2O2 to yield FMA+, the glycan expression level was detected by the reduction current of FMA+at the SECM tip. The cell-surface glycans could thus be in situ imaged by SECM at a single-cell level without peeling the cells from culture dish. Under the optimized conditions, four types of membrane glycan motifs showed statistically distinguishable expression levels. The SECM results for different glycan motifs on adherent single cells were consistent with those estimated by flow cytometric assay. This work provides a reliable approach for in situ evaluation of the characteristic glycopattern of single living cells, and can be applied in cell biologic study based on cell surface carbohydrate expression.