Nanoporous Membrane Based Biosensor For Cell Behaviour Study Via Impedance Spectroscopy.

摘要

A novel poly(ethylene glycol) (PEG) based microchip with nanoporous alumina membrane was developed for the study of human esophageal cancer cells (KYSE 30) in vitro behavior with impedance spectroscopy. Nanoporous alumina membrane was successfully fabricated by a two-step anodization technique while the nanopore size was controlled by applying different anodization voltages. The PEG hydrogel microwells were fabricated using photolithography on nanoporous alumina surface modified with a 3-(Trimethoxysilyl)propyl methacrylate (TPM) monolayer. During the photopolymerization reaction, and hydrogel microwell arrays were covalently bonded to the substrate via the TPM monolayer. In the surrounded areas of the microwells where PEG was UV polymerized, solid hydrogel was covalently bonded with silane-modified membrane and the PEG hydrogel covering layer prevented electrolyte flow through the portions underneath the membrane. The surface modification effect was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle and protein adsorption experiments to confirm the existence of PEG and silane. The diffusion studies for various biomolecules including bovine serum albumin (BSA), insulin and the anti-cancer drug molecule of cisplatin were carried out with the microfabricated membrane array using a mini-diffusion chamber. The diffusion properties of the nanoporous alumina membrane with nanopore size of 20nm and 100nm were studied by a UV-Vis spectrophotometer. The biocompatibility of nanoporous alumina membrane was demonstrated by using two types of cells, rat bone marrow derived mesenchymal stem cells (RMSCs) and human KYSE-30 esophageal squamous epithelial cancer cells. Then, human KYSE-30 esophageal squamous epithelial cancer cells were successfully patterned within the PEG microwells and selective cell adherence on the TPM modified alumina surface inside the microwells was realized. Cell morphology changes due to cells adhesion, spreading, and proliferation were detected by nanoporous membrane based impedance spectroscopy in a real time and non-invasive way. The effects of various anti-cancer drugs of retinoic acid (RA), 5-Fluorouridine (5-FU) and Cisplatin (CDDP) were studied using this nanoporous membrane based cellular array with impedance spectroscopy. The initial concentration effects of 5-Fluorouridine (5-FU) on impedance spectra were also studied with concentrations of 0.1 mg/mL, 0.2mg/mL and 0.5mg/mL. Finally, biochemical control experiments for apoptosis enrichment factor detection under the effect of 5-FU with the concentration of 0.1mg/mL was studied at different treatment time points. The apoptosis enrichment factor results were also compared with impedance spectra and the correlation was found between them. This showed that this new nanoporous membrane based morphology-sensitive electrochemical system could be an effective and sensitive platform to indicate the degree of apoptosis.