Md. Abdul Kafi

Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays

In this paper, a method was introduced for the fabrication of vertically and spatially-controlled peptide nanostructures that enhance cell adhesion, proliferation, spreading on artificial surfaces. The RGD nanostructures with different heights were fabricated on gold surfaces by self-assembly technique through a nanoporous alumina mask composed of nanoscale-controlled pores. Pore diameter and spatial distribution were controlled by manipulating the pore widening time at a constant voltage during the mask fabrication process. Two-dimensional RGD nanodot, three-dimensional RGD nanorod, and RGD nanopillar arrays were carried out using various concentrations of RGD peptide solution, self-assembly times, and pore sizes, which were 74 nm, 63 nm, and 43 nm in diameter, respectively. The fabricated RGD nanodot, nanorod, and nanopillar arrays were utilized as a cell adhesion layer to evaluate the cell adhesion force, adhesion speed, spreading assay, and phosphorylation of cofilin protein in PC12, HeLa, and HEK293T normal cells. Among the three different nanostructures, RGD nanopillar arrays were found to be suitable for cellular attachment, spreading, and proliferation due to the proper arrangement of the RGD motif, which mimics in vivo conditions. Hence, our newly fabricated RGD nanostructured array can be successfully applied as a bio-platform for improving cellular functions and in in vitro tissue engineering.

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells.

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip.

Fabrication of cell chip for detection of cell cycle progression based on electrochemical method.

A new strategy for on-site monitoring of cell cycle progression was proposed using cell chip technology. Cell synchronization has been utilized in intensive cellular research due to the fact that cells in different phases of the cell cycle exhibit different behaviors even when exposed to the same concentrations of drugs or toxicants. However, confirmation of cell cycle arrest in research is usually dependent on fluorescence-assisted cell sorting (FACS), which is laborious, time-consuming, and expensive. In this study, we employed a cell-chip-based electrochemical method to detect the cell-cycle-dependent electrochemical properties of cells. Electron transfer at the cell-electrode interface played a key role in our strategy and accurately reflected the redox activity of the cells in different phases. Rat pheochromocytoma cells were synchronized with thymidine and nocodazole, and well-defined current peaks from cells in the G1/S- and G2/M-phases were significantly different as determined by differential pulse voltammetry. FACS assay and Western blot analysis were used to validate the electrochemical findings. Hence, our cell-chip-based electrochemical method can be a useful tool in determining cell cycle progression easily and economically.

Effects of nanopatterned RGD peptide layer on electrochemical detection of neural cell chip

The cell-based chip is becoming a popular tool for monitoring living cell viability under various conditions. In this study, several biomaterials, such as synthetic Cys-(Arg-Gly-Asp)(4) (C(RGD)(4)), Arg-Gly-Asp-Multi Armed-Cys (RGD-MAP-C) peptide, and poly-L-lysine (PLL) nano-dots were fabricated on the gold surface of a neural cell chip. The material-dependent effects both on electrochemical signal detection in neural cells and on cellular adhesion were analyzed. The nano-dot structures were fabricated through a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). PC12 cells were allowed to attach on several peptide nanopatterned surfaces, and electrochemical tools were applied to neural cells attached on the chip surface. The RGD-MAP-C peptide nanopatterned surface provided the strongest voltammetric signals when the cell was exposed to cyclic voltammetry (CV) and differential pulse voltammetry (DPV) after 48 h of incubation, which may largely be due to an enhanced affinity between cells and the Au surface. Chemical toxicity assessments were conducted in the fabricated cell chip, and they showed negative correlations between neural cell viability and the concentration of chemicals. In conclusion, a nanopatterned RGD-MAP-C layer improved cell-binding affinity to Au substrates and showed sufficient sensitivity for electrochemical detection of cell viability.

Engineered peptide-based nanobiomaterials for electrochemical cell chip

Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

Electrochemical cell chip to detect environmental toxicants based on cell cycle arrest technique

A cell-based chip was recently developed and shown to be an effective in vitro tool for analyzing effect of environmental toxin on target cells. However, common cell chips are inappropriate for the detection of multiple environmental toxins. Here, we fabricated a neural cell chip to detect different cellular responses induced by BPA (bisphenol-A) and PCB (poly chlorinated biphenyl). This approach was based on an electrochemical method using a cell cycle-arrest technique. Neural cells were synchronized at the synthesis phase by treatment with thymidine, which results in a sharp reduction peak when compared to unsynchronized cells. The fabricated chip containing 50% G1/S and 50% G2/M phase cells was used to determine the effects of environmental toxins on neural cancer cells. At the end, the cell-chips could be used to assess both BPA and PCB toxicity that the cells were completely synchronized at the G1/S and G2/M phase. The proposed neural cell chip can be a useful tool for biosensors to evaluate easily and sensitively multiple effects of environmental toxicants on target cells.

Nanoscale fabrication of a peptide layer in cell chip to detect effects of environmental toxins on HEK293 cells

A cysteine-terminated C(RGD)4 peptide film was fabricated on a gold electrode for improving the attachment of cells. The electrochemical signals of cyclic voltammogram from cells on a C(RGD)4 deposited electrode was enhanced from 0.27 to 0.49 μA compared to a bare electrode. The developed cell-based sensor determined the effect of bisphenol-A (BPA) and dichlorodiphenyltrichloroethane (DTT) on the viability of HEK-293 cells by detecting decrease of reduction peaks (1.12–0.15 μA for BPA and 0.81–0.29 μA for DDT) after the treatment of environmental chemicals. This developed system can be a powerful tool for the monitoring of environmental toxicants.

Neural Cell Chip Based Electrochemical Detection of Nanotoxicity

Development of a rapid, sensitive and cost-effective method for toxicity assessment of commonly used nanoparticles is urgently needed for the sustainable development of nanotechnology. A neural cell with high sensitivity and conductivity has become a potential candidate for a cell chip to investigate toxicity of environmental influences. A neural cell immobilized on a conductive surface has become a potential tool for the assessment of nanotoxicity based on electrochemical methods. The effective electrochemical monitoring largely depends on the adequate attachment of a neural cell on the chip surfaces. Recently, establishment of integrin receptor specific ligand molecules arginine-glycine-aspartic acid (RGD) or its several modifications RGD-Multi Armed Peptide terminated with cysteine (RGD-MAP-C), C(RGD)4 ensure farm attachment of neural cell on the electrode surfaces either in their two dimensional (dot) or three dimensional (rod or pillar) like nano-scale arrangement. A three dimensional RGD modified electrode surface has been proven to be more suitable for cell adhesion, proliferation, differentiation as well as electrochemical measurement. This review discusses fabrication as well as electrochemical measurements of neural cell chip with particular emphasis on their use for nanotoxicity assessments sequentially since inception to date. Successful monitoring of quantum dot (QD), graphene oxide (GO) and cosmetic compound toxicity using the newly developed neural cell chip were discussed here as a case study. This review recommended that a neural cell chip established on a nanostructured ligand modified conductive surface can be a potential tool for the toxicity assessments of newly developed nanomaterials prior to their use on biology or biomedical technologies.