Real-time sensing of epithelial cell-cell and cell-substrate interactions by impedance spectroscopy on porous substrates

Abstract

Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (Rb) corresponding to the initial adhesion phase of cells. It is observed that Rb and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions.

Reliability Study of Nanoporous Silicon Oxide Impedance Biosensor for Virus Detection: Influence of Surface Roughness

“…The penetration depth of the solution within the pore is dependent on different parameters like hydrophilicity of the surface, friction (due to viscosity), gravity, applied electric field and the 'pressure forces' originating out of various sources and can be estimated from Computational Fluid Dynamics software Ansys-FLUENT. It has been already reported that for oxidized porous substrates of around 50nm diameter, the penetration depth is around 1µm [20] which indicates that in our fabricated sensor samples of 50-100nm thickness, the solution can reach the pore bottom. Thus the sensing performance is expected to depend primarily on varying surface roughness of the samples.…”

Real-Time Monitoring of Wound Healing on Nano-Patterned Substrates: Non-Invasive Impedance Spectroscopy Technique

“…To ensure that most of the field lines intersect the cellular layer on top, it is required that the medium should not reach the pore bottom. Estimation of medium penetration depth into the pore by computational fluid dynamics software has been reported earlier [18] which suggests the thickness selection of 8 µm and 1 µm for PS1 and PS2 substrates respectively. A distributed electrical model has been proposed for various stages of cell development after wounding.…”

“…There are no reports of non-invasive electrical monitoring of wound healing phenomena on nano/micro structured surfaces. Recently, real time impedance monitoring has been performed on nano/micro porous silicon substrates to quantify the vital cellular attributes like cell-cell junction resistance, cell substrate separation, mean cell radius and others during the growth of keratinocytes (HaCaT) [18]. But there has been no attempt to study the wound healing process electrically even on porous silicon substrates.…”

“…After formation of PS, thermal oxidation has been performed in a dry-wet-dry sequence in an oxidation furnace at 1000°C for 3 hours in 1 µm samples and for 1 hour at 900°C in 100 nm samples so that the pore openings are 500 nm (PS1) and 50 nm (PS2), respectively. The thickness of the PS1 and PS2 samples have been selected as 8 µm and 1 µm respectively to ensure that the majority of the field lines penetrate the cellular layer [18]. Thermal oxidation is required to result in a hydrophilic surface for facilitation of subsequent functionalization and for reliable impedance measurement.…”

“…Metal-coated oxidized PS substrates have been then sterilized with 70% ethanol for 10 minutes followed by drying with nitrogen. A protein layer of poly-l-lysine (PLL) has been coated on the substrates for better cell adhesion, the details of which have been reported earlier [18]. The schematic diagram (cross sectional view) of the PS sensor in presence of cells is shown in Fig.…”