A High-Resolution Microfluidic LAPS Imaging System for 3D Cell Metabolism Monitoring
Authors: Qiu Yong , Liang Tao , Jiang Deming , Liu Xin , Wan Hao , Wang Ping 
Published: July 2021

Light-addressable potentiometric sensor (LAPS) is a kind of semiconductor sensor based on electrolyte-insulating -semiconductor (EIS) structure that is sensitive to surface potential. Due to the light-addressability, it can record the photocurrent signals point to point where the modulated laser beam focused on. Cells encapsuled in hydrogel scaffold to constructed the 3D cell model can form chemical and oxygen gradient thus causing different metabolism state in different spatial position. In this study, a partial thinned Si3N4-LAPS chip integrated with microfluidic channel has been constructed for real-time 3D cell metabolism state monitoring. HepG2 cells are encapsulated in hydrogel to construct 3D liver cancer model. The system consists of sensing component, optical component, precision electronically controlled stage and a homemade software based on LabVIEW. In the bioreactor, cells take in high-glucose medium to secrete acidic metabolites, which leads to a decrease in photocurrent dramatically. With the help of this system, real-time visualization of cell metabolic dynamic process can be achieved. Moreover, medium or anti-cancer drugs can be automatically delivered to the bioreactor with the assistance of microfluidic technology to carry out cell culture and drug efficacy evaluation. This high-resolution photoelectrochemical imaging system has potential to achieve long-term drug screening platform.

Key words Light-addressable potentiometric sensor, 3D cell model, chemical gradient, cell metabolic visualization, drug screening

Introduction

Cellular metabolism is one of the most basic physiological functions of cells to maintain viability. Cells absorb and decompose various nutrients to produce energy necessary for proliferation and differentiation, while secreting acidic metabolites. Glucose, as the main nutrition source of cells, will be decomposed into energy and acidic substance under both aerobic and anaerobic conditions. The acidic products (such as carbon dioxide, lactic acid) will cause the pH decrease in the extracellular microenvironment. Therefore, the extracellular acidification rate (ECAR) can be used to characterized the cellular metabolic level. Nowadays, three-dimensional cell culture model has attracted the attentions of researchers, which can maintain more physiologically relevant microenvironment resemble to in vivo. Among all these models, 3D cell culture integrated with microfluidic chip has the unique advantages to recapitulating the cell microenvironment. Nevertheless, there exists general problems (oxygen concentration, chemical gradient) due to the special property of 3D cell model.Traditional ECIS and MEA system can not detect the spatial distribution of extracellular acidification rate,but laser beam could be focused and irradiated on the designated LAPS area to realize the light-addressable function based on the characteristics of LAPS. So,scanning the light spot along the chip can obtain spatial distribution of current signal to solve this problem.

Method

Si3N4-LAPS chip and backside illumination method were adopted with backside-thinned was thinned. The thickness of the silicon substrate is 500 μm, and the central area is thinned to 100 μm (Fig.1B) to reduce the lateral diffusion of carriers in the semiconductor layer and improve the sign-to-noise ratio while providing sufficient mechanical support.
Before seeding cells, the LAPS chip was cleaned by deionized water, sterilized by UV and 75% ethanol, and incubated with 5 μg/mL gelatin at 4 °C overnight to improve the adhesion ability to cells.
HepG2 cells were cultured in DMEM medium with 10% FBS, 100 U/ml Penicillin and 100 mg/ml Streptomycin. Then cells suspended in the medium were mixed in matrigel and cross-linked in a 37 °C humidified incubator with 5% CO2 for 30 minutes. Drug test is conducted by doxorubicin.
The stage performed grid scanning. In the X-axis direction, the resolution is determined by the stepping distance, and in the Y-axis direction, the motor keeps continuously moving, and the signal is continuously collected. The spatial resolution is determined by the movement rate and the sampling rate.

Results and Conclusions

In order to maintain highly stability of the optical lens group, the optical path is fixed and the sensing chip can move with the stage; the chip is mounted on the stage by an adjustable fixing scaffold, and the laser reflector is fixed on an immovable base. The 680 nm red light emitted by the laser diode is transmitted through a multi-mode fiber and collimated by fiber collimator, then irradiated on the back of the chip from the vertical direction through an aspheric lens (Figure1A).
The horizontal resolution, which was consistent with motor, was step set to 50 μm. The motor speed is 2.5mm/s, the sampling rate is 50k/S, the number of samples per pixel is 2000. The obtained real-time current grayscale image is shown in Figure 1C. The white part (high current value) is the thinning area of the chip, that is, the effective sensitive area; the black part is the non-thinning area, and the gray is the intermediate transition area. The converted pseudo-color image is shown in Figure 1D. The number of pixels of the image is 142×128, and the actual size is 6mm×6mm, indicating that this LAPS scanning imaging system can obtain an imaging resolution of less than 50 μm.
Hanging drop method was conducted to achieve cell 3D culture. The microscope brightfield image of 3D HepG2 cell sphere is shown in figure 1E and green fluorescence image is shown in figure 1F. Then 3D cell sphere were seeded into microfluidic LAPS chip to realize real-time visualization of cell metabolism.

Figure 1