High resolution monitoring of chemotherapeutic agent potency in cancer cells using a CMOS capacitance biosensor

Highlights

• A capacitance-based biosensor for cell-based assays is discussed.

• Device sensitivity is validated with in-incubator time-lapse imaging.

• Results showed the ability to analyze single cell motility and monitor cell health.

• Chemotherapeutic effects on cancer cells were studied.

Abstract

Monitoring cell viability and proliferation in real-time provides a more comprehensive picture of the changes cells undergo during their lifecycle than can be achieved using traditional end-point assays. Particularly for drug screening applications, high-temporal resolution cell viability data could inform decisions on drug application protocols that might lead to better treatment outcomes. We describe a CMOS biosensor that monitors cell viability through high-resolution capacitance measurements of cell adhesion quality. The system consists of a 3 × 3 mm² chip with an array of 16 sensors, on-chip digitization, and serial data output that can be interfaced with inexpensive off-the-shelf components. An imaging system was developed to provide ground-truth data of cell coverage concurrently with data recordings. Results showed the sensor's ability to detect single-cell binding events, track cell morphology changes, and monitor cell motility. A chemotherapeutic assay was conducted to examine dose-dependent cytotoxic effects on drug-resistant and drug-sensitive cancer cell lines. Concentrations higher than 5 μM elicited cytotoxic effects on both cell lines, while a dose of 1 μM allowed discrimination of the two cell types. The system demonstrates the use of real-time capacitance measurements as a proof-of-concept tool that has potential to hasten the drug development process.

Introduction

Cell-based assays can analyze the effects of different materials and growth conditions on cells cultured in vitro. They are a vital tool in the drug discovery process and can be used to quantify the cytotoxic effects of drugs. Most cell-based screening studies require the use of specialized laboratory equipment and reagents, and they provide data through methods that require labeling such as fluorescent, bioluminescent, or colorometric staining. While these screens can provide high-throughput analysis through parallelization of experiments, they generally have high operational costs, can be labor intensive, require sub-sampling of analytes, and may lack specificity (Méry et al., 2017). Furthermore, most provide data at temporally and spatially sparse sampling points or are endpoint assays requiring cell fixation, and so do not provide information on the dynamics of live cell growth and motility.

Real-time monitoring of cell viability over the course of drug exposure and subsequent cell death may provide a more complete picture of the cytostatic and cytotoxic effects of drugs. In particular, profiling cancer cell responses with high-temporal resolution can provide insight into cell death kinetics enabling the development of more beneficial drug dosage regimens and better-targeted therapies (Forcina et al., 2017; Wolbers et al., 2004; Wolpaw et al., 2011). Indeed modern chemotherapeutic regimens generally involve the use of multiple drugs (Frei and Eder, 2003) administered sequentially or in alternation, with the aim of reducing drug resistance, reducing non-specific toxic effects, and maximizing overall efficacy. Computational models have been developed to generate adaptive therapeutic regimes (Gatenby et al., 2009) or select optimum strategies to address issues of tumor heterogeneity (Zhao et al., 2014). Therefore the ability to perform continuous cell measurements in real-time represents a significant advance over current methods of determining drug cytotoxicity and cancer cell susceptibility; replacing current assays with a more rapid and more relevant alternative could hasten the drug development process toward clinical implementation.

Complementary metal-oxide semiconductor (CMOS) technology has been leveraged to develop biosensors to meet this goal by bringing sensing and signal processing electronics into intimate contact with biology. This has the advantages of incorporating the signal transduction and readout pipeline into a single highly integrated platform, thus enabling miniaturization and high-fidelity measurements. Among the different possible sensing modalities for CMOS technologies, impedance and capacitance-based sensors have shown promise in cell viability monitoring. These sensors operate by monitoring the capacitive loading of cells at the cell-chip interface (Hong et al., 2011; Prakash and Abshire, 2005). Several such platforms have been developed for detecting bacterial cells (Couniot et al., 2016), monitoring bacterial growth (Ghafar-Zadeh et al., 2010), and tracking cancer cell proliferation (Laborde et al., 2015; Nabovati et al., 2017; Prakash and Abshire, 2008). A recent study looked at drug screening on chip using a capacitance sensor (Nabovati et al., 2019), comparing cells that were antibiotic-resistant with those that were non-resistant. Results showed a positive correlation with expected outcomes, although the temporal resolution of measurements was on the order of hours, which may limit the ability to observe drug-induced responses over short timespans. Other CMOS sensing modalities have also been studied for drug development, including electrical sensing of action potential variations upon drug dosing (Abbott et al., 2017; Lopez et al., 2018; Park et al., 2018).

In this work, a CMOS-based capacitance biosensor is presented that enables automatic, real-time, and label-free cell tracking to monitor chemotherapeutic agent potency. The device is able to generate measurements of cell-substrate coupling with high spatial, temporal, and amplitude resolution, which in turn can be used to monitor cell adhesion, proliferation, and viability in the presence of a cytotoxic agent. Prior work has presented sensor characterization results and preliminary live cell experiments (Senevirathna et al., 2019, 2018). Here, more comprehensive cell experiments are performed with the addition of time-lapse optical imaging of the sensor surface to verify measurements. This ground-truth validation confirms the operation of the chip and its ability to detect single-cell binding and detachment events. Furthermore, experiments were performed to use the biosensor as a proof-of-concept drug-screening tool. Two human ovarian cancer cell lines were grown on the device, one of which is sensitive to a chemotherapeutic agent and the other resistant. Repeated experiments were performed to monitor cell viability for different concentrations of the drug, and results demonstrate the ability to discriminate the resistant and sensitive cell lines through capacitance measurements. The high-temporal resolution measurements were then used, along with mathematical modeling, to estimate kinetics of cell death by measuring the time lag between drug exposure and death onset, and the rate of cell death. Results showed that cell death is induced faster with increased drug concentration, while the death rate remains unaffected. Capacitance and image data may be found online at http://dx.doi.org/10.21227/9zzd-w936 (Senevirathna et al., IEEE Dataport, 2019).