Real-Time Viscoelasticity Monitoring of Cardiomyocytes Based on Love Wave Biosensor

Abstract

Introduction In addition to electrophysiological signals [1-2], mechanical properties such as contraction and relaxation of cardiomyocytes are also important properties. They are essential for the evaluation of drug efficacy and toxicity, and they can change the viscoelasticity of cells, thus providing the possibility for quantitative detection. The acoustic wave sensors are able to detect this slight change in viscoelasticity, which may not be easily noticed under the microscope in the early stages. At present, it has been reported that QCM-D can be used to detect viscoelastic properties of pre-osteoblast cells [3]. Besides, shear-horizontal surface acoustic wave sensor has been applied for the monitoring of cell spreading and attachment [4]. However, no study has realized the label free, real-time and quantitative detection of cardiac viscoelasticity for drug evaluation based on acoustic wave sensors.Recently, the surface acoustic wave (SAW) sensor has been greatly developed as a well-performed mass and viscoelasticity sensing device for label-free immunoassay. In particular, the Love Wave sensor has shown its advantages in biosensor applications due to its high sensitivity and stability in the liquid phase [5]. Hence, it is a promising device capable of monitoring viscoelastic properties of a population of cells in a label free and real-time way.As shown in Fig.1a, a self-designed Love Wave biosensor was used to monitor the viscoelastic properties of HL-1 cardiomyocytes in this study. Our previous studies have shown that insertion loss (IL) of Love Wave biosensor is a more suitable parameter for the quantitative detection of viscoelasticity than phase. Then, two different drugs were added to observe the effects on the viscoelastic properties of HL-1 cells. Method In this study, the Love Wave biosensor is constructed based on a piezoelectric quartz substrate. Interdigitated transducers (IDTs) with Ti/Au (20/200 nm) are deposited to generate acoustic waves. The input and output IDTs electrodes consist of 50 split-finger pairs with a preset wavelength λ=28 μm, which determines the center frequency of the sensor to be about 152 MHz. The distance between IDTs centers is 200λ and the acoustic aperture is 75λ. Afterwards, the IDTs patterned substrate is guided with a 3 μm SiO2 film deposited by plasma enhanced chemical vapor deposition (PECVD). Au layer (200 nm) is deposited on top of guiding layer to improve the cell attachment on the sensor surface. A polydimethylsiloxane (PDMS) chip (Fig.1b) was designed and used as the cell culturing chamber. The height and volume of this cavity are about 10 mm and 110 μL, respectively. A portable multi-channel detection system was developed to collect both the IL and phase signals of Love Wave biosensors. Then the HL-1 cardiac cells with the density of 50,000 cells/well were seeded on the surface of Love Wave biosensor (Fig.1c). To test the function of the detection system, isoprenaline (ISO) and verapamil (VRP) with different concentrations were used to induce changes in the viscoelastic properties of HL-1 cells. Results and Conclusions In Fig.1c, HL-1 cells attach and grow well on the pre-coated Love Wave biosensor, which demonstrates the biocompatibility of the sensor. The insertion loss signals of sensor after treated by ISO and VRP are shown in Fig.1d. It can be found that the insertion loss of the control group decreases slightly, which may be due to cell proliferation. VRP is a calcium channel antagonist that blocks the L-type calcium channel and prevents calcium influx into the cell, thereby decreasing the cardiomyocyte contraction force. With the addition of VRP solutions, the insertion loss of Love Wave biosensors increases with the increase of VRP concentration within the dosage range tested. In contrast to VRP, ISO is positive inotropic drug. After ISO treatment, HL-1 cells tend to be contractive due to the traction forces. Therefore, the higher the ISO concentration, the tighter the contact of the cells with the sensor substrate. Thus, insertion loss decreases after ISO treatment which changed positively correlated with concentration. Moreover, the optical microscope photos also proved that the cells after ISO treatment seems to be smaller than control group, while the cells after VRP treatment seemed to be larger. According to our preliminary experimental results, the affection on cell viscoelasticity of different drugs can be monitored by recording the insertion loss value of the SAW biosensor. Our results demonstrated that the proposed cell-based SAW biosensor is a potential, convenient and quantitative platform for in vitro cardiac viscoelasticity evaluation, especially in the early stages. It has promising applications in cell monitoring, drug evaluation and many other fields.