Real-Time Monitoring of Platelet Activation Using Quartz Thickness-Shear Mode Resonator Sensors

Abstract

In this study, quartz thickness-shear mode (TSM) resonator sensors were adopted to monitor the process of platelet activation. Resting platelets adhering to fibrinogen-coated electrodes were activated by different concentrations of thrombin (1, 10, and 100 U/mL), and the corresponding electrical admittance spectra of TSM resonators during this process were recorded. Based on a bilayer-loading transmission line model of TSM resonators, the complex shear modulus (G' + jG″) and the average thickness (hPL) of the platelet monolayer at a series of time points were obtained. Decrease in thrombin concentration from 100 to 1 U/mL shifted all peaks and plateaus in G', G″, and hPL to higher time points, which could be attributed to the partial activation of platelets by low concentrations of thrombin. The peak value of hPL was acquired when platelets presented their typical spherical shape as the first transformation in activation process. The G' peak appeared 10 ∼ 20 min after hPL peak, when some filopods were observed along the periphery of platelets but without obvious cell spreading. As platelet spreading began and continued, G', G″, and hPL decreased, leading to a steady rise of resonance frequency shift of TSM resonator sensors. The results show high reliability and stability of TSM resonator sensors in monitoring the process of platelet activation, revealing an effective method to measure platelet activities in real-time under multiple experimental conditions. The G', G″, and hPL values could provide useful quantitative measures on platelet structure variations in activation process, indicating potential of TSM resonators in characterization of cells during their transformation.

Figure 1

Schematic diagram of TSM resonator measurement system. To see this figure in color, go online.

Figure 2

Theoretical model. (a) TLM of TSM resonator. (b) Bilayer-loading model of TSM resonator with adherent platelets. To see this figure in color, go online.

Figure 3

Platelet adhesion. (a) Admittance spectrum set of TSM resonator during platelet adhesion process (adding platelet suspension at time (T) = 0). (Inset, left) Corresponding resonance frequency shift curve extracted from the admittance spectrum set; (inset, right) mean resonance frequency shift curve for all three measurements. (b) Admittance spectrum of TSM resonator with platelet suspension (pink) and with adherent platelet monolayer (black). (Inset) Phase-contrast photo of adherent platelets in 96-well plate taken after 2 h of platelet adhesion process. To see this figure in color, go online.

Figure 4

Platelet activation by thrombin (100 U/mL). (a) Admittance spectrum set of TSM resonator during platelet activation (adding thrombin directly after T = 0) and corresponding resonance frequency shift curve. For clarity, admittance spectra were plotted every 5 min. (b) Resonance frequency shift curves of TSM resonators during platelet activation and phase-contrast photo of activated platelets. (Inset) Phase-contrast photo of activated platelets in 96-well plate taken after 2.5 h of platelet activation process. To see this figure in color, go online.

Figure 5

Characterization of platelet monolayer at a series of time points during platelet activation by thrombin (100 U/mL) (adding thrombin directly after T = 0). (a) Mean ± SE photos of platelets (photos in the first row are a partially enlarged view of corresponding ones in the second row, presenting the characteristic morphologies of platelets at different time points). (b) Storage modulus G′, loss modulus G″, and average thickness h_PL. To see this figure in color, go online.

Figure 6

Typical resonance frequency shift curves of TSM resonators during platelet activation by different concentrations of thrombin (adding thrombin directly after T = 0). To see this figure in color, go online.

Figure 7

Characterization of platelet monolayer at a series of time points during platelet activation by different concentrations of thrombin (adding thrombin directly after T = 0). (a) Storage modulus G′; (b) loss modulus G″; (c) average thickness h_PL. To see this figure in color, go online.