Real-time monitoring of the contractile properties of H9C2 cardiomyocytes by double resonator piezoelectric cytometry.

Testing the mechanical properties of cardiomyocytes plays an important role in the study of the physiological and pathological processes of constant contraction and diastole of the cardiovascular system. However, there is currently no satisfactory and dynamic technology to measure the mechanical properties of cardiomyocytes in a sustained manner, greatly affecting their practical application in clinical diagnosis and treatment evaluation. Herein, a double resonator piezoelectric cytometry (DRPC) technique was employed for dynamic monitoring of H9C2 cardiomyocyte adhesion and the effects of two cardiovascular drugs on the contractile properties of H9C2 cardiomyocytes, i.e., isoprenaline (ISO, a positive inotropic agent) and verapamil (VRP, a negative inotropic agent). Specifically, we used 9 MHz AT and BT-cut bare gold and transparent ITO electrodes and compared their dynamic adhesion to the two electrodes modified with RGD and gelatin respectively versus unmodified to measure the cell generated stress (ΔS) exerted on the quartz crystal surface by a population of cells and the cell viscoelastic index (CVI). We found that the DRPC technique can quantitatively measure the magnitude and direction of the generated forces during the adhesion process of the cells and under the effect of drugs. In conclusion, the technique presented in this study can be used for the simultaneous measurement of cell adhesion, traction force and viscoelasticity of living cells in a noninvasive, dynamic and continuous way, making it an ideal tool for assessing the population contractility of cardiomyocytes and evaluating the efficacy and toxicity of cardiovascular drugs.

A Novel Method in Identifying Pyroptosis and Apoptosis Based on the Double Resonator Piezoelectric Cytometry Technology.

In this study, a double resonator piezoelectric cytometry (DRPC) technology based on quartz crystal microbalance (QCM) was first employed to identify HeLa cell pyroptosis and apoptosis by monitoring cells' mechanical properties in a real-time and non-invasive manner. AT and BT cut quartz crystals with the same frequency and surface conditions were used concurrently to quantify the cells-exerted surface stress (ΔS). It is the first time that cells-exerted surface stress (ΔS) and cell viscoelasticity have been monitored simultaneously during pyroptosis and apoptosis. The results showed that HeLa pyroptotic cells exerted a tensile stress on quartz crystal along with an increase in the elastic modulus (G'), viscous modulus (G″), and a decrease of the loss tangent (G″/G'), whereas apoptotic cells exerted increasing compressive stress on quartz crystal along with a decrease in G', G″ and an increase in G″/G'. Furthermore, engineered GSDMD-/--DEVD- HeLa cells were used to investigate drug-induced disturbance and testify the mechanical responses during the processes of pyroptosis and non-pyroptosis. These findings demonstrated that the DRPC technology can serve as a precise cytomechanical sensor capable of identifying pyroptosis and apoptosis, providing a novel method in cell death detection and paving the road for pyroptosis and apoptosis related drug evaluation and screening.

Quartz Crystal Microbalance Technology Coupled with Impedance for the Dynamic Monitoring of the Cardiomyocyte Beating Function and Drug Screening.

The main sensing techniques used to study myocardial pulsation are electrical impedance sensing (EIS) and by quartz crystal microbalance (QCM). While electrical impedance technology is the gold standard for the study of myocardial pulsation, the clinical application of drugs is being followed up in real time additionally, thus, QCM technology needs to be further developed as a very important class of quality sensor technology. Moreover, the application of EIS, in combination with the QCM, for monitoring myocardial pulsation, has been rarely reported. In this paper, a series of cell growth and adhesion conditions were optimized using rat primary cardiomyocytes, and QCM was used in combination with EIS to monitor the adhesion and the myocardial pulsation ability of the cells in real time. Furthermore, cardiomyocytes that adhered to the QCM and EIS were treated with isoprenaline (ISO), a positive inotropic drug, and verapamil (VRP), a negative inotropic drug. Next, the cell index (CI)-time (T) plots, beating amplitude (BA) and beating rate (BR) of the cardiomyocytes were calculated and changes in these parameters, before and after, dosing were evaluated. The results showed that the QCM technique results were not only consistent with the results obtained with EIS, but also that the QCM technique had a certain degree of sensitivity for the calculation of cardiomyocyte beating. Thus, our findings validate the reliability and validity of the QCM technique for measuring cardiomyocyte beating and drug testing. We hope that further studies would evaluate the application of the QCM technology for clinical use.

An antifouling polymer for dynamic anti-protein adhesion analysis by a quartz crystal microbalance.

Nowadays, the non-specific adsorption of biomolecules is a key issue in numerous fields. Herein, an improved antifouling molecule was synthesized by grafting phenol with oligopoly (ethylene glycol), named (4-(2-(2-ethoxyethoxy) ethoxy) phenol (EEP). An ideal antifouling polymer coating (PEEP) was synthesized by the mechanism of electropolymerization of phenol. Quartz crystal microbalance (QCM), a sensitive mass sensor, was used to dynamically monitor both the modification and anti-protein adhesion (with bovine serum albumin as the model) process. Quantitatively, less proteins were observed to adhere to the modified electrode (277.8 ng for bare GCE and 8.88 ng for the modified GCE). Fourier transform infrared spectrophotometry (FT-IR), scanning electron microscopy (SEM), and electrochemical methods were used to study the coatings in detail. In this study, EEP was synthesized for the electrochemical preparation of an antifouling coating and characterized by QCM and electrochemical methods. The mild preparation environment (lower potential window and in phosphate buffered saline) and one-step method enable potential applications of PEEP in the field of biomaterials and biosensors.

Quartz Crystal Microbalance with Dissipation Monitoring of Dynamic Viscoelastic Changes of Tobacco BY-2 Cells under Different Osmotic Conditions.

The plant cell mechanics, including turgor pressure and wall mechanical properties, not only determine the growth of plant cells, but also reflect the functional and structural changes of plant cells under biotic and abiotic stresses. However, there are currently no appropriate techniques allowing to monitor the complex mechanical properties of living plant cells non-invasively and continuously. In this work, quartz crystal microbalance with dissipation (QCM-D) monitoring technique with overtones (3-9) was used for the dynamic monitoring of adhesions of living tobacco BY-2 cells onto positively charged N,N-dimethyl-N-propenyl-2-propen-1-aminiumchloride homopolymer (PDADMAC)/SiO2 QCM crystals under different concentrations of mannitol (CM) and the subsequent effects of osmotic stresses. The cell viscoelastic index (CVIn) (CVIn = ΔD⋅n/ΔF) was used to characterize the viscoelastic properties of BY-2 cells under different osmotic conditions. Our results indicated that lower overtones of QCM could detect both the cell wall and cytoskeleton structures allowing the detection of plasmolysis phenomena; whereas higher overtones could only detect the cell wall's mechanical properties. The QCM results were further discussed with the morphological changes of the BY-2 cells by an optical microscopy. The dynamic changes of cell's generated forces or cellular structures of plant cells caused by external stimuli (or stresses) can be traced by non-destructive and dynamic monitoring of cells' viscoelasticity, which provides a new way for the characterization and study of plant cells. QCM-D could map viscoelastic properties of different cellular structures in living cells and could be used as a new tool to test the mechanical properties of plant cells.

A high-throughput QCM chip configuration for the study of living cells and cell-drug interactions.

In this study, we present a novel design of interference-free, negligible installation-induced stress, suitable for the fabrication of high-throughput quartz crystal microbalance (HQCM) chips. This novel HQCM chip configuration was fabricated using eight independent yet same-batch quartz crystal resonators within a common glass substrate with eight through-holes of diameter slightly larger than that of the quartz resonator. Each quartz resonator's rim was adhered to the inner part of the through-hole via silicone glue to form the rigid (quartz)-soft (silicone)-rigid (glass) structure (RSRS) which effectively eliminates the acoustic couplings among different resonators and largely alleviates the installation-induced stresses. The consistence of the eight resonators was verified by very similar equivalent circuit parameters and very close response slopes to liquid density and viscosity. The HQCM chip was then employed for real-time and continuous monitoring of H9C2 cardiomyoblast adhesions and viscoelastic changes induced by the treatments of two types of drugs: drugs that affect the cytoskeletons, including nocodazole, paclitaxel, and Y-27632, and drugs that affect the contractile properties of the cells: verapamil and different dosages of isoprenaline. Meanwhile, we compared the cytoskeleton affecting drug-induced viscoelastic changes of H9C2 with those of human umbilical vein endothelial cells (HUVECs). The results described here provide the first solution to fabricate HQCM chips that are free from the limitation of resonator number, installation-induced stress, and acoustic interferences among resonators, which should find wide applications in areas of cell phenotype assay, cytotoxicity test, drug evaluation and screening, etc. Graphical abstract Schematic illustration of the principle and configuration of interference-free high-throughput QCM chip to evaluate and screen drugs based on cell viscoelasticity.

Dynamic cell adhesion and viscoelastic signatures distinguish normal from malignant human mammary cells using quartz crystal microbalance.

During transformation of a normal cell to a cell capable of forming a cancerous growth, cellular morphology, the cytoskeleton, and focal contacts undergo significant changes. These changes should be capable of being characterized via real-time monitoring of the dynamic cell adhesion process and viscoelastic properties of cells. Here, we describe use of the quartz crystal microbalance (QCM) to distinguish the dynamic cell adhesion signatures of human normal (HMEC) versus malignant (MCF-7) mammary epithelial cells. The significantly reduced QCM responses (changes in frequency [Δf] and motional resistance ΔR) of MCF-7 cells compared with those of HMECs mirror the cancer cells' morphological features as observed via optical microscope. We analyzed the initial 2-h cell adhesion kinetics, suggesting cell-cell cooperativity for HMECs and no or weak cell-cell interactions for MCF-7 cells. We propose that changes of the ΔR/Δf ratio, which we term the cell viscoelastic index (CVI), reflect the establishment of cytoskeleton structure and dynamic viscoelastic properties of living cells. The CVI decreases significantly on initiation of cell to surface interactions as cells establish their cytoskeletal structures. During the cell adhesion process, MCF-7 cells were consistently softer, exhibiting up to a 2.5-fold smaller CVI when compared with HMECs.

Electropolymerized tyrosine-based thin films: selective cell binding via peptide recognition to novel electropolymerized biomimetic tyrosine RGDY films.

We have created thin films by cyclic voltammetry (CV) electropolymerizations of the following phenolic functional group-based monomers: phenol; tyrosineamide; the tetrapeptide RGDY-containing the integrin membrane adhesion protein recognition tripeptide RGD; RDGY, a nonsense control tetrapeptide; and 1:3 mixtures of tyrosineamide with the two tetrapeptide monomers. The film formation process and description of the film properties were obtained by repetitive CV cycling using the oscillating quartz frequency shift, Deltaf, and motional resistance shift, DeltaR, parameters obtained with the electrochemical quartz crystal microbalance technique. Only the poly(phenol) film exhibited close chain packing-based self-limiting behavior, where all film synthesis ceased after approximately 7 CV cycles. All other films continued to form by electropolymerization with successive CV cycles out to the maximum cycle number (30 cycles) we measured. All of the films exhibited little energy dissipation behavior. Using the quartz crystal microbalance, we next compared the time course of cell attachment with the washed films and demonstrated that cells bound best to films in the following order: RGDY sense peptide:tyrosineamide films>RDGY nonsense peptide:tyrosineamide films=tyrosineamide films>phenol films. Cell enumeration after washing and trypsinization revealed firm protein-based cell attachment to the underlying extracellular matrix for the RGDY-containing films. These sense peptide films bound and retained two- to fivefold as many cells as the other films, with cells exhibiting a normal morphology. These results suggest the operation of specific cell attachment to the electropolymerized films via the RGD binding site for cellular integrin membrane proteins. The electropolymerization method we studied here forms a cassette system for creating electropolymerized films tailored to specific attachment of different cell types by varying the electropolymerized Y(tyrosine)-containing recognition peptide.

A comparative study of the cytoskeleton binding drugs nocodazole and taxol with a mammalian cell quartz crystal microbalance biosensor: different dynamic responses and energy dissipation effects.

The quartz crystal microbalance (QCM) was used to create piezoelectric whole-cell biosensors utilizing either living endothelial cells (ECs) or the metastatic human mammary cancer cell line MDA-MB-231 adhering to the gold QCM surface under in vitro growth conditions. We utilized the whole-cell QCM biosensors for the detection of the effects of varying concentrations of the microtubule binding drugs taxol and nocodazole by measuring changes in the QCM steady state frequency (Deltaf) and motional resistance (DeltaR), shift values. Using 0.11-50 microM nocodazole, we observed the Deltaf shift values of the biosensors, consisting of 20,000 ECs, to decrease significantly in magnitude (nearly 100%) to a limiting value, in a dose-dependent fashion, over a 5- to 6-h incubation period following drug addition. This effect is consistent with nocodazole's known disruption of intracellular microtubules. On the other hand, 10 microM taxol caused little alteration in Deltaf over the same time period, consistent with its microtubule hyperstabilization effect. When the EC QCM biosensor Deltaf shift values were normalized by the number of ECs found firmly attached to the QCM surface via trypsin removal and electronic counting, the dose curve was shifted to lower nocodazole concentrations, resulting in a more sensitive drug biosensor. The kinetics of the Deltaf decrease with increasing nocodazole concentrations measured by the EC QCM biosensor was found to be similar at all drug concentrations and was well fit by a single first-order exponential decay equation. For all nocodazole doses, t(0.5) was invariant, averaging t(0.5)=0.83+/-0.14 h. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, is disrupted by the addition of nocodazole and this process is sensed by the cell QCM biosensor. This interpretation of the data was confirmed by a fluorescence light microscopy investigation of ECs undergoing treatment with increasing nocodazole doses using a fluorescent antibody to alpha-tubulin. These studies revealed a corresponding loss of the spread morphology of the cells, concomitant with a rearrangement of the extended native microtubules into increasingly large aggregates with the cells eventually lifting from the surface in significant numbers at 50 microM. At 6 microM nocodazole, partial reversibility of the EC QCM biosensor was demonstrated. These results indicate that the EC QCM biosensor can be used to detect and study EC cytoskeleton alterations and dynamics. We suggest the potential of this cellular biosensor for the real-time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment and cellular spreading, regardless of their molecular mechanism of action.

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix.

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.

Electropolymerized films formed from the amphiphilic decyl esters of D- and L-tyrosine compared to L-tyrosine using the electrochemical quartz crystal microbalance.

Using the electrochemical quartz crystal microbalance (EQCM), we compared thin films formed on Pt by electropolymerization of l-tyrosine to that of the amphiphilic monomers, decyl esters of d- and l-tyrosine (DEDT and DELT). Mass build-up and film properties were determined as a function of monomer concentration via frequency, f, motional resistance, R, and charge passage, Q, measurements. Films were found to occur by a combination of monomer electropolymerization and adsorption for DEDT and DELT, but only by electropolymerization for l-tyrosine. This difference in film formation process for the monomers is reflected in the net mass build-up for each film, as represented by calculated df/dQ values. For the adsorbing monomers DEDT and DELT, films possessed concentration dependent df/dQ values, more than 100-fold greater than that for l-tyrosine film formation under equivalent electropolymerization conditions. During the entire film growth process, all three films exhibited no significant energy dissipation properties (DeltaR invariant). Concentration dependent adsorption of significant levels of unpolymerized but self-assembled DEDT and DELT monomers account for the subsequent time dependent mass loss observed from the films maintained in buffer in the absence of monomer. Contact angle measurements demonstrated a pH dependent increase in the surface hydrophilicity of films electropolymerized from the DEDT, DELT, and l-tyrosine monomers but not films formed from phenol and 3-nitrophenol monomers. This behavior is consistent with the monomers' known changes in titration/charge state properties with increasing pH. This study provided insight into the film formation, stability, and surface hydrophilicity resulting from electropolymerization of these related tyrosine based monomers. This information is critical to assessing the utility these films may have in the development of new biomaterials and as biological macromolecule or cell immobilization strategies in biosensors.

Detection of apoptosis and drug resistance of human breast cancer cells to taxane treatments using quartz crystal microbalance biosensor technology.

Taxanes are used for the treatment of many human cancers, as first- and second-line chemotherapeutics. In the course of treatment many patients develop resistance or hypersensitivity to one form of taxane and require a different taxane to rescue the therapeutic benefit of the drug. There is currently no method to reliably predict tumor responses to taxanes prior to therapy or when resistance or hypersensitivity develops. We adapted the quartz crystal microbalance (QCM) biosensor technique to study responses of human mammary epithelial tumor cells to taxanes. Studies indicate that stable frequency and resistance levels are reached at 24 h. Cells in the QCM can then be treated with taxanes and responses monitored in real time via frequency and resistance changes reflecting alterations of cell mass distribution and viscoelastic properties. Distinct shifts in frequency and resistance accurately predicted apoptosis or resistance to treatment, as determined in parallel convention assays. QCM analysis accurately predicted docetaxel was more effective than paclitaxel and MCF-7 cells were more resistant to taxanes compared to MDA-MB-231 cells. These studies suggest "signature" patterns for taxane responsivity could be compared to those of patient biopsy samples to predict therapy outcome prior to treatment for initial therapy or to rescue therapy efficacy.

Quartz crystal microbalance study of endothelial cell number dependent differences in initial adhesion and steady-state behavior: evidence for cell-cell cooperativity in initial adhesion and spreading.

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Deltaf and DeltaR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Deltaf and DeltaR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Deltaf and DeltaR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Deltaf and DeltaR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Deltaf and DeltaR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Deltaf and DeltaR versus trypsinized, attached EC number plot 1 h post-seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post-seeding exhibits a hyperbolic curve shape. This number dependence suggests cell-cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post-addition, steady-state Deltaf and DeltaR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady-state 24 h Deltaf and DeltaR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy-dissipative structure. This is consistent with the time-dependent elaboration of surface contacts in anchorage-dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.