We are designing an assay to evaluate the platelet function in physiological settings, to address the limitation of current assays by activating platelets in a semi-rigid microenvironment with a membrane capacity sensor. The assay measures platelet count, stimulation strength, and activation pathways. This tool offers a comprehensive approach for studying platelet mechanisms during the hemostasis.
Our protocol can evaluate multiple parameters of the platelet in the single assay under physiological conditions. We will focus on improving this protocol and the development of other hemostasis evaluation sensors. Begin by using standard CAD layout software to design the layout of the team membrane capacitance chip, or MCC, for a four-inch silicon wafer substrate for the microfabrication process as described in the manuscript.
For bio-functionalization, clean the sensing electrode of TMCC using oxygen plasma for 45 seconds at 100 watts. Add one Do Decanal solution in 200 proof ethanol to the sample well on the TMCCs. Place the TMCCs in a container filled with dry nitrogen.
Seal the container and wrap it with parafilm for 24 to 48 hours. After two days, rinse the gold surface of TMCC with deionized water and 200 proof ethanol. Dry the TMCCs with nitrogen gas at room temperature.
Add human fibronectin solution in PBS to the sample well of TMCC and incubate at 37 degrees Celsius for two to eight hours. For capacitance sensor setup, use an LCR meter with micro positioners and needle probes to establish electrical contact with the sensor. Employ 3D-printed plastic fixtures to securely place the TMCC and BMCC.
Ensure the bottom fixture is equipped with stoppers on the XY axis to align the TMCC precisely over the BMCC, forming a capacitor. Apply a sinusoidal signal of 0.5 volts at 100 kilohertz with an eight hertz sampling rate. To obtain concentrated platelet-rich plasma, or CPRP, centrifuge the human blood samples.
Transfer the CPRP to a sterile container and conduct the platelet count. For inhibitor studies, incubate the CPRP with a predefined concentration of aspirin in Tyrode's buffer. For the platelet functional assay, assemble TMCC and BMCC in the 3D-printed fixtures.
Measure the baseline capacitance of the assembled MCCs for five minutes then add 45 microliters of CPRP to the sample well in the TMCC and wait for 30 minutes to allow platelets to adhere to the fibronectin-coated electrode in the TMCC. Next, remove 30 microliters of CPRP from the sample well without disturbing the adhered platelets and replenish the well with Tyrode's buffer. After the last wash, add 10 microliters of the agonist solution at the desired concentration and equilibrate the sample for 80 minutes.
Measure the maximum change in capacitance post-30-minute adhesion to determine delta C adhesion. For the activation phase, measure the maximum change in capacitance, referred to as delta C activation. Calculate the slope of the capacitance curve between 200 and 300 seconds after activation to determine S activation.
Perform statistical analysis using analysis of variance with Tukey's post-op test to compare results between groups. Use Shapiro-Wilk goodness of fit to test for normal distribution. A CPRP solution with a predetermined platelet count showed a linear decrease in capacitance during adhesion.
An exponential steady-state decrease was observed after thrombin activation. Delta C adhesion values showed a strong correlation with platelet count across a range of platelet counts, with a significant reduction at 1.3 millimoles aspirin concentration. The rate of exponential decrease in capacitance following platelet stimulation increased with cell concentration.
The magnitude of capacitance loss also grew with higher concentrations, showing a clear upward trend in delta C activation. A similar trend was observed for S activation and platelet count. With increasing aspirin dosage, capacitance, delta C activation, and S activation showed decreasing trends.
A statistically significant difference in S activation was observed between high and medium doses, while no significant difference was found in delta C activation. Capacitance signals for CPRP samples activated with thrombin showed that capacitance reduction became more pronounced with increasing thrombin concentration. Both delta C activation and S activation exhibited a statistically significant trend with thrombin levels.
