Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

We have developed a new method that can adequately capture the biomechanical attributes of thrombosis and also detect prothrombotic abnormalities in humans. To begin, use a silicon wafer to fabricate an SU8 photoresist master mold via standard photolithography based on the design and secure the master mold at the bottom of a 15 centimeter Petri dish with tape. Prepare the polydimethylsiloxane, or PDMS, mixture by mixing the prepolymer base and curing agent from the silicone elastomer kit at a 10 to 1 weight ratio.

Pour the PDMS mixture onto the master mold. Then place the plate containing the PDMS mixture in a vacuum desiccator to degas it. Thermally cure the PDMS mixture by placing it in an incubator set to 75 degrees Celsius for two hours.

After removing the sample from the incubator, carefully cut out the cured PDMS from the master mold, and section the cured PDMS into individual chip units. Using a probe needle, punch holes at the designated locations to create the inlet and outlet. In a chemical hood, use a 75%ethanol wetted task wipe to clean the glass slides and dry them.

Next, using a high frequency generator, treat the glass slides for 30 seconds and the PDMS chips for 20 seconds. Align each chip with a glass slide and gently press the chip onto the glass slide surface to bind it. Now thermally bond the devices by placing them in an oven set to 150 degrees Celsius for 15 minutes.

After bonding, store the devices at room temperature under dust-free conditions. Then, using a pincer, remove the plastic part of the probe needles and bend the metal tubes to 90 degrees to create connectors for the microfluidic devices. After setting up the reactions for conjugating fibrinogen and required antibodies with Alexa Fluor, centrifuge the purification columns at 1, 100 G for two minutes to remove the storage buffer.

After discarding the flow through, load the reaction solution onto the purification column mounted in a compatible collection vial and centrifuge at 1, 100 G for five minutes to harvest the required mix. Using a spectrophotometer, measure the absorbance for the protein and the dye signal. Calculate the protein concentration and the fluorescence-to-protein molar ratio.

Store the dyes at four degrees Celsius in the dark. Add heparin to the Tyrode buffer to a final concentration of 0.32 units per milliliter per 10 milliliters of blood to be collected, and prepare the syringe by aspirating 500 microliters of Tyrode buffer at pH 7.4. After collecting the blood via venipuncture in the heparin-containing syringe, transfer it into a 15 milliliter centrifuge tube and keep it at 37 degrees Celsius.

Dilute Von Willebrand factor monomer to two micrograms per milliliter in PBS. Precoat the microfluidic devices with the diluted von Willebrand factor monomer and incubate them for one hour at room temperature. Now incubate the blood sample with fluorescent sensor set 1 or set 2 for 10 minutes at room temperature.

Next, connect a microfluidic device with inlet and outlet connectors and tubing. Connect the other end of the inlet connector to tubing containing PBS, and then connect the other end of the outlet connector to a syringe. Mount the syringe onto a syringe pump and the microfluidic device onto an inverted microscope.

Manually maneuver the microscope stage to locate the site of stenosis. Fill the microfluidic channel and tubing with PBS using the syringe pump at a flow rate of 0.5 milliliters per minute. Inspect the device to ensure no air bubbles are present around the site of stenosis.

Connect the inlet tubing to the blood sample and perfuse the blood sample through the microfluidic channel using the syringe pump at a flow rate of 0.018 milliliters per minute. In an inverted microscope, set the exposure time and gain for each fluorescence channel in the software. Perform real-time multicolor fluorescence imaging by alternating among the four channels and exciting one fluoro-4 at a time.

Adjust the focus plane to the thrombus and record fluorescent signals at the site of stenosis for 15 to 30 minutes. For data analysis, open the data file using ImageJ version 1.53. Use the SZ 22 fit C channel to identify the contour of the thrombus.

Then using polygon selections, roughly select the area of the thrombus. For each channel, eliminate background signal by selecting image, choosing Adjust, and then selecting Threshold. Finally, measure the area and average signal intensity within the thrombus by selecting Analyze and choosing Measure.

Representative snapshots showed thrombi formed in healthy blood within the stenotic channel with platelets, fibrinogen, von Willebrand factor, and P-selectin visualized using sensor set 1, and platelets, phosphatidylserine, E-positive integrin alpha IIb beta 3, an activated integrin alpha IIb beta three visualized using sensor set 2. A single fluorescence channel image was processed by selecting the thrombus area, removing background signal, and measuring the signal area and average brightness to enable quantitative analysis. Continuous analysis of fluorescent signal intensity over time generated a signal versus time curve for platelet accumulation during thrombus formation.

For each time point, total signal intensities were obtained for four biomarkers in sensor set one and four biomarkers in sensor set two. Thrombus size was calculated and a seven-dimensional thrombus profile was generated. Currently, no bioassay is available to evaluate the prothrombotic tendency of human patients, and our work attempts to fill this gap.

By detecting the biomechanical activity of blood samples, our assay can comprehensively evaluate the prothrombotic attributes of subjects. Our assay can be developed into a clinical test for detecting prothrombotic tendency in patients, and it can also be used to screen next generation antithrombotic drugs.