Name: Dynamic Multiparameter Platelet Function Assessment Using a Capacitive Biosensor (Video) | JoVE Journal
Uploaded: 2025-05-02T12:40:00.000+00:00
Duration: 6 min 32 s
Description: Learn dynamic multiparameter platelet function assessment using a capacitive biosensor in a semi-rigid microenvironment. This method measures platelet count, stimulation strength, and activation pathways for improved hemostasis evaluation.

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5 minutes

The authors express their gratitude to Dr. Moritz Stolla and Dr. Jason Acker for their valuable discussions and technical assistance. They also acknowledge the Biology Imaging Facility at the University of Washington for its infrastructure and support. This work received partial funding from the CoMotion Innovation Fund at the University of Washington (Grant No. 682548, D.Y.G.).

The study proposal was approved by the Human Subjects Division (HSD) at the University of Washington Internal Review Board (UW-IRB; Study ID: STUDY00005211). All volunteer subjects who participated in the study provided written informed consent. The details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Fabrication steps for the membrane capacitive sensor (MCS)
NOTE: The MCS sensor was fabricated utilizing traditional microfabrication techniques. This biosensor was composed of a top (T-) and bottom (B-) membrane capacitance chip (MCC). Briefly, the fabrication steps are shown in Figure 1.
<ol>
	<li>Microfabrication steps for T-MCC (Figure 1A)
	<ol>
		<li>Design the layout of the T-MCC using standard CAD layout software for a 4-inch silicon wafer substrate.</li>
		<li>Transfer the design onto a chromium glass photomask using the standard mask fabrication process29,30.</li>
		<li>On a clean double-side polished silicon substrate (400&#181;m thick), deposit 500-nm-thick of Si3N4 using standard low-pressure chemical vapor deposition (LPCVD) technique31. Spin coat 12 &#181;m of AZ 9260 photoresist at 2000 rpm for 25 s using a manual spin coater.</li>
		<li>Using standard UV photolithography technique32, pattern the photoresist, which involves exposing the photoresist with the chrome glass photomask in a UV aligner, and developing the resist in a developer solution to remove the UV-exposed region of the resist.</li>
		<li>Pattern the underlying 500-nm-thick silicon nitride layer using a reactive ion etching process, which exposes the silicon substrate.</li>
		<li>Remove the exposed 400&#181;m thick silicon substrate using a standard Bosch process: Deep Reactive Ion Si Etch process33.</li>
		<li>Calculate the desired number of etch cycles to remove 400 &#181;m of silicon. 
		NOTE: This will vary depending on the tool that is utilized for the process, as they will influence the etch rate (in this case, it was 1.8 &#181;m/cycle).</li>
		<li>To prevent accidentally etching the Si3N4 layer, stop the dry etching at least 10 &#181;m before (i.e. at 390 &#181;m). Remove the remaining silicon using the traditional highly selective KOH at 80 &#176;C for 5 min.</li>
		<li>Deposit 20 nm of Cr and 150 nm of Au metal layers consecutively using an E-beam evaporation process34 and a shadow mask to fabricate the sensing electrode in the T-MCC.</li>
		<li>At this stage, the T-MCCs in the wafer will be connected by tiny silicon holders. Separate the T-MCCs by carefully puncturing the tiny holders.</li>
	</ol>
	</li>
	<li>Microfabrication steps for B-MCC (Figure 1B)
	<ol>
		<li>On a clean thermally oxidized silicon substrate (400&#181;m thick), deposit Cr/Au (20 nm, 150 nm) metal layers using a shadow mask, similar to step 1.1.9.</li>
		<li>Use standard photolithography32 with backside alignment, followed by a KOH wet-etching step to release the B-MCCs. 
		NOTE: The T- and the B-MCC have 2.5 x 1.5 cm contact pads.</li>
	</ol>
	</li>
</ol>
2. Biofunctionalization of the capacitive sensor
NOTE: Briefly, this step is about coating the T-MCC electrode with Human Fibronectin (Fn) to facilitate platelet attachment onto the sensor.
<ol>
	<li>Prior to biomolecular coatings, clean the sensing electrode with oxygen plasma for 45 s at 100 W.</li>
	<li>Add 1-Dodecanethiol (1 mM) in 200-proof ethanol to the sample well on T-MCCs.</li>
	<li>Place the T-MCCs in a container filled with dry nitrogen, sealed, and wrapped with parafilm for 24-48 h.</li>
	<li>Rinse the gold surface with deionized water and 200-proof ethanol, followed by nitrogen drying at room temperature. At this stage, store the sensors at 2-8 &#176;C in a dry nitrogen atmosphere until the next steps.</li>
	<li>Add Fn solution in phosphate-buffered saline (PBS, 50 &#181;g/mL) to the well 12 h before the measurement and incubate at 37 &#176;C for 2-8 h.</li>
</ol>
3. Capacitance sensor setup
NOTE: Figure 2 represents the photograph of the experimental setup.
<ol>
	<li>Use an LCR meter with micro positioners and needle probes to make electrical contact with the sensor.</li>
	<li>Employ 3D-printed plastic fixtures (see Table of Materials) to securely place the T- and the B-MCC. The bottom fixture is equipped with stoppers on the x-y axis to ensure precise alignment of the T-MCC over the B-MCC to form a capacitor. 
	NOTE: All 3D printers used were from the University of Washington.</li>
	<li>Apply a sinusoidal signal (0.5 V) at 100 kHz and an 8 Hz sampling rate.</li>
</ol>
4. Preparation of citrated platelet-rich-plasma (c-PRP)
NOTE: All blood samples were from volunteers who participated in this research. None of the participants had a previously known platelet abnormality or clotting disorder, and they had not taken any platelet medications, including Non-Steroidal Anti-inflammatory Drugs (NSAIDs), in the two weeks leading up to sample collection. A licensed phlebotomist conducted a blood draw using a 21 G needle to standard 3.2% citrate tubes. The first 1 mL was discarded to avoid tissue factor contamination. Samples were transported in a polystyrene container, and all measurements were performed within 6 h of blood draw.
<ol>
	<li>Centrifuge the whole blood sample at 200 x g for 10 min at 20 &#176;C to obtain c-PRP.</li>
	<li>Transfer the c-PRP to a sterile container and conduct the platelet count. For inhibitor studies, incubate the c-PRP with a predefined concentration of the drug in Tyrode&#39;s buffer (see Results section for details).</li>
</ol>
5. Platelet functional assay
NOTE: The schematic for the presented platelet functional assay is shown in Figure 3.
<ol>
	<li>Assemble the T- and the B-MCC in the 3D-printed fixtures.</li>
	<li>Measure the baseline capacitance measurement for 5 min, then add 45 &#181;L of c-PRP to the sample well in the T-MCC.</li>
	<li>Wait for 30 min for the platelets to adhere to the Fn-coated electrode in the T-MCC.</li>
	<li>Remove 30 &#181;L of the c-PRP carefully without touching the platelets attached to the membrane, and immediately replenish with Tyrode&#39;s buffer. Repeat washing at least 5 times with a 20 s interval to ensure that no additional platelets or macroaggregates land on the sensing electrode after activation.</li>
	<li>Add 10 &#181;L of the agonist solution (Thrombin or ADP) at a desired concentration. Allow 80 min equilibration time.</li>
</ol>
6. Data and statistical analysis for the signal markers
NOTE: Capacitance is measured continuously from steps 5.2-5.5.
<ol>
	<li>For the capacitance signal during the adhesion phase (step 5.3), measure the maximum change in capacitance after the 30 min equilibration time, which is referred to as &#916;&#8203;Cadh.</li>
	<li>Similarly, for the activation phase (step 5.5), measure the maximum change in capacitance, which is referred to as &#916;Cact, and the slope of the curve&#160;between 200-300 s after activation, which is referred to as Sact.</li>
	<li>Use analysis of variance (ANOVA) with Tukey&#39;s post-hoc to compare the results between the groups. Use Shapiro-Wilk Goodness of Fit for normal distribution analysis.</li>
</ol>

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</ol>

The authors declare no competing interests.

This study pioneered a novel capacitance-based method for assessing platelet function, which evaluates both adhesion and post-activation platelet dynamics within a single device, marking the first reported instance of such an approach. The novel experimental protocol introduces a relatively straightforward technique to counteract the impacts of fibrin formation and plasma clotting factors through a wash-out procedure. This results in measurements capable of discerning various factors that affect platelet function. Compar...

Platelets, specialized blood cells, are pivotal in orchestrating the hemostatic response to halt bleeding following injury and in facilitating the healing of blood vessels1. Additionally, they also serve as crucial mediators in thrombosis, a leading cause of thromboembolic disease-related deaths globally2,3,4,5,6. When a vascular injury occurs, platelets undergo a series of complex, regulated, and multi-stage functional processes. These include adhesion to the intimal matrix, an influx of intracellular calcium triggering platelet conformational changes, activation, granule secretion, aggregation, and cytoskeletal contraction, ultimately forming and stabilizing hemostatic plugs to seal the damaged sites and prevent bleeding7,8. Despite significant advancements in antiplatelet drugs and therapeutic strategies9,10,11, thrombosis risk persists. Antiplatelet therapy management presents challenges, including the risk of iatrogenic bleeding, difficulty in achieving antithrombotic efficacy while maintaining hemostasis, and variability in patient responsiveness, including drug resistance12,13.
Although the molecular mechanisms governing platelet response phases are well documented, current methods for testing platelet function remain suboptimal. Traditional laboratory-based tests often fall short as they only assess limited aspects of early-to-mid-stage platelet activity, such as adhesion, aggregation, or clot viscosity7,14,15,16,17,18. This partial analysis can lead to insufficient information. Moreover, these tests do not offer simultaneous, continuous, and rapid assessment of multiple crucial platelet functional elements within a single assay. Consequently, this limitation hampers advancements in both clinical and experimental hematology. Over time, a plethora of impedimetric or capacitive sensors have been developed for various biomedical applications19,20,21,22,23,24,25,26,27,28.
Here, we present the protocol for a multiplexed platelet function assessment using a capacitive biosensor. The proposed approach offers an attractive feature by sensitively monitoring dynamic changes in a broad spectrum of platelet functions at the cellular level. The presented approach utilizes a biosensor comprised of two microchips: a top-silicone chip, which is disposable, featuring a sample well for citrated platelet-rich plasma and a sensing electrode coated with human Fibronectin to facilitate platelet adhesion, alongside a reusable bottom silicon chip housing a reference electrode. Continuous measurement of dynamic capacitance changes during the whole coagulation process, encompassing platelet adhesion, activation, and post-activation, enables sensitive analysis linked to variations in platelet counts, levels of platelet activation, and the inhibition of activation pathways. The clinical feasibility and utility of this method were shown using pertinent human plasma samples, underscoring its potential for robust platelet function assessment in clinical settings.

<table><tbody><tr><td>1-Dodecanethiol</td><td>Sigma-Aldrich, MO, U.S.A</td><td>471364-100ML</td><td>1 mM</td></tr><tr><td>200-proof ethanol</td><td>Sigma-Aldrich, MO, U.S.A</td><td>EX0276-1</td><td></td></tr><tr><td>3D printer</td><td>Shenzhen Creality 3D Technology Co, Ltd.</td><td>Ender-3 V3</td><td></td></tr><tr><td>3D printing material</td><td>HATCHBOX 3D, CA, U.S.A</td><td>3D PLA-1KG-1.75</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate</td><td>Sigma Aldrich, U.S.A</td><td>01905-250MG-F</td><td>ADP</td></tr><tr><td>Aspirin&amp;nbsp;</td><td>Sigma-Aldrich, MO, U.S.A</td><td>A2093-100G</td><td></td></tr><tr><td>Deep Reactive Ion Etching</td><td>Omega Engineering, Inc.</td><td>SPTS Rapier DRIE</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich, MO, U.S.A</td><td>D8418-50ML</td><td>DMSO</td></tr><tr><td>High Vacuum Deposition Systems</td><td>CHA&amp;nbsp;</td><td>SEC-600</td><td></td></tr><tr><td>Human Fibronectin</td><td>Sigma-Aldrich, MO, U.S.A</td><td>CLS356008-1EA</td><td>Fn</td></tr><tr><td>KOH</td><td>Sigma-Aldrich, MO, U.S.A</td><td>P1767-250G</td><td></td></tr><tr><td>LCR meter&amp;nbsp;</td><td>Keithley Instruments, Inc., OH, U.S.A</td><td>Keithley EL 4980AL</td><td></td></tr><tr><td>LCR meter holders</td><td>Signatone Corporation, CA, U.S.A</td><td>SCA-50-4</td><td></td></tr><tr><td>Mask Aligner System</td><td>ABM, U.S.A, Inc.</td><td>ABM/6/350/NUV/DCCD/SA</td><td></td></tr><tr><td>Micro-positioners</td><td>Signatone, CA, U.S.A</td><td>S-725</td><td></td></tr><tr><td>needle probe</td><td>Signatone Corporation, CA, U.S.A</td><td>SCAT5T-4</td><td>12.5 &amp;mu;m radius</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma-Aldrich, MO, U.S.A</td><td>P4474-1L</td><td>PBS, pH 7.4</td></tr><tr><td>Reactive Ion Etching</td><td>Plasma-Therm,U.S.A</td><td>RIE Vision 320</td><td></td></tr><tr><td>silicon substrate</td><td>Wafer World Inc</td><td>SKU# 1766</td><td></td></tr><tr><td>Standard 3.2% citrate tubes</td><td>Tiger Medical, NJ, U.S.A.</td><td>Covidien / Cardinal Health 8881340478 Monoject</td><td></td></tr><tr><td>Thrombin</td><td>Enzyme Research Laboratories, U.S.A</td><td>HT 1002a</td><td></td></tr><tr><td>Ticagrelor</td><td>Sigma-Aldrich, MO, U.S.A</td><td>PHR2788-400MG</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s buffer</td><td>Boston Bioproducts, U.S.A</td><td>BSS-375</td><td></td></tr><tr><td>UV photoresist</td><td>AZ electronic materials, NC, U.S.A.</td><td>AZ 9260</td><td>15um</td></tr></tbody></table>

dynamic multiparameter platelet function assessment using a capacitive biosensor

Dynamic Multiparameter Platelet Function Assessment Using a Capacitive Biosensor

Platelets play a fundamental role in blood clotting through a series of regulated responses, including adhesion, spreading, granular secretion, aggregation, and cytoskeletal contraction. However, current assays are limited to partial analysis of platelet function under non-physiological conditions. Thus, an improved assay that reflects the dynamic and multifaceted nature of platelet function in physiological settings is necessary. In this context, a novel approach is introduced to measure several key parameters related to platelet function in a more physiologically relevant ex vivo semi-rigid microenvironment compared to traditional assays. This method utilizes an advanced electrical biosensor, the membrane capacitance sensor (MCS), which provides unique insights into the clotting process through three distinct readouts. These readouts are highly sensitive to variations in platelet count, stimulation intensity, and specific activation pathways. As a purely electrical sensing platform, the MCS demonstrates significant potential as a diagnostic tool for detecting primary hemostatic function disorders, evaluating the efficacy of therapeutic treatments, and advancing the broader understanding of the roles of platelets in hemostasis and thrombosis.

This study aims to conduct a dynamic assessment of platelet function. Following the protocol described above, the c-PRP solution was prepared, and platelets were seeded onto the Fn-coated electrode in T-MCC. The free-floating platelets were washed out by the washing step, and an agonist was added to activate the attached platelets. Detailed results and discussion can be found in our previous report28.
Figure 4A represents the results for a ...

A protocol for a novel dynamic multiparameter platelet functional assay using a capacitive biosensor is presented here. This approach, designed within a semi-rigid microenvironment to enhance physiological relevance, provides three output parameters sensitive to platelet count, stimulation strengths, and activation pathways.

Platelets play a fundamental role in blood clotting through a series of regulated responses, including adhesion, spreading, granular secretion, ...

dynamic-multiparameter-platelet-function-assessment-using-capacitive

Research

JoVE Journal

Bioengineering

This video demonstrates the method to assess the impact of shear stress on platelets. Antibody treatment induces dissociation and conformational changes in mechanoreceptor complexes, activating the platelets and expressing markers on the surface. These markers are observed via immunostaining and flow cytometry.

The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

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Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

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Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin bond.

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics in Assessing Platelet Function

Multi-color flow cytometry for platelets can identify new platelet subtypes within classical platelet subpopulations, such as resting, aggregatory, procoagulant, and apoptotic platelets. This allows comparison of different subpopulation patterns induced by various platelet agonists. Here, a procedure for establishing a multi-color flow cytometry panel is described in detail.

Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

The present protocol provides a comprehensive set of procedures necessary to analyze procoagulant platelets, which exhibit overlapping features of necrosis, apoptosis, and platelet activation.

Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

This work describes a microfluidic assay that uses shear stress to trigger thrombus formation in blood and characterizes the thrombus by multiple dimensions of readout. The assay can measure the prothrombotic tendency of blood samples, and is thus useful in disease diagnosis, drug discovery, as well as basic mechanistic studies related to thrombosis.

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

Learn dynamic multiparameter platelet function assessment using a capacitive biosensor in a semi-rigid microenvironment. This method measures platelet count, stimulation strength, and activation pathways for improved hemostasis evaluation.

Dynamic Multiparameter Platelet Function Assessment Using a Capacitive Biosensor

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