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Method Article
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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, 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.
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.
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.
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.
3. Capacitance sensor setup
NOTE: Figure 2 represents the photograph of the experimental setup.
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.
5. Platelet functional assay
NOTE: The schematic for the presented platelet functional assay is shown in Figure 3.
6. Data and statistical analysis for the signal markers
NOTE: Capacitance is measured continuously from steps 5.2-5.5.
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 ...
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...
The authors declare no competing interests.
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.).
Name | Company | Catalog Number | Comments |
1-Dodecanethiol | Sigma-Aldrich, MO, U.S.A | 471364-100ML | 1 mM |
200-proof ethanol | Sigma-Aldrich, MO, U.S.A | EX0276-1 | |
3D printer | Shenzhen Creality 3D Technology Co, Ltd. | Ender-3 V3 | |
3D printing material | HATCHBOX 3D, CA, U.S.A | 3D PLA-1KG-1.75 | |
Adenosine 5′-diphosphate | Sigma Aldrich, U.S.A | 01905-250MG-F | ADP |
Aspirin | Sigma-Aldrich, MO, U.S.A | A2093-100G | |
Deep Reactive Ion Etching | Omega Engineering, Inc. | SPTS Rapier DRIE | |
Dimethyl Sulfoxide | Sigma-Aldrich, MO, U.S.A | D8418-50ML | DMSO |
High Vacuum Deposition Systems | CHA | SEC-600 | |
Human Fibronectin | Sigma-Aldrich, MO, U.S.A | CLS356008-1EA | Fn |
KOH | Sigma-Aldrich, MO, U.S.A | P1767-250G | |
LCR meter | Keithley Instruments, Inc., OH, U.S.A | Keithley EL 4980AL | |
LCR meter holders | Signatone Corporation, CA, U.S.A | SCA-50-4 | |
Mask Aligner System | ABM, U.S.A, Inc. | ABM/6/350/NUV/DCCD/SA | |
Micro-positioners | Signatone, CA, U.S.A | S-725 | |
needle probe | Signatone Corporation, CA, U.S.A | SCAT5T-4 | 12.5 μm radius |
Phosphate Buffered Saline | Sigma-Aldrich, MO, U.S.A | P4474-1L | PBS, pH 7.4 |
Reactive Ion Etching | Plasma-Therm,U.S.A | RIE Vision 320 | |
silicon substrate | Wafer World Inc | SKU# 1766 | |
Standard 3.2% citrate tubes | Tiger Medical, NJ, U.S.A. | Covidien / Cardinal Health 8881340478 Monoject | |
Thrombin | Enzyme Research Laboratories, U.S.A | HT 1002a | |
Ticagrelor | Sigma-Aldrich, MO, U.S.A | PHR2788-400MG | |
Tyrode’s buffer | Boston Bioproducts, U.S.A | BSS-375 | |
UV photoresist | AZ electronic materials, NC, U.S.A. | AZ 9260 | 15um |
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