Dynamic Multiparameter Platelet Function Assessment Using a Capacitive Biosensor

Summary

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.

Abstract

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.

Introduction

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 globally^2,3,4,5,6. 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 infl....

Protocol

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). Bri....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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