A microfluidic anti-Factor Xa assay device for point of care monitoring of anticoagulation therapy

Abstract

The development of new point of care coagulation assay devices is necessary due to the increasing number of patients requiring long-term anticoagulation in addition to the desire for appropriate, targeted anticoagulant therapy and a more rapid response to optimization of treatment. The majority of point of care devices currently available for hemostasis testing rely on clot-based endpoints which are variable, unreliable and limited to measuring only certain portions of the coagulation pathway. Here we present a novel fluorescence-based anti-Factor Xa (FXa) microfluidic assay device for monitoring the effect of anticoagulant therapy at the point of care. The device is a disposable, laminated polymer microfluidic strip fabricated from a combination of hydrophobic and hydrophilic cyclic polyolefins to allow reagent deposition in addition to effective capillary fill. Zeonor was the polymer of choice resulting in low background fluorescence (208.5 AU), suitable contact angles (17.5 AE 0.9 ) and capillary fill times (20.3 AE 2.1 s). The device was capable of measuring unfractionated heparin and tinzaparin from 0-0.8 U ml À1 and enoxaparin from 0-0.6 U ml À1 with average CVs < 10%. A linear correlation was observed between the device and the fluorescent assay in the plate for plasma samples spiked with UFH, with an R 2 value of 0.99, while correlations with tinzaparin and enoxaparin resulted in sigmoidal responses (R 2 ¼ 0.99). Plasma samples containing UFH resulted in a linear correlation between the device and a standard chromogenic assay with an R 2 value of 0.98, with both LMWHs resulting in sigmoidal relationships (R 2 ¼ 0.99).

A fully integrated microfluidic device for point of care monitoring of antithrombotics

“…The adaptation of many of these assays to POC devices was instigated by the significant need for more rapid analysis of coagulation status in emergency room settings. 16 While these devices are commonly used and there is a demand for them, there is certainly scope for the development of novel devices that could improve upon conventional testing methods. The first move away from standard clot-based testing began in the 1960s with the development and introduction of peptide substrates specific for coagulation proteins such as thrombin and plasmin.…”

“…The results of this study have been published previously. 16 In the second stage of development, the aim was to develop a fully integrated device with all reagents incorporated on chip.…”

“…We have developed a three layered hybrid chip, comprised of native hydrophobic Zeonor®, treated hydrophilic Zeonor® and a PSA spacer that forms the channel. 16 The assay is incorporated onto the chip and works on the basis of the anti-Xa principle outlined above. Using fluorescence as the method of detection, the assay is capable of measuring therapeutic concentrations of heparin in calibration plasmas from 0-1 U/mL and in plasma samples from patients on heparin therapy.…”

Low-cost origami fabrication of 3D self-aligned hybrid microfluidic structures

“…In addition to this, it is a chemically resistant and malleable material, and it can be chemically functionalised to perform surface assays (Gandhiraman et al 2010). Moreover, this thermoplastic polymer has low degree of impurities, becoming a biocompatible material for biological, medical and environmental applications (RW.ERROR -Unable to find reference:138, BenitoLopez et al 2014, Fuchiwaki and Nagai 2014, Harris et al 2013, Moral-Vico et al 2015, Tachibana et al 2015.…”

“…A large number of papers can be found in literature explaining the fabrication of COP microfluidic devices where PSA was used for bonding or sealing de device in applications such as the development of a device for monitoring anticoagulants at the point of care (POC) (Harris et al 2013) or in the fabrication of optofluidic valves (Garcia-Cordero et al 2010). Nevertheless in all their fabrication protocols the different layers of COP and PSA were xurographied individually and stuck together manually or through a mold before bonding.…”