Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs

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Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs Bincy Jose, Peter McCluskey, Niamh Gilmartin, Martin Somers, Dermot Kenny, Antonio Joseph Ricco, Nigel J. Kent, and Lourdes Basabe-Desmonts Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03540 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on March 2, 2016

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Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs Bincy Jose,a Peter McCluskey,a Niamh Gilmartin,a Martin Somers,a Dermot Kenny,b Antonio J. Ricco,*,a Nigel J. Kent,c Lourdes Basabe-Desmonts*,a,d,e a b

Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Ireland

Biomedical Diagnostics Institute (BDI), Royal College of Surgeons in Ireland, Dublin, Ireland c

d

Dublin Institute of Technology, Dublin, Ireland

BIOMICs Research Group, Lascaray Ikergunea Research Center, Univ. Basque Country, Euskal Herriko Unibertsitatea UPV EHU, Vitoria, Spain e

Basque Foundation of Science, IKERBASQUE, Bilbao, Spain

ABSTRACT' We report the development of a microfluidic device for the rapid assay in whole blood of interfacial platelet-protein interactions indicative of the efficacy of antiplatelet drugs—e.g., aspirin and Plavix, two of the world’s most widely used drugs—in patients with cardiovascular disease (CVD). Because platelet adhesion to surface-confined protein matrices is an interfacial phenomenon modulated by fluid shear rates at the blood/protein interface, and because such binding is a better indicator of platelet function than platelet self-aggregation, we designed, fabricated, and characterized the performance of a family of disposable, self-powered microfluidic chips with well-defined flow and interfacial shear rates suitable for small blood volumes (≤ 200 µL). This work demonstrates that accurate quantification of cell adhesion to protein matrices, an important interfacial biological phenomenon, can be used as a powerful diagnostic tool in those with CVD, the world’s leading cause of death. To enable such measurements, we developed a simple technique to fabricate single-use self-powered chips incorporating shear control, “SpearChips”. These parallel-plate flow devices integrate on-chip vacuum-driven blood flow, using a pre-degassed elastomer component to obviate active pumping, with microcontact-printed arrays of 6-µm-diameter fluorescently-labeled fibrinogen dots on a poly(cycloolefin) base plate as a means to quantitatively count platelet-protein binding events. The use of SpearChips to assess in whole blood samples the effects of GPIIb/IIIa and 1" " ACS Paragon Plus Environment

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P2Y12 inhibitors—two important classes of “antiplatelet” drugs—is reported.

INTRODUCTION" The continuing revolution in microtechnologies, combined with unprecedented control of thinfilm (bio)materials properties, is spurring the development of biomedical diagnostic tests that are easy to use, require only compact, comparatively simple instrumentation to operate, and provide answers in a short time period, often at the medical point of care (POC).1 Such tests, most of which depend critically upon interfacial phenomena, have major implications for improved accuracy and efficiency in the diagnosis and monitoring of patients, and are beginning to spur the development of more efficient personalized therapies as well. We are designing and developing a range of POC-appropriate devices, a number of them focused on diagnosis, treatment, and monitoring of cardiovascular disease (CVD) and the drugs that treat it. Antiplatelet drugs such as aspirin and P2Y12 inhibitors (clopidogrel (Plavix), prasugrel, and ticagrelor) reduce cardiovascular events in patients with established CVD.2 Recent advances in platelet function testing have identified patients who respond suboptimally to both types of drug,3,4 yet the efficacy of these drugs is not routinely monitored, demonstrating a need for an easy-to-use test to directly assess drug effect upon platelet function in each patient. Such a test would facilitate clinical decisions in support of personalized treatments that could more successfully reduce the number of adverse events. Currently-available POC systems for platelet function using whole blood, including ICHOR-Plateletworks® and Accumetrix VerifyNow®, require large sample volumes and/or complex cartridges and have not proven to predict adverse cardiovascular outcomes; both of these assays monitor platelet function indirectly by measuring platelet aggregation,5,6 rather than directly by characterizing platelet-protein interactions. Because the interfacial process of platelet adhesion to a protein matrix is an early step in the complex platelet physiological response, variants of which lead to hemostasis as well as arterial occlusion, the direct evaluation of platelet-protein adhesion may be preferable to measuring platelet aggregation as a means to evaluate platelet function.5,7 Within living organisms with circulatory systems, fluid shear forces often play critical roles in interfacial phenomena, including cell-protein binding; some biological interactions at interfaces occur only over a specific range of 2" " ACS Paragon Plus Environment

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shear rates.

Having tools to control shear rate at a well-defined interface that accurately

simulates the relevant physiological interface is therefore essential in order to understand and apply fundamental interfacial science to biomedical measurements, including those that underlie diagnostic devices. We recently reported a rapid, accurate methodology to quantify platelet adhesion to protein matrices:8 a printed microarray of 6-µm protein dots patterned on < 1 mm2 of transparent substrate binds platelets from whole blood, one platelet per dot. After a defined interaction time, some number of individual fibrinogen dots are occupied by individual platelets, the dot array occupancy (DAO) being a direct measure of platelet adhesion. This individual platelet assay (iPA) offers the basis for a new type of POC platelet function test: its size and simplicity make the iPA a strong candidate for integration in a disposable microfluidic device to measure platelet adhesion to protein matrices,9 providing information about platelet function in a matter of minutes. Such a test would enable not only personalized monitoring of the effect of the drugs taken by CVD patients, but it would also facilitate a study of the efficacy of platelet adhesion inhibitors as a component of large clinical studies. The development of a disposable, inexpensive, low-sample-volume microfluidic platform to measure platelet adhesion presents several challenges given the fluidic characteristics of whole blood and the details of the adhesion process of platelets to surface-confined proteins: first, platelet adhesion to protein matrices is modulated by fluid shear rate;1,10 second, the ideal microfluidic platform should provide a flow of blood from a single sample-loading step without user intervention. The original iPA utilized a so-called rocking table (near-zero interfacial shear) to bring platelets and proteins into contact, requiring large volumes of blood (~ 1 mL) sloshing in petri dishes in a laboratory setting. The near-zero shear condition limited studies to those proteins and platelet receptors that interact at low shear rates. We therefore set out to implement the iPA measurement in a more physiologically realistic environment, wherein shear rates are controlled and can be higher when appropriate; with application to POC diagnostics in mind, we also sought to automate the analytical process and to reduce significantly the required volume of blood. Our goal was a simple tool to routinely measure platelet adhesion to a variety of proteins, including those interacting at both low and 3" " ACS Paragon Plus Environment

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high interfacial shear rates. Control of shear rate requires combined control of flow velocity and the appropriate geometry in the device’s microfluidic channel(s). One approach to achieve this is implemented by the standard laboratory tool to measure shear-mediated platelet-surface interactions under defined flow conditions, the parallel-plate flow (PPF) chamber. The PPF chamber produces near-constant shear force over an area sufficient to image multiple cell-protein interaction events. This constant shear at the interface of the flowing sample and the bottom surface of its flow path is obtained in part by using a high-aspect-ratio channel (cross sectional height-to-width ratio of 1:30).11 In contrast to ideal POC devices, however, the conventional PPF setup utilizes tubing and a syringe pump to control flow, and it often uses many milliliters of blood per assay,11,12 resulting in a bulky and costly measurement process that requires specialized personnel—i.e. a phlebotomist. In order to eliminate the need for active pumps and simplify operation, we recently developed a self-powered microfluidic platform for whole-blood analysis using degas-driven flow (DDF).13 DDF platforms incorporate a quantity of previously vacuum-degassed elastomer, typically poly(dimethylsiloxane) (PDMS).14,15

This elastomer gradually reabsorbs air upon

removal from vacuum—e.g., by tearing open a vacuum package in which it has been stored— thus providing vacuum-based pumping for up to tens of minutes.13,16-20 Flow rates in DDF devices depend, among many other parameters, on the total surface area of the PDMS microchannel walls responsible for air flow.18,21 Thus, for a given cross-sectional area, a longer microchannel typically induces more rapid flow due to a higher pressure differential (better vacuum) pulling the fluid forward.

In previous reports, we showed for the first time the

possibility of controlling the flow rate of liquids inside DDF devices by controlling the chip design.21 To date, this extraordinary feature of DDF devices, self-powered flow-rate control, has been little investigated, nor has it been applied to the development of application-specific devices.15 Herein we demonstrate how to apply the design and control of a dynamic biological interfacial process to the development of a potential POC system via the implementation of new disposable “self pumping” microfluidic platelet function testing devices. These SpearChips build 4" " ACS Paragon Plus Environment

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on our published iPA by adding and integrating (a) autonomy from external pumping as well as flow-rate-control capabilities, both conferred by degas-driven flow; (b) low-aspect-ratio-channel PPF chambers that enable unidirectional defined-shear-rate flow using ~2 – 6 drops (100 – 200 µL) of whole blood; (c) patterns of protein dots, the platelet occupancy of which is read out using our DAO approach, that are microcontact printed on a poly(cycloolefin) plate that forms the base of the microfluidic device. SpearChips enable rapid evaluation of an interfacial process, namely cell adhesion to a protein matrix under flow, which is a biological interfacial phenomenon of broad general relevance.

EXPERIMENTAL'DETAILS' Methods'and'Materials. All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise indicated. 4.5 mL Vacutainers® including dried 3.2% sodium citrate were purchased from Becton Dickenson UK Limited (Oxford, UK); adenosine-5´-diphosphate (ADP) was obtained from BioData (Horsham, PA, USA); abciximab (ReoPro) was purchased from Eli Lilly and Co. (Indianapolis, IN, USA); cangrelor was from The Medicines Company (Parsippany, NJ, USA). Device' Fabrication. Poly(dimethylsiloxane) (Dow Corning Sylgard 184) was purchased from Farnell (Farnell Ireland, Dublin, Ireland). Poly(cycloolefin) (PCO) sheets, 150 µm thick, were obtained from Ibidi GmbH (Munich, Germany); 1.5 mm-thick poly(methylmethacrylate) (PMMA) was purchased from Radionics (Ireland); 50 µm- double-sided pressure-sensitive adhesive (PSA) laminate, Ar-CARE®92712, were purchased from Adhesives Research (Limerick Ireland). Human fibrinogen (95% clottable and plasminogen depleted) was purchased from Calbiochem (Merck, KGaA, Darmstadt, Germany) and Cy3-labeled bovine serum albumin (BSA) was purchased from ChemQuest Limited (Wilmslow, UK). Protein' Patterning' on' Poly(cycloolefin)' Sheet. 3-Aminopropyltriethoxysilane (APTES)functionalized PCO sheets were cut to size to serve as SpearChip base plates (75 mm x 25 mm, 150 µm thickness). A 200 µg/mL fibrinogen solution was prepared in 1x phosphate-buffered saline (PBS) containing Cy3-labeled BSA (25 µg/mL) to aid in the visualization of the fibrinogen 5" " ACS Paragon Plus Environment

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dots on the slide, and either drop coated or microcontact printed onto the slide surface. 50 µL of the fibrinogen solution was drop-coated onto a PCO device base plate and the remaining solution was removed from the sheet after incubation at room temperature for one hour. The fibrinogencoated slide was then “blocked” (i.e., any regions of the surface susceptible to protein adsorption were covered) by immersion in 1% (w/v) BSA solution in PBS for 60 min at least at room temperature.

The fibrinogen solution was patterned as dots on the slide surface using

microcontact printing as described previously.8 Briefly, patterned PDMS stamps were fabricated by pouring a 10:1 (v/v) mixture of Sylgard 184 elastomer and curing agent over a patterned silicon-wafer master. After curing for 1 hr in an oven at 70 ºC, the PDMS was removed from the master and the PDMS stamps were “inked” with 50 µL of the fibrinogen solution for 30 min. The excess fibrinogen solution was then removed from the PDMS stamp using a pipette and the stamp was dried with flowing nitrogen. The stamp was then placed in contact with the APTEStreated PCO sheet for 5 min. After printing, the entire sheet was blocked with 1% BSA in PBS for 60 min at least. Fabrication'of'SpearChips The chip outline, designed in AutoCAD, was cut into a 50-µmthick sheet of double-sided PSA (protected by liner films on both sides) using a vinyl cutter (Graphtec GB Ltd., Wrexham, UK). One of the protective liners was then removed and the PSA placed in a plastic petri dish that served as the mold for curing the PDMS that forms the top of the microfluidic network (see Supporting Information (SI) for details). The petri dish and PSA surfaces do not adhere well to PDMS, obviating the need for hydrophobic pretreatment of the mold. After curing, the PDMS slab is taken apart form the PSA-glass mold and assembled onto a thin (150 µm) PCO substrate, which provides leak-free sealing to the PDMS without irreversible bonding; it also provides excellent clarity, biocompatibility, and low autofluorescence. Note that the sealing of the PDMS chip to the plastic substrate is very important: any significant leak in the sealing interface will reduce or in the worst case eliminate the DDF. SpearChips should be used within 3 minutes of removal from a vacuum container or package, a limitation that requires appropriate design of the assay process sequence. Blood'Collection'and'Sample'Preparation. Venous blood was collected from healthy adult volunteers who had not ingested any drugs known to interfere with platelet function for at least 2 6" " ACS Paragon Plus Environment

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weeks prior to phlebotomy. Blood was collected by venipuncture through a 19-gauge needle into a 4.5-mL Vacutainer® including 3.2% (v/v) sodium citrate. The citrated whole blood was either loaded directly onto the device or incubated with anti-platelet drugs. The binding of platelets to fibrinogen was prevented, when desired, by incubating various concentrations (0, 2, 2.5, 3, 4, 5, and 6 µg/mL) of abciximab with the citrated blood for 15 min at 37 °C before loading onto the device. In order to assess the effect of anti-platelet drugs, citrated whole blood was incubated with a final concentration of 10 µM cangrelor for 15 min at 37 °C. To modulate the extent of platelet-fibrinogen binding, the agonist ADP was added to a final concentration of 20 µM to the cangrelor-treated and untreated whole blood prior to loading onto the device. Platelet'Adhesion'to'ProteinHFunctionalized,'PreHDegassed'SpearChips. Protein-patterned SpearChips were prepared for use by placing them in an evacuated container (pressure < 1 kPa) for at least 60 min to evacuate the air and water vapor dissolved in the PDMS slab. The time between removal of the chip from vacuum and sample introduction was always 1 minute to ensure reproducible, maximum flow. 100 – 200 µL of blood was loaded by pipet into the inlet well of the SpearChip (Figure 1), effectively blocking the flow path between the network of microchannels and external air, the consequence of which is the gradual development of vacuum inside the microchannels as the air they contain is absorbed by the previously evacuated PDMS. This reduced pressure (which is insufficient to cause any noticeable deflection or deformation of the PDMS microchannels within the slab) draws the blood over the protein interaction region and towards the DDF pumping zone (Fig. 1). After the appropriate flow time to enable platelet binding (see Results and Discussion), the PCO bottom layer was removed and washed with PBS and the captured platelets fixed with 3.7% (v/v) paraformaldehyde (PFA) in PBS. The fixed platelets were labelled with anti-CD41 (1 µg/mL in PBS containing 1% BSA, anti-CD41 mouse monoclonal antibody (clone P2) was obtained from Immunotech, Marseilles, France) for 1 h at room temperature. After washing with PBS, the platelets were then labelled with goat anti-mouse Alexa Fluor 488-labelled antibody (4 µg/mL in PBS containing 1% BSA; antibody obtained from Invitrogen, Carlsbad, CA, USA) or with goat anti-mouse fluorescein isothiocyanate (FITC)-labelled antibody (4 µg/mL in PBS containing 1% BSA, antibody was obtained from Sigma-Aldrich, St Louis, MO, USA ) for 30 7" " ACS Paragon Plus Environment

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min at room temperature in the dark. After washing with PBS and deionized H2O the platelets were imaged. Fluorescence microscopy images were obtained using an inverted microscope (Olympus IX81) equipped with a CCD camera (Hamamatsu C4742-80-12AG) and a xenon lamp as light source. Images were collected with a 20X objective (excitation filter BP492/18; emission light was collected through a filter cube, U-MF2, Olympus). The surface coverage of Alexa Fluor 488-labeled platelets (see above for labeling details) was calculated (following blood contact with the surface as described in the Results and Discussion), in the case of the entire surface of the device having been coated with fibrinogen, using software written in MATLAB 2008b (Mathworks, Natick, MA, USA) based upon fluorescence intensity of platelets relative to background.

Software was written in C++ to calculate the percentage occupancy when

fibrinogen was microcontact printed on the surface of the device. An image of the protein array in each frame’s Cy3 channel (fibrinogen on the surface) was compared to a corresponding image of adhered platelets in the Alexa 488 channel (labeled platelets on the surface). Approximately 1000 fibrinogen dots were evaluated for each sample and, by determining the presence or absence at each dot of a platelet, the percentage of occupied dots was calculated. Shear' rate' calculations. The platelet binding to fibrinogen described above requires movement of blood across the protein surface at relatively low shear rates.7,8 In fact, our research shows that the precise shear rate is not critical; rather, it is the presence of sufficient blood movement to prevent red cells from accumulating by sedimentation on the surface and thereby blocking platelet access, together with red cell / platelet collisions that promote platelet surface adhesion: our studies show far lower rates of platelet adhesion when either (a) blood flow is negligible, allowing red cells to sediment or (b) red cells are absent (as in platelet-rich plasma). Therefore, although homogeneous flow of a Newtonian fluid would be necessary to calculate shear rate precisely13 with Equation (1), we use it nonetheless to make a rough approximation of shear rate at the device surface (!): ! =!

!! ! !! !

,

(1)

where Q is the volumetric flow rate, a is the channel height, and ! is the channel width. Higher 8" " ACS Paragon Plus Environment

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fidelity is not critical, as the focus here is on understanding if shear rates within an approximate range relevant to platelet adhesion can be deliberately modulated in self-powered microfluidic devices. Accordingly, the approximate shear rate in the degas-driven-flow device (SpearChip) was estimated using images acquired using software developed within the Microsoft .NET framework and the DirectX framework using C++. A Microsoft LifeCam Studio 1080p webcam was positioned above the chip to acquire images of fluid flow. Once a sequence of frames (images) had been acquired, they were individually masked and a binary matrix was created, wherein zero represents a single pixel of background and one a single pixel of fluid. By subtracting the timestamped matrices for time t = n from t = n – 1, the number of pixels the fluid moved, in the direction of flow, was determined from frame to frame. Using the known channel height and width (specified for each SpearChip design in the Results and Discussion section), this number of pixels was converted to volumetric flow rate (!), which was used in Eq. 1 to estimate shear rate.

RESULTS'AND'DISCUSSION' The design of the SpearChips, Figure 1, implements high-aspect-ratio channels that enable shear control by eliminating edge effects, typical of PPF chambers used for platelet studies. SpearChips comprise a PDMS slab with PSA-defined microchannels and a thin (150 µm) bottom transparent substrate of PCO. SpearChips have two distinct zones: a single 1.5-mm-wide channel with 1:30 aspect ratio (channel height of 50 µm) runs from the inlet to just beyond the analysis zone; this channel then divides into multiple parallel channels that constitute the DDF pumping zone. In human blood circulation, platelets undergo a wide range of shear rates from as low as 100 s-1 in venous circulation to over 2000 s-1 in the arterial circulation (and higher still near partial arterial occlusions).22 Platelet adhesion to protein matrices is a shear-dependent process: platelet transient adhesion/translocation to/on von Willebrand factor occurs primarily in the upper range of shear rates > 800 s-1, while platelet static adhesion to fibrinogen occurs to the greatest extent at shear rates bellow 300 s-1.23 Based on our previous experience with DDF devices,13,21 9" " ACS Paragon Plus Environment

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the first generation of SpearChips (“SpearChip_Gen1”) were designed with a target shear rate of less than 300 s-1 in order to focus on platelet adhesion to a fibrinogen matrix.

Figure 1. (A) Photos of a SpearChip loaded with blood showing self-powered flow within the device. (a) Blood loaded in the inlet well flows first across the analysis zone, then (b) enters the DDF pumping zone, flowing towards the ends of the parallel channels. The analysis zone comprises a channel of aspect ratio 1:30 (1.5 mm width, 50 µm height) and the DDF pumping zone comprises multiple parallel microchannels (1 mm width, 50 µm height) that are “self evacuating” due to the PDMS having been previously degassed. (B) Schematic showing the fabrication of a SpearChip, which includes a PDMS slab with drafted microchannels and a 150 µm-thick bottom transparent substrate of PCO, typically functionalized with a protein matrix at the position of the analysis zone and BSA blocked elsewhere. A family of SpearChip_Gen1 devices was fabricated to implement a range of PDMS areas 10" " ACS Paragon Plus Environment

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to drive DDF by varying the number of channels in the DDF pumping zone: 2, 4, 6, and 8 pumping channels (all 50 µm x 1.0 mm) provided 190, 294, 403, and 503 µm2 of PDMS area exposed to the microfluidic network of the chip (denoted Chip 1, 2, 3, and 4, respectively, in Figure 2). To initiate DDF, the SpearChip_Gen1 devices were placed in a vacuum container for at least 60 min to evacuate the air dissolved in the PDMS slab. The time between removal of the chip from vacuum and sample introduction (“idle time”) was kept at 1 minute to ensure reproducible, maximum flow.

(Hosokawa et al. suggest that idle times < 5 min provide

maximum and reproducible flow.16)

A video clip showing self-powered fluid flow in the

SpearChip_Gen1 is provided in the SI, Video 1. The volumetric flow rate (!) was calculated by imaging the progress of the fluid front over 7 min of whole blood flow in the SpearChip_Gen1 devices using a webcam positioned above the chip. Once the volumetric flow rate was determined, the wall shear rate (!) was estimated from Eq. 1, with the caveats regarding accuracy noted in the Experimental section. As Figure 2 shows, each chip design induces a different flow rate; the shear rates in these devices change with time in a reproducible and predictable manner. Importantly, all four chips have the same channel width and depth in the region where analysis is conducted (prior to branching), meaning that a and b in Eq. 1 remain constant and only the surface area of the channel pumping zone varies.

While this strategy is effective in determining the design

parameters needed for a desired interfacial shear rate, the relationship between pumping zone surface area and flow rate is not analytically predetermined, hence the need to characterize several different pumping zone surface areas. As expected, the chip with the largest PDMS area, Chip 4, containing 8 channels in the pumping zone, provided the highest flow rates and therefore the highest shear rates (Eq. 1). The maximum initial shear rate of ~120 s-1 gradually diminished to 65 s-1 after 7 min. A similar trend in the time-dependent decrease of flow and calculated shear rates is observed in the other three designs (Chips 1, 2, 3). The flow rates ranged from 120 nL/s (Chip 4, t = 0) to 10 nL/s (Chip 1, t = 7 min). Inter-chip (n = 3) reproducibility was good, with average standard deviations less than 10% of the median value. A video clip showing filling of the three different chip designs is provided in the SI, Video 2. "

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Figure 2. (A) SpearChip_Gen1 designs and dimensions; devices have a range of microchannel surface areas as noted. Increasing the number of channels in the DDF zone increases the total area of PDMS channel walls responsible for the in-channel vacuum that drives fluid flow. (B) Flow rates versus time for whole blood flowing in the SpearChip_Gen1 devices. (C) Shear rates obtained using the various SpearChip_Gen1 designs. Error bars represent ±1 standard deviation for measurements from 3 chips of each design (where the error bars are not visible, twice the standard deviation is less than the size of the symbol).

PlateletHprotein' adhesion' and' assay' of' drug' efficacy' using' SpearChips. To evaluate platelet adhesion to protein matrices in SpearChips, fibrinogen was drop-coated onto the bottom PCO substrates in the analysis zones (Figure 1B) of the SpearChip_Gen1 designs denoted Chips 1 – 4 (Figure 2). After degassing the chips, 100 µL of whole blood were loaded at each inlet; the blood flowed spontaneously through the analysis zone towards the DDF zone. After 1, 3, 5, 7, and 10 minutes of flow, the chips where disassembled and washed with PBS; platelets were then fixed with 3.7% paraformaldehyde (PFA) in PBS and stained with a fluorescent probe. Fluorescence microscopy images were collected with a 20x objective for each disassembled chip; the percentage platelet surface coverage was used as an indication of platelet adhesion to the fibrinogen surface. Figure 3 shows the time-dependent binding curves of platelets adhering to the surface. The majority of adhesion occurs after 200 s, with increasing numbers of platelets adhering over time for all four SpearChip_Gen1 designs. Platelet adhesion rate increased from Chip 1 to Chip 4 (Figure 3): the highest binding rate was obtained for the chip with the highest range of shear rates. This difference in shear-rate behavior among the 4 chips provides important fundamental insight into the shear-dependent adhesion of platelets on a contiguous fibrinogen surface: it shows that a range of shear rates below 60 s-1 provide very similar, low adhesion rates. To promote more effective interfacial interaction with the fibrinogen surface and thereby obtain higher adhesion rates, higher shear rates are needed. To evaluate the use of SpearChips as monitors of antiplatelet drug efficacy, the effect of the GPIIb/IIIa inhibitor abciximab was first characterized via platelet binding to a fibrinogen matrix 13" " ACS Paragon Plus Environment

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on SpearChip_Gen1 chips. Abciximab (ReoPro) is normally administered intravenously to CVD patients as a bolus after a percutaneous intervention. It is a monoclonal antibody that blocks the GPIIb/IIIa receptor, which binds fibrinogen to platelets. The Chip 4 design was chosen because it induced the highest platelet adhesion rate for the time of analysis. The experiment was run in triplicate with blood from three healthy donors; for each, three sets of five chips were loaded with whole blood aliquots pre-incubated with increasing concentrations of abciximab, from 0 to 6 µg/mL. Platelet adhesion was measured as described above onto the drop-coated fibrinogen in the analysis zone.

Figure 3. (A) Platelet adhesion to drop-coated fibrinogen SpearChip_Gen1 Chip 4, depicted by fluorescence images at 20x magnification, showing adherent FITC-labeled platelets after 1, 3, and 5 min of whole blood flow. (B) Time-dependent adhesion of platelets on drop-coated fibrinogen SpearChip_Gen1, Chips 1 to 4. Error bars represent ± 1 standard deviation of three replicates. 14" " ACS Paragon Plus Environment

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Figure 4 shows that for all three donors, platelet adhesion decreased with increasing abciximab concentration; complete inhibition was achieved at 4 µg/mL. The results also show a degree of natural variability among the three donors, despite which all three demonstrate total inhibition of binding beginning at the same concentration of abciximab.

Figure 4.

Platelet adhesion to drop-coated fibrinogen on SpearChip_Gen1 Chip 4 after 7

minutes of flow. Whole blood samples from 3 donors were pre-incubated with a range of concentrations of abciximab, as indicated on the abscissa. Error bars represent ± 1 standard deviation of three replicates per concentration for each donor. Based on the results of the preliminary study presented in Figures 3 and 4, we sought to demonstrate an assay of the efficacy of oral antiplatelet drugs using the iPA-SpearChip by integrating our previously developed iPA8 system onto the PCO substrate of the SpearChips via microcontact printing an array of 6-µm-diameter fibrinogen dots. The adhesion of platelets to the on-chip protein array was characterized using untreated whole blood. No adhesion of platelets was observed on SpearChip_Gen1 devices when the homogeneous matrix of fibrinogen was replaced by the 6-µm fibrinogen dot array. We believe this is because, during the process of adhesion of platelets to a protein surface, the platelet on and off rates are 15" " ACS Paragon Plus Environment

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influenced, among other factors, by the probability of a platelet encountering an unoccupied binding site and by the forces “dragging” adhered platelets off the substrate after initial adhesion to the protein surface (platelets are typically bound by multiple receptor-protein bonds and therefore are likely to be more susceptible to removal soon after initial adhesion, when the number of bonds is smaller). The first factor is related to the percentage of the substrate surface covered by the binding protein—fibrinogen in this case—while the dragging force is related to the interfacial shear forces generated by the flow inside the microchannel at the surface. For the same flow rate, the probability of platelets encountering fibrinogen-coated areas in a given time was much lower for the dot array (which covers only 28% of the available surface) than for a homogenously coated surface, where the effective density of platelet binding sites is the 100 % of the surface. Based on these results, the SpearChip was redesigned specifically for this measurement. Increasing the flow rate should increase the number of platelet-protein collisions per unit time, increasing the on-rate and, presumably, leading to more adhesion events. On the other hand, keeping the same microchannel dimensions and increasing the flow rate would also lead to a higher shear force at the surface, with a tendency to increase the off rate. Thus, our goal was to maintain a shear rate similar to the SpearChip_Gen1 devices despite an increase in blood flow rate. The new design, iPA-SpearChip, therefore includes a greater microchannel surface area in the DDF zone to produce a higher flow rate, and a higher aspect ratio in the flow path of the analysis zone in order to maintain a similar shear rate to the previous generation, Figure 5A. Four different chips were chips were fabricated with increasing widths of the flow path in the analysis zone. All the chips had the same DDF zone design, nine channels 2 mm in width and 50 µm high. Only the region around the analysis zone differed between Chips 5 – 8. Figure 5 shows the iPA-SpearChip design and flow-rate characterization. The flow rates generated in these chips were similar to one another because the design of the DDF zone was the same for all four devices. In contrast, the changes in aspect ratio of the flow path over the analysis area affected the shear stress generated at the surface of each chip in that region, with the highest aspect ratio (Chip 8) resulting in the lowest shear rate. See SI, Video 3 to see whole 16" " ACS Paragon Plus Environment

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blood flowing into three iPA-SpearChip devices.


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Figure 5. (A) Design and dimensions of iPA-SpearChip devices. All chips had similar total areas of the PDMS DDF microchannels. (B) Flow rates versus time of whole blood flowing in the iPA-SpearChip devices. (C) Shear rates versus time of whole blood flowing in the iPASpearChip devices, calculated in the analysis zone of each chip.

Next, the iPA-SpearChip family was evaluated as a monitoring tool for the efficacy of various clinical P2Y12 inhibitors, which include clopidogrel (Plavix®), prasugrel, ticagrelor, and cangrelor. Because clopidogrel is a pro-drug that must be converted by metabolism to the active drug while cangrelor is administered in its active form, the latter was chosen for initial in-vitro studies. ADP induces platelet aggregation mainly by activating P2Y12 receptors.

Abciximab

inhibits ADP-induced platelet aggregation (Figure 6). We have previously shown that individual platelets bind singly to individual 6-µm fibrinogen dots, the dot array occupancy (DAO) being a direct measure of platelet adhesion (Figure 6A). Additionally, platelet aggregates do not adhere to the 6-µm fibrinogen dots.8,9 It should therefore be possible to monitor the effect of cangrelor by measuring platelet adhesion on the iPA-SpearChip. Whole blood samples of 200 µL, pre-incubated with buffer, or 20 µM ADP, or 20 µM ADP and 10 µM cangrelor, were introduced to iPA-SpearChips (Chip 8 design) and flowed across the 6-µm fibrinogen dot arrays for nine minutes. A significant decrease of DAO was observed when the blood was pre-treated with ADP (DAO = 7%) (due to formation of platelet aggregates decreasing the availability of single platelets to bind to the dots) compared to untreated whole blood (DAO = 59%); this effect was largely negated by the addition of cangrelor to the ADPtreated blood (DAO = 50%). These results are promising for the application of iPA-SpearChips to asses the efficacy of P2Y12 inhibitors.


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Figure 6. (A) Cartoon showing the integration of the iPA assay on a SpearChip to form an iPASpearChip. A fibrinogen dot array on the PCO bottom plate of the chip is used as protein matrix. Whole blood is loaded through the inlet of the device and flows via DDF over the fibrinogen micropattern.

Individual fibrinogen dots are occupied by individual platelets; dot array

occupancy is a direct measurement of platelet adhesion statistics. (B) Schematic representation of the P2Y12 assay. ADP-induced aggregation of platelets results in a lower adhesion rate, but if the ADP effect is mitigated by a P2Y12 inhibitor, the level of platelet adhesion is similar to normal, even in the presence of ADP. (C) Fluorescence images at 20x magnification of 6-µm 19" " ACS Paragon Plus Environment

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Cy-3-labeled fibrinogen-dot arrays (red dots) on the surface of the iPA-SpearChip (Chip 8 design) and of FITC-labeled platelets (green dots) adhering to the fibrinogen dot arrays after 9 min of flow of the sample across the protein pattern; prior to introduction to the chip, the whole blood sample was incubated with (i) buffer, (ii) 20 µM ADP (“Cangrelor (–)”), or (iii) 20 µM ADP plus 10 µM cangrelor (“Cangrelor (+)”). White bars indicate 50 µm in all images. (D) DAO values indicative of the effects of ADP and ADP + cangrelor measured with an iPASpearChip. Error bars represent one standard deviation for three separate blood draws in each case.

SUMMARY'and'CONCLUSIONS' Inspired by the parallel-plate flow chamber, and with the ambition of simplifying our assay setup to make an accessible tool to measure interfacial platelet adhesion, we developed the SpearChip concept, an integrated combination of interfacial platelet assay, dot array occupancy, and degasdriven flow. We thoroughly explored flow control on DDF devices and determined in detail for the first time how to adjust the surface area of the PDMS microchannels in the pumping zone, while keeping the critical dimensions of the cell-protein interaction channel constant, to set the flow rate in DDF devices in order to provide a desired interfacial fluid shear rate at the measurement

location.

Previously-reported

DDF

devices,

fabricated

with

classical

photolithography techniques and based upon channels of small cross-sectional area (0.0025 to 0.025 mm2) and low aspect ratio (1:1 to 1:4)13,16-18,24 display low flow rates that would not provide the appropriate range of fluid shear rates to allow binding of sufficient numbers of platelets in a matter of minutes to obtain meaningful counting statistics.

In contrast, our

SpearChips incorporate microchannels of 0.075 mm2 cross section and 1:30 aspect ratio, enabling larger sample volumes at ~ten times higher flow rates than previously reported devices; they also provide a uniform shear rate free of edge effects over a convenient measurement area. Despite the higher flow rate, 100 – 200 µL of blood (~2 – 6 drops) is sufficient for an assay. Control of chip design parameters, channel aspect ratios, and the total volume and exposed surface area of degassed PDMS enable the manipulation of both shear forces at the channel walls and the total flow rate, something unprecedented in self-powered microfluidics. Such control enabled us to demonstrate for the first time an assay of the efficacy of antiplatelet 20" " ACS Paragon Plus Environment

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drugs as measured by interfacial platelet adhesion, a shear-dependent process carried out on single-use “self-powered” (assuming use of standard vacuum packaging) disposable devices. SpearChips were used to monitor the effect of abciximab, a GPIIb/IIIa inhibitor, on whole blood samples from healthy donors. Interestingly, the results showed noticeable though not unlikely differences among three healthy donors, demonstrating the importance of personalized monitoring of drug effects in order to prevent adverse occlusive or bleeding events. Integration of our clinically proven iPA assay with the SpearChip enabled quantitative assay of the effect of a P2Y12 inhibitor, a class of drug widely administered to CVD patients. A limitation of the current methodology for the development of SpearChips is the fact that the flow rate is intimately linked to the chip design. To avoid this disadvantage, i.e. to separate design for the desired assay from the means for providing the appropriate flow and shear rates, we are currently developing a modular approach, using detachable and tunable DDF PDMS micropumps.

Nonetheless, the methodology described here provides application-specific

bioassay devices, advantageous for reproducibility in high-volume applications. The straightforward fabrication techniques presented here for self-powered microfluidic devices with integral protein dot arrays should be accessible to many laboratories: devices can be kept in vacuum packaging (or a vacuum desiccator) prior to use, and they could be used for a variety of cell-protein interfacial binding/adhesion studies. We believe that the combination of degas-driven flow, microfluidic design, and an integrated “digital adhesion” assay will pave the way for the development of enhanced biomedical devices that enable rapid analysis at the point of care.

ASSOCIATED'CONTENT' Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR'INFORMATION' 21" " ACS Paragon Plus Environment

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Corresponding Authors *[email protected]; [email protected] Notes The experimental work reported here was carried out at the Biomedical Diagnostics Institute, Dublin, Ireland. !

ACKNOWLEDGMENTS' The material reported in this paper is based upon works supported by the Science Foundation Ireland, Grant No.10/CE/B1821. We gratefully acknowledge helpful discussions with Prof. Luke P. Lee of the University of California at Berkeley.


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A"

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B"

C"


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PDMS area 2 (mm )

X (mm)

iPA-SpearChip Design No.

4 5

Chip 5 Chip 6

1379 1432

6

Chip 7

1478

7

Chip 8

1530

!


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Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs

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