OA18581A - Microfluid chip-based, universal coagulation assay. - Google Patents

Abstract

A microfluidic, chip-based assay device has been developed for measuring physical properties of an analyte (particularly, whole blood or whole blood derivatives). The technologies can be applied to measure clotting times of whole blood or blood derivatives, determine the effects of anticoagulant drugs on the kinetics of clotting/coagulation, as well as evaluate the effect of anticoagulant reversal agents. These technologies can additionally be used to optimize the dosage of anticoagulation drugs and/or their reversal agents. The assay is independent of the presence of anticoagulant; clotting is activated by exposure of the blood sample in the device to a glass (or other negatively charged material such as oxidized silicon) surface, which activates the intrinsic pathway and can be further hastened by the application of shear flow across the activating materials surface. The absence of chemical activating agents and highly controlled and reproducible micro-environment yields a point of care universal clotting assay.

Description

MICROFLUIDIC CHIP-BASED, UNIVERSAL COAGULATION ASSAY

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of and priority to U.S. Provisional Application No. 62/048,183, filed September 9,2014, ail of which is Incorporated by reference in its entirety.

FIELD OF THE INVENTION

The présent invention relates to a point of care microfluidic chip-based universal coagulation assay device and reader that can be used to measure the global coagulation status of normal healthy, coagulation Impaired, anticoagulated, and anticoagulant reversed patients.

BACKGROUND OF THE INVENTION

Coagulation (clotting) is the process by which blood changes from a liquid to a gel. It potentlally results In hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The mechanism of coagulation involves activation, adhesion, and aggregation of platelets along with conversion of fibrinogen to fibrin, which deposits and matures into a robust network. Disorders of coagulation are disease states which can resuit in bleeding or obstructive clotting (thrombosis).

Coagulation begins very quickly after an Injury to the blood vessel has damaged the endothélium linlng the vessel. Exposure of blood to the space under the endothélium initiâtes two categories of processes: changes in platelets, and the exposure of subendothilial tissue factor to plasma Factor VII, which ultimately leads to fibrin formation. Platelets immediately form a plug at the site of injury; this Is called primary hemostasis. Secondary hemostasis occurs simultaneously: additional coagulation factors or clotting factors beyond Factor VII, respond in a complex cascade to form fibrin strands, which strengthen the platelet plug.

The coagulation cascade of secondary hemostasis has two pathways which lead to fibrin formation. These are the contact activation pathway (also known as the Intrinslc pathway), and the tissue factor pathway (also known as the extrinsic pathway). It was previously thought that the coagulation cascade consisted of two pathways of equal importance jolned to a common pathway. It Is now known that the primary pathway for the initiation of blood coagulation is the tissue factor pathway. The pathways are a sériés of reactions, in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein co-factor are actlvated to become active components that then catalyze the next reaction In the cascade, ultimately resulting in cross-linked fibrin. Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form.

The coagulation factors are generally serine proteases which act by cleaving downstream proteins. There are some exceptions. For example, FVIII and FV are glycoprotéine, and Factor XIII Is a transglutaminase. The coagulation factors circulate as Inactive zymogens. The coagulation cascade Is classlcaily divided Into three pathways. The tissue factor and contact activation pathways both activate the final common pathway of factor X, thrombin and fibrin.

Tissue factor pathway (extrinsic)

The main rôle of the tissue factor pathway is to generate a thrombin burst, a process by which thrombin, the most important constituent of the coagulation cascade in terms of its feedback activation rôles, is reieased very rapidly. FVIIa circulâtes in a higher amount than any other activated coagulation factor. The process includes the following steps:

Following damage to the biood vessel, FVIi leaves the circulation and cornes into contact with tissue factor (TF) expressed on tissue-factor-bearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVlia).

TF-FVila activâtes FIX and FX.

FVII is itself activated by thrombin, FXIa, FXII and FXa.

The activation of FX (to form FXa) by TF-FVila Is almost Immediately inhibited by tissue factor pathway inhibitor (TFPi).

FXa and its co-factor FVa form the prothrombinase complex, which activâtes prothrombin to thrombin.

Thrombin then activâtes other components of the coagulation cascade, including FV and FVIII (which activâtes FXI, which, in tum, activâtes FIX), and activâtes and releases FVili from being bound to vWF.

FVilla is the co-factor of FIXa, and together they form the tenase compiex, which activâtes FX; and so the cycle continues. (Tenase is a contraction of ten and the suffîx -ase used for enzymes.)

Contact activation pathway (intrinsic)

The contact activation pathway begins with formation of the primary complex on collagen by high-molecuiar-weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor). Prekallikrein Is converted to kallikrein and FXII becomes FXila. FXIIa converts FXI into FXIa. Factor Xia activâtes FIX, which with its co-factor FVIila form the tenase complex, which activâtes FX to FXa. The minor rôle that the contact activation pathway has in initiating clôt formation can be Illustrated by the fact that patients with severe deficiencies of FXII, HMWK, and prekallikrein do not hâve a bleeding disorder. Instead, contact activation system seems to be more involved in inflammation.

Coagulation Assays

Several techniques, Including clot-based tests, chromogenic or color assays, direct chemical measurements, and ELISAs, are used for coagulation testing. Of these techniques, clot-based and chromogenic assays are used most often. Whereas clotting assays provide a global assessment of coagulation function, chromogenic tests are designed to measure the level or function of spécifie factors.

Clot-based assays are often used for évaluation of patients with suspected bleeding abnormalities and to monitor anticoagulant therapy. Most of these tests use citrated plasma, which requires tens of minutes for préparation and typically requires hours to days to recelve results In a hospital setting. The end point for most clotting assays Is fibrin clôt formation.

Prothrombln Time (PT) Is performed by adding a thromboplastin reagent that contains tissue factor (which can be recombinant In origln or derived from an extract of brain, lung, or placenta) and calcium to plasma and measuring the clotting time. The PT varies with reagent and coagulometer but typically ranges between 10 and 14 seconds. The PT Is prolonged with deficiencies of factors Vil, X, and V, prothrombln, or fibrinogen and by antibodies directed against these factors. This test also is abnormal In patients with inhibitors of the fibrinogen-tofibrin reaction, including high doses of heparin and the presence of fibrin dégradation products. Typically, PT reagents contain excess phospholipid so that nonspecific inhibitors (ie, lupus anticoagulants), which react with anionic phospholiplds, do not prolong the clotting time. The PT Is most frequently used to monitor warfarin therapy. PT measurements are not comparable between devices or centers and most warfarin clînics develop their own normal patient range, which Is non-transferrable and highly spécifie to the exact reagents présent In the spécifie assay used.

The activated Partial Thromboplastin Time (aPTT) assay is performed by first adding a surface actîvator (eg, kaolin, celite, ellagic acid, or silica) and diluted phospholipid (eg, cephalin) to citrated plasma. At the point of care, aPTT can also be measured in whole blood typically using similar chemical activating agents. The phospholipid in this assay is called partial thromboplastin because tissue factor Is absent. After incubation to allow optimal activation of contact factors (factor XII, factor XI, prekallikrein, and high-molecular-weight kininogen), calcium Is then added, and the clotting time Is measured. aPTT measurements are not comparable between devices or hospltals and most clinical laboratories develop their own normal patient range, which is non-transferrable and highly spécifie to the exact reagents présent in the spécifie assay used.

Although the clotting time varies according to the reagent and coagulometer used, the aPTT typically ranges between 22 and 40 seconds. The aPTT may be prolonged with deficiencies of contact factors: factors IX, VIII, X, or V; prothrombln; or fibrinogen. Spécifie factor Inhlbitors, as well as nonspecîfic Inhîbîtors, may also prolong the aPTT. Fibrin dégradation products and anticoagulants (eg, heparin, direct thrombln Inhlbitors, or warfarin) also prolong the aPTT, although the aPTT Is less sensitive to warfarin than Is the PT.

The thrombin clotting time (TOT) Is performed by adding excess thrombln to plasma. The TCT Is prolonged In patients with low fibrinogen levels or dysfibrinogénémie and in those with elevated fibrin dégradation product levels. These abnormalities are commonly seen with disseminated Intravascular coagulation. The TCT is also prolonged by heparin and direct thrombin inhlbitors.

The activated clotting time (ACT) is a point-of-care whoie-blood clotting test used to monitor high-dose heparin therapy or treatment with bivalirudin. The dose of heparin or bivalirudin required in these settings Is beyond the range that can be measured with the aPTT. Typically, whole blood Is collected into a tube or cartridge containing a coagulation activator (eg, celite, kaolin, or glass particles) and a magnetic stir bar, and the time taken for the blood to clôt is then measured. The reference value for the ACT ranges between 70 and 180 seconds. The désirable range for anticoagulation dépends on the Indication and the test method used. The ACT does not correlate well with other coagulation tests.

For the ecarin clotting time (ECT), venom from the Echis carinatus snake is used to convert prothrombln to meïzothrombln, a prothrombln intermediate that is sensitive to inhibition by direct thrombin Inhibitors. The ECT cannot be used to detect states of disturbed coagulation and Is useful only for therapeutic drug monitoring. This assay is insensitive to heparin because steric hindrance prevents the heparin-antithrombln complex from ïnhibîting meïzothrombin. Because ecarin also activâtes the noncarboxylated prothrombin found in plasma of warfarin-treated patients, leveis of direct thrombin inhibitors can be assayed even with concomitant warfarin treatment. Although the ECT has been used in preclinical research, the test has yet to be standardized and is not widely available.

Anti-factor Xa assays are used to measure levels of heparin and low-molecular-weight heparin (LMWH). These are chromogenic assays that use a factor Xa substrate onto which a chromophore has been linked. Factor Xa cleaves the chromogenic substrate, releasing a colored compound that can be detected with a spectrophotometer and is directly proportional to the amount of factor Xa présent. When a known amount of factor Xa Is added to plasma containing heparin (or LMWH), the heparin enhances factor Xa inhibition by antithrombin rendering less factor Xa available to cleave the substrate. By correlating this resuit with a standard curve produced with known amounts of heparin, we can calculate the heparin concentration In the plasma. The use of anti-Xa assays requlres the knowledge of which anticoagulant the patient Istaking In order to use the appropriate calibrator and cannot be used to monitor anti-lla anticoagulant thérapies.

Anticoagulant drugs ln clinical use include warfarin, heparins (unfractionated heparin and LMWH), and direct thrombin inhibitors (bivalirudin, hirudin, and argatroban).

Warfarin is effective for primary and secondary prévention of venous thromboembolism; for prévention of cardioembolic events In patients with atrial fibrillation or prosthetic heart valves; for prévention of stroke, récurrent infarction, or cardiovascular death ln patients with acute myocardial infarction: and for the primary prévention of acute myocardial Infarction in high-risk men. Because of the variability In the anticoagulant response to warfarin, which reflects genetic variations in metabolism and environmental factors such as médications, diet, and concomitant Illness, regular coagulation monitoring and dosage adjustment are required to maintain the International Normalized Ratio (INR) within the therapeutic range. Heparins are Indirect anticoagulants that activate antithrombin and promote its capacity to inactivate thrombin and factor Xa. To cataiyze thrombin Inhibition, heparin binds both to antithrombin via a high-affinity pentasaccharide sequence and to thrombin. in contrast, to promote factor Xa inhibition, heparin needs only to bind to antithrombin via its pentasaccharide sequence. Heparin molécules containing <18 saccharide units are too short to bind to both thrombin and antithrombin and therefore cannot cataiyze thrombin inhibition. However, these shorter heparin fragments can cataiyze factor Xa inhibition, provided that they contaîn the pentasaccharide sequence. The anticoagulant response to heparin is unpredictable because of variable nonspecific binding to endothélial cells, monocytes, and plasma proteins. Because of this variable anticoagulant response, coagulation monitoring is routinely performed when heparin is glven in greater than prophylactic doses. The aPTT is the test most often used to monitor heparin. Unfortunately, aPTT reagents vary in their responsiveness to heparin, and the aPTT therapeutic range différa, depending on the sensitivity of the reagent and the coaguiometer used for the test. The aPTT has proved more difficult to standardîze than the PT, and the commonly quoted therapeutic range of 1.5 to 2.5 times the control value often leads to systematic administration of subtherapeutic heparin doses. The evidence supporting the concept of an aPTT therapeutic range that predicts efficacy and safety (with respect to bleeding) is somewhat tenuous. Approximately 25% of patients require doses of heparin of >35 000 U/d to obtain a therapeutic aPTT and are cailed heparin résistant. Most of these patients hâve therapeutic heparin levels when measured with the anti-Xa assay, and the discrepancy between the 2 tests is the resuit of high concentrations of procoagulants such as fibrinogen and factor VIII, which shorten the aPTT. Although the aPTT response is linear with heparin levels within the therapeutic range, the aPTT becomes immeasurabie with higher heparin doses. Thus, a less sensitive test of global anticoagulation such as the ACT Is used to monitor the level of anticoagulation In patients undergoing percutaneous coronary interventions or aortocoronary bypass surgery.

LMWH is derived from unfractionated heparin by chemicai or enzymatic depolymerization. LMWH has gradually replaced heparin for most indications. LMWH is typicalîy administered in fixed doses when given for prophylactic purposes or in weight-adjusted doses when given for treatment. Pitfalls in the monîtoring of LMWH by anti-factor Xa levels include poor comparability between commercially avaîlabie anti-Xa chromogenic assays, différences in ratios of anti-Xa to anti-lla among the various LMWH préparations, and the importance of timing of blood sampling in relation to dosing. Although the aPTT may be prolonged with high doses of LMWH, this assay is not used for monîtoring. No clinicalîy avaîlabie point of care assay to date Is avaîlabie for the monîtoring of the millions of patients administered LMWH.

Direct thrombîn inhibitors bind dîrectiy to thrombin and block the interaction of thrombin with its substrates. Three parentéral direct thrombin inhibitors hâve been licensed for limited indications in North America. Hirudin and argatroban are approved for treatment of patients with heparin-induced thrombocytopenia, whereas bivalirudin is licensed as an alternative to heparin in patients undergoing percutaneous coronary intervention (PCI). Hirudin and argatroban requîre routine monîtoring. The TCT is too sensitive to small amounts of hirudin and argatroban to be used for this purpose. Although the ACT has been used to monitor the higher doses of direct thrombin inhibitors required in interventional settings, it does not provide an optimal linear response at high concentrations. The aPTT is recommended for therapeutic monîtoring; however, each direct thrombin inhibitor has its own dose response, and the sensitivity of the test to drug levels varies between aPTT reagents. When hirudin therapy is monitored wîth the aPTT, the dose is adjusted to maîntain an aPTT that is 1.5 to 2.5 times the control, whereas for argatroban, the target aPTTis 1.5 to 3 times control (but not to exceed 100 seconds). The aPTT appears less usefui in patients requiring higher doses of direct thrombin Inhibitor in cardiopuimonary bypass procedures because this test becomes less responsive at increasing drug concentrations. The ECT appears to be usefui for both low and high concentrations of direct thrombin inhibitors and Is less affected by interfering substances than the aPTT. However, as stated above, it is not routinely avaîlabie. The responsiveness of the INR to different drug concentrations differs with assay reagent and with the type of direct thrombin inhibitor. This feature compiicates the transitioning of patients with heparin-induced thrombocytopenia from argatroban to vitamin K antagonists.

As is clear from the foregoing, clotting, and inhibition of clotting, Is a complex process. The type of anticoagulant can give mlsleading and dangerous results if determined using the wrong clotting assay. This créâtes a potentially disastrous scénario when an anticoagulated patient arrives at an emergency room without information as to medicines he Is on, as well as the condition being treated. Sometimes It Is Impossible to wait for further diagnostics to détermine the anticoagulant or disorder causing prolonged bleeding. The need for a rapid, accurate, and universal test for clotting, especially a point of care (“POC) test, Is well known; options, however, are extremely limited.

It is therefore an object of the présent invention to provide a rapid, accurate and universal test for clotting.

it is a further object of the présent invention to provide a point of care test for clotting.

It Is a stili further object of the présent Invention to provide a test that Is accurate, reproduclble, easy to operate, and requires a very small amount of sample.

SUMMARY OF THE INVENTION

A microfluidic, chip-based assay device has been developed for measuring physical properties of an analyte (in particular, whole blood or whoie blood dérivatives). The technologies can in particular be applied to measure clotting times of whole blood or blood dérivatives, to détermine the effects of anticoagulant drugs on the kinetics of clotting/coagulation, as well as to evaluate the effect of anticoagulant reversai agents. These technologies can additionally be used to optimize the dosage of anticoaguiation drugs and/or their reversai agents. The assay Is independent of the presence of anticoagulant since clotting is activated by exposure of the blood sample In the device to a glass (or other negativeiy charged material such as oxidized silicon) surface, which activâtes the Intrinsic pathway and can be further hastened by the application of shear flow across the activating materials surface. The absence of chemical activating agents and highly controlled and reproducible microenvironment yieids a point of care universal clotting assay.

The sample is handled In a microfluidic system. The volume of sampie introduced into the testing chamber Is In the nano-, micro-, or milliiiter range (most preferably 1-10 microliters). The sample Is Introduced into the collection well directly from a blood sample or the Individuel (such as from a finger stick), in one embodiment, the sample, such as blood or plasma, is collected and transferred to the heated microdevice immediately after collection by syringe into a no-additive red-topped tube or capillary tube. Clotting in the samples, preferably In duplicate, Is initiated by exposure of the blood or plasma to the glass surface within the device and the sampie exposed to means for analysis of clotting. The blood sample is then drawn from the collection well into the testing chamber either passively by capillary action or with a pump, which induces sheer and exposes the blood sample to the activating materials surface, while deiivering a geometricaliy controlled amount of the blood sample into the testing chamber, The microfluidic system along with Integrated électrodes, heater structures or other parts of sensors or actuators ls deslgned to be disposable. The microfluidic System is Inserted Into an analytical re-usable housing (referred to herein as a reader) that is part of an analysis instrument, which connecte the microfluidic System and provides fluidic, electrical, optical and thermal interfaces for measuring clotting and transmïtting the time and characteristics of the measurement to an extemal reader, monitor, or recorder.

Clotting Is assessed by a change ln viscosity, optical transmission, electrical Impédance, and/or pressure. In the preferred embodiment, clotting Is detected through measurement of blood Impédance through integrated électrodes and/or through measurement of optical transmission using Inffared (IR) LEDs and photodiodes, respectively. An integrated thermal résistive heater/cooler structure such as a solid state heat pump or Peltier cooler keeps the blood sample at a defined température, most preferably approximately 37”C (body température), and ensures repeatability and comparability of measurements. As fibrinogen converts to fibrln, the iR absorbance increases until It peaks In a measurable fashlon. The détermination of whole blood clotting time Is made on or about the peak of IR absorbance. After completion of measurements, the microfluidic chip containing the blood sample is removed from the reader and discarded.

The development of clotting can be monitored by measuring electrical impédance. The development of fibrin Increases electrical Impédance until it peaks in a measurable fashlon. On or about the peak of electrical Impédance, the détermination of whole blood clotting time Is made. Whole blood clotting time can be measured by IR absorption and electrical impédance measured alone or simultaneously as a basis of comparison. The electrical impédance and IR absorption curves are essentially coïncident, and provide confirmation via Independent measurement modes.

The microfluidic System Is fabricated through application of mïcrotechnologies and processing and bonding of wafers made of silicon, glass or other suitable materials. The microfluidic System can also be fabricated through alternative means, for example, through application of soft lithography technologies or through génération of reader parts (made, for example, of plastic or a different suitable, substantially IR-opaque material) that may be used alone or In combination with mlcropattemed chips to form a microfluidic System. The microfluidic System typically consiste of an Inlet, an outlet and one or more chambers that are connected through channels, which range In length from tens of microns to millimeters, with heights and depths In the tens of microns to hundreds of microns range.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 ta a schematic of the coagulation cascade.

Figure 2A is a cross-sectional view of one embodiment of the microfluidic device in which blood is pipetted into one chamber, and the chip Is then Inserted Into packaging.

Figure 2B Is a cross-sectional view of an embodiment of the microfluidic device where blood can be introduced through chamber side; sipper sticks out of reader, similar to blood glucose measurement

Figure 3A is a view of the open version of the two sample chamber devices.

Figure 3B is a view of the closed of the two sample chamber devices. Figures 4A-4H are views of the chambers showing the chambers, connecting channels, electrical contact pads, and thermistors.

Figure 5 Is a perspective view from the top of the bottom of the closed version reader.

Figure 6 is a cross-sectional view of the side of the bottom of the closed version reader.

Figure 7 is a perspective cross-sectional view of the top of the closed version reader showing the sample assaying device in place.

Figure 8 Is a perspective cross-sectional view from the top of the top of the closed version reader.

Figure 9A is a perspective cross-sectional view from the top of the bottom part of the open version reader.

Figure 9B is a perspective cross-sectional view from the side of the bottom part of the open version reader.

Figure 10A Is a perspective cross-sectional view from the side and bottom of the top of the open version reader.

Figure 10B is a perspective cross-sectional view from the side and top of the top of the open version reader.

Figure 11 is a schematic of the system, showing a box containing the single use disposable assay chambers, the reader, and the connections to a computer processor and monitor.

Figures 12A-12F are graphs of the impédance (12A, 12C, and 12E) and infrared transmission (12B, 12D, 12F) over time in minutes for control no anticoagulant, 300 ng edoxaban anticoagulant/mi blood, and 300 ng edoxaban and clraparantag (PER977; an anticoagulant reversai agentymi of blood, respectively.

DETAILED DESCRIPTION OF THE INVENTION

I. Définitions

Microfluldics

Microfluidics Is a highly interdisciplinary field, drawing from engineering, physics, chemistry, biochemlstry, mlcro-/nanotechnology, and biotechnology. Smali volumes of liquid, ranging from femto- to mllliliters are typically handled In a microfluidic System. The methods for fabrication of a microfluidic System typically allow for Intégration of sensors and/or actuators, so that liqulds can be effectively transported, manipulated and analyzed Inside the microfluidic System. Interfaces between the microfluidic System and Its environment enable implémentation of extemai mechanisms for transport, manipulation and analysis.

Micro-/Nanotechnolog!es

Micro’/Nanotechnoiogies are typically used to fabricate mlcrosystems, Including microfluidic Systems. Micro-/nanotechnologles typically enable génération of structures with dimensions in the micro - or nanometer range. Such technologies can be based on silicon wafer processing technologies, orlglnaliy developed for fabrication of Integrated electronic circuits.

Capillary action

A convenient way of loading a liquid sample Into a microfluidic System Is by capillary action. A sample collection port Is wetted with the sample and the sample Is effectively drawn into the narrow, hydrophiitc channels and chambers of the microfluidic System by capillary forces.

Anticoagulant

An anticoagulant Is a substance that interfères with the ability of blood to dot. Administered as a therapeutic drug, an anticoagulant can, for example, help reduce or prevent the occurrence of potentially health- and/or life-threatening emboli or thrombl.

Anticoagulant reversai agent

An anticoagulant reversai agent can be administered as a therapeutic drug in order to reverse partially or fuliy the effect of an anticoagulant. Restoration of the capacity of the blood to clôt can be iife-saving, for example, a patient who takes an anticoagulant and is experiencing a severe injury can be treated with a reversai reagent for restoration of blood ciotting capacity and prévention of excessive blood loss.

Open” and Closed” Devlces

An “open’ device has a channei to the outslde from an Interior chamber, allowing for direct access of blood into the chamber. A closed device has no exterior channels, and is filled before being sealed to the outside except for small hoies associated with sensors, électrodes, LEDs, and other éléments utilized In assessing ciotting within the chamber. The 'open version’ of the microfluidic System Is designed so that the chip can be Inserted Into its packaging first, be heated up, but still be accessible (open). A sample can be loaded Into the System through wetting of the side port, with the chip residing already in its packaging. The sample will be drawn into the chip by capillary forces.

The ’closed version’ of the mlcrofluldic System wîil not be accessible to the user after It is placed into its packaging. A sample has to be loaded into the System prior to placement of the chip into its packaging through wetting of or pipetting into one of the back side ports.

II. Device

Embodiments of the microfluidic chip and its reader are shown In Figures 2-10, with the System including both parts reflected by Figure 11. As indicated in Figures 2A and 2B, microfluidic chips 10 are fabricated through anisotropic wet etching of silicon wafers and subséquent thermal oxidation, isotropie wet etching of PYREX® (a clear, low-thermalexpansion borosilicate glass) wafers, sputter déposition of thln métal films onto both wafers through stencils, anodic bonding of silicon and PYREX® wafers, and subséquent séparation of single chips by wafer dicing.

The microfluidic chips can be fabricated by alternative means, using any method that is suited to generate microfluidic structures and any negatively charged material that is suited to activate the blood clottîng cascade.

The cross-sectional dimensions and geometries of the chambers 12a and 12b and the channel 14 connecting the chambers can be modified, and the number of chambers can be varied. The surface to volume ratio will overall influence the clottîng time. Access to the chambers is direct (“closed*, Figure 2B), prior to sealing of the chamber device, or via a side channel 16 that allows access from the exterior of the microchambers.

As shown by Figure 3A and 3B, the chips are designed to enable:

- Heating through backside résistance or electricai heater structure (on underside of chambers 12a and 12b)

- Température measurement for heating control through top side (outer) thermistor 18

- Clottîng détection through air pressure measurements

- Clottîng détection through measurement of Impédance across blood sample through embedded électrodes 20a and contact pads 20b

- Clottîng détection through optlcal measurements

As shown in Figures 4A-4H, résistance structures 22 are deposited onto the back of a siiicon wafer 24 (to form résistive heating structures 32, Figures 4G, 4H), onto the front side 26 of the silicon wafer (to form électrodes 20a, 20b for impédance measurements and thermistors 18, respectiveiy, at the floor of each chamber), and onto the front side of the PYREX® wafer (to form thermistors 18 on top of one or both chambers 12a, 12b). The heater and the thermistor can be external to the ‘chip, and can be integrated into the reader structure. If the walls of the cavity Into which the chip Is placed are of significantly greater mass than the chip, hîghly thermally conductive, and form an almost complété surround, then the cavity approxlmates a “black body and the chip must corne to thermal equilibrium with the cavity. If the chip Is in contact with the cavity, or closely spaced, the equilibrium time constant can be very short. This can be established by pyrometer measurement during development. This should reduce the complexity of the disposable part of the system, and the cost. Connecting channel 14 and entry port 36 can be etched using potassium hydroxide (KOH) into the silicon wafer 26.

The device in Figure 2B, 3B (called ‘closed device') has two entry ports 28a, 28b etched through the silicon part 30. A sample can be pipetted Into one of the ports 28a, 28b before the device 10 is Inserted Into a sealed packaging. The device In Figures 2a, 3b has one entry port 28b etched through the silicon part and one sideway entry port 16, realized as channel etched into the PYREX® (called 'open device'). In the cases of a silicon substrate, the surface wave structure can be directly integrated into the microfluidic design by weil understood microfabrication techniques. An open device chip (Figures 2A, 3A) can be Inserted into a sealed packaging and/or reader first, with the edge with the sideway port sticking out of the reader. Wetting of the sideway port 16 wiil then resuit in sample being drawn into the chamber 12a by capillary action.

The combined heater/cooler control system and the thermistor can be extemal to the 'chip', and can be integrated Into the reader structure. If the walls of the cavity, into which the chip is place, are of significantly greater mass than the chip, highly thermally conductive, and form an almost complété surround, then the cavity approximates a 'black body* and the chip must corne to thermal equilibrium with the cavity. If the chip is In contact with the cavity, or closely space, the equilibrium time constant can be very short. This can be established by pyrometer measurement during development. This would reduce the complexity of the disposable part of the system and (hopefully) the cost

Chip packages or‘readers’ as shown in Figures 5-10, provide electrical, optical and fluidic interfaces to the chip. A reader consiste of a bottom (Figures 5,6,9A, 9B) and a top (Figures 7, 8,10A, 10B) part that are manufactured by high précision 3D printing, molding, machining, or other fabrication processes. Both parts are joined and pressed against each other by locking métal dowel pins that fit into holes 56. The reader can include means for display, storage of Information, and a communications capability.

Figures 5 and 6 show the bottom reader part 50 for the closed device chip from two different angles. Channeis 52a, 52b, located In recess 54, inside the reader connect chambers 2 and 1, respectively, to the chip entry ports. On one side of the reader, a solenoid valve (not shown) can be attached to the reader at channel ends 58 to control the connection between the reader channel and a barbed tube connecter that Is screwed Into the bottom of the reader. On the opposing side, a pressure sensor (not shown) can be attached to the reader at holes 60 to monîtor the pressure appîied to the reader channel and chip entry port. Each entry port 62a, 62b is controlled/monitored by its own solenoid valve/pressure sensor secured at holes 70a, 70b, 70c, and 70d. The bottom reader part exhibits a recess 54 for the microfluidic chip. Small vertical holes 64a, 64b hold pogo pins to contact the heater structure at the bottom of the chip. A hole 66 at the center of the reader part holds an IR LED chip in a métal can reader, which passes along light path 68.

The IR components can be molded or integrated into a “chipstrate form.

Although shown as a single point optical measurement, a multipoint optical measurement could be used.

Figures 7 and 8 show the top reader part 80 for the closed device chip from two different angles. The large (for example, 5 mm) hole 82 in the center holds an IR photodiode chip (not shown) in a métal can reader that directs light through hole 86. Three small (for example, less than one mm) vertical holes 84 hold three pogo pins (not shown) to contact three électrodes for Impédance measurements (one common ground electrode for both chambers and one counter electrode in each chamber), so that impédance measurements can be carried out in both chambers. Two other vertical holes 88 hold pogo pins to contact the thermlstor on top of the chip. Other embodiments of these devices are known and readily available for the same fonction. IR LED and photodiode are placed so that they Interrogate the 1 mm diameter center région of one chamber.

The chip reader itself Is attached to the cover of a project box 100 that contains electronic circuitry, valves, and pumps needed to perform automated measurements, as shown In Figure 11. The project box 100 Is connected to a PC 102 where measurements are controlled by a LabView program and processor 110, and results shown on monltor 104.

Figures 9A and 9B show the bottom reader part 120 for the open device chip from two different angles. Dowel pîn holes 122 are used to secure the device. Channels 124 inside the reader connect chambers 2 and 1, respectively, to the chip entry ports. On one side of the reader, a solenoid valve (not shown) can be attached to the reader at channel ends to control the connection between the reader channel and a barbed tube connector that Is screwed Into the bottom of the reader. On the opposing side, a pressure sensor (not shown) can be attached to the reader at holes 126a, 126b to monltor the pressure applied to the reader channel and chip entry port. Each entry port Is controlled/monitored by Its own solenoid valve/pressure sensor secured at holes. The bottom reader part exhibits a recess 128 for the microfluidic chip. A hole 130 at the center of the reader part holds an IR LED chip in a métal can reader.

Figures 10A and 10B show the top reader part 140 for the open device chip from two different angles. The large (for example, 5 mm) hole 142 In the center holds an IR photodiode chip (not shown) In a métal can reader that directs light through hole 144. Three small (for example, less than one mm) vertical holes 146 hold three pogo pins (not shown) to contact three électrodes for impédance measurements (one common ground electrode for both chambers and one counter electrode in each chamber), so that impédance measurements can 5 be carried out in both chambers. Two other vertical holes 148 hold pogo pins to contact the thermlstor on top of the chip. IR LED and photodiode are placed so that they interrogate the 1 mm diameter center région of one chamber.

Although described with reference to Interrogation of both chambers with one beam, It Is possible to Interrogate both chambers using separate beams. One beam Is shown for

Illustration purposes only.

The chip reader is attached to the cover plate of a project box as described above using screw holes 150, as shown in Figure 11.

A. Surfaces for Activation of Blood Clotting

The mlcrofluldic system Is fabricated so that the Introduced blood or plasma sample Is In contact with a glass surface, the top part of the microfluidic chip and/or the surface of the bottom part of the microfluidic chip, formed of a material such as PYREX® or thermally oxidized silicon, such as amorphous SiO₂, silicon oxide, and silicon nitride. The glass serves to activate the clotting cascade without use of additional chemical or biological reagents. Activation Is either achieved through mere contact of the sample with the glass surface or through active movement (for example, through an extemally applied air pressure puise) of the sample along the glass surfaces inslde the microfluidic system.

The microfluidic system Is fabricated so that the Introduced blood or plasma sample Is in contact with glass surfaces (or other negatively charged surfaces), which serve to activate the clotting cascade without use of additional chemical or biological reagents. Glass surfaces are generated through use of glass wafers and oxidized silicon wafers, respectively, for fabrication of microfluidic Systems. Altematively, glass surfaces can be realized through use of glass chips that are Integrated In reader parts that form a microfluidic system, through déposition of glass onto the Inner surfaces of the microfluidic system (for example, through use of spin-on glass products) or through Intégration of small objects with glass surfaces (for example, glass microbeads) Inside the microfluidic system. Additionally glass surfaces can be Introduced by the oxidation of silicon surfaces.

B. Electrical Characteristics of Deposlted Métal Thln Films

Deposited métal thln films can be formed of chromlum adhesion layers (approximately 20 nm thick) and gold top layers (approximately 50 nm thick for inner électrodes for impédance measurements, 100 nm thick for thermistors and 150 nm thick for heater structures). Springloaded pogo pins in the plastic reader were used to realize electrical contacts to ali thin film électrodes on the microfluidic chip. Typical résistances between two pins connected to either end of a métal film test structure, with approximate iength of 2 mm and width of 1 mm, are 1.8 Ohm, with pins contacting métal films for heater structures, 2.7 Ohm, contacting métal films for thermistors, and 22 Ohm, contacting métal films for Inner électrodes for Impédance measurements. The markedly higher résistance between inner electrode pins Is llkely due to a thinner gold layer and possibly contact dégradation during anodic bonding at approximately 300’C. Heater structures and thermistors are deposited after anodic bonding.

For measurement of température coefficients of résistances of deposited métal thin films, a chip was used that exhibited resistor/thermlstor structures Instead of open circuit électrodes for impédance measurements. The chip was inserted Into Its reader and heated up inslde an oven. Electrical résistances of heater, thermistor and inner electrode reslstors were measured at different températures and température coefficients of résistances a were calculated:

- heater thln film:

- thermistors thln film:

S - Inner electrode thln film :

a =0.0016 K'¹ a =0.00115 K'¹ a = 0.000108 K'¹.

C. Internai Electrodes for Sample Posltloning

Apart from Impédance measurements, integrated électrodes can also be used to detect the presence of the analyte, such as fibrin, In the microfluidic system and/or to track movement of the analyte, for example, due to extemally applied air pressure puises. Such détection and 10 tracklng can be used to initiate the analysis procedure once the analyte is added to the microfluidic system, to position the analyte at a spécifie location within the microfluidic system, and to move it repeatedly back and forth between defined locations, respectively.

Although exemplified with référencé to two chambers and two électrodes, multiple électrodes can be used to confirm filling of multiple chambers, at the site of measurement of 15 clotting or at a point prior to the chamber where the clotting is measurement, such as doser to the lnlet.

Repeated movement of whole blood or blood plasma along glass surfaces can be applied to Increase activation of the blood, to accelerate blood clotting and/or to decrease measurement times.

D. Intégration of filter structures

Mechanical filter structures can be Integrated into the microfluidic chip, so that only blood plasma Is arriving at the analysis chambers. The filters can be realized as array of micropillars or as microchanneis etched Into silicon or glass. This way, plasma (without the use of an anticoagulant such as sodium citrate or EDTA) can be produced in situ and very quîckiy tested 25 In the same manner as whole blood, without red blood cell interférence In the analysis. The mlcropillar arrays can be arranged in offset patterns to inhibit red blood cell transit, whiie mlnlmlzing the probability of “plugging an excessive fraction of the available channei cross section.

E. Means for Clotting Détection

A variety of modalities can be applied to détermine blood clotting times.

Viscosity

The viscosity of the blood can serve as a measure to characterize clotting times. Two general principles may be applied to yield a direct or Indirect measure for the viscosity. The sample can be moved through a channel with known geometry. Viscosity can be measured by 35 tracklng the rate of pénétration through a long channel either optically or by imaglng or multiple beam “check points or electrically by multiple electrode Impédance sensors. The viscosity may be measured Indirectly, for example through measurement of the distance the sample has travelled Inslde a channel during a spécifie time Interval, the sample volume that has been displaced during a spécifie time Internai, or the change In driving force during a spécifie time Interval (for example, if the sample Is moved by a pressurized volume of trapped air, the change In air pressure can serve as an Indirect measure for sample volume displacement). Alternatively, objects can be moved through the blood sample, for example, driven through electrostatic or magnetic forces. Tracking of the object movement can yield an Indirect measure for the sample viscosity.

The viscosity of the analyte can be detected through movement of the analyte Inslde the microfluidic System through a pressurized, entrapped air volume. Air can be pressurized, for example, through electric air pumps that are connected to the microfluidic System. Pressurized air can be entrapped through closure of solenoid valves connected to the microfluidic System. Decreaslng pressure of the entrapped airat one entry port of the chip Indicates movement of the analyte. Knowledge of the geometry of the microfluidic system and the magnitude of the applied pressure allows calculation of analyte viscosity and détection of viscosity changes (for example, a viscosity increase due to clotting In case of blood).

Clôt détection by viscosity monitoring Involves measuring differentîal pressure across an on-chip Inlet and outlet, connected to fluîdic ports on the reader (made air/fluid tight using o-ring seals).

The viscosity of the analyte can be detected through movement of the analyte inside the microfluidic System through a pressurized, entrapped air volume. Air can be pressurized, for example, through electric air pumps that are connected to the microfluidic System. Pressurized air can be entrapped through closure of solenoid valves connected to the microfluidic system. Decreaslng pressure of the entrapped air at one entry port of the chip Indicates movement of the analyte. Knowledge of the geometry of the microfluidic system and the magnitude of the applied pressure allows calculation of analyte viscosity and détection of viscosity changes (for example, a viscosity Increase due to clotting In case of blood).

Impédance

Clotting of a sample can be related to Its electrical impédance, or Its complex résistance when an electric current or voltage Is applied. The electrical impédance results from ohmlc résistances as weli as capacitive components of the sample. The electrical Impédance can, for example, be measured through électrodes that are directly Integrated in the microfluidic chip or Integrated in the reader that forms, together with other parts, a microfluidic system. Electrodes can be partially In direct contact with the sample or separated from the sample only through a thin Insulator (with a thickness ranging from nanometers up to hundreds of mlcrometers).

Electrodes and conductor Unes may be formed as pattemed thin métal films that are deposited onto substrates forming the mlcrofluldic system. Electrodes and conductor lines can also be realized through intégration of pattemed métal sheets or films that are Inserted and sandwlched between reader parts, if semlconductor wafers are used to fabricate microfluidic chips, the semlconductor material itself can contain Integrated électrodes fabricated by diffusion, implantation, etching, micro machlnlng, or any combination of appropriate techniques similar to the techniques used in Integrated circuit or other micro device production. The Integrated électrodes can be used to measure directly electrical properties of the anaiyte (for example, résistance, capacitance, Impédance). Suitable electronlc circuits may also be used to translate anaiyte changes (and related changes In electrical properties) into measureable electric voltages, currents, frequencies or other suitable parameters. Parts can also be inserted during construction by 3D printing, or Integrated directly. For example, a resistor heating element would be fabricated by a doped channel In a semiconductor substrate (by Ion beam implantation, for example).

Clôt détection by impédance monitoring Is accomplished by inserting the chip Into the reader, maklng contact between gold pads (connected to the on-chip électrodes) and Pogo pins in the reader from which the electrical signal Is read by an LCR meter, for example, or any other appropriate measurement system.

For détection of clotting through measurement of Impédances, a sample Is loaded Into the chip, the chip Is Inserted into its reader, and the pogo pins connecting to the internai impédance électrodes connected via Kelvin clip leads to a QuadTech 1920 LCR meter. The magnitude and the phase of the complex impédance of the blood sample were recorded at 15 second intervals. Measurements at 100 Hz, 1kHz, 10 kHz, and 100 kHz showed characteristics peaks or plateaus of either the magnitude or the phase or both. The peaks or plateaus Indlcated a measure for the clotting time.

Referring te Figures 3A-3B, for impédance measurements, the extemal LCR meter applies an AC voltage (20 mV RMS) between the two électrodes 20b, 20a on each side of the chamber 12a, and measures the electrical current between the électrodes. The magnitude and phase of impédance are then computed and the clotting caiculated.

Optlcal Properties

Optical properties of the sample can be related te clotting events. Light with wavelength in the range of 500 nm te 10,000 nm (preferably 1,300 nm) can be used to llluminate the sample through the microfluidic and chip reader. The following parameters can be used to track clotting: transmitted, reflected and scattered light. If a cohérent light source Is used, polarizatlon may be used as additional parameter.

Clôt détection by IR transmission is performed by Inserting the chip Into the reader and measuring infrared transmission across the thîckness of the chip (through the glass, fluid-fill chamber, and underlying silicon). This is accomplished via placement of an IR source (LED) above the chip and photodiode detector aligned immediately below.

For optical clôt détection, IR LED and photodiode are inserted Into their reader parts as described above. A blood sample is pipetted into the chip and the chip placed Into the reader. The sample Is continuously llluminated with IR light at 1,300 nm. At time intervals of typically 100 ms, the voltage drop across a 1 MOhm resistor caused by the photocurrent of the photodiode was recorded. Voltages typically measured several volts. Clotting of the sample caused the transmitted light and the photocurrent to vary over time. Characteristic peaks of the transmitted light curve Indicated a measure for the clotting time.

The continuous Illumination measurement Is presented as a simple illustration. More sophlsticated measurement techniques may be used. For example, If the IR emitter were lliumlnated for 50% duration, at a répétition rate of 1 khz, and a synchronous detector were used to process the photodetector output, followed by a 1 second Intégration period, the signal to noise ratio In the above example could be Improved by as much as thirty-foid. In addition, active signal processing would allow processing of much smaller signais, permitting a relatively low Impédance termination of the photodetector, lowering the intrinsic noise, and canceiing drift. Ambient electrical noise sensitivity would be substantially reduced.

Acoustic propertîes

Measurement of Sound propagation In the sample or along the sample surface can serve as an additional measure for clotting. Extemal ultrasound transducers can be used to measure the time It takes ultrasound to travel through the sample. Additionaliy, surface acoustic wave devices can be used to measure acoustic propertîes of the sample and to detect clotting.

III. Methods of Making Devices

In addition to standard processes such as photolithography, spécial technologies such as anodic bonding or potassium hydroxide anisotropic wet etching of silicon wafers can be applied to form microsystems. Apart from standard ultraviolet light lithography, techniques such as direct laser writing microablation or érosion, électron beam lithography, or focused ion beam milling can be used to define micro- or nanometer-sized structures. Soft lithography Is a related way to fabricate mîcrofluidic Systems and Is based on génération of microstructures or -patterns, for example, through standard photolithography techniques, and subséquent use of these patterns In molding/casting processes. Elastomeric material such as polydlmethylsiloxane (PDMS) are typically used for génération of microfluidic system by soft lithography. Structured films generated by soft lithography can be attached to each other or to any other structured or non-structured substrate to form complex microfluidic Systems. Furthermore, other technologies such as drilling, miliing, molding, or 3D printing, may be used alone or in combination with other micro-/nanotechnologîes to fabricate microsystems.

IV. Sample Collection

Blood Collection

In most cases, Individuels to be tested will présent at a clinic or a hospital, posslbly with unknown status as to treatment with anticoagulants. Blood can be obtained by the use of a syringe, a lancelet, or directly from a blood containing line. Due to the use of the alternative clotting pathway In which clotting is activated using a glass type surface, the blood may contain anticoagulants such as warfarin, heparin, low molecular weight heparin, factor lia inhibitors, factor Xa inhibitors, and other factor inhibiting or factor impaired blood.

Warfarin and related 4-hydroxycoumarin-containing molécules decrease blood coagulation by inhibiting vitamin K epoxide reductase, an enzyme that recycles oxldized vitamin K, to its reduced form after it has participated in the carboxylation of several blood coagulation proteins, mainly prothrombin and factor VII. Warfarin does not antagonize the action of vitamin K_b but ratherantagonizes vitamin Ki recycling, depleting active vitamin K Thus, the pharmacologie action may always be reversed by fresh vitamin K. When administered, these drugs do not anticoagulate blood immediately. Instead, onset of their effect requires about a day before remaining active clotting factors hâve had time to naturally disappear in metabolism, and the duration of action of a single dose of warfarin is 2 to 5 days. Reversai of warfarin's effect when it is dîscontinued or vitamin Ki is administered, requires a similar time.

Heparin is a compound occurring in the liver and other tissues that inhibits blood coagulation. A suifur-containing polysaccharide, it is used as an anticoagulant in the treatment of thrombosis. Low molecular weight heparin, a more highly processed product, is useful as it does not require monitoring by aPTT coagulation parameter (it has more predictable plasma levels) and has fewer sîde effects. However, in emergency bîeeding situations the ability to monitor LMWH is a significant unmet clinical need as no point of care assay is clinically accepted for LMWH anticoagulant monitoring.

Drugs such as rivaroxaban, apixaban and edoxaban work by inhibiting factor Xa directly (unlike the heparins and fondaparinux, which work via antithrombin activation).

Another type of anticoagulant is the direct thrombin inhibitor. Current members of this class include the bivalent drugs hirudin, lepirudin, and bivalirudin; and the monovalent drugs argatroban and dabigatran.

The sample can be tested as blood or as plasma. Plasma can be prepared by filtration or centrifugation. Additionally, additional glass surface area can be added to one or more of the microfluidic channels by the introduction of glass beads Into the channel using for example a double depth chip or ln-channel bead packing.

Other Blologlcal Samples

The device can be used with other types of samples that are activated with exposure to glass.

V. Methods of Use

The samples are collected and administered into the device. The means for determining clotting are started as the sample Is placed into the device. Results are compared to standard results for uncoagulated samples, typically from pooled plasma or pooled blood, or by reference to the clotting time at Initiation of treatment, as in the case where an individual Is administered anticoagulant, or a therapeutic to neutralize the anticoagulant and restore more normal blood clotting.

The présent invention will be further understood by reference to the following nonlimiting examples.

Example 1. Démonstration of On-chip Heating

A chip was used to demonstrate the effect of the integrated heater (or integrated heater/cooler; preferably a solid state heat pump or ’Peltier cooler*) structure. A 12 V DC voltage was applied to the heater reslstor on the back of the sîlicon part of the microfluidic chip. Résistances of thermistors were measured inside each chamber (Inner thermlstors, on the front silicon surface) and on top of each chamber (outer thermistors, on top of the PYREX®) before application of a heater voltage and during heating. Local températures Increase due to heating were calculated using measured résistances and température coefficients of résistances as reported earlier. Room température was approximately 27’C. Average local température increases after approx. 2 min of unregulated heating were:

- outer thermistors: ΔΤ = 20.3 K

- inner thermistors: ΔΤ = 23.8 K.

Example 2: Measurement of Blood Clotting

Matériels and Methods

Blood was harvested from a patient using commercially available lancing devices. 10 pL of biood were obtained and pipetted into an Eppendorf tube. Saline was used as a buffer solution. The blood sample was mixed in the Eppendorf tube through up and down plpetting five times with one of the following reagents:

- 1 pL of buffer solution (called sham control),

- 1 pL of buffer solution containing the anticoagulant edoxaban at a concentration of

300 ng/mL,

- 1 pL of buffer solution containing the anticoagulant edoxaban and the anticoagulant reversai agent PER977, both at a concentration of 300 ng/mL.

Edoxaban is a commercially available anticoagulant. PER977 (ciraparantag) is an investîgational drug that Is designed to reverse the effect of edoxaban. Immediately after mixing, 2.5 pL of each blood sample was pipetted into a closed device chip. The chip was Inserted Into Its reader, and both IR light and impédance measurements were Immediately recorded at room température (approximately 27^eC). Heating of the blood sample were omitted.

Results were obtained both by IR and viscosity impédance.

Results

As évident from Figures 12A-12F, IR (Figures 12B, 12D, and 12F) and impédance measurements (12A, 12C, and 12E) correlate well with each other. Both measurements show for the sham control a characteristic peak around 2 minutes that Is indicative of the sample clotting time. Addition of the anticoagulant edoxaban shifts this peak to approximately 4 minutes. In addition to the peak, the edoxaban curves show a characteristic local minimum around 12 minutes. Addition of the anticoagulant reversai agent PER977 to a blood sample containing edoxaban shifts the peak In each curve back to 2 minutes and suppresses the occurrence of a local minimum around 12 minutes. These measurements Indicate the clottingdelaying effect of edoxaban and the reversai of this effect through additional administration of PER977.

Modifications and variations of the devices, Systems and methods of use thereof will be évident to those skilled in the art from the foregoing detailed description and are Intended to corne within the scope of the appended daims.

Claims (29)

1. A test microchip for measuring clotting in a blood or plasma sample, the test microchip comprising

An inlet for the blood or plasma sample, the inlet communicating with one or more microchannels having a length between tens of microns and

5 millimeters, each microchannel comprising one or more test chambers, each microchannel having a defined volume between nanoliters and milliliters , . the one or more microchannels each comprising at least one anionically charged surface which activâtes clotting of the blood or plasma sample upon entry of the blood or plasma sample into the 10 ' one or more microchannels or test chamber, wherein the test chamber is formed of a material which allows changes in viscosity, ’ impédance, acoustic properties, or optical properties in the blood or plasma in the test chamber, ¹ wherein the test microchip is insertable into a reader, the reader

15 comprising, a detector which détermines changes in viscosity, impédance, acoustic properties, or optical properties in the blood or plasma sample in a test chamber which are indicative of clotting, a température control regulating the température of the test

20 chamber, and output conveying the clotting time determined from the time of activation of the blood or plasma sample to the time of the change in viscosity, impédance, acoustic properties, or optical propertiesin the test chamber indicative of clotting.

2. The test microchip of claim 1 wherein the test microchip comprises a single microchannel for measuring clotting.

3. The test microchip of claim 1 wherein the test microchip is fabricated through anisotropic wet etching of silicon wafers and subséquent thermal oxidation, isotropie wet etching of dear, low-thermal-expansion borosilicate glass wafers, sputter déposition of thin métal films onto wafers through

JO stencils, anodic bonding of silicon and clear, low-thermalexpanslon borosilicate glass wafers, or germanium or gallium and subséquent séparation of single chips by wafer dicing.

4. The test microchip of claim 1 wherein the detector in the reader comprises an IR LED and photodiode for measuring optical changes in the test chamber of the test microchip.

5. The test microchip of claim 1 wherein the test microchip comprises électrodes or pattemed métal sheets or films.

6. The test microchip of claim 5 wherein comprising électrodes which can be used to directly measure résistance, capacitance, or impédance.

7. The test microchip of claim 1 wherein the microchip comprises a métal pattemed doped semiconductor substrate fabricated into a resistor heating element.

8. A microassay device for measuring clotting in a blood or plasma sample from an individual, the device comprising a test microchip, the test microchip comprising

An inlet for the blood or plasma sample, the inlet communicating with one or more microchannels having a length between tens of microns and millimeters, each microchannel comprising one or more test chambers, each microchannel having a defined volume between nanoliters and milliliters, the one or more microchannels each comprising at least one anionically charged surface which activâtes clotting of the blood or plasma sample upon entry of the blood or plasma sample into the one or more microchannels or test chamber, and the test chamber is formed of a material which allows changes in viscosity, impédance, acoustic properties, or optical properties in the blood or plasma in the test chamber, wherein the test microchip is inserted into a reader, the reader comprising a detector which détermines changes in viscosity, impédance, acoustic properties, or optical properties in the blood or plasma sample in the test chamber which are indicative of clotting, a température control regulating the température of the test chamber, and output conveying the ciotting time determined from the time of activation of the sample to the time of the change in viscosity, impédance, acoustic properties, or optical properties in the test chamber indicative of ciotting.

5

9. The device of claim 8 wherein the output from the reader communicates with a monitor or display or information storage.

10. A method for measuring ciotting time comprising introducing a blood or plasma sample into a test microchip inserted into a reader for measuring ciotting in a blood or 10 plasma sample, the test microchip comprising

An inlet for the blood or plasma sample, the inlet communicating with one or more microchannels having a length between tens of microns and millimeters, each microchannel comprising one or more 15 test chambers, each microchannel having a defined volume between nanoliters and milliliters, the one or more microchannels each comprising at least one anionically charged surface which activâtes ciotting of the blood or plasma sample upon entry of the blood or plasma sample Into the one or more 20 microchannels or test chamber, wherein the test chamber is formed of a material which allows changes in viscosity, impédance, acoustic properties, or optical properties in the blood or plasma sample to be measured in the test chamber, and

25 the reader comprising a detector which détermines changes in viscosity, impédance, acoustic properties, or optical properties in the blood or plasma sample in the test chamber which are indicative of ciotting, a température control regulating the température of the test 50 chamber, and output conveying the ciotting time determined from the time of activation of the blood or plasma sample to the time of the change in viscosity, Impédance, acoustic properties, or optical properties in the test chamber indicative of ciotting.

11. The method of claim 10 comprising recording the clotting time.

12. The method of claim 10 wherein the sample is obtained from a perso n with anticoagulated blood.

13. The method of claim 12 wherein the type of anticoagulation, if any, in the sample Is unknown.

14. The method of claim 12 wherein the sample is blood anticcagulated with an anticoagulant that affects blood so that clotting time cannct be measured with Partial Thromboplastin Time (aPPT).

15. The method of any one of claims 10-14 wherein transmission through the sample is measured to determined time to peak clotting time.

16. The method of claim 15 wherein the measurement is made with light at between 1150 and 1700 nm,

17. The method of claim 15 wherein the measurement is made with light at a wavelength of 1300 nm.

18. The method of any one of claims 10-16 wherein the température control heats the sample to body température for measurement of clotting time. '

19. The test microchip of claim 1 wherein the charged surface is glass or oxidized silica.

20. The test microchip of claim 1 comprising résistive heating structures.

21. The test microchip of claim 1 wherein the volume of the test chamber is between about one and ten microliters.

22. The test microchip of claim 1 comprising multiple microchannels and test chambers.

23. The test microchip of claim 1 comprising électrodes for détermination of impédance, résistance or capacitance.

24. The test microchip of claim 1 wherein the test microchip is single use.

5

25. The microassay device of claim 8, wherein the reader comprises an integrated heater for controlling the température of the test chamber in the inserted test microchip.

26. The microassay device of claim 8 comprising electrical, optical or fluidic interfaces with the test microchip.

10

27. The microassay device of claim 8 comprising an infrared source and an infrared detector, positioned to detect change in viscosity in the test chamber of the inserted test microchip. ’

28. The microassay device of claim 8 wherein clotting is measured by the reader from changes through the test chamber of the test microchip by

15 changes in résistance.

29. The microassay device wherein clotting is measured by the reader using acoustic transducers.