Development of a paper-based lateral flow prothrombin assay.

Disorders of haemostasis result in both excessive bleeding and clotting and are a major global cause of morbidity and mortality, particularly in the developing world. A small number of simple tests can be used to screen and monitor for such dysfunctions, one of which is the prothrombin time (PT) test and associated International Normalisation Ratio (INR). PT/INR is routine in hospital laboratories in developed countries, and can also be performed using point-of-care instruments. However, neither of these approaches is appropriate in low-resource settings. Significant interest has grown in paper-based devices to form the basis of simple and low-cost assays that may have the potential for application in such environments. This study describes the development of a simple, low-cost, paper-based lateral flow prothrombin assay. The assay employed wax printing on chromatography paper to define test channels, with deposition of thromboplastin reagent and calcium chloride onto the resulting strips. These were placed in a test housing and measurement of the flow rates of deposited plasma samples were performed in triplicate. The flow dynamics of the assay was optimised according to the type of paper substrate used, the nature and quantity of the thromboplastin reagent, the amount of calcium chloride required, and the volume of sample employed. An optimised assay configuration demonstrated a dynamic range of 6 mm between normal and factor-deficient plasmas. The assay showed good correlation with laboratory-based PT assay (Yumizen G200) in artificial plasmas in the 9.8 to 36 s range (r2 = 0.8112). The assay also demonstrated good dynamic range and correlation in patient plasma samples in comparison with hospital PT, with a range of 9.8 to 45 s (r2 = 0.7209).

Paper-Based Lateral Flow Device for the Sustainable Measurement of Human Plasma Fibrinogen in Low-Resource Settings.

Fibrinogen concentration is a major determinant of both clotting and bleeding risk. Clotting and bleeding disorders cause extensive morbidity and mortality, particularly in resource-poor and emergency settings. This is exacerbated by a lack of timely intervention informed by measurement of fibrinogen levels under conditions such as thrombosis or postpartum haemorrhage. There is an absence of simple, rapid, low-cost, and sustainable diagnostic devices for fibrinogen measurement that can be deployed in such environments. Paper-based analytical devices are of significant interest due to their potential for low-cost production, ease of use, and environmental sustainability. In this work, a device for measuring blood plasma fibrinogen using chromatography paper was developed. Wax printing was used to create hydrophobic structures to define the test channel and sample application zone. Test strips were modified with bovine thrombin. Plasma samples (22 µL) were applied, and the flow rate was monitored over 5 min. As the sample traversed the strip, clotting was induced by the conversion of soluble fibrinogen to insoluble fibrin. The flow rate and distance travelled by the sample were dependent on fibrinogen concentration. The device was able to measure fibrinogen concentration in the range of 0.5-7.0 ± 0.3 mg/mL (p < 0.05, n = 24) and had excellent correlation with laboratory coagulometry in artificial samples (r2 = 0.9582, n = 60). Devices were also stable at 4-6 °C for up to 3 weeks.

Biomedical Diagnostics Enabled by Integrated Organic and Printed Electronics.

Organic and printed electronics integration has the potential to revolutionize many technologies, including biomedical diagnostics. This work demonstrates the successful integration of multiple printed electronic functionalities into a single device capable of the measurement of hydrogen peroxide and total cholesterol. The single-use device employed printed electrochemical sensors for hydrogen peroxide electroreduction integrated with printed electrochromic display and battery. The system was driven by a conventional electronic circuit designed to illustrate the complete integration of silicon integrated circuits via pick and place or using organic electronic circuits. The device was capable of measuring 8 µL samples of both hydrogen peroxide (0-5 mM, 2.72 × 10-6 A·mM-1) and total cholesterol in serum from 0 to 9 mM (1.34 × 10-8 A·mM-1, r2 = 0.99, RSD < 10%, n = 3), and the result was output on a semiquantitative linear bar display. The device could operate for 10 min via a printed battery, and display the result for many hours or days. A mobile phone "app" was also capable of reading the test result and transmitting this to a remote health care provider. Such a technology could allow improved management of conditions such as hypercholesterolemia.

Measurement of the viscoelastic properties of blood plasma clot formation in response to tissue factor concentration-dependent activation.

The coagulation of blood plasma in response to activation with a range of tissue factor (TF) concentrations was studied with a quartz crystal microbalance (QCM), where frequency and half width at half maximum (bandwidth) values measured from the conductance spectrum near resonant frequency were used. Continuous measurement of bandwidth along with the frequency allows for an understanding of the dissipative nature of the forming viscoelastic clot, thus providing information on the complex kinetics of the viscoelastic changes occurring during the clot formation process. Using a mathematical model, these changes in frequency and bandwidth have been used to derive novel QCM parameters of effective elasticity, effective mass density and rigidity factor of the viscoelastic layer. The responses of QCM were compared with those from thromboelastography (TEG) under identical conditions. It was demonstrated that the nature of the clot formed, as determined from the QCM parameters, was highly dependent on the rate of clot formation resulting from the TF concentration used for activation. These parameters could also be related to physical clot characteristics such as fibrin fibre diameter and fibre density, as determined by scanning electron microscopic image analysis. The maximum amplitude (MA) as measured by TEG, which purports to relate to clot strength, was unable to detect these differences.

Development of a hydrogen peroxide sensor based on screen-printed electrodes modified with inkjet-printed Prussian blue nanoparticles.

A sensor for the simple and sensitive measurement of hydrogen peroxide has been developed which is based on screen printed electrodes (SPEs) modified with Prussian blue nanoparticles (PBNPs) deposited using piezoelectric inkjet printing. PBNP-modified SPEs were characterized using physical and electrochemical techniques to optimize the PBNP layer thickness and electroanalytical conditions for optimum measurement of hydrogen peroxide. Sensor optimization resulted in a limit of detection of 2 × 10(-7) M, a linear range from 0 to 4.5 mM and a sensitivity of 762 µA ∙ mM(-1) ∙ cm(-2) which was achieved using 20 layers of printed PBNPs. Sensors also demonstrated excellent reproducibility (<5% rsd).

Wholly printed polypyrrole nanoparticle-based biosensors on flexible substrate.

Printing has been widely used in the sensor industry for its speed, low cost and production scalability. In this work we present a wholly-printed polypyrrole (PPy) based biosensor produced by inkjet printing bioinks composed of dispersions of PPy nanoparticles and enzymes onto screen-printed carbon electrodes. Two enzymes, horseradish peroxidase (HRP) or glucose oxidase (GoD) were incorporated into the PPy nanoparticle dispersions to impart biosensing functionality and selectivity into the conducting polymer ink. Further functionality was also introduced by deposition of a permselective ethyl cellulose (EC) membrane using inkjet printing. Cyclic voltammetry (CV) and chrono-amperometry were used to characterize the response of the PPy biosensors to H2O2 and glucose. Results demonstrated the possibility of PPy based biosensor fabrication using the rapid and low cost technique of inkjet printing. The detection range of H2O2 was found to be 10 µM-10 mM and for glucose was 1-5 mM.

Point of care monitoring of hemodialysis patients with a breath ammonia measurement device based on printed polyaniline nanoparticle sensors.

A device for measuring human breath ammonia was developed based on a single use, disposable, inkjet printed ammonia sensor fabricated using polyaniline nanoparticles. The device was optimized for sampling ammonia in human breath samples by addressing issues such as variations in breath sample volume, flow rate, sources of oral ammonia, temperature and humidity. The resulting system was capable of measuring ammonia in breath from 40 to 2993 ppbv (r(2 )= 0.99, n = 3) as correlated with photoacoustic laser spectroscopy and correlation in normal human breath samples yielded a slope of 0.93 and a Pearson correlation coefficient of 0.9705 (p < 0.05, n = 11). Measurement of ammonia in the breath of patients with end-stage kidney disease demonstrated its significant reduction following dialysis, while also correlating well with blood urea nitrogen (BUN) (r = 0.61, p < 0.01, n = 96). Excellent intraindividual correlations were demonstrated between breath ammonia and BUN (0.86 to 0.96), which demonstrates the possibility of using low cost point of care breath ammonia systems as a noninvasive means of monitoring kidney dysfunction and treatment.

Direct measurement of ammonia in simulated human breath using an inkjet-printed polyaniline nanoparticle sensor.

A sensor fabricated from the inkjet-printed deposition of polyaniline nanoparticles onto a screen-printed silver interdigitated electrode was developed for the detection of ammonia in simulated human breath samples. Impedance analysis showed that exposure to ammonia gas could be measured at 962 Hz at which changes in resistance dominate due to the deprotonation of the polymer film. Sensors required minimal calibration and demonstrated excellent intra-electrode baseline drift (≤1.67%). Gases typically present in breath did not interfere with the sensor. Temperature and humidity were shown to have characteristic impedimetric and temporal effects on the sensor that could be distinguished from the response to ammonia. While impedance responses to ammonia could be detected from a single simulated breath, quantification was improved after the cumulative measurement of multiple breaths. The measurement of ammonia after 16 simulated breaths was linear in the range of 40-2175 ppbv (27-1514 µg m(-3)) (r(2)=0.9963) with a theoretical limit of detection of 6.2 ppbv (4.1 µg m(-3)) (SN(-1)=3).

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

The development of new point of care coagulation assay devices is necessary due to the increasing number of patients requiring long-term anticoagulation in addition to the desire for appropriate, targeted anticoagulant therapy and a more rapid response to optimization of treatment. The majority of point of care devices currently available for hemostasis testing rely on clot-based endpoints which are variable, unreliable and limited to measuring only certain portions of the coagulation pathway. Here we present a novel fluorescence-based anti-Factor Xa (FXa) microfluidic assay device for monitoring the effect of anticoagulant therapy at the point of care. The device is a disposable, laminated polymer microfluidic strip fabricated from a combination of hydrophobic and hydrophilic cyclic polyolefins to allow reagent deposition in addition to effective capillary fill. Zeonor was the polymer of choice resulting in low background fluorescence (208.5 AU), suitable contact angles (17.5°± 0.9°) and capillary fill times (20.3 ± 2.1 s). The device was capable of measuring unfractionated heparin and tinzaparin from 0-0.8 U ml(-1) and enoxaparin from 0-0.6 U ml(-1) with average CVs < 10%. A linear correlation was observed between the device and the fluorescent assay in the plate for plasma samples spiked with UFH, with an R(2) value of 0.99, while correlations with tinzaparin and enoxaparin resulted in sigmoidal responses (R(2) = 0.99). Plasma samples containing UFH resulted in a linear correlation between the device and a standard chromogenic assay with an R(2) value of 0.98, with both LMWHs resulting in sigmoidal relationships (R(2) = 0.99).

The modelling of blood coagulation using the quartz crystal microbalance.

Blood is a clinically-important analytical matrix that is routinely selected for disease monitoring. Having a clear understanding of the mechanisms involved in blood coagulation is a key consideration in haemostasis, with modern clinical practices requiring rapid, miniaturised and informative diagnostic platforms to reliably study changes in viscoelasticity (VE). Oscillatory transducers such as the Quartz Crystal Microbalance (QCM) have considerable potential in this area, provided that they present simple, linear rheometric readings which can be adequately analysed and interpreted. Hence, integrating QCM data obtained in the laboratory with mathematical modelling of acoustic interactions between quartz crystal surfaces and coagulating blood is an important consideration for modelling thrombus formation. Here, we provide a comprehensive overview of experimental and theoretical applications currently being employed to monitor and model the VE properties of coagulating blood when applied to a QCM resonator, with key emphasis on data modelling and interpretation.

Evaluation of a silver-based electrocatalyst for the determination of hydrogen peroxide formed via enzymatic oxidation.

Silver paste electrodes modified with lyotropic phases formed from dodecyl benzenesulphonic acid and KCl were used as the reductant in the determination of the hydrogen peroxide released from the enzymatic reaction of glucose oxidase with glucose and oxygen. The response of the modified electrode to hydrogen peroxide reduction (-0.1 V vs. Ag/AgCl) was shown to suffer from interference resulting from co-localization of enzyme and substrate at the electrode surface. This interference was eradicated by the introduction of a perm-selective membrane in the form of cellulose acetate. This further facilitated immobilization of the enzyme while allowing diffusion of the generated peroxide to the electrode. The resulting configuration was shown to be capable of the analytical determination of glucose.

Effects of four commercially available factor Xa proteins on the fluorogenic anti-factor Xa assay when monitoring unfractionated heparin.

Four commercially available factor Xa (FXa) reagents were evaluated in a fluorogenic anti-FXa assay. The four reagents - of which three were of human origin and the fourth was bovine - were compared in terms of the resulting assay dynamic ranges, lag times, coefficient of variation and R2 values, as well as their sensitivity to unfractionated heparin within the therapeutic range of 0-1.2 U/ml. Similar performance of reagents in the fluorogenic anti-FXa assay was observed independent of the source of the reagent or its physical state, which may assist in the standardization of coagulation assays in clinical settings.

Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays.

INTRODUCTION: Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the 'gold standard' assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity. MATERIALS AND METHODS: Commercial plasmas were spiked with pharmacologically relevant concentrations (0-1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7µM fluorogenic substrate, in addition to 6µl of 100mM CaCl(2) and 44µl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed. RESULTS: The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0-1 U/ml with CVs of<5% for UFH and tinzaparin and CVs<9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits. CONCLUSIONS: This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.

Comparison of a fluorogenic anti-FXa assay with a central laboratory chromogenic anti-FXa assay for measuring LMWH activity in patient plasmas.

INTRODUCTION: Low molecular weight heparins (LMWHs) are used worldwide for the treatment and prophylaxis of thromboembolic disorders. Routine laboratory tests are not required due to the predictable pharmacokinetics of LMWHs, with the exception of pregnant patients, children, patients with renal failure, morbid obesity, or advanced age. Anti-Factor Xa (anti-FXa) plasma levels are most often employed in the assessment and guidance of accurate dosing in these patient cohorts. MATERIALS AND METHODS: A LMWH calibration curve was generated using citrated human pooled plasma spiked with pharmacologically relevant concentrations (0-1.2U/ml) of two low molecular weight heparins; enoxaparin and tinzaparin. Least squares analysis determined the best curve fit for this set of data which returned low sum of squares (SS) values for the log linear fit with an R(2) value of 0.98. 30 patient samples were tested in the fluorogenic assay and concentrations were determined using the log linear regression equation and correlated with a standard chromogenic assay used for heparin monitoring. RESULTS: A statistically significant correlation was found between the fluorogenic and the chromogenic anti-FXa assays for 30 patient samples, with a slope of 0.829, offset of 0.258 and an R(2) value of 0.72 (p<0.0001). CONCLUSIONS: In the study presented here, a fluorogenic anti-FXa assay was correlated with a standard laboratory chromogenic anti-FXa assay using samples from patients on LMWH therapy. Significant correlations between the values derived by the fluorogenic and chromogenic anti-FXa assays were found for the patient cohort tested in this study.

Quantification of unfractionated heparin in human plasma and whole blood by means of novel fluorogenic anti-FXa assays.

Novel and sensitive plate-based fluorogenic anti-factor Xa (FXa) assays were investigated to quantify unfractionated heparin (UFH) in human plasma and whole blood within the therapeutic ranges of 0-1.6 U/mL and 0-0.8 U/mL, respectively. Two fluorogenic anti-FXa assay methods were defined for low (0-0.6 U/mL) and high (0.6-1.2 U/mL) pharmacologically relevant UFH concentration ranges in pooled human plasma. In both cases significant differences were observed at intervals of 0.2 U/mL (P<0.05). The semi-logarithmic plots of the calibration curves in the low and high UFH range were both fitted to linear regressions with correlation coefficients of 0.96 and >0.99, respectively. The assay was also optimized for whole blood which was capable of differentiating UFH concentrations at intervals of 0.2 U/mL (P<0.05) in the range of 0-0.4 U/mL. The statistically different results were fitted to a linear regression with a correlation coefficient of >0.99. The results obtained in this study could assist diagnostic laboratories towards improved monitoring of UFH therapy.

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor.

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.

Development of a fluorescent method for detecting the onset of coagulation in human plasma on microstructured lateral flow platforms.

Microfluidic devices and microsystems have been used to develop blood coagulation monitoring devices for point of care diagnostic use. However, many of them suffer from inherent variability and imprecision, partly due to the fact that they only detect changes in bulk clotting properties and do not reflect the microscopic nature of blood coagulation. This work demonstrates microstructured lateral flow platforms used in combination with fluorescently labelled fibrinogen to detect microscopic clot formation. Plasma samples applied to platforms modified with coagulation activation reagents and fluorescent fibrinogen produced changes in fluorescence intensity due to incorporation of the fluorophore into the forming microclots. It was found that the change in the distribution of the fluorescence within the sample over time was an excellent predictor of the onset of coagulation, which could be used to determine the clotting time. The impact of various assay parameters was optimised and the assay was shown to be capable of measuring the effect of heparin concentration on blood clotting time from 0 to 1.5 U mL(-1).

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Fluorescence-based blood coagulation assay device for measuring activated partial thromboplastin time.

The measurement of blood clotting time is important in a range of clinical applications such as assessing coagulation disorders and controlling the effect of various anticoagulant drug therapies. Clotting time tests essentially measure the onset of clot formation which results from the formation of fibrin fibers in the blood sample. However, such assays are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. This work describes a clotting time measurement assay which uses a fluorescent probe to very precisely detect the onset of fibrin clot formation. It uses a microstructured surface which enhances the formation of multiple localized clot loci and which results in the abrupt redistribution of the fluorescent label at the onset of clot formation in both whole blood and plasma. This methodology was applied to the development of an activated partial thromboplastin time (aPTT) test in a lateral flow microfluidic platform and used to monitor the effect of heparin dosage where it showed linearity from 0 to 2 U/mL in spiked plasma samples (R(2)=0.996, n = 3), correlation against gold standard coagulometry of 0.9986, and correlation against standard hospital aPTT in 32 patient samples of 0.78.

Development of a fluorescent anti-factor Xa assay to monitor unfractionated and low molecular weight heparins.

Fluorogenic assays have many potential advantages over traditional clot-based and chromogenic assays such as the absence of interference from a range of factor deficiencies as well as offering the possibility of assays in platelet rich plasma or whole blood. A fluorogenic anti-factor Xa (anti-FXa) assay has been developed for the determination of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), namely enoxaparin and tinzaparin, and the synthetic heparinoid danaparoid, in commercial human pooled plasma. The assay was based on the complexation of heparin-spiked plasmas with exogenous FXa at a concentration of 4nM in the presence of 0.9microM of the fluorogenic substrate methylsulfonyl-D-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Pooled plasma samples were spiked with concentrations of anticoagulants in the range 0-1.6U/ml. The assay was capable of the measurement of UFH and danaparoid in the range 0-1U/ml, and enoxaparin and tinzaparin in the range 0-0.8 and 0-0.6U/ml, respectively. Correlation coefficients generated by linear regression of the log/lin data analysis were between 0.93 and 0.96 for the anticoagulants tested. Assay percentage coefficients of variation were typically below 7%.