CHEMILUMINESCENT BIOSENSOR FOR DETECTING COAGULATION FACTORS

Abstract

A chemiluminescent biosensor for detecting a coagulation factor in a blood sample including: a fluorogenic substrate for the coagulation factor, where the fluorogenic substrate includes a fluorescent dye; and a quencher conjugated with the fluorogenic substrate. The biosensor rapidly and accurately detects a coagulation factor in a blood sample including whole blood or plasma, thereby useful for minimizing or eliminating any reversal effect of anticoagulants.

Claims

1. A biosensor for detecting a coagulation factor in a blood sample comprising: a fluorogenic substrate for the coagulation factor, wherein the fluorogenic substrate includes a fluorescent dye; and a quencher conjugated with the fluorogenic substrate.

2. The biosensor of claim 1, wherein the coagulation factor is coagulation factor IIa or Xa.

3. The biosensor of claim 1, wherein the blood sample is plasma or whole blood.

4. The biosensor of claim 1, wherein the blood sample is 1 to 1,000-fold diluted plasma or whole blood.

5. The biosensor of claim 1, wherein the fluorescent dye is at least one selected from the group consisting of 2-aminobenzoyl (Abz), N-methyl-anthraniloyl (N-Me-Abz). 5-(dimethylamino)naphthalene-1-sulfonyl (Dansyl), 5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid (EDANS), 7-dimethylaminocoumarin-4-acetate (DMACA), 7-amino-4-methylcoumarin (AMC), (7-methoxycoumarin-4-yl)acetyl (MCA), rhodamine, rhodamine 101, rhodamine 110 and resorufin.

6. The biosensor of claim 1, wherein the fluorescent dye emits light when: the fluorescent dye dissociates from the fluorogenic substrate by a hydrolysis reaction between the coagulation factor and the fluorogenic substrate; and the fluorescent dye interacts with high-energy intermediate formed from 1, 1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent.

7. The biosensor of claim 1, wherein the 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent comprises an ODI and H.sub.2O.sub.2.

8. The biosensor of claim 1, wherein the quencher is at least one selected from the group consisting of 2,4-Dinitrophenyl (DNP), N-(2,4-Dinitrophenyl)ethylenediamine (EDDnp), 4-Nitro-phenylalanine, 3-Nitro-tyrosine, para-Nitroaniline (pNa), 4-(4-Dimethylaminophenylazo)benzoyl (DABCYL) and 7-Nitro-benzo[2,1,3]oxadiazol-4-yl (NBD).

9. A method of monitoring a coagulation factor in a blood sample, the method comprising: mixing and reacting the biosensor of claim 1 with a blood sample including a coagulation factor in a buffer; adding a 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent to the reacted mixture; and measuring CL intensity.

10. The method of claim 9, wherein the reaction time between the blood sample and the fluorogenic substrate in the biosensor at room temperature (212 C.) or 37 C. is 10 seconds to 120 minutes.

11. The method of claim 9, wherein measuring CL intensity is performed for 1 to 10 seconds after adding the ODI-CL reagent.

12. The method of claim 9, wherein the coagulation factor is coagulation factor IIa or Xa.

13. The method of claim 9, wherein the blood sample is plasma or whole blood

14. The method of claim 9, wherein the buffer is selected from the groups consisting of PBST, PBS, TBST and TBS.

15. A method of quantifying a coagulation factor in a blood sample, the method comprising: mixing and reacting the biosensor of claim 1 with a blood sample including a coagulation factor in a buffer; adding 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent to the reacted mixture; measuring CL intensity; and comparing the intensity of CL with a standard intensity.

16. A kit for quantifying a coagulation factor in a blood sample, the kit comprising: the biosensor of claim 1; and a container.

17. The kit of claim 16 further comprising: a buffer; and 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A shows the role of Xa and IIa in the blood coagulation cascade.

[0018] FIG. 1B is a diagram for the hydrolysis reaction between protease and substrate conjugated with chromophore or fluorescent dye.

[0019] FIG. 1C shows a reaction mechanism of 1,1 -Oxalyldiimidazole chemiluminescence (ODI-CL), where L is luminophore under the ground state and L* is luminophore under the excited state.

[0020] FIG. 1D is a graph showing a rapid ODI-CL spectrum.

[0021] FIG. 2A is a graph showing relative CL intensities in the absence and presence of the coagulation factor IIa (5 nM) or Xa (5 nM).

[0022] FIG. 2B depicts chemiluminescent resonance energy transfer (CRET) in the absence of biomarker such as factors IIa and Xa in plasma.

[0023] FIG. 2C depicts ODI-CL reaction in the presence of fluorogenic substrate and protease enzyme.

[0024] FIG. 2D shows hydrolysis reaction between the fluorogenic substrate and coagulation factor IIa or Xa.

[0025] FIG. 2E shows ODI-CL reaction in the presence of a fluorescent dye (e.g., AMC) formed from the hydrolysis reaction between fluorogenic substrate and coagulation factors (e.g., IIa, Xa)

[0026] FIG. 3A is a graph showing the effect of plasma in the presence of coagulation factor IIa using a specific substrate conjugated AMC in PBS.

[0027] FIG. 3B is a graph showing the effect of plasma in the presence of coagulation factor Xa using a specific substrate conjugated AMC in PBS.

[0028] FIG. 3C is a graph showing the selection of buffer for the quantification of coagulation factor IIa (6.8 nM) in 10% human plasma.

[0029] FIG. 3D is a graph showing the selection of buffer for the quantification of coagulation factor Xa (10 nM) in 10% human plasma.

[0030] FIG. 4A is a graph showing the calibration curves for the quantification of coagulation factor IIa with the rapid biosensor with ODI-CL detection.

[0031] FIG. 4B is a graph showing the calibration curves for the quantification of coagulation factor Xa with the rapid biosensor with ODI-CL detection.

[0032] FIG. 4C is a graph showing the correlations (N=10) between ODI-CL and fluorescence detection for the quantification of coagulation factor IIa in human plasma. (The error range of each value were 4-7%).

[0033] FIG. 4D is a graph showing the correlations (N=10) between ODI-CL and fluorescence detection for the quantification of coagulation factor Xa in human plasma. (The error range of each value were 4-7%).

[0034] FIG. 5A is a graph showing the effect of incubation time in the absence and presence of coagulation factor IIa in whole blood.

[0035] FIG. 5B is a graph showing the effect of incubation time in the absence and presence of coagulation factor Xa in whole blood.

[0036] FIG. 5C is a graph showing the enhancement of relative CL intensity with the extension of reaction (incubation) time between IIa fluorogenic substrate and coagulation factor IIa in whole blood (N=5).

[0037] FIG. 5D is a graph showing the enhancement of relative CL intensity with the extension of reaction (incubation) time between Xa fluorogenic substrate and coagulation factor Xa in whole blood (N=5).

[0038] FIG. 6A is a graph showing the calibration curves capable of rapidly quantifying trace levels of coagulation factor IIa in whole blood using the biosensor with ODI-CL detection.

[0039] FIG. 6B is a graph showing the calibration curves capable of rapidly quantifying trace levels of coagulation factor Xa in whole blood using the biosensor with ODI-CL detection.

[0040] FIG. 6C is a graph showing the selectivity and specificity of Xa fluorogenic substrate conjugated with AMC.

[0041] FIG. 6D is a graph showing the selectivity and specificity of IIa fluorogenic substrate conjugated with AMC.

[0042] FIG. 7 is a graph showing the effect of plasma in ODI-CL reaction in the presence of AMC (12.5 M) as a luminophore (fluorescent dye). Each sample was prepared as a mixture (volume ratio=1:1) of AMC in TBST and a certain % concentration of human plasma diluted with H.sub.2O.

[0043] FIG. 8 is a graph showing the relative CL intensity of AMC (25 M) in four different buffer solutions.

[0044] FIG. 9 is a graph showing CL.sub.3.4/CL.sub.0 over different reaction (incubation) time for the quantification of IIa (3.4 nM) in 10% human plasma.

[0045] FIG. 10 is a graph showing the dilution effect for the quantification of Xa in human whole blood. Reaction time of the diluted whole blood and fluorogenic substrate is 2, 4, and 10 min.

[0046] FIG. 11 is a graph showing the effect of components in whole blood in ODI-CL reaction in the presence of AMC (25 M) as a luminophore (fluorescent dye).

[0047] FIG. 12A is a graph showing the specificity and selectivity of IIa fluorogenic substrate applied to develop the biosensor with ODI-CL detection. Concentration: [IIa]=28.6 ng/ml, [Glucose]=10.sup.6 ng/ml, [Hemoglobin]=10.sup.6 ng/ml, [HSA]=10.sup.6 ng/ml, [IgG]=10.sup.6 ng/ml.

[0048] FIG. 12B is a graph showing the specificity and selectivity of Xa fluorogenic substrate applied to develop the biosensor with ODI-CL detection. Concentration: [Xa]=10.8 ng/ml, [Glucose]=10.sup.6 ng/ml, [Hemoglobin]=10.sup.6 ng/ml, [HSA]=10.sup.6 ng/ml, [IgG]=10.sup.6 ng/ml.

DETAILED DESCRIPTION

[0049] According to an embodiment of the present invention, a biosensor is provided for detecting a coagulation factor in a blood sample, the biosensor comprises: a fluorogenic substrate for the coagulation factor, wherein the fluorogenic substrate includes a fluorescent dye; and a quencher conjugated with the fluorogenic substrate. The fluorescent dye emits light when the fluorescent dye is dissociated from the fluorogenic substrate by a hydrolysis reaction between the coagulation factor and the fluorogenic substrate, and when the fluorescent dye interacts with high-energy intermediate formed from 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent. The 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent may comprise an ODI and H.sub.2O.sub.2.

[0050] The fluorescent dye used in the fluorogenic substrate may be at least one of 2-aminobenzoyl (Abz), N-methyl-anthraniloyl (N-Me-Abz), 5-(dimethylamino)naphthalene-1-sulfonyl (Dansyl), 5-[(2-aminoethyeamino]-naphthalene-1-sulfonic acid (EDANS), 7-dimethylaminocoumarin-4-acetate (DMACA), 7-amino-4-methylcoumarin (AMC), (7-methoxycoumarin-4-yl)acetyl (MCA), rhodamine, rhodamine 101, rhodamine 110 and resorufin. In this specification, AMC is used as an example, but other fluorescent dye can be used alone or in combination with each other.

[0051] The quencher used in the biosensor may be at least one of 2,4-Dinitrophenyl (DNP), N-(2,4-Dinitrophenyl)ethylenediamine (EDDnp), 4-Nitro-phenylalanine, 3-Nitro-tyrosine, para-Nitroaniline (pNa), 4-(4-Dimethylaminophenylazo)benzoyl (DABCYL) and 7-Nitro-benzo[2,1,3]oxadiazol-4-yl (NBD).

[0052] The coagulation factor of the present invention can be any type of coagulation factor that is involved in the blood coagulation cascade. Among the various coagulation factors, factor IIa (thrombin) and factor Xa are preferable.

[0053] As shown in FIG. 2A, a fluorescent dye (e.g., AMC) in a fluorogenic substrate for the coagulation factor IIa (or Xa) does not emit light in an ODI-CL detection system in the absence of the coagulation factor IIa (or Xa). This is because the fluorescent dye (AMC; luminophore (L)) is excited by the high-energy intermediate formed from the reaction between the ODI and H.sub.2O.sub.2 transfer energy to the quencher (Q) conjugated with the fluorogenic substrate due to the chemiluminescent resonance energy transfer (CRET) as shown in FIG. 2B. FIG. 2A shows that relative CL intensity in the presence of the coagulation factor (5 nM) is much higher than that in the absence of the coagulation factor. The results can be illustrated by the reaction scheme shown in FIG. 2C. The fluorogenic substrate was separated by the hydrolysis reaction of the fluorogenic substrate and the coagulation factor. Thus, the fluorescent dye (luminophore) not bound with the quencher can emit light in the ODI-CL reaction. FIG. 2D shows that a fluorescent dye (AMC) is separated as a result of the hydrolysis reaction between the coagulation factor IIa (or Xa) and a specific fluorogenic substrate for the coagulation factor. In the example shown in FIG. 2E, AMC excited by the high-energy intermediate (X) formed from ODI-CL reaction can emit (blue) light.

[0054] An exemplary structure of a IIa specific fluorogenic substrate and an Xa specific fluorogenic substrate are shown TABLE 1 below.

TABLE-US-00001 TABLE 1 Fluorogenic substrate IIa Xa Formula Benzoyl-Phe-Val-Arg- CH.sub.3SO.sub.2-D-CHA-Gly-Arg- AMC, HCl AMC, AcOH Physical Form Lyophilized Lyophilized Fluorescent dye Chemical Structure [00001] [00002] Mw (g mol.sup.1) 681.78 679.8 Ex/Em Wavelength (nm) 342/440 342/440 K.sub.m (M) 3.90 220 k.sub.cat (s.sup.1) 42.7 162.0 k.sub.cat/K.sub.m (s.sup.1 M.sup.1) 10.9 0.74

[0055] In the present invention, the blood sample can be either plasma or whole blood. The blood sample can be used as is, or 1 to 1,000-fold diluted. The effect of using plasma in ODI-CL reactions using AMC (12.5 M) as a fluorescent dye is shown in FIG. 7. As shown, the relative CL intensity of AMC in 0.110% plasma (10 to 1000-fold dilution) was constant within a statistically acceptable error range (<5%). The relative CL intensity in 100% plasma was lower than those in 0.110% plasma because some components in human plasma may act as an inhibitor or quencher in ODI-CL reactions.

[0056] FIGS. 3A and 3B show the sensitivity of ODI-CL emitted in the biosensor depending on the concentration of the plasma. The signal/background ratio (CL.sub.IIa/CL.sub.0 or CL.sub.Xa/CL.sub.0) was enhanced when the composition of human plasma was diluted. Thus, the biosensor using 10 to 1000-fold diluted human plasma can be more sensitive than that using 100% human plasma. Additionally, the signal/background ratio in 10% human plasma was about 50% lower than that in 0.1% human plasma. These results indicate that the limit of detection (LOD=3) for the biosensor operated with 10-fold diluted human plasma may be as low as or slightly higher than that for the biosensors generated with 1,000-fold diluted human plasma. is the standard deviation of background measured in the absence of the coagulation factor IIa or Xa.

[0057] In the present invention, peptides specific to the coagulation factor included in the biosensor may react with a coagulation factor in a buffer. The buffer can be any one of Phosphate buffered saline with Tween-20 (PBST), Phosphate buffered saline (PBS), Tris buffered saline with Tween-20 (TBST) and Tris buffered saline (TBS). As shown in FIG. 8, the light emitted from AMC (25 M) in TBS is brighter than those in other buffer solutions. The results imply that TBS is the best buffer solution of ODI-CL biosensor capable of rapidly quantifying trace levels of AMC formed from the hydrolysis reaction between the coagulation factor IIa (or Xa) and a specific substrate conjugated with AMC shown in FIG. 2D. However, FIGS. 3C and 3D indicate that the best buffer for the hydrolysis reaction between the coagulation factor IIa and the substrate conjugated with AMC is TBST, whereas PBS is the best buffer for the hydrolysis reaction between the coagulation factor Xa and the substrate. These results indicate that the yield of AMC formed from the hydrolysis reaction between the coagulation factor and a specific substrate conjugated with AMC is dependent on the type of buffer solution. For example, FIG. 3C shows that the relative CL intensity measured after the 2-min hydrolysis reaction in TBST is the strongest. Thus, the results shown in FIGS. 8 and 3C indicate that the concentration of AMC formed from the 2-min hydrolysis reaction in TBST is higher than those in the other buffer solutions. In other words, the hydrolysis reaction in TBST is faster than those in the other buffer solutions. As another example, FIG. 3D shows that the best buffer solution for the quantification of the coagulation factor Xa using the biosensor with ODI-CL detection is PBS because the concentration of AMC formed after the 2-min hydrolysis reaction in PBS is higher than those in PBST, TBS, and TBST. Based on the results, TBST may be preferable for monitoring/quantifying IIa in a human sample while PBS may be preferable for monitoring/quantifying Xa in a human sample.

[0058] In the present invention, the reaction (hydrolysis) time between the blood sample and the fluorogenic substrate in the biosensor at room temperature (212 C.) or 37 C. may be controlled in the range of approximately 10 seconds to 120 minutes. Preferably, the reaction (hydrolysis) time may be controlled to be 1-30 minutes, and most preferably, 1-4 minutes. FIG. 9 shows that the sensitivity of the biosensor with ODI-CL detection is dependent on the incubation time necessary for the hydrolysis reaction between the coagulation factor and the substrate conjugated with AMC. With an increase in the hydrolysis reaction (incubation) time, CL.sub.3.4/CL.sub.0 was enhanced. CL.sub.3.4/CL.sub.0 calculated with relative CL intensities measured after a 4-minute incubation was similar to that after a 5-minute incubation. Thus, it is possible to have the reaction (hydrolysis) time for quantifying coagulation factor IIa in 10% human plasma as 4 minutes using the biosensor with ODI-CL detection and the substrate conjugated with AMC in TBST. Additionally, in accordance with the exemplary embodiments of the present invention in FIG. 9, a 1-minute incubation time is also possible. This is because 3.4 nM detected after a 1-minute incubation (hydrolysis) is lower than the normal range (1015 nM) in 10% human plasma even though it is expected that the sensitivity of biosensor operated with a 1-minute incubation may not be as good as that generated after 4 minutes incubation. Thus, the biosensor with ODI-CL detection is possible for the rapid quantification of the coagulation factor IIa with a short incubation (14 min) of the mixture of 10% human plasma and substrate conjugated with AMC in TBST.

[0059] The reaction (hydrolysis) time is also applicable to the biosensor capable of sensing the coagulation factor Xa in 10% human plasma in PBS. The following table shows a normalized intensity of ODI-CL and fluorescence (conventional) for quantifying factor Xa in 10% human plasma.

TABLE-US-00002 TABLE 2 Normalized Normalized X.sub.a ODI-CL Intensity Fluorescence Intensity (nM) (2-min incubation) (30-min incubation) 0 2.51 4.22 0.02 3.46 4.92 0.05 3.94 4.08 0.11 4.42 4.22 0.27 5.26 5.63 0.62 6.57 6.61 1.45 8.48 9.14 3.37 11.11 13.50 7.87 17.68 20.68 18.37 27.12 39.24 42.86 48.51 63.01 100.00 100.00 100.00 *The error range of each value measured with ODI-CL or fluorescence detection was 3~7%. **The excitation and emission wavelengths for the fluorescence measurement were 342 and 440 nm.

[0060] As shown in TABLE 2, the biosensor with ODI-CL detection is much more sensitive than a conventional sensor with fluorescence detection. ODI-CL was able to detect 0.02 nM X.sub.a with only a 2-min incubation period under ambient conditions, whereas the fluorescence detection could not sufficiently sense 0.11 nM X.sub.a even with the 30-min incubation due to the high background generated while operating light source. The sensitivity of the biosensor with the fluorescence detection, a conventional method, was used to compare with the biosensor with ODI-CL detection. (https://www.mybiosource.com/prods/Assay-Kit/Factor-Xa/datasheet.php?products_id.84634).

[0061] According to another embodiment of the present invention, a method for monitoring/quantifying a coagulation factor in a blood sample by using a biosensor as described above. The method includes mixing and reacting the biosensor with a blood sample including a coagulation factor in a buffer; adding a 1,1-oxalyldiimidazole chemiluminescence (ODI-CL) reagent to the reacted mixture; and measuring CL intensity. The reaction (hydrolysis) time between the blood sample and the fluorogenic substrate in the biosensor at room temperature (212 C.) or 37 C. may be 10 seconds to 120 minutes, and the measuring CL intensity may be performed for 1 to 10 seconds after adding the ODI-CL reagent.

[0062] With a 2-min incubation of the coagulation factor IIa (and Xa) and a substrate conjugated with AMC, as shown in FIGS. 4A and 4B, the biosensor with ODI-CL detection can rapidly quantify trace levels of IIa and Xa with wide linear calibration curves. The dynamic range of linear calibration curves for quantifying factor IIa was as wide as 0.3 to 27.2 nM. The LOD of the biosensor capable of analyzing factor IIa was as low as 104 pM. Also, the dynamic range of linear calibration curves for the analysis of factor Xa was as wide as 0.25 to 20 nM. The LOD of the biosensor was as low as 44 pM. Using the biosensor with excellent linear calibration curves as in FIGS. 4A and 4B, the present invention achieves the accurate, cost-effective, and rapid quantification of a coagulation factor with just a 10-fold diluted plasma sample instead of 10010,000 times diluted plasma as in the case of previous biosensors. FIGS. 4C and 4D show a good correlation between a biosensor with ODI-CL detection and a conventional biosensor with fluorescence detection. These results indicate that a biosensor with ODI-CL detection with a 2-min incubation of the mixture (e.g., a specific fluorogenic substrate, factor IIa or Xa) may be a cost-effective, rapid, and easy-to-use diagnostic method for quantifying coagulation factors.

[0063] The following TABLE 3 shows that a biosensor with ODI-CL detection according to exemplary embodiments of the present invention can quantify coagulation factors IIa and Xa with good accuracy, precision, and recovery. Thus, a biosensor according to the present invention can quantify factors IIa and Xa in human plasma with a statistically acceptable reproducibility far more rapidly than conventional biosensors.

TABLE-US-00003 TABLE 3 (Accuracy, precision, and recovery for the all-in-one Biosensor with ODI-CL detection for the quantization of IIa and Xa in human plasma (N = 5)) Sample 1 Sample 2 Expected Measured Recovery Factor (nM) (nM) (nM) (nM) (%) IIa 6.8 27.6 17.2 16.52 0.94 95.9 8.5 14.0 11.25 11.94 0.68 105.8 Xa 5.0 20.0 12.5 12.79 0.43 101.6 2.5 17.5 10.0 9.42 0.52 94.2

[0064] Analyses of Factors IIa and Xa in Whole Blood

[0065] A biosensor according to exemplary embodiments of the present invention can be used with whole blood as the sample. FIG. 10 shows the application of the biosensor to a whole blood sample (where the whole blood sample was 1040 times diluted with deionized H.sub.2O), which again indicates that a biosensor with ODI-CL detection can rapidly quantify trace levels of coagulation factors in 10-fold diluted whole blood similar to the quantification of factors IIa and Xa in 10-fold diluted plasma. FIGS. 5A and 5B also show that the factor IIa (or Xa) substrate conjugated with AMC is so stable in negative sample not containing IIa (or Xa) that the relative CL intensity (background) measured after three different incubation times of the mixture (e.g., negative sample and IIa (or Xa) substrate conjugated with AMC) was constant within the statistically acceptable error range (<5%). The strength of the light emitted in the patient sample (e.g., 10-fold diluted whole blood) containing trace levels of IIa and Xa proportionally increased with the extension of the incubation time. The relative CL intensity of the sample, spiked IIa (2.5 nM) or Xa (5 nM) in the patient sample, was higher than that of the pure patient sample as well being dependent on the incubation time.

[0066] As shown in FIGS. 5C and 5D, the relative CL intensity of a whole blood sample was different from those of the other four whole blood samples because IIa and Xa concentrations in each whole blood sample were different. However, the relative CL intensity of each whole blood sample was proportionally enhanced with the extension of the incubation time. Thus, FIGS. 5C and 5D indicate that a biosensor with ODI-CL detection can rapidly quantify trace levels of IIa (or Xa) in the patient sample (e.g., 10-fold diluted whole blood) with statistically acceptable precision and reproducibility.

[0067] As shown in FIG. 11, the relative CL intensity of AMC in the 10% whole blood was about 50% lower than those in 0.1 and 1% whole blood, whereas the relative CL intensity of AMC in the 10% plasma was the same as those in 0.1 and 1% plasma (See FIG. 7). Also, the relative CL intensity of AMC in 100% whole blood was about 60-fold lower than those in 0.1 and 1% whole blood. The results shown in FIG. 11 indicate that the quantum efficiency of AMC emitted in ODI-CL reaction is decreased by some inhibitors existing in 10 and 100% whole blood samples. Based on the results shown in FIGS. 7 and 11, it is expected that human whole blood may contain some components capable of acting as a strong inhibitor in ODI-CL reaction. In order to overcome the disadvantages associated with using a whole blood sample in ODI-CL reactions, a 4-min incubation of the mixture (e.g., IIa (or Xa) substrate conjugated with AMC and 10-fold diluted whole blood sample) was selected for the development of a highly sensitive biosensor with ODI-CL detection capable of rapidly diagnosing and preventing bleeding, thrombosis, and stroke. Thus, preferably, the reaction (hydrolysis) time for the quantification of IIa and Xa in whole blood may be set 2 times longer than that in plasma.

[0068] The linear calibration curves of FIGS. 6A and 6B indicate that a biosensor with ODI-CL detection, and with the 4-min incubation of the mixture, can rapidly quantify IIa and Xa in patient whole blood. LODs of the biosensor capable of quantifying IIa and Xa were as low as 66 and 18 pM in whole blood. LODs of the biosensor in whole blood were lower than those in plasma, as shown in TABLE 4, because the 4-min reaction (hydrolysis) time applied in the biosensor in whole blood is longer than the 2-min reaction (hydrolysis) time selected in the biosensor in plasma. These results indicate that the sensitivity of the biosensor with ODI-CL detection is dependent on the reaction time for the hydrolysis reaction between protease enzyme (e.g., factors IIa, Xa) and substrate conjugated with AMC. Thus, it is expected that the LOD of the biosensor with ODI-CL detection may vary depending on the incubation time for the hydrolysis reaction between a fluorogenic substrate and the coagulation factor IIa (or Xa) in plasma or whole blood.

TABLE-US-00004 TABLE 4 Sample Analytical Dynamic Method Factor type time (min) range (nM) LOD (pM) References Self-powered IIa Plasma 30 1-100 410 (Jung et al. triboelectric aptasensor 2016)* Bio-dots/AuNPs IIa Serum 60 0-35 1,050 (Kuang et al. nanosensor with FRET 2016)** detection Aptasensor with IIa Serum/ 150 0-0.05 0.01 (Hao and Zhao fluorogenic substrate plasma 2016)*** Cyclic peptide coated IIa plasma 180 0.005-0.2 5 (Zhao and Gao magnetic bead with 2015)**** fluorogenic substrate Colorimetric assay IIa Plasma 90 0.001-0.1 0.2 (Chen et al. with chromogenic 2010)***** substrate Fluorometric Assay kit Xa Plasma 30-60 0-4.4 444 (MyBiosource) ****** Biosensor with ODI- IIa Plasma 2 0.3-27.2 104 This research CL detection Biosensor with ODI- IIa Whole 4 0.21-10 66 This research CL detection blood Biosensor with ODI- Xa Plasma 2 0.25-20 44 This research CL detection Biosensor with ODI- Xa Whole 4 0.06-5 18 This research CL detection blood *Jung, Y.K., Kim, K.N., Baik, J.M., Kim, B.-S., 2016 Self-powered triboelectric aptasensor for label-free highly specific thrombin detection. Nano Energy 30, 77-83. **Kuang, L., Cao, S.P., Zhang, L., Li, Q.H., Liu, Z.C., Liang, R.P., Qiu, J.D., 2016. A novel nanosensor composed of aptamer bio-dots and gold nanoparticles for determination of thrombin with multiple signals. Biosens Bioelectron 85, 798-806. ***Hao, L.H., Zhao, Q., 2016. Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate. Microchim Acta 183(6), 1891-1898. **** Zhao, Q., Gao, J., 2015. Sensitive and selective detection of thrombin by using a cyclic peptide as affinity ligand. Biosens Bioelectron 63, 21-25. ***** Chen, C.K., Huang, C.C., Chang, H.T., 2010. Label-free colorimetric detection of picomolar thrombin in blood plasma using a gold nanoparticle-based assay. Biosens Bioelectron 25(8), 1922-1927. ******(https://www.mybiosource.com/prods/Assay-Kit/Factor-Xaidatasheet.php? products_id=841634)

[0069] TABLE 4 shows that the sensitivity of a biosensor with ODI-CL detection, capable of quantifying IIa and Xa in plasma and whole blood, is as low as other methods operated with 10100 fold diluted human samples such as serum and plasma.

[0070] The fluorogenic substrate for the coagulation factors IIa and Xa having a fluorescent dye (AMC) have good specificity and selectivity. FIGS. 12A and 12B show that the fluorogenic substrate of the present invention does not react with other main proteins (e.g., Glucose, Hemoglobin, HSA, IgG) existing in a whole blood. The relative CL intensity of biosensor in the absence and presence of main proteins may be enhanced with the extension of the incubation time because a whole blood (e.g., 10%) contains trace levels of IIa and Xa.

[0071] Also, FIGS. 6C and 6D are related to experiments that test whether the fluorogenic substrates for the coagulation factors IIa and Xa conjugated with AMC can specifically and selectively react with active IIa or Xa in the presence of anticoagulants. FIGS. 6C and 6D show that the fluorogenic substrates for the coagulation factors IIa and Xa conjugated with AMC applied in the biosensor have good specificity and selectivity. IIa substrate conjugated with AMC can specifically interact with active IIa not bound with IIa anticoagulant (e.g., Dabigatran) while Xa fluorogenic substrate can selectively react with active Xa not bound with Xa anticoagulant (e.g., Rivaroxaban). Thus, the biosensor confirmed that trace levels of active IL are present in patient whole blood with Dabigatran as shown in FIG. 6C. The relative CL intensity measured after the reaction between Xa and Xa substrate conjugated with AMC in patient whole blood with Dabigatran was strong because Xa doesn't bind with Dabigatran (FIG. 6C). As shown in FIG. 6D, the biosensor confirmed that the concentration of active Xa in patient whole blood with Rivaroxaban is very low. Also, FIG. 6D shows that the relative CL intensity measured after the reaction of IIa and IIa substrate conjugated with AMC was strong in patient whole blood in the presence of Xa anticoagulant. In conclusion, the results shown in FIGS. 6C and 6D indicate that the biosensor operated with the substrates conjugated with AMC can be applied to prevent bleeding, thrombosis, and stroke with excellent selectivity and specificity.

[0072] TABLE 5 shows that the accuracy, precision, and recovery of the biosensor for whole blood are as good as those for human plasma.

TABLE-US-00005 TABLE 5 (Accuracy, precision, and recovery for the all-in-one Biosensor with ODI-CL detection for the quantization of IIa and Xa in whole blood (N = 5)) Sample 1 Sample 2 Expected Measured Recovery Factor (nM) (nM) (nM) (nM) (%) II.sub.a 0.5 1.5 1.0 0.95 0.05 95.9 1.5 3.5 2.5 2.66 0.18 106.8 X.sub.a 0.8 2.0 1.4 1.35 0.09 96.4 1.4 4.2 2.8 2.63 0.20 93.9

[0073] Accordingly, a biosensor with ODI-CL detection according to exemplary embodiments of the present invention rapidly quantify the coagulation factors IIa and Xa in whole blood with acceptable reproducibility as compared to conventional biosensors.

[0074] Additionally, TABLE 6 shows that the concentrations of IIa and Xa in whole blood quantified using the biosensor with ODI-CL detection are the same as those determined using the conventional method with fluorescence detection within the statistically acceptable error range.

TABLE-US-00006 TABLE 6 (Quantification of IIa and Xa in whole blood using the biosensor with ODI-CL detection and conventional method with fluorescence detection (N = 3)) Biosensor with Conventional method with ODI-CL detection fluorescence detection (4-min incubation) (30-min incubation) IIa Xa IIa Xa Sample 1 0.98 (0.05) 0.63 (0.04) 0.95 (0.06) 0.67 (0.05) Sample 2 1.60 (0.09) 1.29 (0.06) 1.54 (0.11) 1.20 (0.09) Sample 3 2.85 (0.12) 2.22 (0.11) 2.99 (0.16) 2.03 (0.14)

[0075] A biosensor and a method of using a biosensor as described above may be provided in the form of a kit. In one embodiment of the present invention, the kit includes the above-described biosensor and a container. The kit may further include a buffer and an ODI-CL reagent (e.g., ODI and H.sub.2O.sub.2).

[0076] Accordingly, the present invention provides a cost-effective biosensor with ODI-CL detection which can be applied as a new device for rapid coagulation testing. The fluorescent dye (Luminophore) can be formed from the rapid reaction between coagulation factors (e.g., Xa) and a specific fluorogenic substrate. The intensity of light emitted with the addition of ODI-CL reagents (e.g., ODI, H.sub.2O.sub.2) in the solution was proportionally enhanced with the increase of the coagulation factor concentration in blood sample (e.g., plasma, whole blood). It is expected that the wide dynamic range of the biosensor with ODI-CL detection can diagnose and monitor bleeding and clotting in patients with statistically acceptable accuracy, precision, and reproducibility. In addition, the analytical procedure of the biosensor with ODI-CL detection is rapid and simple because sample pretreatment, time-consuming multiple incubations and washings aren't necessary. In conclusion, the concepts and principle of the biosensor with ODI-CL detection of the present invention can be widely applied for the early diagnosis and rapid monitoring of human diseases such as cancer, cardiac ailments, and infectious diseases (e.g., HIV, SARs, Zika virus).

EXAMPLES

[0077] The experiments described in this specification were conducted with the following materials and procedures.

[0078] Chemicals and Materials

[0079] Thrombin from human plasma (coagulation factor IIa, 100 UN) and fluorogenic substrate of thrombin (Benzoyl-Phe-Val-Arg-AMC, HCl, 25 mg) were purchased from Sigma-Aldrich. Factor Xa (human) native protein was purchased from Invitrogen. Fluorogenic substrate of factor Xa (CH.sub.3SO.sub.2-D-CHA-Gly-Arg-AMC, AcOH) was purchased from Cryopep. AMC, as a fluorescent dye (fluorophore), is 7-Amino-4-methylcoumarin. Normal plasma lyophilized with pooled human dornors (1 g) was purchased from LEE Biosolution. Bis (2,4,6-trichlorophenyl) oxalate (TCPO) and 4-methylimidazole (4 MImH) were purchased from TCI America. 3 and 30% H.sub.2O.sub.2 were purchased from VWR. Deionized H.sub.2O (HPLC grade), Ethyl acetate, Isopropyl alcohol, and high concentration of PBS (pH 7.4, 20), TBS (pH 7.410), PBST and TBST were purchased from EMD. 8-well EIA/RIA strip-well plate was purchased from Costar. Human plasma and whole blood were provided by Meritus Medical Center, IIagerstown, Md., USA.

[0080] Confirmation of the Chemical Reaction Between Coagulation Factor IIa or Xa and a Specific Fluorogenic Substrate Using ODI-CL Detection

[0081] Experiment 1: Background of Fluorogenic Substrate Only in the Absence of Factor IIa or Xa in ODI-CL Reaction (FIG. 2A)

[0082] Each fluorogenic substrate (5 mg/ml) was dissolved in DMSO as a stock solution. The stock solution was stored in a freezer (80 C.). The working solution of fluorogenic substrate (5 g/ml) diluted in PBS (pH 7,4) was prepared before conducting the experiment. Each working solution (10 l) was injected into a borosilicate test tube (12 mm75 mm). The tube was inserted into the detection area of the luminometer (Lumat LB 9507, Berthold, Inc) with two syringe pumps. 100 mM H.sub.2O.sub.2 (25 l) dissolved in isopropyl alcohol was dispensed through the first syringe pump of the luminometer. With the addition of ODI (25 l) using the second syringe pump, we investigated whether each fluorogenic substrate can emit light in the absence of factor IIa or Xa. With this procedure, we were able to determine the background of fluorogenic substrate in the absence of factor IIa and Xa in ODI-CL reaction.

[0083] Experiment 2: CL Emission of AMC Formed from the Reaction of Fluorogenic Substrate and Coagulation Factor (FIG. 2A)

[0084] Each coagulation factor (IIa or Xa, 5 nM) was prepared in 10-fold diluted plasma with deionized H.sub.2O. Each fluorogenic substrate (5 g/ml) was prepared in PBS. The mixture of factor IIa (50 l) and fluorogenic substrate (50 l) of factor IIa in a strip-well was incubated for 2 minutes under ambient condition. Also, the mixture of factor Xa (50 l) and flurogenic substrate (50 l) of factor Xa in a strip-well was also incubated for 2 minutes under ambient condition. After the incubation, each mixture (10 l) was inserted into a borosilicate test tube. H.sub.2O.sub.2 (25 l) and ODI (25 l) were consecutively dispensed through two syringe pumps of the luminometer to measure relative CL intensity of light emitted in the tube.

[0085] Experiment 3: Sensitivity of Fluorescence and ODI-CL for the Quantification of Coagulation Factor (TABLE 2)

[0086] 12 standards (0-10 nM) of factor Xa in 10% human plasma were prepared. Fluorogenic substrate (5 g/ml) of factor Xa was prepared in PBS. Each standard solution (50 l) was mixed with fluorogenic substrate (50 l) in a strip-well. The mixture was incubated for 2 minutes under ambient condition. After the incubation, relative CL intensity of each sample was measured using the luminometer operated with the same method described in Experiments 1 and 2. In order to measure fluorescence intensity of each sample, the mixture in the strip-well was incubated for 30 min under ambient condition. After the incubation, the strength of fluorescence emitted in the strip-well was measured with a microplate reader (Infinite M 1000 of Tecan, Inc.). Finally, the sensitivity of ODI-CL detection for the quantification of coagulation factor was compared with that of fluorescence detection.

[0087] Experiment 4: Quantification of Coagulation Factors in Human Plasma Using the Biosensor with ODI-CL Detection (TABLE 3)

[0088] Standards of factor IIa and Xa were prepared with 10% plasma diluted with deionized H.sub.2O. Unknown samples were prepared with 100% plasma. Then, each sample was 10-fold diluted in deionized H.sub.2O. Each standard or sample (50 l) was dispensed into a strip-well containing fluorogenic substrate. The mixture in the strip-well was incubated for 2 minutes under ambient condition. The fluorogenic substrate of factor IIa (5 g/ml) was prepared in TBST. Also, the fluorogenic substrate of factor Xa (5 g/ml) was prepared in PBS. After the incubation, light emitted from each mixture with the addition of ODI CL reagents was measured for 2 sec using the luminometer.

[0089] Experiment 5: Quantification of Coagulation Factors in Human Whole Blood Using the Biosensor with ODI-CL Detection (TABLE 5)

[0090] Standards of factor IIa and Xa were prepared with 10% whole blood diluted with deionized H.sub.2O. Unknown whole blood samples were 10-fold diluted in deionized H.sub.2O. Each standard or sample (50 l) was dispensed into a strip-well containing fluorogenic substrate. The mixture in the strip-well was incubated for 4 minutes under ambient condition. The fluorogenic substrate of factor IIa (25 g/ml) was prepared in TBST. Also, the fluorogenic substrate of factor Xa (25 g/ml) was prepared in PBS. After the 4-min incubation, light emitted from each mixture with the addition of ODI-CL reagents was measured for 2 sec using the luminometer.

[0091] Experiment 6: Correlation Between Biosensor with ODI-CL Detection and Conventional Method with Fluorescence Detection for the Quantification of IIa and Xa in 10-Fold Diluted Plasma and Whole Blood (TABLE 4)

[0092] In order to confirm the correlation between the biosensor with ODI-CL detection and the conventional method with fluorescence detection, the concentrations of IIa and Xa in 10-fold diluted plasma or whole blood (e.g., standards, samples) were determined with a microplate reader with fluorescence detection (Infinite M 1000, Tecan, Inc). The concentrations of fluorogenic substrates of IIa and Xa for the quantification of IIa and Xa using the conventional method were the same as those using the biosensor with ODI-CL detection described in Experiments 4 and 5. Each standard or sample (50 l) was mixed with fluorogenic substrate (50 l) in a black well. The black well-plate (96 well, Greiner Bio-One) containing various mixtures, was inserted into the microplate reader with fluorescence detection and incubated for 30 min at room temperature. After the incubation, the relative intensity of fluorescence emitted from each well was measured at 440 nm emission wavelength (excitation wavelength: 342 nm). After determining the concentrations of samples in plasma and whole blood using the conventional method, they were compared with those that used the biosensor with ODI-CL detection to confirm the correlation between the new and conventional methods.

[0093] Analysis of Experimental Data

[0094] All experimental results observed in this specification were analyzed using the statistical tools of Microsoft Excel and SigmaPlot 12.5 (Systat software, Inc.).

[0095] It is to be understood that the above-described biosensor and method are merely illustrative embodiments of the principles of this disclosure, and that other compositions and methods for using them may be devised by one of ordinary skill in the art, without departing from the spirit and scope of the invention. It is also to be understood that the disclosure is directed to embodiments both comprising and consisting of the disclosed parts.