OPTICAL SENSORS

Abstract

An optical sensor is used to detect a target in a sample liquid. The optical sensor has an optical sensing layer including, in the same layer, a fluorescent reporter, a fluorescent reference, and an optical isolating reagent. The optical sensing layer has an outer surface that contacts the sample liquid and an opposing surface through which a light source irradiates the optical sensing layer with excitation light, and a detector detects fluorescence emitted from within the optical sensing layer. The optical isolating reagent reduces the amount of (i) excitation light that passes through the optical sensing layer and reaches the sample liquid and (ii) background fluorescence that is emitted within the sample liquid and passes back through the optical sensing layer to the detector. Accordingly, the optical reporter and optical reference fluorescence can be detected with higher signal to noise ratios than in the absence of the optical isolating reagent.

Claims

1.-12. (canceled)

13. An optical sensor for detecting a target present in a sample fluid, comprising: a detection chamber configured to receive the sample fluid; a polymeric optical sensor medium that (i) is disposed within the detection chamber and has an inner surface configured to contact sample fluid present in the detection chamber, (ii) is at least semi-permeable to the target, (iii) comprises a luminescent reagent that is covalently bound to a polymer of the polymeric optical sensing layer and is configured to generate an optical signal indicative of the presence of the target within the optical sensor medium when the optical sensor medium is irradiated with excitation light from an excitation source, and (iv) comprises an optical reference that is covalently bound to a polymer of the polymeric optical sensing layer and is configured to generate an optical signal generally independent of the presence of the target within the optical sensor medium when the optical sensor medium is irradiated with light from an excitation source, and (v) has a transmittance of about 20% or less at a wavelength of maximum intensity of the excitation light.

14. The optical sensor of claim 13, wherein the optical sensor medium has a thickness in the dry state, of about 75 μm or less along an optical axis of the excitation light from the excitation source.

15. The optical sensor of claim 13, wherein the optical sensor medium has a thickness in the dry state, of at least about 20 μm along an optical axis of the excitation light from the excitation source.

16. The optical sensor of claim 13, wherein the detection chamber comprises a first wall that is transparent at a wavelength of maximum intensity of the excitation light, the optical sensor medium has an outer surface disposed against an inner surface of the first wall of the detection chamber, and an optical axis of the excitation light passes through the first wall into the optical sensor medium.

17. The optical sensor of claim 13, wherein the luminescent reagent comprises a first moiety configured to interact with the target, a second moiety comprising a fluorescent moiety, and a linker by which the luminescent reagent is covalently bound to the polymer and wherein the linker is disposed between the first moiety and the second moiety or is disposed on the first moiety.

18. The optical sensor of claim 17, wherein the first moiety is configured to chelate a target.

19. The optical sensor of claim 13, wherein the optical isolating reagent comprises carbon black, carbon nanostructures, and/or a non-fluorescent dye.

20. (canceled)

21. The optical sensor of claim 13, wherein the sample fluid comprises blood, plasma, or serum.

22. (canceled)

23. The optical sensor of claim 13, wherein the target is selected from the group consisting of Ca++, K+, Na+, or H+, creatinine, lactate, and glucose.

24. A method of detecting a target present in a sample fluid, the method comprising: contacting a first surface of a polymeric optical sensor medium with the sample fluid, the optical sensor medium being at least semi-permeable to the target and comprising a luminescent reagent covalently bound to a polymer of the polymeric optical sensor medium and configured to generate an optical signal indicative of the presence of the target within the optical sensor medium when the optical sensor medium is irradiated with excitation light from an excitation source and an optical reference covalently bound to a polymer of the polymeric optical sensing layer configured to generate an optical signal generally independent of the presence of the target within the optical sensor when the optical sensor medium is irradiated with excitation light from an excitation source; irradiating the optical sensor medium with the excitation light along an optical axis that passes into the optical sensor medium through a second surface of the optical sensor medium not in contact with the sample fluid, the optical axis being oriented toward the first surface of the optical sensor medium; and absorbing at least about 80% of the excitation light within the optical sensor medium prior to the excitation light reaching the first surface of the optical sensor medium.

25.-28. (canceled)

29. The method of claim 24, wherein essentially all of the detected fluorescence arises from the luminescent reagent or the optical reference in the optical sensing layer with the optical isolating reagent.

30. The method of claim 24, wherein the target is selected from the group consisting of Ca++, K+, Na+, or H+, creatinine, lactate, and glucose.

31. The method of claim 24, wherein the optical isolating reagent comprises carbon black, carbon nanostructures, and/or a non-fluorescent dye.

32. The method of claim 24, wherein the optical sensing layer has a thickness, in the dry state, of about 75 μm or less.

33. The method of claim 24, wherein the optical sensing layer has a thickness, in the dry state, of at least about 20 μm.

34. The method of claim 24, wherein the luminescent reagent comprises a first moiety configured to interact with the target, a second moiety comprising a fluorescent moiety, and a linker by which the luminescent reagent is covalently bound to the polymer and wherein the linker is disposed between the first moiety and the second moiety or is disposed on the first moiety.

35. The method of claim 34, wherein the first moiety is configured to chelate a target.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1 is a side view of an optical sensor of the invention;

[0051] FIG. 2 is a graph of fluorescence emitted by a sodium ion optical reporter and an optical reference at different sodium ion concentrations; and

[0052] FIG. 3 is a graph of the ratio of the fluorescence of the optical reporter signal S and the optical reference signal S.sub.ref obtained from the fluorescence spectra of FIG. 2.

DETAILED DESCRIPTION

[0053] With reference to FIG. 1, an optical sensor 10 is configured to determine the presence and/or amount of a target present in a sample liquid 20. Sensor 10 includes an optical sensing layer 12 disposed within a detection chamber 14 defined by walls 16 and 18. Detection chamber wall 18 includes an optically transmissive window 20. Optical sensing layer 12 has a secured surface 22 secured with respect to an inner window surface 24 of optically transmissive window 20 and an exposed sensor surface 26 exposed to sample liquid within detection chamber 14. Optically transmissive window is an optically transparent support for the optical sensing layer 12.

[0054] Optical sensing layer 12 is a semi-permeable polymer matrix and includes, disposed therein, an optical reporter 36, an optical reference 38, and an optical isolating reagent 40. Optical reporter 36 is configured to emit fluorescence when irradiated with excitation light falling within an excitation wavelength band of the optical reporter. Optical reference 38 is configured to emit fluorescence when irradiated with excitation light falling within an excitation wavelength band of the optical reference. The excitation wavelength band of optical reporter 36 and the excitation wavelength band of optical reference 38 may substantially overlap, e.g., be the same, or may have essentially no overlap so that light within the excitation wavelength band of one does not efficiently excite fluorescence from the other. The fluorescence emission range of the optical reporter 36 and optical reference 38 may substantially overlap, e.g., be the same, or may have essentially no overlap so that light detected within a fluorescence emission band of one can be distinguished from light detected within a fluorescence emission band of the other. Typically, the excitation wavelength bands of the optical reporter and optical reference are different and/or the fluorescence emission bands of the optical reporter and optical reference are different so that the reporter and reference responses can be readily distinguished.

[0055] Optical sensor 10 also includes a light source 28 and a detector 32. Light source 28 irradiates optical sensing layer 12 through window 20 with excitation light 30 falling within the excitation wavelength band of the optical reporter 36 and falling within the excitation wavelength band of the optical reference 38. Detector 32 detects fluorescence 34 through window 20 emitted from each of the irradiated optical reporter 36 and optical reference 38 residing within optical sensing layer 12. Window 20 acts as a support for optical sensing layer 12 and permits the excitation and detection of luminescence from within optical sensing layer 12, but window 20 does not otherwise participate in the determination of a particular target.

[0056] Optical isolating reagent 40 attenuates light at wavelengths falling within the excitation wavelength band(s) and/or the fluorescence emission band(s) of the optical reporter and the optical reference. For example, the average transmittance of sensing layer 12 for light impinging upon sensing layer 12 from light source 28 within the excitation wavelength bands of one or both of optical reporter 36 and optical reference 38 may be about 50% or less, about 25% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less over a distance dl corresponding to the thickness of sensing layer 12. The average transmittance of sensing layer 12 for light impinging upon sensing layer 12 from light source 28 within the excitation wavelength bands of one or both of optical reporter 36 and optical reference 38 may be at least about 0.5%, at least about 1%, at least about 2%, or at least about 3% over a distance dl corresponding to the thickness of sensing layer 12. Alternatively, or in combination, the average transmittance of sensing layer 12 for light detected by detector 32 within the fluorescence emission bands of one or both of optical reporter 36 and optical reference 38 may be about 50% or less, about 25% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less over distance dl corresponding to the thickness of sensing layer 12. The average transmittance of sensing layer 12 for light detected by detector 32 within the fluorescence emission bands of one or both of optical reporter 36 and optical reference 38 may be at least about 0.5%, at least about 1%, at least about 2%, or at least about 3% over a distance dl corresponding to the thickness of sensing layer 12.

[0057] Because the optical isolating reagent reduces the amount of excitation light that passes through the optical sensing layer into the sample liquid, the amount of background fluorescence generated within the sample liquid is reduced. In addition, optical isolating reagent attenuates the amount of such background fluorescence within the fluorescence emission bands of the optical reporter and optical reference that passes through the optical sensing layer and reaches the detector. The optical isolating reagent also attenuates some excitation light that would have otherwise reached the optical reporter and optical reference and some of the fluorescence emitted therefrom that would have otherwise reached the detector. However, because the optical sensing layer is semi-permeable with respect to the target, the target diffuses through exposed sensor surface 26 into the optical sensing layer where it reacts with the optical reporter. Therefore, the average pathlength within the optical sensing layer for excitation light reaching the optical reporter and optical reference is shorter than the average pathlength for excitation light passing through the sensing layer into the sample liquid. Therefore, the excitation light is attenuated to a lesser extent before reaching the optical reporter and optical reference than for the sample liquid. The average pathlength within the optical sensing layer for fluorescence emitted by the optical reporter and optical reference is also shorter than the average pathlength for fluorescence emitted within the sample liquid and passing through the optical sensing layer and out of the second surface 22 before being detected by detector 32. Therefore, the fluorescence emitted by the optical reporter and optical reference is attenuated to a lesser extent before reaching the detector than for fluorescence generated within the sample liquid. As a result, the background fluorescence is attenuated to a greater extent than the optical reporter fluorescence so that the signal to noise ratio is increased as compared to the absence of the optical isolating agent, e.g., carbon black.

[0058] The optical isolating reagent of the optical sensing layer, e.g., carbon black, may be stabilized. The use of the stabilized optical isolating reagents can improve rheological properties such as viscosity, sedimentation, flow and/or dispensing behavior. For example, the optical isolating reagent may be stabilized using a non-ionic dispersant or slightly anionic dispersant in a concentration of 0.1% to 1% by weight. Exemplary non-ionic dispersants or slightly anionic dispersants include alcohol alkoxylates such as alcohol ethoxylates, polymeric ethoxylated non-ionic wetting reagents, modified polymers with pigment affinity groups, and/or oxirane, phenyl-, polymer with oxirane, monoalkyl ethers. Exemplary commercial products include Tego® 760W, Tego® 761W, Tego® 755W, and Tego® 650. As another example, the optical isolating reagent may be stabilized using a surface modified carbon black from 0.5% to 1% by weight that incorporates hydrophilic groups for solvent affinity. Examples of such dispersants include alcohols, C13-C15, branched and/or linear, ethoxylated. Exemplary commercial products include PX Kappa and OE430W from Orion Engineered Carbons (Sennengerberg, Luxembourg), and XSL from Kremer Pigments (New York, N.Y.). As yet another example, the optical isolating reagent may be stabilized using pre-stabilized carbon black dispersion from 0.5% to 1% by weight. Exemplary pre-stabilized carbon black dispersions may be stabilized with, e.g., ethylene glycol and surfactants. Suitable commercial products include Aquablack 8367 and Aquablack 8386 from ChromaScape (Independence, Ohio).

EXAMPLES

Examples 1 and 2 Refer to the Following Diagram

[0059] ##STR00010##

Example 1: Synthesis of 3,6-dihydroxy-9H-xanthen-9-one (1)

[0060] 2,2′,4,4′-Tetrahydroxybenzophenone (5.0 g, 20.3 mmol) was dispensed in ten equal portions into glass culture tubes sealed loosely with a rubber septum and heated to 210° C. on a heat block. The reaction liquefied then re-solidified upon heating overnight. The tubes were cooled and the solids suspended in MeOH and combined. The volume was reduced to approximately 30 mL by rotary evaporation and the solids were collected by filtration and rinsed with a little MeOH then dried in vacuo to give (1) (3.78 g, 82%) as a brown solid. mp>250° C. (R.sub.f=0.46, 2:8 hexanes:EtOAc). Observed [M+H].sup.+=229.0 m/z (calc'd for C.sub.13H.sub.9O.sub.4 229.2 m/z).

Example 2: Synthesis of 3,6-bis((tert-butyldimethylsilyl)oxy)-9H-xanthen-9-one (2)

[0061] 3,6-dihydroxy-9H-xanthen-9-one (1) (2.0 g, 8.76 mmol) from Example 1 and imidazole (1.79 g, 26.3 mmol) were dissolved in anhydrous DMF (44 mL) and treated with TBS-Cl (2.91 g, 19.3 mmol) and stirred vigorously at rt. After 4 hrs, the mixture was diluted with EtOAc and washed with 1N HCl (×3), H.sub.2O, brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was dissolved in CHCl.sub.3 and purified by flash column chromatography on SiO.sub.2 eluted with 95:5 hexanes:EtOAc. The resulting solids triturated with a small volume of hexanes, collected by filtration, and dried in vacuo to give compound 2 (3.42 g, 85%) as a white crystalline solid. mp 152-153° C. (R.sub.f=0.43, 9:1 hexanes:EtOAc). .sup.1HNMR (400 MHz, CDCl.sub.3): δ 0.28 (s, 12H), 1.01 (s, 18H), 6.83 (s, 2H), 6.85 (dd, J=9.2, 2.2 Hz, 2H), 8.20 (ddd, J=9.2, 1.4, 1.4 Hz, 2H). .sup.13C NMR (10 MHz, CDCl.sub.3): δ −4.21, 18.42, 25.70, 107.49, 116.57, 117.73, 128.34, 157.89, 161.51, 175.88.

Examples 3 and 4 Refer to the Following Diagram

[0062] ##STR00011##

Example 3: Synthesis of (±)-2-((4-allyl-2-methoxyphenoxy)methyl)oxirane (3)

[0063] Eugenol (7.4 g, 45.1 mmol), ±-ephichlorohydrin (10.6 mL, 135 mmol), and Bu.sub.4NHSO.sub.4 (766 mg, 2.26 mmol) were dissolved in 1,4-dioxane (23 mL) and treated with freshly prepared 5M NaOH (27 mL) and heated in a pre-heated 80° C. oil bath for 30 min. The mixture was then cooled on ice and partitioned between EtOAc and sat. NaHCO.sub.3. The aqueous was extracted once with EtOAc and the combined extract was washed with sat. NaHCO.sub.3, brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 15.fwdarw.20% EtOAc in hexanes to give (3) (7.5 g, 75%) as a colorless oil. (R.sub.f=0.27, 8:2 hexanes:EtOAc). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 2.73 (dd, J=4.8, 2.7 Hz, 1H), 2.89 (dd, J=4.8, 4.2 Hz, 1H), 3.33 (br d, J=6.7 Hz, 1H), 3.36-3.41 (m, 1H), 3.38 (s, 3H), 4.02 (dd, J=11.4, 5.5 Hz, 1H), 4.21 (dd, J=11.4, 3.6 Hz, 1H), 5.04-5.07 (m, 1H), 5.07-5.11 (m, 1H), 5.95 (dddd, J=16.9, 10.2, 6.7, 6.7 Hz, 1H), 6.69-6.74 (m, 2H), 6.87 (d, J=8.1 Hz, 1H).

Example 4: Synthesis of (±)-1-(4-allyl-2-methoxyphenoxy)-3-(2-hydroxyethoxy)propan-2-ol (4)

[0064] (±)-2-((4-allyl-2-methoxyphenoxy)methyl)oxirane from Example 3 (7.5 g, 34 mmol) was dissolved in anhydrous THE (34 mL) and anhydrous ethylene glycol (34 mL) and cooled to 0° C. then treated with BF.sub.3.Et.sub.2O. The mixture was stirred for 30 min, then the cooling bath was removed and stirring continued for an additional 5 hrs. The mixture was diluted with EtOAc and washed with sat. NaHCO.sub.3(×3), brine (×2), dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 1.fwdarw.5% MeOH in CH.sub.2Cl.sub.2 to give (4) (7.47 g, 78%) as a colorless, viscous oil. (R.sub.f=0.43, 9:1 hexanes:EtOAc). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.30 (br s, 2H), 3.33 (br d, J=6.68 Hz, 2H), 3.60-3.63 9 m, 2H), 3.65 (d, J=5.8 Hz, 1H), 3.69 (dd, J=10.1, 4.0 Hz, 1H), 3.72-3.76 (m, 1H), 3.84 (s, 3H), 4.02 (dd, J=9.8, 6.5 Hz, 1H), 4.05 (dd, J=9.6, 4.5 Hz, 1H), 4.15-4.22 (m, 1H), 5.04-5.06 (m, 1H), 5.06-5.10 (m, 1H), 5.94 (dddd, J=16.9, 10.2, 6.7, 6.7 Hz, 1H), 6.69-6.73 (m, 2), 6.85 (d, J=8.6 Hz, 1H).

Examples 5-9 Refer to the Following Diagram

[0065] ##STR00012## ##STR00013##

Example 5: 2,2′-((((2-methoxy-5-(trifluoromethyl)phenyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-ol) (5)

[0066] A 150 mL pressure bottle was charged with 2-methoxy-5-(trifluoromethyl)aniline (972 mg, 5.09 mmol. Caution: stench chemical.), KI (1.27 g, 7.64 mmol), CaCO.sub.3 (560 mg, 5.60 mmol), and 2-(2-chloroethoxy)ethanol (1.27 mL, 7.64 mmol) and heated to 150° C. under N.sub.2 atmosphere overnight. The reaction was cooled, vented, and additional CaCO.sub.3 (1.0 g) and 2-(2-chloroethoxy)ethanol (2 mL) was introduced and the mixture re-heated to 150° C. After 6 hrs, the mixture was cooled and diluted with 1N HCl then washed with Et.sub.2O (×2). The combined wash was back-extracted with 1N HCl once and the combined aqueous was basified with K.sub.2CO.sub.3, shaken with EtOAc, and filtered through Celite. The organic phase was collected and the aqueous extracted twice more with EtOAc. The combined extract was washed with brine, dried over Na.sub.2SO.sub.4, and concentrated to give (5) (2.3 g, >100%) of an orange colored oil which was used without further purification.

Example 6: 2,2′-((((4-bromo-2-methoxy-5-(trifluoromethyl)phenyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-ol) (6)

[0067] The aniline compound (5) from Example 5 (2.3 g crude, 5.09 mmol) was dissolved in DMF, cooled to 0° C., and treated with solid N-bromosuccinimide (906 mg, 5.09 mmol). After 5 min, additional NBS (75 mg) was added. After an additional 5 min, the reaction mixture was poured into 1N HCl (75 mL) and washed with Et.sub.2O (2×50 mL). The combined wash was back-extracted with 1N HCl (25 mL) and the combined aqueous was neutralized with K.sub.2CO.sub.3 and extracted with EtOAc (×3). The combined extract was washed with brine (×2), dried over Na.sub.2SO.sub.4, and concentrated to give (6) as a brown oil which was used without further purification.

Example 7: ((((4-bromo-2-methoxy-5-(trifluoromethyl)phenyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate) (7)

[0068] The diol compound (6) from Example 6 (2.5 g crude, 5.09 mmol) was dissolved in anhydrous DCM (10 mL), cooled to 0° C., and treated with tosyl chloride (2.13 g, 11.2 mmol), Et.sub.3N (1.77 mL, 12.7 mmol), and DMAP (62 mg, 0.51 mmol). The cooling bath was removed and the mixture was stirred at rt for 75 min then diluted with EtOAc and washed with sat. NaHCO.sub.3 (×3), brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 20.fwdarw.40% EtOAc in hexanes to give (7) (3.21 g, 84% over 3 steps) as a faintly yellow oil. (R.sub.f=0.39, 6:4 hexanes:EtOAc). Observed [M+H].sup.+=754.3/756.3 m/z (1:1) (calc'd for C.sub.30H.sub.36BrF.sub.3NO.sub.9S.sub.2 754.1 m/z).

Example 8: 5-((4-allyl-2-methoxyphenoxy)methyl)-16-(4-bromo-2-methoxy-5-(trifluoromethyl)phenyl)-1,4,7,10,13-pentaoxa-16-azacyclooctadecane (8)

[0069] The 1,5-diol compound (4) from Example 4 (1.20 g, 4.25 mmol) and the ditosylate compound (7) from Example 7 (3.21 g, 4.25 mmol) were combined and co-evaporated once from anhydrous THF, dried briefly in vacuo, then redissolved in anhydrous THE (10 mL). A 100 mL round bottom flask fitted with a reflux condenser was flame dried then charged with anhydrous THE (30 mL), and NaH (1.02 g, 25.5 mmol) that had been washed twice with hexanes immediately prior. The mixture was heated to reflux and the solution of compounds (4) and (7) was added dropwise over a period of 30 min. After an additional 4 hrs, the mixture was cooled to rt and carefully poured into a mixture of EtOAc and sat. NH.sub.4Cl. The organic phase was collected and the aqueous extracted twice more with EtOAc. The combined extract was dried over MgSO.sub.4, amended with 5% vol MeOH, and filtered through a pad of basic alumina. The filtrate was concentrated and purified by flash column chromatography on SiO.sub.2 eluted with 20-50% EtOAc in hexanes+2% vol Et.sub.3N to give (8) (777 mg, 26%) as a faintly fellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.32 (br d, J=6.6 Hz, 2H), 3.46 (br dd, J=12.5, 6.1 Hz, 4H), 3.52-3.80 (m, 18H), 3.82 (s, 3H), 3.85 (s, 3H), 3.96-4.04 (m, 1H), 4.04-4.10 (m, 2H), 5.94 (dddd, J=16.8, 10.1, 6.7, 6.7 Hz, 1H), 6.66-6.72 (m, 2H), 6.82-6.89 (m, 1H), 7.05 (s, 1H), 7.32 (s, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 39.9, 52.2, 52.5, 56.06, 56.07, 69.7, 69.8, 69.9, 70.0, 70.3, 70.7, 70.9, 71.10, 71.11, 71.7, 78.0, 109.4, 111.6, 112.7, 114.3, 115.7, 117.6, 118.8, 119.6, 119.7, 120.7, 122.1, 122.4, 124.1, 124.8, 130.7, 133.5, 137.8, 139.1, 146.9, 149.7, 155.0. .sup.19F NMR (101 MHz, CDCl.sub.3): δ −61.0. Observed [M+H].sup.+=692.4/694.4 m/z (1:1) (calc'd for C.sub.31H.sub.42BrF.sub.3NO.sub.8 692.2 m/z).

Example 9: 9-(4-(5-((4-allyl-2-methoxyphenoxy)methyl)-1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)-5-methoxy-2-(trifluoromethyl)phenyl)-6-hydroxy-3H-xanthen-3-one (9)

[0070] The aryl bromide compound (8) of Example 8 (150 mg, 0.217 mmol) was dissolved in MeOH and filtered through a pad of basic alumina and the filtrate was concentrated, co-evaporated twice from 2-Me-THF, and dried thoroughly in vacuo. The residue was redissolved in anhydrous 2-Me-THF (1.1 mL) and cooled in to −116° C. (N.sub.2/EtOH) and tert-butyllithium (285 μL, 0.541 mmol, 1.9M in pentane) was added via the cold sidewall. The pale orange suspension was stirred cold for 10 min. Xanthone 2 (119 mg, 0.26 mmol) was dried by gently warming in vacuo then dissolved in anhydrous 2-Me-THF (1 mL) and added slowly to the aryllithium intermediate via the cold sidewall. A dark red color developed. The mixture was stirred cold for 10 min, then the cooling bath was removed and the mixture was stirred at rt for 10 min which caused the color to fade to orange. A mixture of TFE (1.1 mL) and 2N HCl (1.1 mL) was added which caused the color to dissipate, liberating an intermediate green color, and ultimately a bright red/orange color. The mixture was stirred for 10 min then diluted with 0.5M HCl and washed with Et.sub.2O (×2). The ethereal wash was back-extracted with two small portions of 0.5M HCl and the combined aqueous was neutralized with Na.sub.2CO.sub.3 then back-extracted with EtOAc (×3). The extract was dried over MgSO.sub.4 and concentrated, and the residue was purified by flash column chromatography on SiO.sub.2 eluted with 2.fwdarw.12% MeOH in CH.sub.2Cl.sub.2+2% vol Et.sub.3N to give (9) (59.4 mg, 33%) as an orange film. Observed [M+H].sup.+=824.2 m/z (calc'd for C.sub.44H.sub.49F.sub.3NO.sub.11 824.3 m/z).

Example 10: Preparation of a Sodium Ion (Na.SUP.+.) Sensing Layer

[0071] Example 10 refers to the following diagram:

##STR00014##

[0072] An optical reporter having a terminal vinyl group is reacted with two vinyl monomers in a solution of 2% w/v azobisisobutyronitrile (AIBN) in THE at 70° C. The resulting hydrophilic polymer between about 0.1% to 1% by weight optical reporter, which is covalently bound to the polymer. The polymer is semipermeable with respect to sodium ion.

Example 11: Preparation of an Optical Sensor for the Determination of Sodium Ion

[0073] A mixture is prepared containing: (i) 10% by weight hydrophilic urethane polymer; (ii) 1% by weight carbon black; (iii) 5% by weight of the polymer of Example 10; and (iv) the remainder a 90:10 EtOH:H2O solution. The polymer is applied as a thin layer within the detection zone of a microfluidic strip such as a microfluidic strip disclosed in the '946 application or '325 application. The applied polymer is dried for 3 minutes at 65° C., in which time substantially of the solvent solution evaporates and leaves behind an optical sensing layer within the detection zone of the microfluidic strip.

[0074] In use, the microfluidic strip is inserted into a reader, such as one described in the '946 application or '325 application. A liquid sample, e.g., of blood, is applied to the strip. The reader operates the strip to introduce the sample into the detection zone whereupon sodium ion present in the sample diffuses into the optical sensing layer therein and interacts with the optical reporter residing within the layer. The reader has a light source that irradiates the optical sensing layer with excitation light causing the optical reporter to emit fluorescence indicative of the presence and/or amount of sodium ion present in the liquid sample. The carbon black attenuates the excitation light passing through the optical sensing layer into the sample liquid within the detection zone thereby reducing the amount of background fluorescence generated from the sample liquid. In addition, the carbon black attenuates the amount of background fluorescence that is generated before it passes through the optical sensing layer and reaches the detector of the reader. The carbon black also attenuates some excitation light before it reaches the optical reporter and some of the fluorescence emitted by the optical reporter. However, because the sodium ion diffuses into the polymer layer, the average pathlength within the optical sensing layer of such excitation light and fluorescence is less than average pathlength for light traveling through the optical sensing layer into the liquid and for background fluorescence emitted within the liquid and traveling through the optical sensing layer out of the detection zone. Therefore, the background fluorescence is attenuated to a greater extent than the optical reporter fluorescence so that the signal to noise ratio is increased as compared to in the absence of the carbon black.

Example 12: Preparation of a Sodium Ion (Na.SUP.+.) Sensing Layer Including an Optical Reporter and an Optical Reference

[0075] Example 12 refers to the following diagram:

##STR00015##

[0076] An optical reporter and an optical reference each having a terminal vinyl group are reacted with two vinyl monomers in a solution of 2% w/v azobisisobutyronitrile (AIBN) in THE at 70° C. The resulting hydrophilic polymer between about 0.1% to 1% by weight each of the optical reporter and optical reference, which are covalently bound to the polymer. The polymer is semipermeable with respect to sodium ion.

Example 13: Preparation of an Optical Sensor with Internal Reference for the Determination of Sodium Ion

[0077] A mixture is prepared containing: (i) 10% by weight hydrophilic urethane polymer; (ii) 1% by weight carbon black; (iii) 5% by weight of the polymer of Example 12; and (iv) the remainder a 90:10 EtOH:H2O solution. The polymer is applied as a thin layer within the detection zone of a microfluidic strip such as a microfluidic strip disclosed in the '946 application or '325 application. The applied polymer is dried for 3 minutes at 65° C., in which time substantially of the solvent solution evaporates and leaves behind an optical sensing layer within the detection zone of the microfluidic strip.

Example 14: Calibration Spectra and Calibration Curve for a Sodium Sensor

[0078] The mixture of Example 13 was applied to a surface of an optical flow cell and dried as per Example 13. The flow cell was inserted into a fluorimeter configure to irradiate the dried sensing layer with excitation light at 490 nm to excite fluorescence from the optical reporter and with excitation light at 550 nm to excite fluorescence from the optical reference. The fluorescence with each excitation light was measured upon exposing the sensing layer to solutions containing different sodium ion concentrations (FIG. 2). The optical reporter emits a fluorescence signal “S” indicative of the presence and/or amount of sodium ion present in the liquid sample. The excitation light causes the optical reference to emit a fluorescence signal “S.sub.Ref” the intensity and amount of which is insensitive to the presence and/or amount of sodium ion or concomitant species present in the sample liquid. Accordingly, the optical reporter fluorescence can be normalized by the optical reference fluorescence (S/S.sub.ref) to correct for variations in the system thereby increasing the accuracy and precision of the sodium ion determination (FIG. 3). The carbon black attenuates the signals, including the background fluorescence and optical reference and optical reporter fluorescence increasing the signal to noise ratio.

Examples 15 to 25 Refer to the Following Diagram

[0079] ##STR00016## ##STR00017## ##STR00018##

Example 15: 1-(2-methoxyethoxy)-2-nitrobenzene (21)

[0080] 1-Fluoro-2-nitrobenzene (10 g, 71 mmol) and 2-methoxyethanol (6.5 g, 85 mmol) were dissolved in DMF:THF (30:1, 70 mL) and NaH dispersion (3.4 g, 85 mmol) was added portionwise over approximately 15 min. After an additional 15 min, the reaction was quenched by careful addition of H.sub.2O then diluted with EtOAc and washed with H.sub.2O (×1), K.sub.2CO.sub.3 (×2), brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was taken up in MeOH and washed with three small portions of hexanes. The methanolic phase was concentrated and distilled in vacuo to give compound (21) (11.1 g, 79%) as an orange oil. Bp 120-130° C., 1 torr. (R.sub.f=0.15, 8:2 hexanes:EtOAc). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.45 (s, 3H), 3.78-3.82 (m, 2H), 4.23-4.28 (m, 2H), 7.04 (ddd, J=8.1, 7.4, 1.0 Hz, 1H), 7.11 (dd, J=8.5, 1.0 Hz, 1H), 7.52 (ddd, J=8.5, 7.4, 1.7 Hz, 1H), 7.83 (dd, J=8.1, 1.7 Hz, 1H).

Example 16: 2-(2-methoxyethoxy)aniline (22)

[0081] Nitro compound 21 (11.1 g, 56.1 mmol) was dissolved in MeOH (140 mL). A slurry of Pd/C in MeOH was prepared under N.sub.2 atmosphere and added to the substrate solution. The vessel was evacuated and backfilled with H.sub.2 several times then stirred vigorously for 21 hrs. The mixture was filtered through Celite and the cake carefully rinsed with MeOH. The filtrate was concentrated and purified by flash column chromatography on SiO.sub.2 eluted with 20.fwdarw.40% EtOAc in hexanes to give compound (22) (8.48 g, 90%) as a pale brown oil. (R.sub.f=0.38, 6:4 hexanes:EtOAc). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.45 (s, 3H), 3.74-3.78 (m, 2H), 3.86 (br s, 2H), 4.12-4.17 (m, 2H), 6.69-6.77 (m, 2H), 6.79-6.84 (m, 2H). Observed [M+H].sup.+=168.3 m/z (calc'd for C.sub.9H.sub.14NO.sub.2 168.1 m/z).

Example 17: 2,2′-((2-(2-methoxyethoxy)phenyl)azanediyl)bis(ethan-1-ol) (23)

[0082] A 150 mL pressure bottle was charged with aniline 22 (8.48 g, 50.7 mmol), KI (8.42 g, 50.7 mmol), CaCO.sub.3 (45.6 mmol), and 2-chloroethanol (10.2 mL, 152 mmol) and the apparatus was purged with N.sub.2, sealed, and heated to 110° C. for 20 hr. The mixture was diluted with 1N HCl and washed with two small volumes of Et.sub.20. The combined wash was back-extracted with 1N HCl once. The combined aqueous phase was basified with conc. NH.sub.40H at 0° C. and extracted with CH.sub.2Cl.sub.2 (×5). The combined extract was washed with 1M NaOH, brine, dried over Na.sub.2SO.sub.4, then diluted with 0.1 volumes of MeOH filtered through a thin pad of SiO.sub.2 rinsing with 9:1 CH.sub.2Cl.sub.2:MeOH. The filtrate was concentrated to give compound (23) (12.3 g, 95%) as a pale brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.14-3.18 (m, 4H), 3.44 (s, 3H), 3.45-3.51 (m, 4H), 3.61 (br s, 2H), 3.74-3.78 (m, 2H), 4.11-4.15 (m, 2H), 6.92 (dd, J=8.2, 1.4 Hz, 1H), 6.99 (ddd, J=7.6, 7.6, 1.4 Hz, 1H), 7.13 (ddd, J=8.1, 7.5, 1.7 Hz, 1H), 7.22 (dd, J=7.8, 1.7 Hz, 1H). Observed [M+H]*=256.2 m/z (calc'd for C.sub.13H.sub.22NO.sub.4 256.1 m/z).

Example 18: 2-(2-methoxyethoxy)-N,N-bis(2-(2-nitrophenoxy)ethyl)aniline (24)

[0083] Compound 23 (5.49 g, 21.5 mmol) and 1-fluoro-2-nitrobenzene were dissolved in anhydrous DMF:THF (30:1, 27 mL) and NaH dispersion (1.89 g, 47.3 mmol) was added portionwise over a period of approximately 15 min. An exotherm resulted which was not mitigated. After 2 hrs, the reaction was carefully quenched with H.sub.2O then partitioned between EtOAc and H.sub.2O and the aqueous phase extracted twice more with EtOAc. The combined extract was washed with 5% K.sub.2CO.sub.3 (×3), brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 30.fwdarw.40% EtOAc in hexanes to give compound (24) (9.9 g, 92%) as a yellow colored oil that crystallized upon standing. Observed [M+H].sup.+=498.2 m/z (calc'd for C.sub.25H.sub.28N.sub.3O.sub.8 498.2 m/z).

Example 19: N,N-bis(2-(2-aminophenoxy)ethyl)-2-(2-methoxyethoxy)aniline (25)

[0084] bis-Nitro compound 24 (4.98 g, 10 mmol) was dissolved in THF:EtOH (1:1, 50 mL). A slurry of Pd/C in EtOH was prepared under N.sub.2 atmosphere and added to the substrate solution. The mixture was warmed to 50° C. and hydrazine hydrate 4.9 mL was added dropwise, addition of which initially caused effervescence. Following addition, the mixture was warmed to gentle reflux for 12 hrs then cooled to rt and filtered through Celite carefully rinsing with THF. The filtrate was concentrated and the residue was treated with 5% Na.sub.2CO.sub.3 (50 mL) and extracted with EtOAc (3×30 mL). The combined extract was washed with brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 50% EtOAc in hexanes to give compound (25) (3.35 g, 76%) as a faintly brown colored viscous oil. (R.sub.f=0.31, 1:1 hexanes:EtOAc). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.39 (s, 3H), 3.70-3.76 (m, 6H), 4.09-4.16 (m, 6H), 6.63-6.70 (m, 4H), 6.72-6.80 (m, 4H), 6.87-6.92 (m, 2H), 6.94-7.00 (m, 1H), 7.08-7.12 (m, 1H). Observed [M+H].sup.+=438.2 m/z (calc'd for C.sub.25H.sub.32N.sub.3O.sub.4 438.2 m/z).

Example 20: 22-(2-(2-methoxyethoxy)phenyl)-9,10,21,22,23,24-hexahydro-5H,12H,20H-dibenzo[h,q][1,4,10,16]tetraoxa[7,13,19]triazacyclohenicosine-6,13(7H,14H)-dione (26)

[0085] bis-Aniline compound 25 (3.35 g, 7.66 mmol) and 3,6-dioxa-1,8-dioic acid were dissolved in anhydrous DMF (20 mL) and added via syringe pump over a period of 90 min to a stirred suspension of EDC.HCl (7.34 g, 38.3 mmol), DMAP (936 mg, 7.66 mmol), and KBF.sub.4 (964 mg, 7.66 mmol) in anhydrous DMF (130 mL) at 40° C. The mixture was stirred for 12 hr then concentrated to approximately one third the initial volume and partitioned between EtOAc and half-saturated NaHCO.sub.3. The aqueous phase was extracted with EtOAc and the combined extract was washed with sat. NaHCO.sub.3 (×2), brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 1.fwdarw.3% MeOH in CH.sub.2Cl.sub.2 to give compound (26) (2.51 g, 57%) as a pale yellow oil that solidified upon standing. (R.sub.f=0.58, 94:6 CHCl.sub.3:MeOH). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.41 (s, 3H), 3.70-3.74 (m, 2H), 3.84 (t, J=5.7 Hz, 4H), 3.93 (s, 4H), 4.09 (t, J=5.7 Hz, 4H), 4.08-4.12 (m, 2H), 4.15 (s, 4H), 6.75 (d, J=7.8 Hz, 1H), 6.76 (d, J=7.9 Hz, 1H), 6.86-6.91 (m, 2H), 6.91-7.01 (m, 5H), 7.03-7.07 (m, 1H), 8.35 (dd, J=7.6, 1.8 Hz, 1H), 9.20 (br s, 1H). Observed [M+H].sup.+=580.3 m/z (calc'd for C.sub.31H.sub.38N.sub.3O.sub.8 580.3 m/z).

Example 21: 22-(4-bromo-2-(2-methoxyethoxy)phenyl)-9,10,21,22,23,24-hexahydro-5H,12H,20H-dibenzo[h,q][1,4,10,16]tetraoxa[7,13,19]triazacyclohenicosine-6,13(7H,14H)-dione (27)

[0086] Aniline compound 26 (2.47 g, 3.75 mmol) was dissolved in DMF (21 mL) and treated with NBS (741 mg, 4.16 mmol) with vigorous stirring at rt. After 10 min, additional NBS (82 mg, 463 μmol) was added. After an additional 10 min, the reaction mixture was diluted with EtOAc and washed with Na.sub.2CO.sub.3 (×2), brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 0.5.fwdarw.3% MeOH in CH.sub.2Cl.sub.2 to give compound 27 (2.97 g, greater than theory) as a brown oil which crystallized upon standing. (R.sub.f=0.37, 99:1 CHCl.sub.3:MeOH). Observed [M+H].sup.+=658.4/660.4 m/z (1:1) (calc'd for C.sub.31H.sub.37BrN.sub.3O.sub.8 658.2 m/z).

Example 22: 22-(4-bromo-2-(2-methoxyethoxy)phenyl)-6,7,9,10,13,14,21,22,23,24-decahydro-5H,12H,20H-dibenzo[h,q][1,4,10,16]tetraoxa[7,13,19]triazacyclohenicosine (28)

[0087] bis-Amide compound 27 (2.97 g, approx. 4.26 mmol) was dissolved in anhydrous THE (43 mL) and treated with BH.sub.3.Me.sub.2S and the mixture was heated to gentle reflux under N.sub.2 atmosphere for 14 hrs. The mixture was then cooled and quenched by dropwise addition of 3N HCl (21 mL) then returned to reflux for 30 min and again cooled. The solution was rendered basic by the addition of 10M NaOH and extracted with EtOAc (×3). The combined extract was washed with brine (×2), dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by flash column chromatography on SiO.sub.2 eluted with 30.fwdarw.50% EtOAc in hexanes to give compound 28 (2.33 g, 87%) as a faintly yellow heavy oil. (R.sub.f=0.21, 7:3 hexanes:EtOAc). Observed [M+H].sup.+=630.2/632.3 m/z (1:1) (calc'd for C.sub.31H.sub.41BrN.sub.3O.sub.6 630.2 m/z).

Example 23: Dioxo-2.2.3-cryptand (29)

[0088] bis-Aniline compound 28 (2.33 g, 3.69 mmol) and anhydrous pyridine (654 μL, 8.12 mmol) were dissolved in anhydrous DCM (10 mL). Separately, 3,6-dioxaoctan-1,8-dioic acid chloride (873 mg, 4.06 mmol) was dissolved in anhydrous DCM (10 mL). These two solutions were added via syringe to a 250 mL round bottom flask containing anhydrous THF (74 ml) over a period of approximately 6 hours. The turbid mixture was stirred at rt overnight then diluted with EtOAc and washed with sat NaHCO.sub.3 (×3), brine, dried over MgSO.sub.4, and concentrated. The residue was purified by flash chromatography on SiO.sub.2 eluted with 0.fwdarw.1% MeOH in CH.sub.2Cl.sub.2 with 1% Et.sub.3N to give compound 29 (1.2 g, 42%) as a white foam. Observed [M+H].sup.+=772.3/774.3 m/z (1:1) (calc'd for C.sub.37H.sub.47BrN.sub.3O.sub.10 772.2 m/z).

Example 24: 2.2.3-cryptand (30)

[0089] Diamide compound 29 (1.2 g, 1.55 mmol) was dissolved in anhydrous THE (16 mL) and BH.sub.3.Me.sub.2S (15.5 mmol, 1.47 mL) was added via syringe. The mixture was warmed to 60° C. and stirred for 2 hours. A white precipitate was observed after 1 hour. The reaction was cooled to 0° C. and carefully quenched by dropwise addition of 1N HCl (25 mL) then re-heated to 60° C. for 30 min. The reaction was again cooled to 0° C. and the pH adjusted above 10 by addition of solid NaOH. The mixture was extracted with CHCl.sub.3 (×3), and the combined extract was washed with brine, dried over Na.sub.2SO4 and concentrated. The residue was dissolved in DCM and purified by flash chromatography on SiO.sub.2 eluted with 0.fwdarw.1% MeOH in DCM and 1% Et.sub.3N to give 30 (1.1 g, 95%) as a foam. Observed [M+H].sup.+=743.9/746.0 m/z (1:1) (calc'd for C.sub.37H.sub.51BrN.sub.3O.sub.8 743.3 m/z).

Example 25: 2.2.3-Cryptand K.SUP.+ Sensor B (31)

[0090] Bromide compound 30 (0.208 mmol, 155 mg) was dissolved in DCM:MeOH (9:1) and filtered through a pipet column of basic alumina then concentrated and further dried in vacuo. The residue was co-evaporated twice from anhydrous 2-methyl-THF then dried in vacuo. The residue was redissolved in anhydrous 2-Me-THF (1.0 mL) and cooled in to −116° C. (N.sub.2/EtOH) and tert-butyllithium (275p L, 0.52 mmol, 1.9M in pentane) was added via the cold sidewall. The suspension was stirred cold for 10 min. Xanthone (116 mg, 0.250 mmol) was dried by gently warming in vacuo then dissolved in anhydrous 2-Me-THF (1 mL) and added slowly to the aryllithium intermediate via the cold sidewall. A dark red color developed. The mixture was stirred cold for 15 min, then the cooling bath was removed and the mixture was stirred at rt for 20 min. A mixture of TFE (1 mL) and 2N HCl (1 mL) was added. The mixture was stirred for 10 min then diluted with 0.5M HCl and washed with 1:1 EtOAc:Hexanes. The aqueous was adjusted to pH 4 and back-extracted with EtOAc (×5). The extract was purified by prep HPCL (10.fwdarw.100% ACN+0.1% HCO.sub.2H over 40 min, 20 ml/min, Clipeus C18 20×250 mm, 10 μm) to give compound 31 (88 mg, 48%) as a green film. Observed [M+H].sup.+=876.6 m/z (calc'd for C.sub.50H.sub.58N.sub.3O.sub.11 876.4 m/z).

Example 26: Preparation of a Potassium Sensor with Internal Reference

[0091] The monomers HEMA and AMP were purified by passing through alkaline aluminum oxide (Acros basic, Brockmann I, for chromatography, 50-200 μm, 60 A, ACROS Organics) to remove inhibitor. The ratio between the monomer to Al.sub.2O.sub.3 was 10 mL/1 g. The purified monomers were kept at 4° C. for short time use (within one week), or −20° C. for long term use. Prior to polymerization, the monomers were brought to room temperature.

[0092] A first block polymer of poly(HEMA) containing a reference dye was produced as follows. To a 20 mL vial, were added: an internal reference dye, Rhodamine 594 methacrylate monomer (click chemistry tools, MW: 646.76, 3.00 μmol, 1.94 mg), (E)-2,2′-(diazene-1,2-diyl)bis(2-methylpropanenitrile) (AIBN, MW: 164.21, 3.9 mg, 0.024 mmol), 2-cyanobutan-2-yl-4-chloro-3,5-dimethyl-1H-pyrazole-1-carbodithioate (chain Transfer Reagent MW: 0.144 mmol, 41.4 mg), 2-hydroxyethyl methacrylate (sigma: 128635-500 g; MW: 130.14, 6.00 mmol, 781 mg) and N, N-dimethylformamide (DMF) (3 mL). A magnetic stir bar was added and the vial sealed with a rubber cap. The sealed vial was purged with argon gas. The vial was heated to 70° C. and stirred for 20 hrs at 400 rpm. This formed the first block of poly(HEMA) containing the internal reference rhodamine 594. The vial was cooled down to room temperature.

[0093] A second block containing AMP and an Oregon Green® dye was synthesized as follows. To the first block of poly(HEMA) was added: AIBN (MW: 164.21, 3.9 mg, 0.024 mmol), 2-(2,7-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((2-(methacryloyloxy)ethyl)carbamoyl)benzoic acid monomer (MW: 523.44; 0.017 μmol, 52.0 μg, stock solution 10 μL), N-acryloylmorpholine (AMP) (Sigma-Aldrich® catalog no. 448273-250 mL, 9.0 mmol, 1.27 g), DMF (2 mL). The vial was resealed, purged with argon for 30 minutes. The mixture was heated to 70° C. and stirred for 20 hrs at 400 rpm.

[0094] After cooling the mixture to room temperature, 16 mL methanol was added to the flask. The polymer was precipitated by adding 250 ml of methyl tert-butyl ether by using syringe (12 ml). After stirring for 30 mins, the stir bar was removed. The top clear solution was poured in a Buchner's filter funnel (60 ml), then the polymer suspension placed together in one flask, and stirred for another for 10 mins. The remain suspended solution was filtered out using vacuum filtration. The white-pink product was washed with 100 mL of diethyl ether. The product was placed with the funnel in a vacuum oven at 50° C. overnight. Finally, ˜2.0 g white-pale pink product was obtained.

Example 27: Preparation of a Calibration Medium Including Two Different Fluorescent Compounds

[0095] 600 mgs of solvent (20% ethanol and 80% water) were added to 400 mgs of the polymer from Example 26 in a glass vial with an appropriate sized stir bar to agitate at a rate of 500 rpm at 50° C. hot plate overnight. When the resulting formulation was completely dissolved into an uniform solution, free of debris, carbon black was added to create a final concentration of 0.2% carbon black by weight. The mixture was stirred until ready for use.

Example 28: Preparation of a Calibration Layer Including Two Different Fluorescent Compounds

[0096] A Vermes microdispenser (Vermes Microdispensing, Holzkirchen, Germany) was used to dispense 4 spots of approximately 500 nanoliters each into an open channel of each of 5 different microfluidic devices (each without a cover layer). After dispensing, the spots were dried in a drier. Then the cover layer was laminated over each open channel to seal the channel.

[0097] The fluorescence intensity of the spots was determined using the configuration shown in FIG. 1 with respect to the optical sensor. The CV of the fluorescence intensities across all 5 strips was 1%. When the identical calibration layers were formed but without using carbon black, the CV of the fluorescence intensities across all 5 strips was 4.7%.

INCORPORATION BY REFERENCE

[0098] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0099] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.