Patent Grant
7670844
A field of the invention is biofluid analysis. Other exemplary fields of the invention include medical diagnosis, medical test systems, and medical laboratory processes.
Blood and blood plasma viscosity may become abnormal due to a variety of pathologic conditions. While the viscosity of full blood is primarily determined through hematocrit, changes in viscosity are observed in conjunction with various diseases, primarily those associated with altered protein levels. For example, infection, infarction, hypertension, or diabetes may alter plasma viscosity, which in turn may complicate the condition. Protein-induced hyperviscosity may lead to an elevated risk of atherosclerosis. Additionally, one of the adverse effects of smoking is elevated plasma viscosity, which may be the link between cigarette consumption and cardiovascular disease.
Non-pathogenic conditions may also influence blood rheology, including bed rest (e.g., associated with prosthetic implants), pregnancy and aging. Viscosity changes during the aging process may also be attributed to indirect effects, such as age-related changes in habits (e.g., increased smoking or lack of exercise).
Accordingly, using plasma viscosity as a diagnostic tool to allow early detection of diseases is advantageous. Even where there is no direct causal relationship between disease and plasma viscosity, measurement of plasma viscosity nonetheless becomes a crucial element in sever hemorrhage when hemodilution leads to dramatically lowered hematocrit and plasma viscosity plays an important role. Blood expansion with high-viscosity fluids enhances capillary perfusion and thus tissue oxygenation.
Accordingly, for many related purposes, including viscosity-related research, diagnosis of cardiovascular disease, or monitoring of the blood resuscitation process, a viscometer that is capable of fast serial measurement with low volumes is desirable. Mechanical devices for the measurement of fluid viscosity are presently available, but they continue to pose disadvantages such as slow measurements, lack of robustness, the need for meticulous cleaning and susceptibility to operator errors. The most widely used conventional mechanical devices are: the falling-ball viscometers (wherein the time required to produce a result usually measures from between 1 minute and 20 minutes), which have high susceptibility toward uncleanliness and the occurrence of solid particles or air in the fluid; flow-resistance viscometers, such as capillary or Oswald-type viscometers, which need high volumes of fluid and require measurement times in the order of minutes; and cone-and-plate viscometers, which have the same disadvantages of the flow-resistance viscometers.
Additionally, a class of molecules known as molecular rotors have been used to probe fluid viscosity in vitro. Molecular rotors are fluorescent molecules with a quantum yield that is dependent on the free volume—and thus the viscosity—of their environment. Molecular rotors form a twisted internal charge transfer (TICT) complex upon excitation. These molecules have a preferred de-excitation pathway of internal conversion through rotation about a C═C double bond. This internal rotation rate depends on the viscosity of the solvent, leading to a viscosity-dependent quantum yield. For example, 9-(2,2-dicyanovinyl)-julolidine (DCVJ) has been used to probe fluid viscosity in vitro by dissolving the DCVJ in a fluid having a viscosity to be measured, and subsequently observing the fluorescence emission. However, because the rotors are dissolved in the fluid to be observed, the applications are limited to in vitro applications.
The instant invention is directed to viscosity sensors for both in vitro and in vivo applications, and is more specifically directed to the use of molecular rotors disposed on a solid surface for use as a viscosity probe for in vivo applications as well as in vitro applications. In a particular embodiment of the invention, a fiber optic tip has molecular rotors adhered to it. The tip may be shaped and sized, for example, to be inserted into animal or human vessels having a predetermined circumference. The fiber optic tip may form the end of an optical waveguide that can guide both excitation light and responsive emissions for viscosity testing. The responsive emissions are analyzed for example by a computer that determines viscosity values based upon the acquired responsive emissions.
Another embodiment of the invention includes adhering molecular rotors to one or more walls of a glass receptacle, which may then be filled with a fluid having a viscosity to be measured. Measuring the fluorescence emission from the receptacle-bound molecular rotors is indicative of viscosity.
Molecular rotors are fluorescent molecules with a quantum yield that is dependent on the free volume—and thus the viscosity—of their environment. Molecular rotors form a twisted internal charge transfer (TICT) complex upon excitation. These molecules have a preferred de-excitation pathway of internal conversion through rotation about a C═C double bond. This internal rotation rate depends on the viscosity of the solvent, leading to a viscosity-dependent quantum yield.
Molecular rotors have been successfully used to probe cell membrane viscosity and blood plasma viscosity in vitro. However, measurement requires that a defined amount of the fluorescent dye (the molecular rotor material) be dissolved in a sample fluid at known concentrations. Variations in dye concentration, different light absorption coefficients of the measured fluid as well as turbidity of the fluids needs to be compensated for. In addition, a molecular rotor that needs to be dissolved in the fluid is precluded from in vivo measurements, which prevents their use in viscosity analyses of biofluids such as blood plasma.
Embodiments of the invention provide for molecular rotors that are placed on a solid surface, such as any glass or polymer substrate that may be pre-functionalized (e.g., quartz, polystyrene or silicate glass) to create a probe that may then be used for in vivo as well as in vitro viscosity measurements. Such a probe has virtually unlimited potential for viscosity measurement applications, for example either in vivo or in vitro biofluid viscosity monitoring and measurement, industrial viscosity monitoring (e.g., in the food industry), organic solvents, aqueous solutions, and aqueous colloid solutions (e.g., the food and/or soft drink industry).
For example, in a first preferred embodiment, molecular rotors are adhered to a fiber optic probe, which may then be used as an in vivo probe of blood or biofluid viscosity via fluorescence intensity measurements. In a second preferred embodiment, molecular rotors are adhered to one or more walls of a glass cuvette, and once the cuvette is filled with a sample, the viscosity of a boundary layer of the fluid in the cuvette can be measured via fluorescence intensity measurements. In both preferred embodiments, the molecular rotors are covalently bound to the solid surface. Additionally, while the invention contemplates inclusion of a virtually unlimited number of molecular rotors, several exemplary molecular rotors will be shown and described.
Rotor-Covered Fiber Optic Probe for In Vivo Measurements:
A device for in vivo use is shown in
(a) etch a new tip on the fiber optic with hydrofluoric acid 40% solution, approximately 4 hours total, rinse in distilled water. Peel away the polymer coating to expose the tip using a razor blade and remove the cladding.
(b) wash tip in hydrochloric acid and methanol, then in sulfuric acid. Rinse and dry.
(c) sonicate tip for 6 to 8 minutes in 5% solution (v/v) of 3-aminopropyl-triethoxysilane (ABS from Signam cat no. A-3646) in absolute ethanol.
(d) bake the fiber tip for 1 minute at 120 degrees C.
(e) prepare working solution of 9-(2-carboxy-2-cyanovinyl)-julolidine-NHS (CCVJ-NHS) in 50 MM sodium bicarbonate solution pH 8.5, the final concentration of dye is 0.1 μg/μl with 10% DMSO present.
(f) incubate the tip of the fiber optic in the working solution of CCVJ-NHS for at least 1 hour. Sonicate in DMSO to remove excess dye.
The tip may be catheterized and inserted into blood vessels in vivo. Due to the low incidence angle, excitation light is refracted out of the tip, and problems with reflected or scattered incident light are expected to be minimized. Additionally, a pulsed light source in combination with gated signal acquisition will be used to reduce influences of environmental light. Thus, a patient with a probe inserted does not need to stay in a low-light environment. The tip is also preferably autoclaved for sterility.
Fluorescent molecular rotors belong to the group of twisted intramolecular charge-transfer complexes that possess free rotary vinyl groups in their molecules. Each molecular rotor is composed of two subgroups, which include an electron-donor group and an electron acceptor group. Both groups are linked by at least one single bond, so that the two groups are able to rotate freely against each other. Photoexcitation leads to an electron transfer from a donor group to an acceptor group. Relaxation can either occur through radiation (fluorescence) or intramolecular rotation (thermally induced nonfluorescent relaxation). The preferred relaxation mechanism, intramolecular rotation, is reduced in solvents with low free volume. Therefore, the quantum yield of a molecular rotor increases with decreasing free volume of the solvent. Free volume and viscosity are related, which links quantum yield directly to the viscosity of the microenvironment. The relationship between fluorescence quantum yield (φ) and the viscosity (η) of the solvent has been derived analytically and experimentally and is known as the Förster-Hoffmann equation:
log φ=C+χlog η
Thus, a virtually unlimited number of molecular rotors may be used in conjunction with the instant invention. For example, several exemplary acceptor groups include any motif that contains an OH, CN, COOH or its carboxylic esters and amides, such as esters of n-ethylene glycol attached to a vinyl (C═C), while several exemplary donor groups include an N atom that is not bound in a cyano (CN) manner, such as julolidine and amino-benzenes. The “opened” structures and the three julolidine rings are especially advantageous.
For the instant embodiment as well as additional embodiments discussed herein, it is anticipated that derivation of quantum yield may be obtained via both lifetime measurement as well as continuous-wave fluorescence. As illustrated by the Förster-Hoffmann-equation, viscosity and quantum yield are related, so measurement of quantum yield is desirable. In addition to being proportional to quantum yield, emission intensity also depends on excited-state lifetime, from which quantum yield may be computed. Thus, using advanced instrumentation capable of lifetime measurement, quantum yield may be measured via lifetime analysis as well as via continuous-wave fluorescence.
Specific exemplary molecules that may act as molecular rotors are illustrated in
In an exemplary probe of the invention, a solid surface (e.g., quartz glass or polystyrene) is activated, and reacts with 8-(2-carboxy-2-cyanovinyl)julolidine, N-succimidyl ester. Thus, the rotors are covalently linked to the surface. In general, the invention is directed to molecular rotors bonded upon a surface that can be placed in a biofluid. Some particular applications will now be discussed to illustrate additional aspects and variations of the invention.
Preparation of the preferred fiber optic tip may be based on commercially available polymeric supports, frequently employed in combinatorial chemistry. Polystyrene-based polymers, such as compound 10 in
While the invention contemplates use of any solid-bound rotor having a polar group, exemplary rotors include solid-bound polar rotors having one of the structures illustrated in
R1 would preferably be used as a reference moiety, wherein one R1 group is preferably fluorescent but not viscosity-sensitive. Intensity could be accurately calibrated by computing a ratio of rotor fluorescence to a reference fluorescence.
Viscosity-Sensitive Glass Surfaces for Both In Vivo and In Vitro Applications:
While it is contemplated that the invention may be used with receptacles such as glass or polymer tubes or microchannels, another preferred embodiment of the invention uses a glass cuvette, wherein one or more walls of a glass cuvette are covered with rotor molecules. As with the fiber optic probe, preparation of the glass cuvette and subsequent attachment of the molecular rotor(s) on the glass could be achieved on prefunctionalized silicate glass as illustrated in
The viscosity of the boundary layer of any fluid in the cuvette can be measured using the principle of operation of a solid-bound molecular viscometer. Since the light path does not cross the fluid, it becomes independent of turbidity. While it is contemplated that as many as four walls of the cuvette may be treated, for exemplary purposes, the second preferred embodiment will be shown and described as having a single treated wall. For example, where one wall of a cuvette is treated, the cuvette will include one viscosity-sensitive wall. The fluid to be measured will then be filled into the cuvette and the fluorescence intensity measured. The measurement options are: a) comparative measurement to determine absolute viscosity using standard fluids; and b) timecourse measurement to determine viscosity changes over time.
More particularly,
Alternatively, a beamsplitter assembly is used to couple excitation light into the fiber and to collect emission light. A dichroic mirror is used to direct blue light from a light source onto the dichroic mirror and to reflect the blue light into the fiber. At a tip of the fiber, the excitation evanescent wave excites the rotors attached to the surface of the tip. As the emission light, which is the non-reflecting wavelength of the dichroic mirror, exits the fiber inside the measurement device, it passes the dichroic mirror. Scattered or otherwise leaked excitation light is further filtered with a filter positioned in front of the detector (photomultiplier tube, or PMT). The detector then quantitatively determines emission intensity.
Both viscosity-sensitive glass surfaces and rotor-covered fiber optic probes will have a wide range of applications in viscosity measurement (relative) and viscosity monitoring in the chemical and processing industry with the emphasis on fast read-outs and real-time measurements. There is a high demand for real-time viscometry in the biological and medical disciplines, specifically blood resuscitation medicine, and for clinical research as well. This is particularly valid for the in vivo probe of the invention. The use of disposable elements (disposable tips, disposable cuvettes) promotes the ease and simplicity of the application.
Proof of Principle and Experimental Results:
Glass surfaces have been covered with molecular rotors and measurements performed. A distinct emission peak matching that of free molecular rotors indicates the presence of covalently bound rotors on the surface. This fluorescence is resistant against different solvents (water, various alcohols). The glass slide exhibits this fluorescence when immersed in water. The presence of dextran in water increases the intensity of the emission peak, as illustrated in
log φ=C+χlog η
From the plot of log intensity over log viscosity, it can clearly be seen that CCVJ emission intensity closely follows the Förster-Hoffmann relationship, while fluorescein does not show significant changes of intensity with viscosity. Thus, it is clear that immobilized molecular rotors retain their viscosity-sensitivity, and the value of solid-state fluorescent biosensors with molecular rotors linked to fiberoptics is evident.
Experiments: Materials, Methods and Results
Reagents obtained as follows: Fresh frozen human plasma was obtained from Interstate Blood Bank, INC., CCVJ from Helix Research, and CCVJ-TEG was synthesized. Fluoroscopy grade dimethyl sulfoxide (DMSO) was obtained from Sigma, Pentaspan (10% pentastarch in 0.9% NaCl, avg. MW 260 000) from B. Braun, Hetastarch (6% in 0.9% NaCl, avg. MW 670 000) from Abbott Laboratories. Dextran (80 000 average MW) from Sigma was used to prepare 5% solution in physiologic saline. Fluorescence spectroscopy was performed with samples in 4 mL clear methacrylate cuvettes using Spex Fluoromax-3. Absorbance measurements were made with a Beckman DU 520 spectrophotometer. Indices of refraction were taken with ATAGO R-5000 hand refractometer.
Fresh frozen plasma was thawed prior to use, then centrifuged at 180 g for 15 minutes and filtered through 75 mm 0.22 μm SFCA filter to remove cryoprecipitate. Post-processing protein concentration was determined by refractometry and confirmed by Lowry method. Crystalline dye was dissolved in DMSO to make 20 mM stock solutions of CCVJ and CCVJ-TEG. Sample viscosity was modulated by varying the amount of unstained plasma mixed with the starch plasma expanders dextran, hetastarch, and pentastarch. Plasma prestained with CCVJ or CCVJ-TEG (67 μM) was added in equal amounts to samples to minimize mixing errors. Final sample dye concentration was 13.3 μM. Three milliliters of each sample were transferred to 4 ml cuvettes and capped. All measurements were performed at room temperature (24° C.).
Calibration sets consisting of 5 different viscosities and 5 replicates each were made according to
Physical viscosity measurements for plasma mixtures were made with Brookfield DV-III+ cone-plate viscometer with CPE-40 spindle. Samples of 0.5 ml were measured at discrete shear rates: 150, 300, 450, 600, and 750 s−1, (20, 40, 60, 80, and 100 rpm) in ramp-up, ramp-down series. Viscosity measurements were taken at 80 rpm (600 s−1). This shear rate was chosen to remain within instrument accuracy limits, and to reduce any apparent non-Newtonian effects on results. While noticeable instrument fluctuations as well as apparent shear-thinning effects existed at shear rates of 300 s−1 and below, no visible influence of shear rate on measured viscosity existed at 450 s−1 and above. Reference viscosity 0ref was computed for each plasma-plasma expander combination using Eq. 1:
where ηi is the viscosity of the individual component as measured by cone-plate viscometer and wi is the fractional amount of n=2 components, plasma and expander. The equation was verified and found to excellently described the plasma/expander mixtures, as represented in
The fluorescence emission intensity of each sample was measured from 470-530 nm at excitation wavelength of 440 nm for CCVJ, and 460 nm for CCVJ-TEG. Excitation and emission slits were 2 nm with integration time of 0.5 s. Peak intensity per sample was computed by averaging the emission intensity in a 5 nm window centered on the emission peak. Using the known relationship between viscosity η and quantum yield Φ of a molecular rotor, Equation 2:
log Φ=C+χlog η
which was solved for η, which yielded the fluorescence-based vicsosity ηF, as in Equation 3:
ηF=(κ·lν
The constant κα 10−C relates quantum yield and dye concentration and ν=l/χdescribes the viscosity sensitivity of the molecular rotor. Constants κ and ν were determined from linear regression of log intensity over log viscosity for each plasma-expander calibration set. Deviation d of ηF from ηref was computed using the following Equation 4:
Fluorescence intensities of the test set samples were measured in the same manner as the calibration set. For each sample, ηF was computed according to Eq. 3, using the constants κ and ν derived from the calibration set of plasma and pentastarch mixtures. The cone-plate rheometer was also used to measure sample viscosities. Deviation from ηref was calculated for both ηF and rheometer results using Eq. 4.
Absorption measurements at 440 nm and 490 nm were used to normalize dye concentration between calibration set and individual test samples. A standard concentration factor was determined from the calibration set by linear regression of absorbance at 440 nm (A440) versus absorbance at 490 nm (A490), where the slope (χ) is related to plasma absorbance, and the intercept (β) is directly related to dye concentration. A normalization factor (Δ) was calculated for each sample according to Equation 5:
Normalized intensity was determined by multiplying measured sample intensity by the factor Δ.
Results
Cone-Plate Rheometer
Physical viscosity values of human blood plasma at the shear rates used are represented in
Refraction
Indices of refraction varied from 1.349 for pure plasma to 1.348 for pentastarch solution. According to Lakowicz, the correction factor is (ηi/ηF)2, where ηi is the refractive index of the sample and ηF is the refractive index of air. This would lead to a difference of less than 0.15% of fluorescence intensity values between plasma and pentastarch solution. The influence of refractive index change was neglected in spectroscopic measurements.
Absorbance
Molecular Rotor Fluorescence
Both CCVJ and CCVJ-TEG dissolved completely in the plasma mixtures. Plasma autofluorescence did not significantly affect fluorescence measurements at the dye concentration used in this study. Average wavelength of peak emission was 476 nm for CCVJ and 498 nm for CCVJ-TEG at excitation wavelength of 440 nm and 460 nm, respectively, with less than 5 nm deviation.
For the test set, viscosity determined by fluorescence ηF of dissolved CCVJ in plasma-pentastarch mixtures was compared with values of ηref as determined by Eq. 1 for the plasma-pentastarch calibration set (
While various embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.
Various features of the invention are set forth in the following claims.
Pursuant to 35 U.S.C. §119(e), this application is entitled to the benefit of U.S. Provisional Application Ser. No. 60/537,679, filed Jan. 20, 2004.
This invention was made with Government support under Contract Numbers NIH IR21 RR018399, NIH HL040696 and NIH HL062354 awarded by the National Institutes of Health. The Government has certain rights in the invention.
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| 5384079 | Bur et al. | Jan 1995 | A |
| 6531097 | Vojnovic et al. | Mar 2003 | B1 |
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| Number | Date | Country |
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| WO2005073697 | Aug 2005 | WO |
| Number | Date | Country | |
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| 20060084177 A1 | Apr 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60537679 | Jan 2004 | US |
