WO2016081727A1 - Flow cells and methods of measuring an absorption spectrum of a sample - Google Patents

Abstract

One aspect of the invention provides a flow cell including: a light source adapted and configured to generate light; at least one of a capillary and a capillary holder adapted and configured to hold a capillary, wherein: the capillary is adjacent to the light source; and the capillary is at least substantially aligned, but perpendicular with the light generated by the light source such that at least a portion of the light generated by the light source strikes a capillary-sample medium boundary at an angle larger than the critical angle with respect to the normal of an inner surface of the capillary to achieve total internal reflection within a wall of the capillary; and a light collector positioned to collect at least a portion of light reflected off of the capillary-sample medium boundary.

Description

FLOW CELLS AND

METHODS OF MEASURING AN ABSORPTION SPECTRUM OF A SAMPLE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application Serial

No. 62/082,443, filed November 20, 2014. The entire content of this application is hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Precise measurement of the absorbance spectrum of highly scattering and highly absorbing liquids such as whole blood is impossible using known methods without radical dilution.

SUMMARY OF THE INVENTION

One aspect of the invention provides a flow cell including: a light source adapted and configured to generate light; at least one of a capillary and a capillary holder adapted and configured to hold a capillary, wherein: the capillary is adjacent to the light source; and the capillary is at least substantially aligned, but perpendicular with the light generated by the light source such that at least a portion of the light generated by the light source strikes a capillary- sample medium boundary at an angle larger than the critical angle with respect to the normal of an inner surface of the capillary to achieve total internal reflection within a wall of the capillary; and a light collector positioned to collect at least a portion of light reflected off of the capillary- sample medium boundary.

This aspect of the invention can have a variety of embodiments. The light source and the light collector can be substantially aligned. The capillary or the capillary holder can be offset from the light source and the light collector. The offset can be between about 10 microns and about 20 microns.

The light source can include one or more selected from the group consisting of: an optical fiber, a white light source, and a light-emitting diode. The light source can include one or more selected from the group consisting of: an optical fiber, a monochromator, and a spectrometer. The capillary can have a substantially circular profile. The capillary can be a microfluidic capillary. The capillary can have an inner volume of about 0.25 uL. The capillary can have an inner diameter of about 100 microns. The capillary can have an outer diameter of about 500 microns.

Another aspect of the invention provides a method of measuring an absorption spectrum of a sample. The method includes: loading the sample within the capillary of the flow cell as described herein; introducing light in the first optical fiber; collecting light from the second optical fiber; and analyzing a difference between a spectrum of the introduced light and the collected light.

This aspect of the invention can have a variety of embodiments. The method can further include correlating the difference with one or more known absorption spectra. The method can further include identifying a composition or a property of the sample based on a correlation with one or more known absorption spectra.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the

accompanying drawing figures wherein like reference characters denote corresponding parts throughout the several views.

FIG. 1 depicts a flow cell according to an embodiment of the invention.

FIG. 2 depicts a method of measuring an absorption spectrum of a sample according to an embodiment of the invention.

FIG. 3 depicts equipment for administering a flow through a spectrometer cell according to an embodiment of the invention. It was designed and successfully used as an example of application of the spectrometer measuring spectra of the fluid sample flowing through the spectrometer cell.

FIG. 4 depicts the absorbance spectra of whole blood as measured in a 100 μιη capillary according to an embodiment of the invention. The singular wave decomposition fit procedure allows calculation of the level of deoxygenation of the blood sample with high precision.

FIG. 5 depicts a system for injecting sodium dithionite into a capillary of blood. FIG. 6 depicts the kinetics of level of deoxygenation for different flow rates of sodium dithionite (0-10 μί/ηιίη) injected into blood flow (100 μί/ηιίη) using the system depicted in FIG. 5.

FIG. 7 depicts the kinetics of level of deoxygenation for different flow rates of sodium dithionite once the combined blood and sodium dithionate flow is arrested using the system depicted in FIG. 5.

FIG. 8 depicts the kinetics of level of deoxygenation for different flow rates of sodium dithionite (0-10 μί/ηιίη) injected into blood flow (100 μί/ηιίη) using the system depicted in FIG. 5 when the time of travel is increased to 250 second by moving the measurement point further downstream from the injection point.

FIG. 9 depicts a mixing device include a bead chamber (500 μιη ID tube filled

with 120 μιη glass beads).

FIG. 10 depicts the kinetics of level of deoxygenation for different flow rates of sodium dithionite (0-10 μί/ηιίη) injected into blood flow (100 μί/ηιίη) using the system depicted in FIG. 5 and the mixing device depicted in FIG. 9.

FIG. 11 depicts the kinetics of level of deoxygenation for different flow rates of sodium dithionite (0-10 μί/ηιίη) injected into blood flow (100 μί/ηιίη) using the system depicted in FIG. 5 and the mixing device depicted in FIG. 9 once the combined blood and sodium dithionate flow is arrested.

FIG. 12 depicts a spectrometer prototype according to an embodiment of the invention.

DEFINITIONS

The instant invention is most clearly understood with reference to the following definitions:

As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. As used in the specification and claims, the terms "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," and the like.

Unless specifically stated or obvious from context, the term "or," as used herein, is understood to be inclusive.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).

DETAILED DESCRIPTION OF THE INVENTION

Precise measurement of the absorbance spectrum of highly scattering and absorbing liquids such as whole blood, milk, and crude oil is impossible using known methods without radical dilution. Such measurements of the absorption spectrum are of great importance especially when combined with other measurements (e.g., rheological). For example, complex disperse fluid flows along capillaries and parameters of flow are measured as functions of time. Currently, there are no known methods of correlating absorption spectra to other properties of the undiluted fluid.

Additionally (as in the case of whole blood), particles in solutions often have high extinction coefficients. For example, one red blood cell absorbs as much as 90% of light at 420 nm.

Using whole blood as an example, conventional methods require lysing of the red blood cells (e.g., by adding water to the blood to increase the osmotic pressure of the red blood cells), followed by centrifugation and dilution. This not only dilutes the sample, increases time and cost, and requires milliliters of sample, but also exposes the hemoglobin within the red blood cells to changes in oxygenation as well as bonding to other gases). There is only one instrument on the market that enables measurement of the absorbance spectrum of blood without lysing of red blood cells; however, this instrument still requires 4,000 fold dilution of the blood. Flow Cell

Referring now to FIG. 1, a cross-sectional view of a flow cell 100 is depicted. The flow cell 100 includes a light source 102, a capillary 104 adapted and configured to receive a sample 106, and a light collector 108. The light source 102, the capillary 104, and the light collector 106 are arranged so that light 110 from the light source 102 strikes the capillary-sample medium boundary at an angle larger than the critical angle with respect to the normal of the inner surface of the capillary 106 to achieve total internal reflection within the wall of the capillary 104 before being collected by the light collector 108.

The light source 102 can be any source of light. In one embodiment, the light provided is "white light," i.e., light that is perceived to be similar to daylight and has a balanced mixture of various colors across the visible spectrum. Such light can be provided by one or more light- emitting diodes (LEDs).

In some embodiments, the light is collimated light having rays that are parallel and spread minimally. Such light can be produced using a pinhole or slit in front of a light source. In another embodiment, one or optical fibers {e.g., multi-mode optical fibers) can be utilized to transmit and aim the light in a desired orientation with regard to the capillary 104 and the light collector 108. Optical fibers can include cladding 112 having a lower index of refraction that radially surrounds the optical fiber to promote total internal reflection within the optical fiber.

The capillary 104 can be a cylinder having a sufficiently small cross-section to induce capillary or wicking flow of a sample fluid 106 into the capillary 104. For example, the capillary 104 can have a circular cross-section. Capillaries 104 fabricated from materials such as borosilicate, clear fused quartz, and synthetic fused silica are commercially available from sources such as VitroCom of Mountain Lake, New Jersey and Drummond Scientific Company of Broomall, Pennsylvania. For example, VitroCom offers capillary tubes having circular cross- sections with internal diameters (IDs) of 0.05 mm, 0.10 mm, 0.15 mm, 0.20 mm, 0.30 mm,

0.40 mm, 0.50 mm, 0.60 mm, 0.70 mm, 0.80 mm, 0.90 mm, 1.00 mm, 1.50 mm, 2.00 mm, and the like and lengths of 100 mm, 300 mm, 600 mm, and the like.

Capillary 104 can be replaceable in order to prevent cross-contamination between samples. Accordingly, any of the structures or relationships described herein should be construed to encompass a capillary holding device (e.g., a larger cylinder such as a capillary or tubing or a clamp sized to receive and hold a capillary 104 in a desired location). Light detector 108 can include one or more components adapted and configured collect and/or analysis the light reflected off of the capillary-sample medium boundary. As with the light source 102, light detector 108 can take many forms. In one embodiment depicted in FIG. 1 , an optical fiber can collect and transmit the reflected light. In another embodiment, a pinhole or slit can be used to select the desired ray(s) of reflected light. One or more lenses can be utilized to focus the desired light. A monochromator and/or a spectrometer can be utilized to analyze the reflected light. Suitable spectrometers include the USB series (e.g., the USB4000) available from Ocean Optics, Inc. of Dunedin, Florida. Data can be collected and/or displayed (e.g., in real time) using a computer programmed with software such as LAB VIEW® software available from National Instruments Corporation of Austin, Texas.

FIG. 1 depicts a particularly advantageous arrangement of the light source 102, capillary 104, and light collector 108 in which the light source 102 and light collector 108 are substantially aligned along an axis 1 14 that is offset from a midline 1 16 of the capillary 104 (which is perpendicular to the rays generated by light source 102). In other embodiments, the light source 102 can be centered on the midline 1 16 of the capillary 104 and the light

collector 108 can be offset. Without being bound by theory, it is believed that any arrangement in which a center of the light source 102, capillary 104, and light collector 108 are in sufficient proximity so that light reflected off of the capillary-sample medium boundary and received by the light collector 108 will facilitate the desired measurement of the absorption spectrum of the sample 106 received within the capillary 104. Again, without being bound by theory, it further believed that many such arrangements will form a triangle with vertices 'A' at the center of the light source 102, 'B' at the location of reflection of the ray of interest at the capillary-sample medium boundary, and 'C at the center of the light collector.

The flow cell 100 can include one or more adjustment devices that enable adjustment of one or more components 102, 104, 108 (e.g., a component that is offset from the others) in order to obtain a spectrum of interest. Suitable adjustment devices include set screws. In another embodiment, an electronic micromanipulator can be adapted, configured, and/or programmed to move one or more components 102, 104, 108 until an optimal spectrum is obtained by the light collector 108. The optimal spectrum can be determined by a user or can be selected by a computer using appropriate software including one or more optimization algorithms. Method of Measuring Absorption Spectrum of a Sample

FIG. 2 depicts a method of 200 of measuring an absorption spectrum of a sample.

In step S202, a sample is loaded within capillary of a flow cell, e.g., a flow cell 100 as described herein. The sample can be loaded by contacting the capillary with the sample so that the sample is wicked into the capillary by capillary action. The sample need not be prepared in any way and can be a nano liters volume {e.g., 1 nL)

Although method 200 and flow cell 100 can be applied to measure the absorption spectrum of any fluid including liquids and gases, method 200 and flow cell 100 are particularly useful for measuring the absorption spectrum of highly scattering and absorbing liquids such as suspensions (heterogeneous mixtures containing solid particles that are sufficiently large for sedimentation) and emulsions (a mixture of two or more liquids that are normally immiscible). Exemplary suspensions include blood, which includes solid particles such as hemoglobin.

Exemplary emulsions include dairy products such as milk, cream, and the like.

In step S204, light is introduced through the sidewall of the capillary.

In step S206, reflected light is collected.

In step S208, a difference between a spectrum of the introduced light and a spectrum of the collected light is analyzed. For example, the difference can be correlated between one or more known absorption spectra. For example, oxygenated hemoglobin and deoxygenated hemoglobin exhibit markedly different absorption spectra. Likewise, various proteins have different absorption spectra. For example, this difference can be utilized to identify the presence of proteins that are harmful {e.g., poisonous) in and of themselves or associated with harmful pathogens. Example of proteins that can be identified using absorption spectra include caseins, beta-lactoglobulin, immunoglobulins {e.g., antibodies), serum albumin, and alpha-lactalbumin in milk. Other examples include melamine, patulin, and other toxins that may be present in foods such as milk, peas, rice, wheat protein isolates, juices, and the like.

The absorption spectrum can also be quantitatively compared to one or more known spectra in order to quantify the relative concentrations within a sample. For example, an absorption spectrum that approximates the average of the absorption spectrum of oxygenated hemoglobin and the absorption spectrum of deoxygenated hemoglobin can be assumed to be about 50% oxygenated. In some embodiments, at least the collection of the absorption spectrum can be completed within 15 seconds of loading of the sample. Current prototypes quantified a complex mixture of differently liganded hemoglobins with precision of 4% percent using standard 0.25 μΐ, capillaries.

Working Example #1

Referring now to FIG. 12, a working prototype of a spectrometer was built and used for extensive data collection. A white 1 watt LED with THORLABS® F810SMA collimator was used as an illumination source and an OCEAN OPTICS® USB4000 spectrometer was used as a detector.

Working Example #2

Referring now to FIGS. 3-11, embodiments of the invention were used provide rapid micro-measurement of deoxygenation levels of blood. Deoxygenated red blood cells (RBCs) lose their flexibility and obstruct flow through the microcirculation. Embodiments of the invention can measure the degree of deoxygenation.

FIG. 3 depicts equipment for administering a flow through a spectrometer cell according to an embodiment of the invention. Two syringe pumps mixed flows of whole blood and deoxygenation agent (dithionite) in a mixer. The mixture was then passed through a capillary where the optical spectra were measured 1,000 times per second. The constant flow of blood was then mixed with the flow of sodium dithionite. The flow was stopped and the spectra of blood was measured as function of time, processed, and a level of deoxygenation was calculated in real time (1,000 times per second).

Referring now to FIG. 4, the absorbance spectra of whole blood were measured with the described method in a 100 μιη capillary. The data were collected from partially deoxygenated blood samples. The colored lines are the result of fitting with a linear combination of two known spectra of oxygenated and deoxygenated hemoglobin. The fitting parameter gives 15/75 and 98/2 as a ratio of the components.

For the reference spectra, the capillary was filled with water. The fitting procedure used the well-known singular value decomposition algorithm.

Intensity of detected light correlate with the number of scattering objects in the fluid sample. This enables the calculation of relative concentration of red blood cells.

FIG. 5 depicts a system for injecting sodium dithionite into a capillary of blood. Referring now to FIG. 6, the kinetics of level of deoxygenation was measured for different flow rates of sodium dithionite (0-10 μί/ηιίη) being injected into blood flow

(100 μί/ηιίη) as depicted schematically in FIG. 5. Distance from mixing point and measurement cap was 7 cm (10 sec). The data are extremely unstable especially for the low level of deoxygenation.

Stop-flow technique data support the hypothesis that the non-homogeneous distribution of the oxygen in the blood cause the instability of the data. The equilibrium reached in few minutes once the flow is arrested as depicted in FIG. 7 in which the time is shown on the x axis relative to the point when flow is stopped and the labels on each curve represent the

concentration of sodium dithionate introduced.

Solving the following diffusion equations on the right for distance between droplets of the dithionite yields a distance of 200 μιη .

δώ δ²ώ

— = D— - dt dx²

x²

t ^¾ 2

This calculated distance is in general agreement with the size of the injector needle used, which had a diameter D = 0.0002 cm²/sec.

Referring now to FIG. 8, a longer distance to the measurement point improves mixing. By increasing the time of the travel to 250 seconds, the sample is effectively incubated, which significantly improves the homogeneity of the blood and stability of the signal.

Referring now to FIG. 9, a bead chamber (500 μιη ID tube filled with 120 μιη glass beads) was used as a mixing device. The blood and dithionite flow through the beads in a second, but with effective mixing and deoxygenation corresponding to 10-minute waiting in the stop-flow experiment described herein as depicted in FIG. 10.

To confirm the homogeneous blood deoxygenation, Applicant repeated the stop-flow experiment again as depicted in FIG. 11. Stability of the signal supports great mixing efficiency for the whole range of deoxygenation levels.

The bead chamber used here as mixer is optimized for measurement of the number of rigid cells in the blood based on resistance to the flow. EQUIVALENTS

Although preferred embodiments of the invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

Claims

1. A flow cell comprising:

a light source adapted and configured to generate light;

at least one of a capillary and a capillary holder adapted and configured to hold a capillary, wherein:

the capillary is adjacent to the light source; and

the capillary is at least substantially aligned, but perpendicular with the light generated by the light source such that at least a portion of the light generated by the light source strikes a capillary-sample medium boundary at an angle larger than the critical angle with respect to the normal of an inner surface of the capillary to achieve total internal reflection within a wall of the capillary; and

a light collector positioned to collect at least a portion of light reflected off of the capillary-sample medium boundary.

2. The flow cell of claim 1, wherein:

the light source and the light collector are substantially aligned; and

the capillary or the capillary holder is offset from the light source and the light collector.

3. The flow cell of claim 2, wherein the offset is between about 10 microns and about 20 microns.

4. The flow cell of claim 1, wherein the light source includes one or more selected from the group consisting of: an optical fiber, a white light source, and a light-emitting diode.

5. The flow cell of claim 1, wherein the light source includes one or more selected from the group consisting of: an optical fiber, a monochromator, and a spectrometer.

6. The flow cell of claim 1, wherein the capillary has a substantially circular profile.

7. The flow cell of claim 1 , wherein the capillary is a micro fluidic capillary.

8. The flow cell of claim 1, wherein the capillary has an inner volume of about 0.25 uL.

9. The flow cell of claim 1, wherein the capillary has an inner diameter of about 100 microns.

10. The flow cell of claim 1, wherein the capillary has an outer diameter of about 500 microns.

11. A method of measuring an absorption spectrum of a sample, the method comprising: loading the sample within the capillary of the flow cell of claim 1 ;

introducing light in the first optical fiber;

collecting light from the second optical fiber; and

analyzing a difference between a spectrum of the introduced light and the collected light.

12. The method of claim 11, further comprising:

correlating the difference with one or more known absorption spectra.

13. The method of claim 12, further comprising:

identifying a composition or a property of the sample based on a correlation with one or more known absorption spectra.