WO2012079076A2

WO2012079076A2 - Methods and systems for distributing a plurality of particles within a viscoelastic specimen

Info

Publication number: WO2012079076A2
Application number: PCT/US2011/064412
Authority: WO
Inventors: Richard Superfine; Jeremy Cribb; Stephen Norris
Original assignee: University of North Carolina at Chapel Hill
Current assignee: University of North Carolina at Chapel Hill
Priority date: 2010-12-10
Filing date: 2011-12-12
Publication date: 2012-06-14
Anticipated expiration: 2013-06-10
Also published as: WO2012079076A3

Abstract

Methods and systems for distributing a plurality of particles within a viscoelastic specimen are disclosed. According to one example, a method includes drawing, into a doping device, a particle solution that includes a plurality of particles suspended in a buffer fluid. The method further includes adhering the particles to a surface of the doping device by evaporating the buffer fluid from the particle solution and drawing, into the doping device, a viscoelastic specimen that causes the particles to dislodge from the surface of the doping device and become suspended within the viscoelastic specimen. The method also includes measuring the movement of the particles suspended within the viscoelastic specimen to determine at least one rheological property of the viscoelastic specimen.

Description

METHODS AND SYSTEMS FOR DISTRIBUTING A PLURALITY OF PARTICLES WITHIN A VISCOELASTIC SPECIMEN

DESCRIPTION

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/421 ,973, filed December 10, 2010; the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter described herein relates to methods and systems for measuring physical properties of fluid specimens. More particularly, the subject matter described herein relates to methods and systems for distributing a plurality of particles within a viscoelastic specimen.

BACKGROUND

Currently, microrheology of a viscous fluid sample is measured by tracking probe particles (i.e., microparticles or nanoparticles) that are introduced into test specimen sample via dilution. Dilution is a process that involves adding particles suspended in a buffer fluid to a viscoelastic specimen solution for the purpose of safely distributing the particles throughout the viscoelastic specimen. This addition of the buffer fluid, however, can dilute the specimen sample solution and change the viscoelastic properties of the specimen, and thus adversely affect the ability to measure the viscoelastic properties of the specimen. The problem is magnified if the specimen volume is small, as is desirable in human clinical applications.

Accordingly, there exists a need for methods and systems for distributing a plurality of particles within a viscoelastic specimen.

SUMMARY

According to one aspect, the present subject matter described herein includes a method for distributing a plurality of particles within a viscoelastic specimen. For example, the method includes drawing, into a doping device, a particle solution that includes a plurality of particles suspended in a bufferfluid. The method further includes adhering the particles to a surface of the doping device by evaporating the buffer fluid from the particle solution and drawing, into the doping device, a viscoelastic specimen that causes the particles to dislodge from the surface of the doping device and become suspended within the viscoelastic specimen. The method also includes measuring the movement of the particles suspended within the viscoelastic specimen to determine at least one rheological property of the viscoelastic specimen.

According to one aspect, the present subject matter described herein includes a system for dispersing particles in a viscoelastic specimen. For example, the system comprises a plurality of particles and a buffer fluid for suspending the plurality of particles. The system also includes a doping device for drawing the plurality of particles into itself, for allowing evaporation of the buffer fluid such that the particles adhere to a surface of the doping device, and for drawing in a viscoelastic specimen and dislodging the particles from the surface, wherein the dislodged particles are suspended within the viscoelastic specimen. The system further includes a measurement device for measuring the movement of the particles suspended in the viscoelastic specimen to determine at least one rheological property of the viscoelastic specimen.

According to one aspect, the present subject matter described herein includes a doping device that includes a housing having at least one sidewall forming an opening and a plurality of particles distributed on an inner surface of the sidewall through the drawing of a buffer fluid containing the particles through the opening and the evaporation of the buffer fluid, wherein the opening is configured to draw in a viscoelastic specimen that dislodges the particles into the viscoelastic specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the subject matter described herein will now be explained with reference to the accompanying drawings, wherein like reference numerals represent like parts, of which: Figure 1 is a diagram illustrating an exemplary method for distributing a plurality of particles within a viscoelastic specimen according to an embodiment of the subject matter described herein;

Figure 2 is a diagram illustrating an exemplary pipette used to distribute particles within a viscoelastic specimen according to an embodiment of the subject matter described herein;

Figure 3A is a diagram illustrating the distribution of particles within a particle solution contained in an exemplary pipette tip according to an embodiment of the subject matter described herein;

Figure 3B is a diagram illustrating the adhering of particles on the side walls of an exemplary pipette tip according to an embodiment of the subject matter described herein;

Figure 3C is a diagram illustrating the dispersion of particles in a viscoelastic solution from the according to an embodiment of the subject matter described herein;

Figure 4 is a diagram illustrating the distribution of particles within a viscoelastic specimen contained in a pipette tip according to an embodiment of the subject matter described herein; and

Figure 5 is a block diagram illustrating an exemplary rheological measurement system according to an embodiment of the subject matter described herein.

DETAILED DESCRIPTION

In accordance with the subject matter disclosed herein, systems and methods are provided for distributing particles within a specimen for rheological measurement. The subject matter disclosed herein is directed to a reduced dilution technique for dispersing particles throughout a viscoelastic specimen or sample of interest, thereby enabling the measurement of viscoelasticity properties of the specimen, rather than the combination of a specimen and a buffer solution. The technique may be applied to materials of biological or clinical origin, such as developing or dissolving blood clots, mucus, or sputum, and the like. Similarly, the technique may utilize various doping modalities such as capillary tubes, pipette tips, and microfluidics systems. In one embodiment, the present subject matter uses very small specimen volumes (e.g., micro- volumes) and can be implemented in a variety of forms that would be applicable to high throughput systems, point of care applications, and patient diagnosis. In one embodiment, essential components (e.g., pipette tips) may be made disposable so that sterility can be maintained and there would be no risk of contaminating the specimen.

In one embodiment of the present subject matter, a small amount of particles may be mixed with a buffer fluid to create a particle solution. As used herein, a particle may range from about 10 nanometers to about 100 micrometers in size. In one embodiment, a particle may be shaped as a sphere (e.g., a microsphere), a rod, a bead e.g., a microbead), a cone, and the like. Particles may also be a particle constructed of any material, such as a metallic, ferromagnetic, ferroelectric particle. Alternatively, particles may be constructed from a non-metallic material, such as polystyrene, that may be impregnated with a magnetic material. In one embodiment, particles may be coated with a non-reactive material that prevents the particle from chemically reacting (e.g., sticking or fusing together) with other particles or container surfaces. One such non-reactive material includes polyethylene glycol (PEG). Particles may also be constructed with various surface chemistries, such as a carboxyl (i.e., COOH) surface chemistry, Tosyl-activated surface chemistry, an amine surface chemistry, and the aforementioned a polyethylene glycol (PEG) surface chemistry.

After being mixed, the particle solution may be drawn up into a doping device, such as a pipette tip or capillary tube. The doping device may subsequently be placed in an oven or allowed to air dry in order to evaporate the buffer fluid portion of the contained particle solution. After the evaporation process is completed, the remaining dried particles are lightly adhered to the inner surface of the doping device. Next, a viscoelastic specimen (e.g., that will ultimately be measured for rheological properties) is drawn into the pipette or capillary tube containing the particles. As used herein, a viscoelastic specimen may include any viscous material that may require its rheological properties tested or measured. An exemplary viscoelastic specimen may include a biological fluid specimen, such as a blood specimen, a deoxyribonucleic acid (DNA) specimen, a synovial fluid specimen, a saliva specimen, an expectorant specimen, a pus specimen, a tear specimen, a peritoneal fluid specimen, a pleural fluid specimen, and a mucus specimen (e.g. sputum, ocular fluid, sinus fluid, and cervical fluid).

In one embodiment, an air suspension that contains a plurality of particles may be sprayed into a doping device, such as a pipette tip or capillary tube. By spraying an air suspension into the doping device, the particles may become adhered to the inner side walls of the doping device without having to utilize a particle solution and evaporate a buffer fluid portion (as mentioned above).

As the specimen drawn into the doping device encounters the adhered particles, the viscoelastic specimen's bulk dislodges the particles from the doping device's inner surface. This process effectively disperses and distributes the particles throughout the specimen without damaging its viscoelastic network or structure. The present subject matter is disclosed in further detail below.

Figure 1 is a flowchart illustrating an exemplary method 100 for distributing particles within a viscoelastic specimen according to an embodiment of the subject matter described herein. In block 102, a particle solution is prepared. In one embodiment, a solution containing a plurality of particles suspended in a buffer fluid is created. For example, an ethanol-based buffer may be combined in a container with a particle mixture comprising a plurality of particles residing in a solution. In one embodiment, the buffer fluid to particle mixture ratio may be 5:1. For example, if 6 microliters of particle mixture is used, 30 microliters of ethanol should be added to achieve this exemplary ratio. The resulting particle solution may be lightly mixed to attain suitable particle distribution.

In block 104, the particle solution is drawn into a doping device. In one embodiment, the particle solution may be drawn into a pipette tip or a capillary tube. An exemplary pipette used to draw up the particle solution is illustrated in Figure 2. Specifically, Figure 2 depicts a pipette 200 (i.e., an Eppendorf pipette) that is composed of a pipette body 202 and a pipette tip 204, which is attached at one distal end of the pipette body 202. A measurement dial 206 may also be positioned on the opposing distal end of pipette body 202. In one embodiment, pipette tip 204 may be a disposable and detachable pipette tip that facilitates the drawing of a fluid specimen or solution, such as a particle solution. Measurement dial 206 may be used to accurately set the volume of fluid to be drawn into pipette tip 204. Figure 3A illustrates a close-up view of pipette tip 304 (which is not unlike pipette tip 204 depicted in Figure 2) containing the drawn particle solution 306. Specifically, Figure 3A illustrates a small amount of particle solution 306 is drawn into a tip 304 of a clean pipette (e.g., doping device with a housing having at least one sidewall forming an opening).

Returning to Figure 1 , the buffer fluid in the pipette tip is evaporated in block 106. In one embodiment, the buffer fluid portion of the particle solution is evaporated, thus leaving behind the plurality of particles. In one embodiment, the pipette tip containing the particle solution may be placed in an oven or incubator (e.g., for 20 minutes at 80 °C) to facilitate the evaporation of the buffer fluid. In another embodiment, the pipette tip may be air dried. In Figure 3B, pipette tip 304 may be placed in an oven and the buffer fluid portion of the particle solution 306 is allowed to evaporate. Notably, the drying process enables the particles to become lightly associated (e.g., lightly adhered to, but not permanently bound) with the inner pipette tip surface. As shown in Figure 3B, the dry particles 308 that remain from the evaporation process lightly attach with the inner surface sidewall of pipette tip 304. Although Figure 3B depicts the dry particles 308 on two sides of pipette tip 304 for sake of clarity, it is understood that particles 308 are adhered to the entire circumference of the inner sidewall of pipette tip 304. In one embodiment, the inner surface wall of the doping device (e.g., the pipette tip) may be coated with a non-reactive substance (e.g., a PEG coating) if the particles do not possess a non-reactive surface chemistry.

In block 108, a fluid specimen is drawn into the pipette tip. In one embodiment, as shown in Figure 3C, a viscoelastic specimen is drawn into pipette tip 304 which causes the particles to detach from the inner wall of pipette tip 304 and become suspended within the viscoelastic specimen. Notably, the pulling/drawing of the viscoelastic specimen into pipette 304 applies a shear force of sufficient magnitude to dislodge and disperse the particles throughout the specimen fluid. The specimen fluid containing the suspended particles is shown in Figure 3C as viscoelastic specimen 310. In one embodiment, pipette tip 304 is dry and cooled to room temperature before a specimen of interest (e.g., a viscoelastic specimen without particles) is pulled into pipette tip 304. In one embodiment, subsequent delivery of a payload (i.e., viscoelastic specimen 310) contained inside pipette tip 304 to a new container (i.e., a specimen well or a test tube) may provide improved particle distribution within specimen 310.

In block 110, at least one rheological property of the viscoelastic specimen is determined. In one embodiment, the movement of the particles suspended within the viscoelastic specimen is measured using a measurement device, such as a rheometer. For example, a magnetic force or field may be applied to the viscoelastic specimen containing the suspended particles. The applied force may cause the suspended particles, which may comprise or include ferromagnetic material, to move within the viscoelastic specimen. The degree of movement may be captured by an optical tracking subsystem of the rheometer. The movement data and the viscoelastic specimen material data may subsequently be processed to determine at least one rheological property (e.g., viscoelasticity) associated with the viscoelastic specimen.

Figure 4 is a diagram illustrating the distribution of particles within a viscoelastic specimen according to an embodiment of the subject matter described herein. Specifically, Figure 4 illustrates a plurality of cross-sectional views 400-404 of a doping device, such as pipette tip 204, at the various times and stages. For example, at time t₀, cross-section 400 is shown to contain a particle solution (not unlike particle solution 306 in Figure 3) that contains a plurality of particles suspended in a buffer fluid. Pipette tip cross-section 401 depicts, at time t| , the buffer fluid portion of the particle solution beginning to evaporate (e.g., via being placed in an oven or allowing to air dry). Also shown in cross-section 401 is a number of particles adhering to the inner surface wall at the top section of the pipette. Pipette tip cross-section 402, at time t₂, depicts further evaporation of the buffer fluid portion of the particle solution in the pipette and even more particles lightly adhering to (i.e., becoming lightly associated with) the inner surface of the pipette tip. As shown in pipette tip cross-section 403, at time t3, the entire buffer fluid portion of the particle solution has evaporated, thereby causing all of the particles to become lightly adhered to the inner surface of the pipette tip. Cross-section 403 also corresponds to Figure 3B. Lastly, pipette tip cross-section 404, at time , is shown to contain a viscoelastic specimen that is drawn into the pipette tip. Notably, the pulling/drawing of the viscoelastic specimen into the pipette tip has dislodged the dried particles (shown in cross section 403) from the inner surface and thereby caused the particles to be suspended within the viscoelastic specimen. Cross-section 403 also corresponds to Figure 3C.

After being drawn into the doping device (e.g., a pipette tip or the capillary tube) to distribute the particles, the viscoelastic specimen may be utilized to perform rheological measurements. For example, the suspended particles may be imaged either inside a capillary tube directly or be imaged in a separate specimen chamber/container after being dispense by a pipette tip. In one embodiment, the present subject matter may utilize square capillary tubes so that optical microscopy techniques can be performed within the capillary tube itself in order to observe the motion of the suspended particles (i.e., cylindrical capillary tubes can create measurement inaccuracies due to refraction). Alternatively, it may be of interest to subsequently dispense a viscoelastic specimen with suspended particles into a second chamber or container prior to performing a rheological measurement. Thus, a pipette tip with loaded dried particles may be a desirable methodology. Furthermore, subsequent delivery of a payload (i.e., the specimen with particles) inside a pipette tip to a new container may ensure even better mixing of the particles within the specimen.

The present subject matter is also scalable since the utilization of preloaded doping devices may allow for the rapid inoculation, e.g., using microfluidics channels/systems, of many microrheology samples. Dispensing viscoelastic specimens in an efficient manner is often desired when performing any high-throughput type measurements. Quick specimen deployment also mitigates sources of noise or error for measurements where evaporation is a key issue (e.g., using mucus as a specimen) when determining rheological properties.

Figure 5 is a block diagram illustrating an exemplary rheological measurement system 500 according to an embodiment of the subject matter described herein. In one embodiment, system 500 includes a control and measurement subsystem 502, a multiforce application subsystem 506, an imaging and tracking optical subsystem 508, and a specimen container 504.

In one embodiment, specimen container 504 may be a capillary tube or a container, such as a petri dish or a specimen well(s) in a microtiter plate, that receives viscoelastic specimen dispensed from a pipette tip. Alternatively, specimen container 504 may be the doping device described herein after the specimen has been introduced into the doping device (e.g., a square-sided capillary tube containing a viscoelastic specimen). In one embodiment, multiforce application subsystem 506 for actuating the particles comprises a magnetic drive block that is capable of producing forces of significant magnitude (e.g., forces greater than 10 nanoNewtons) directed toward specimen container 504 and applying magnetic force (i.e., paramagnetic or diamagnetic force depending on whether paramagnetic or diamagnetic particles are used). The forces may be produced in multiple dimensions and excitation frequencies.

Imaging and tracking subsystem 508 may include, but is not limited to, an optical system that measures scattered light to detect movement of the actuated particles, an imaging system including a camera that images each specimen container or group of specimen containers (e.g., wells in a microtiter plate), and/or a pick up coil that measures amplitude and phase of a current produced by the motion of the actuated particles in container 504 (or a plurality of containers). For example, imaging and tracking subsystem 508 may include a single specimen imaging system with a robotic stage that can position the specimen container over a microscope objective. Alternatively, Imaging and tracking subsystem 508 may include an array based system that is capable of imaging several specimen containers simultaneously. The recorded images may be used to track the particle position and the like. Control and measurement subsystem 502 may be configured to measure the mechanical properties of the specimen 504 based on the measured movement of the particles under the applied force. For example, control and measurement subsystem 502 may measure viscoelastic properties of the specimen containing a distributed plurality of particles based on how much or how easily the particles move under the applied force. Control and measurement subsystem 502 may also be employed to perform several kinds of measurements, either simultaneously with the application of force produced by subsystem 506 or after the force sequence has been applied.

It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. A method for distributing a plurality of particles within a viscoelastic specimen, the method comprising:

drawing, into a doping device, a particle solution that includes a plurality of particles suspended in a buffer fluid;

adhering the particles to a surface of the doping device by evaporating the buffer fluid from the particle solution;

drawing, into the doping device, a viscoelastic specimen that causes the particles to dislodge from the surface of the doping device and become suspended within the viscoelastic specimen; and

measuring the movement of the particles suspended within the viscoelastic specimen to determine at least one rheological property of the viscoelastic specimen.

2. The method of claim 1 wherein the particles include PEGylated particles.

3. The method of claim 2 wherein each of the PEGylated particles comprises the shape of a rod, a cone, a bead, or a sphere.

4. The method of claim 1 wherein the doping device comprises a pipette tip.

5. The method of claim 1 wherein the doping device comprises a capillary tube.

6. The method of claim 1 wherein the surface of the doping device or the particles are coated with a substance to reduce chemical reactions between the surface and the particles.

7. The method of claim 6 wherein the substance comprises polyethylene glycol (PEG).

8. The method of claim 1 wherein the specimen includes at least one of: a blood specimen, a deoxyribonucleic acid (DNA) specimen, a saliva specimen, an expectorant specimen, a pus specimen, a tear specimen, a synovial fluid specimen, a peritoneal fluid specimen, a pleural fluid specimen, and a mucus specimen.

9. The method of claim 1 wherein evaporating the buffer fluid includes incubating the doping device or air drying the doping device.

10. The method of claim 1 wherein the particles comprise at least one of: a metallic material, a magnetic material, a ferroelectric material, and a polystyrene material.

11. The method of claim 1 wherein drawing the viscoelastic specimen into the doping device includes drawing the viscoelastic specimen into the doping device in order to apply a shear force that dislodges a plurality of the particles attached to the inner surface of the doping device.

2. The method of claim 1 comprising discharging the viscoelastic specimen containing the plurality of particles from the doping device into a container to further disperse the plurality of particles throughout the viscoelastic specimen.

13. The method of claim 1 wherein the at least one rheological property of the viscoelastic specimen includes the viscoelasticity of the viscoelastic specimen containing the particles.

14. A system for dispersing particles in a viscoelastic specimen, the system comprising:

a plurality of particles;

a buffer fluid for suspending the plurality of particles; a doping device for drawing the plurality of particles into itself, for allowing evaporation of the buffer fluid such that the particles adhere to a surface of the doping device, and for drawing in a viscoelastic specimen and dislodging the particles from the surface, wherein the dislodged particles are suspended within the viscoelastic specimen; and a measurement device for measuring the movement of the particles suspended in the viscoelastic specimen to determine at least one rheological property of the viscoelastic specimen.

15. The system of claim 14 wherein the particles include PEGylated particles.

16. The system of claim 15 wherein each of the PEGylated particles comprises the shape of a rod, a cone, a bead, or a sphere.

17. The system of claim 14 wherein the doping device comprises a pipette tip.

18. The system of claim 14 wherein the doping device comprises a capillary tube.

19. The system of claim 14 wherein the surface of the doping device or the particles are coated with a substance to reduce chemical reactions between the surface and the particles.

20. The system of claim 19 wherein the substance comprises polyethylene glycol (PEG).

21. The system of claim 14 wherein the specimen includes at least one of: a blood specimen, a deoxyribonucleic acid (DNA) specimen, a saliva specimen, an expectorant specimen, a pus specimen, a tear specimen, a synovial fluid specimen, a peritoneal fluid specimen, a pleural fluid specimen, and a mucus specimen.

22. The system of claim 14 wherein the buffer fluid is evaporated via incubating the doping device or air drying the doping device.

23. The system of claim 14 wherein the particles comprise at least one of: a metallic material, a magnetic material, a ferroelectric material, and a polystyrene material.

24. A doping device comprising:

a housing having at least one sidewall forming an opening; and a plurality of particles distributed on an inner surface of the sidewall through the drawing of a buffer fluid containing the particles through the opening and the evaporation of the buffer fluid, wherein the opening is configured to draw in a viscoelastic specimen that dislodges the particles into the viscoelastic specimen.