DESC:SYSTEM AND METHOD FOR MICROFLUIDIC BASED LASER ASSISTED BOVINE SPERM SEPARATION

BACKGROUND
[001] The invention relates generally to systems and methods for bovine sperm separation using Microfluidic based Laser assisted Bovine Sperm Separation (MLBSS) technology. The MLBSS technology is based on Hydrodynamic focusing technique which is widely implemented in microfluidic chips.
[002] Bovine sperm sex sorting is of importance for the dairy farmers around the world. The main problem of non-productive male cattle is a drain on their resources. Moreover, having only female calves ensures next generation of cows for dairy operations. Currently, bovine sperm sexing technology is monopolistic and very expensive. Bovine sperm sexing for female sperms increases the farmer incomes by over 200% which prevents agrarian distress.
[003] The current exploration on sexed semen is mainly focused to achieve calf of desired sex. Sexed semen is widely available now and many dairy producers are using it to obtain more (and better) healthier calves. Moreover, the current system involves higher cost per dose and is available for use only once. In addition, the second breeding is the limiting factor for wide acceptability among dairy producers. The economic benefits of the use of sexed semen are different for every dairy farm. In particular, the sexed semen is obtained from fractions of semen for X-bearing (female) and the Y-bearing (male) sperm and is further been modified from the natural mix through sorting and selection, which is based on sorting by flow cytometry for DNA content of sperm (between X having of about 3.8% higher DNA content compared to and Y bearing sperm) using DNA-binding fluorescent dye.
[004] The current technology uses charged plates after orientation of sperms. Subsequently after detection, the sperms are separated by a fluorescence-activated cell sorter. Moreover, at present, the method implemented is approximately of about 90% accurate but is not available to poor countries. Due to electrical field and shear forces the fertility of sexed sperm is typically lower compared with conventional sperm. However, the throughput is also very poor and time consuming.
[005] Currently, the technologies implemented for sorting of sperms by sex include several procedures such as flow cytometry or cell sorting. The flow cytometry involves sorting each sperm cell individually. Sorting of sperms is based on content of DNA in X-bearing sperm which is slightly higher as compared to Y-bearing sperm. After collecting the semen, the semen is processed and stained using Hoechst 33342 DNA-binding dye. For sorting sperms, each sperm cell is placed in an individual droplet. The difference in fluoresce of the droplets when exposed to a laser, differentiates X from Y-bearing sperm. After sex determination, the charge is placed on the droplet and the charged deflector sorts the sperm into one of three collection vessels X, Y, and waste. Sperms that are damaged, oriented incorrectly, or cannot be “read” are discarded. Approximately, about 25% of each ejaculate is sorted at X and another about 25% is sorted as Y. Sorting accuracy can be adjusted, but most companies sort for 90% accuracy. After sorting, the concentration of sperm in the collection vessels is diluted. Sorted sperm must be concentrated by centrifugation which results in additional loss of sperm numbers.
[006] Therefore, a system and method is required that provides an affordable bovine sperm separation, without an additional wastage of sperms numbers and in particular provides the required purity of about 90 percent.
DRAWINGS
[007] FIG. 1 is a block diagram illustrating an ecosystem of microfluidic flow analyzer device for bovine sperm separation, implemented according to aspects of the present technique.
[008] FIG. 2 is a schematic diagram illustrating an example microfluidic flow analyzer device implemented according to aspects of the present technique;
[009] FIG. 3 illustrates an example configuration of a hydrodynamic two dimensional focusing with different flow rates thereby different widths implemented according to aspects of the present technique;
[0010] FIG. 4 illustrates an example graphical representation of the hydrodynamic two dimensional focusing with different sample flow rates thereby different widths implemented according to aspects of the present technique;
[0011] FIG. 5 illustrates an example graphical representation of correlation of first two interrogation points designed for the lag time measurement implemented according to aspects of the present technique;
[0012] FIG. 6 illustrates an example graphical representation of flow control in microfluidic device implemented according to aspects of the present technique;
[0013] FIG. 7 illustrates an example graphical representation of scatter plot for amplitude of SSC vs FSC and histogram of amplitude of events for FSC and SSC implemented according to aspects of the present technique;
[0014] FIG. 8 illustrates an example graphical representation of separation of two different cell types (BV2 and U937) from the FSC-SSC detection implemented according to aspects of the present technique;
[0015] FIG. 9 illustrates an example graphical representation of fluorescence signal detected from cells stained with Cy5 and Alexa Fluor 660 implemented according to aspects of the present technique;
[0016] FIG. 10 illustrates an example graphical representation of fluorescence signal detected from cells stained with Cy5 and unstained cells implemented according to aspects of the present technique;
[0017] FIG. 11 illustrates package optimization of microfluidic analyzer implemented according to aspects of the present technique;
[0018] FIG. 12 illustrates an electrical analog board integrating the lasers and detectors onto a single board implemented according to aspects of the present technique; and
[0019] FIG. 13 illustrates packaging designed in CAD software implemented according to aspects of the present technique.

DETAILED DESCRIPTION
[0020] Embodiments of the present technique disclose a miniaturized instrument with increased functionality and accurate information of bovine cells using a microfluidic device. This microfluidic device implements 2D focusing, flow monitoring/control, coupled opto-electronic system, embedded lens fiber, gated avalanche photodiode for high-sensitivity detection and gated data analysis.
[0021] In one embodiment, the identification and sorting of bovine sperm is done using FDA approved dye Hoechest 33342 or (other approved dyes) for identification of sperms on the basis of DNA content between X and Y chromosome in male and female sperms. The sorted X sperms would be final product and Y sperms would be killed by photo ablation by using laser device with ranges around 1050- 1350 nm.
[0022] Microfluidic based Laser assisted Bovine Sperm Separation (MLBSS) chip disclosed herein is a miniature flow analyzer implemented in lab-on-chip form, combining principles of optics, flow cytometry, microfluidics device fabrication, optoelectronics, data acquisition and analysis to allow rapid cell analysis and quantification. After minimal preparation, the sample flows through a microfluidic device in which laser detects (specifically detects) the amount of DNA content in the cells. Data is collected and processed on a small electronics board. The identification and sorting of female sperms from bovine or farm animals using a microfluidic device is described in further details below.
[0023] FIG. 1 is a block diagram illustrating an ecosystem 10 of microfluidic flow analyzer device for bovine sperm separation, implemented according to aspects of the present technique. The system 10 includes a microfluidic device 12, a micro-controller unit 14, computer 15, a single photon counting module (SPCM) 16, a plurality of detector’s 18-A and 18-B, and one or more laser devices 19-A through 19-C. Each component is described in further details below.
[0024] Microfluidic device 12 implements Microfluidic based Laser assisted Bovine Sperm Separation (MLBSS) technology. MLBSS is based on hydrodynamic focusing technique which is widely used in flow cytometers for accurate analysis of cells. The hydrodynamic focusing technique makes sure that micro particles flow in single file and therefore reduces the chance of missing any micro particles. The system is designed to include an optoelectronics system and is fiber coupled which will miniaturize the whole system. The manner in which a microfluidic device 12 is implemented for the bovine sperm separation is described below.
[0025] In one embodiment, when the sample flows through the microfluidic device 12, the first two lasers (1550nm) as represented by reference numeral 19-A are used to estimate the speed of the sample, the second laser (360nm) as represented by the reference numeral 19-B is used to identify the sperm cell and the third laser (1064nm) as represented by reference numeral 19-C is used to ablate the cells. Each of these signals are detected by the respective detectors (as represented by reference numeral 18-A and 18-B) on the other side, and the signals are sent to the micro-controller unit 14. In one example embodiment, the detector’s 18-A and 18-B are InGaAs detectors.
[0026] The micro-controller unit 14 is implemented to control laser source operation mode whether continuous or modulated mode via GPIO/PWM signal. Further, the data acquisition of detector 18-A and 18-B output signals and data logging of same is done by the micro-controller unit 14. The micro-controller unit 14 facilitates the triggering of 1064 nm laser source (as represented by reference numeral 19-C) if needed. Further, the micro-controller unit 14 is also responsible for transmission of data from board to a computer 15 for data analysis.
[0027] In one embodiment, the lensed fiber would be used for illumination and collection of light by replacing free space lenses. Lensed fiber is placed in the source side and is utilized to focus the light from where particles shall pass. Further, the detector side with higher NA (numerical aperture) lensed fiber helps to collect large amount of scatter/fluorescent light from particles. The flow control would be done by developing an algorithm of correlation of time delayed in optoelectronics signal which will provide reliable hydrodynamic focusing. This algorithm will also help to find timing of each particles at any point of fluidic channel which will pass through it. This instrument and chip would be used for identification. Further, the sorting would be done using FDA approved dye Hoechest 33342 or (other approved dyes) for identification of sperms on the basis of DNA content between X and Y chromosome in male and female sperms.

Design optimization
[0028] FIG. 2 is a schematic diagram illustrating an example microfluidic flow analyzer device implemented according to aspects of the present technique. In one embodiment, the microfluidic flow analyzer device 20 is implemented for bovine sperm separation using Microfluidic based Laser assisted Bovine Sperm Separation (MLBSS) technology.
[0029] The design optimization of the microfluidic analyzer device is described herein. The microfluidic flow analyzer device 20 is configured to have a single sheath inlet which eventually controls the sample fluid width. In one embodiment, the microfluidic flow analyser device 20 includes three optical interrogation points and an ablation point along the flow of the sample. The first two interrogation points (as shown as FR1 and FR2) are designed for the lag time measurement between two interrogation point of cells/particles flowing. At the third interrogation point, the same cell is designed to undergo 360 nm laser illumination where it generates fluorescence signals. Further, at the ablation point, the microcontroller unit 14 will trigger 1064 nm laser to turn ON based on fluorescence detector output as compared to set threshold. The manner in which the fluid testing of the microfluidic device 12 is implemented for the bovine sperm separation is described below.

Fluid testing
[0030] FIG. 3 illustrates an example configuration 30 of a hydrodynamic two dimensional focusing with different flow rates thereby different widths implemented according to aspects of the present technique. In one example embodiment, FIG. 3 illustrates the focusing of sample fluid (middle stream) into different widths where Deionized Water (DI) water is used as the sheath fluid and a solution of food dye as the sample. The width of the sample stream is controlled by selecting the sheath flow rate. The same experiment for fluid testing as described in FIG, 3 is carried out with different sample flow rates. The results of the experiment for fluid testing with different flow rates are illustrated in FIG.4 below.
[0031] FIG. 4 illustrates an example graphical representation 40 of hydrodynamic two dimensional focusing with different sample flow rates thereby different widths implemented according to aspects of the present technique. The graphical representation 40 provides assistance to choose the combination of flow rates for different sized beads/cells. In one embodiment, the FIG 4 shows sample widths as a function of sheath flow rate.
[0032] FIG. 5 illustrates an example graphical representation 50 of flow control implemented according to aspects of the present technique. In particular, the FIG. 5 represents correlation of FR1 & FR2. As mentioned in FIG. 1, the FR1 & FR2 are first two interrogation points (as shown as FR1 and FR2) designed for the lag time measurement between two interrogation point of cells/particles flowing.
[0033] FIG. 6 illustrates an example graphical representation 60 of flow control in microfluidic device implemented according to aspects of the present technique. In one embodiment, the detection signal is detected from FR1 and FR2 which is at +5 degree and -5 degree. Further, by apply cross-correlation on FR1 and FR2, the data gives information related to time lag between them. The flow control is tested with different flow rate of sample.
Testing: Design of data interface/display
[0034] FIG. 7 and FIG. 8 illustrate the analysis and results of the design of data interface/display of the microfluidic analyzer device respectively.
[0035] FIG. 7 illustrates an example graphical representation 70 of scatter plot for amplitude of SSC vs FSC and histogram of amplitude of events for FSC and SSC: Sample U937 cells implemented according to aspects of the present technique.
[0036] FIG. 8 illustrates an example graphical representation 80 of separation of two different cell types (BV2 and U937) from the FSC-SSC detection. In one embodiment, the optimization of signal pickup was done with beads as well as cells. Simultaneous detection of forward scatter and side scatter were done at 5 degree and 45 degree respectively. The algorithm was optimized by using beads as well as with cells. The data interface/display has been designed to give the scatter plot of SSC amplitude versus FSC amplitude, along with the individual histograms.

Testing with fluorescently tagged commercial cell lines
[0037] FIG. 9 illustrates an example graphical representation 90 of fluorescence signal detected from cells stained with Cy5 and Alexa Fluor 660 implemented according to aspects of the present technique. The signal level is increased by increasing the concentration of antibody per cell.
[0038] In one embodiment, the fluorescence detection by the system has been optimized with fluorescence tagged commercial cell lines. The optimization has been done by using the sample taken in a cuvette. IgG cells which do not express fluorescence were used as a control for the measurements. It was observed that, the signal level increased when the antibody concentration was increased from 100 ng to 2 µg for the Alexa Fluor 660 dye. The Fluorescence signal from Cy5 at 125 ng of antibody concentration was found comparable to the signal from 1 - 2 µg antibody stained with Alexa Fluor 660.
[0039] FIG. 10 illustrates an example graphical representation 100 of Fluorescence signal detected from cells stained with Cy5 and unstained cells implemented according to aspects of the present technique. In one example embodiment, the optimization is done in microfluidic device and tested by running several run. The combination and placement of source and detection fiber was about 45 degree. The stained cells were used with Cy5 and unstained cells were used as a sample. Further, a comparison of the fluorescence intensity of stained and unstained cells was carried out.
Optimization: Package optimization.
[0040] FIG. 11-A illustrates a microfluidic analyzer 110-A implemented according to aspects of the present technique. FIG. 11-B illustrates a microfluidics 110-B employed in the system described herein implemented according to aspects of the present technique. FIG. 11-C illustrates size comparison of Guava easycyte flow cytometer and Microfluidic analyzer 110-C implemented according to aspects of the present technique. The overall system was reduced to a size of 40 cm x 23 cm x 10 cm encased in a plastic.
[0041] FIG. 12 illustrates an electrical analog board 120 integrating the lasers and detectors onto a single board implemented according to aspects of the present technique.
[0042] FIG. 13 illustrates packaging 130 designed in CAD software implemented according to aspects of the present technique. In one embodiment, all the components for optics fluidics and electronics have been designed to arrange inside the box efficiently for the final packaging. The packaging is designed to be two layers of components with proper plates to support them. The top layer consists mainly of the fluidics, optics and related electronics. Whereas the bottom layer consists of the integrated analog board, development board, power supply etc. The extra length of the optical fibers are managed to keep at the base plate with properly spooled and clipped. The sample, sheath and the outlet fluids will be kept outside, attached to the enclosure.
[0043] In one embodiment, the present disclosure describes development of validated methods for analysis of quality and function of sperms with advanced flow cytometry and imaging. The system described herein is aimed at to achieve approximately about 90% purity of female bovine sperms. Further, the system yield is of approximately about 30 % of female sperms from single ejaculate which is sufficient for 400 doses for Artificial insemination. In addition, the target is to achieve about 90 % chances of pregnancy by in vitro fertilization. Moreover, the production of about 1-1.5 million Microfluidic based Laser assisted BSS (MLBSS)-chips can be achieved in one year.
[0044] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions.
[0045] It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0046] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
[0047] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present.
[0048] For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.
[0049] In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.).
[0050] It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
[0051] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
[0052] As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art.
,CLAIMS:CLAIMS:

1. A microfluidic laser assisted cell separator system, the microfluidic laser assisted cell separator system comprising:
a microfluidic device configured to introduce sample cells to the process;
a plurality of lasers configured to illuminate each sample cell;
a plurality of detectors configured to capture scattered light and/or emitted fluorescence from each sample cell;
a micro controller unit configured to:
receive signals from detectors;
convert photons to photo electrons; and
convert analog signal to digital signal; and
a computer configured to receive data from the micro controller unit for data analysis.

2. The microfluidic laser assisted cell separator system of claim 1, wherein the microfluidic device comprising:
a central core channel through which fluid containing sample cells are injected.

3. The microfluidic laser assisted cell separator system of claim 1, wherein the plurality of lasers correspond to plurality of interrogation points and ablation points, with each such points used for specific action.

4. The microfluidic laser assisted cell separator system of claim 1, wherein the plurality of detectors corresponds to plurality of lasers.

5. The microfluidic laser assisted cell separator system of claim 3, wherein the interrogation point is where the laser and the sample cell intersect resulting in light scatter or fluorescence emission.

6. The microfluidic laser assisted cell separator system of claim 1, wherein the micro controller unit is configured to:
derive electrical signals from forward scatter;
derive electrical signals from side scatter; and
derive electrical signals from fluorescence emission.

7. The microfluidic laser assisted cell separator system of claim 1, wherein the computer is configured to receive the data from the micro controller unit and perform data analysis.

8. The microfluidic laser assisted cell separator system of claim 7, wherein data analysis involves combining forward scatter data, side scatter data and fluorescence intensity data and forming multiple histograms and dot plots.

9. A microfluidic laser assisted cell separator system, configured to separate or isolate the desired cells, wherein:
the microfluidic laser assisted cell separator system uses multiple parameters to identify and separate X- bearing sperm cell from Y-bearing sperm cells.