Microfluidic device for bovine sperm cell sex sorting

Abstract

The present disclosure relates to a microfluidic device (100) comprising a base member (110) defined with an inlet conduit (112) and an outlet conduit (114), a magnet (140) positioned below the base member (110) The base member (110) includes a plurality of microchannels (116) and is configured to trap a biological sample (200) bounded with silica-coated magnetite nanoparticles under the influence of magnetic field of the magnet (140). The base member (110) further includes a plurality of partition walls (118) defined with a plurality of microgrooves (118a) fluidly interconnecting the adjacent microchannels. The plurality of microgrooves (118a) is configured to selectively allow flow of the processed biological sample to the outlet conduit (114) of the device (100). The biological sample collected at the outlet (114b) exhibit a sorting efficiency of 81% for X sperm cells (200X) and 19% for Y sperm cells (200Y).

Description

[0001] TECHNICAL FIELD

[0002]

[0001] Present disclosure relates to microfluidic devices and methods for the sorting of bovine sperm cells, and particularly to the microfluidic device and method for the manipulation of the bovine sperm cells or other biological samples.

[0003] BACKGROUND OF THE DISCLOSURE

[0004]

[0002] The information in this section merely provides background information related to the present disclosure and may not constitute prior art(s) for the present disclosure.

[0005]

[0003] A sperm sex sorting is one of the most studied fields for over past three decades and there is no reason to doubt the fact that some worthwhile progress has been made over these years. Yet we face the issues like low productivity and lower conception rates. With the increasing population, the livestock industries are facing an immense pressure to increase their productivity without compromising their quality. This raises the need for development of an alternative modern approach for solving the unmet issues pertaining to the increasing market demand.

[0006]

[0004] The sperm sex sorting technology is used to separate sperm cells into those carrying the X chromosome and those carrying the Y chromosome which in turn decides the sex of the offspring. The sex sorting of sperm cells offers significant implications in various applications in animal and reproductive contexts. Selective breeding of desired gender is not only favourable in breeding programs and managing herd composition but also impacts in increased productivity and efficient farming practices. It is also crucial in maintaining genetic diversity of endangered species by increasing the breeding of particular gender. Sperm sorting also becomes more valuable in understanding the genetics and developmental biology.

[0007]

[0005] Preselection of sex before conception has been one of the objectives pursued by scientists and breeders for many years. The current technique used for sperm sexing separates spermatozoa containing the X or Y chromosome based on each cell DNA content. However, this technique exposes spermatozoa to high-pressure speed flow, DNA fluorescent stain, and UV light, factors that may affect sperm quality. The aim of this study was to test a new technique to isolate spermatozoa carrying the X chromosome by means of magnetic nanoparticles (MNPs). Over the years, numerous efforts have been dedicated to developing methods for separating X- and Y-bearing sperm populations, enabling the production of offspring of a desired sex in farm animals for commercial reasons or for medical purposes in humans, such as addressing genetic sex-linked disorders. Microfluidic devices and systems are widely employed for processing and analysing tiny fluid samples. The integration of multiple components within a single microfluidic device has resulted in advanced and versatile analytical systems that can be applied to various tasks, such as cell sorting and protein synthesis. Key microfluidic operations that enhance the performance of these applications include mixing, filtering, metering, pumping, reacting, sensing, and regulating the heating and cooling of fluids within the device.

[0008]

[0006] There are several traditional techniques for separating sperm that typically rely on differences in shape, size, motility patterns, and surface charge between X and Y-bearing sperm cells. However, these methods often lack reproducibility and precision, raising concerns about their reliability. One widely used sorting technique in assisted reproduction is multiple gradient centrifugation. When combined with the swim-up method, this approach can enhance chromatin integrity, motility, and viability of sperm cells. Nonetheless, due to doubts about its reproducibility and accuracy, this method is considered unreliable.

[0009]

[0007] Therefore, there is an immense need to develop a simple and cost effective microfluidic device for segregation of the X chromosome and the Y chromosome contained into a biological sample. The present disclosure is directed to overcome one or more limitations stated above or any other limitations associated with the prior art.

[0010] SUMMARY OF THE DISCLOSURE

[0008] The one or more shortcomings of the prior art are overcome by a microfluidic device for processing a biological sample as claimed, Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.

[0011]

[0009] In one non-limiting embodiment of the present disclosure, the microfluidic device comprising a base member defined with an inlet conduit and an outlet conduit positioned opposite to the inlet conduit. The inlet conduit is adapted to receive the biological sample, and the outlet conduit is configured to selectively eject the processed biological sample. The device comprising a magnet which is positioned below the base member. The magnet is configured to attract the biological sample bounded with silica-coated magnetite nanoparticles flowing over an upper surface of the base member. The base member includes a plurality of microchannels defined in an array on the upper surface of the base member. The plurality of microchannels is configured to trap the biological sample bounded with the silica-coated magnetite nanoparticles under the influence of magnetic field of the magnet. The base member further includes a plurality of partition walls separating the plurality of microchannels. Each partition wall is defined with a plurality of microgrooves fluidly interconnecting the adjacent microchannels. The plurality of microgrooves is configured to selectively allow flow of the processed biological sample to the outlet conduit of the device.

[0012]

[0010] In an embodiment of the present disclosure, the processed biological sample bounded with the silica-coated magnetite nanoparticles and collected at the outlet conduit is Y sperm cells.

[0013] [OH] In an embodiment of the present disclosure, the Y sperm cells are retrieved from the outlet conduit, when the magnet is removed from the base member of the device.

[0012] In an embodiment of the present disclosure, the processed biological sample unbounded with the silica-coated magnetite nanoparticles and collected at the outlet conduit is X sperm cells.

[0014]

[0013] In an embodiment of the present disclosure, the device comprises a magnet holder disposed below the base member and the magnet holder is adapted to removably receive the magnet.

[0015]

[0014] In an embodiment of the present disclosure, the device comprises a glass panel disposed between the base member and the magnet holder.

[0016]

[0015] In an embodiment of the present disclosure, the plurality of microchannels is arranged in a direction orthogonal to a longitudinal direction of the device.

[0017]

[0016] In an embodiment of the present disclosure, the plurality of microgrooves is defined in a non-parallel configuration and are configured to provide a passage between the adjacent microchannels.

[0018]

[0017] In an embodiment of the present disclosure, a length of the plurality of microchannels is in the range of 8 mm to 32 mm, and a width of the plurality of microchannels is in the range of 0.3 mm to 1.8 mm.

[0019]

[0018] In an embodiment of the present disclosure, the length of the plurality of microgrooves is in the range of 0.1 mm to 0.9 mm, and the width of the plurality of microgrooves is in the range of 12 pm to 55 pm.

[0020]

[0019] In an embodiment of the present disclosure, the biological sample is pretreated with the silica-coated magnetite nanoparticles before being received in the inlet conduit of the device.

[0021]

[0020] In an embodiment of the present disclosure, each microgroove of the plurality of microgrooves is defined having a cross sectional shape of circular, semi- circular, rectangular, square, pentagonal, hexagonal, heptagonal or combinations thereof.

[0022]

[0021] In one non-limiting embodiment of the present disclosure, a method for processing the biological sample comprising steps of synthesizing silica-coated magnetite nanoparticles, collecting gel-free biological samples, diluting the collected gel-free semen samples to a concentration of 40-60 million cells / ml, adding the silica-coated magnetite nanoparticles to the biological sample in a concentration of 3 to 5 magnetic nanoparticles per sperm and mixing for the homogenization of the nanoparticles through the biological sample to get the mixture of the biological sample containing the silica-coated magnetite nanoparticles, subjecting the biological sample containing the magnetite nanoparticle to a microfluidic device at a controlled flow rate, trapping of the biological sample bounded with the silica-coated magnetite nanoparticles in a plurality of microgrooves using a magnet, and collecting a biological sample containing unbounded sperm cells from an outlet.

[0023]

[0022] In one non-limiting embodiment of the present disclosure, a method of synthesizing the silica-coated magnetite nanoparticles comprising steps of dissolving iron (III) chloride hexahydrate and sodium acetate trihydrate in Polyethylene glycol (PEG) under constant stirring @500 rpm for 30 mins at 50°C under dark in a reaction dish, purging of a reaction dish with Nitrogen gas for 5 minutes and tightly sealed and heated up to 300°C for Ihour, washing precipitated nanoparticles with ethanol and deionised water DI water and subjecting to dry overnight, collecting the precipitated nanoparticles, coating of silica to the nanoparticles through reverse microemulsion, dispersing of magnetic nanoparticles in ethanol, adding of the magnetic nanoparticles to cyclohexane and IGEPAL CO- 520 and mixing for 30 minutes, adding the above nanoparticle mixture to (3- Aminopropyl)ti ethoxy silane (APTES), Tetraethyl orthosilicate (TEOS) and Ammonium hydroxide mixture and mixing until the particles precipitate, and washing the particles precipitate with ethanol and the deionised water DI water.

[0023] It is to be understood that the aspects and embodiments of the disclosure described above may be used in any combination with each other. Several of the aspects and embodiments may be combined together to form a further embodiment of the disclosure.

[0024]

[0024] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0025] BRIEF DESCRIPTION OF FIGURES

[0026]

[0025] The novel features and characteristics of the disclosure are set forth in the description. The disclosure itself, however, as well as a preferred mode of use, further objectives, and advantages thereof, will best be understood by reference to the following description of an illustrative embodiment when read in conjunction with the accompanying drawings. One or more embodiments are now described, by way of example only, with reference to the accompanying drawings wherein like reference numerals represent like elements and in which:

[0027]

[0026] Figure 1 illustrates a perspective view of a microfluidic device, in accordance with an embodiment of the present disclosure;

[0028]

[0027] Figure 2 illustrates a top side view of the microfluidic device showcases X sperm cells and Y sperm cells bounded to silica coated magnetic nanoparticles, in accordance with an embodiment of the present disclosure;

[0029]

[0028] Figure 3 illustrates a top side view of the microfluidic device showcases flow of the X sperm cells passed through a plurality of microgrooves and the Y sperm cells bounded to silica coated magnetic nanoparticles trapped over a plurality microchannels, in accordance with an embodiment of the present disclosure;

[0029] Figure 4 illustrates a magnified view of the plurality of microchannels and the plurality of microgrooves of the microfluidic device of Figure 1, in accordance with an embodiment of the present disclosure;

[0030]

[0030] Figure 5 illustrates the microfluidic devises having different size of the plurality of microgrooves, in accordance with an embodiment of the present disclosure;

[0031]

[0031] Figures 6(a) to 6(k) illustrates a method of fabrication of the microfluidic device of Figure 1, in accordance with an embodiment of the present disclosure; and

[0032]

[0032] Figure 7 illustrates a method of preparation of silica-coated magnetite nanoparticles to be mixed with the biological sample, in accordance with an embodiment of the present disclosure.

[0033]

[0033] Skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the drawings may be exaggerated relative to other elements to help to improve understanding of embodiments of the present disclosure.

[0034] DETAILED DESCRIPTION

[0035]

[0034] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the Figures and will be described in detail below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure as defined by the appended claims.

[0035] Before describing detailed embodiments, it may be observed that the novelty and inventive step that are in accordance with the present disclosure reside in a microfluidic device for processing of bovine sperm cells or other biological samples. It is to be noted that a person skilled in the art can be motivated from the present disclosure and modify the various constructions of the microfluidic device. However, such modification should be construed within the scope of the present disclosure. Accordingly, the drawings are showing only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

[0036]

[0036] In the present disclosure, the term “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment or implementation of the present subject matter described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0037]

[0037] The terms “comprises,” “comprising,” or any other variations thereof, are intended to cover a non-exclusive inclusions, such that a device that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such setup or device. In other words, one or more elements in an assembly or system proceeded by “comprises. . . a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device / apparatus.

[0038]

[0038] The terms like “at least one” and “one or more” may be used interchangeably or in combination throughout the description.

[0039]

[0039] A sperm cell or sperm or, also known as a spermatozoa. The present disclosure pertains to Bovine sperm cells. Further, the present disclosure is also pertaining to other biological samples, without limiting the scope of the invention. The bovine sperm cells are the male reproductive cells found in bulls and other male cattle. These sperm cells are crucial for fertilizing the female’s egg (oocyte) or ovum during reproduction, leading to the conception of a calf. The spermatozoa has a head, midpiece, and tail. The head contains a nucleus with densely coiled chromatin fibers, surrounded anteriorly by a thin, flattened sac called the acrosome, which helps the sperm cell penetrate the egg. The midpiece contains mitochondria that provide energy for the tail. The tail also known as a flagellum, this whip-like tail propels the sperm cell towards the egg. The sperm cells are produced in the seminiferous tubules of testicles. When the sperm cell’s membrane fuses with egg’s membrane, the sperm nucleus enters the egg. The sperm cells and the eggs DNA combine to create a new individual. Another function of the sperm cell is to determine the sex of the future baby. Depending on the chromosomal distribution that takes place during meiosis, the sperm cells possess either X sperm cells, may also call as X chromosomes (female sex) and Y sperm cells, may also called as Y chromosomes (Male sex).

[0040]

[0040] The separation of the X chromosome sperm cells and the Y chromosome sperm cells for the purpose of preselection of offspring has significant implications both in animal production and in human reproductive medicine. The separation of the X chromosome and the Y chromosome is performed by using zeta potential of the sperm cells. Zeta potential refers to the membrane charge that develops between a solid and liquid surface. Dominguez et al., 2018 study showed that the Y chromosome have a difference in membrane potential called the zeta potential of - 16mV whereas the X chromosome have the zeta potential of -20m V. As a result of this, the Y chromosome form complexes with magnetic nanoparticles more easily than X chromosome.

[0041]

[0041] Reference will now be made to the exemplary embodiments of the disclosure, as illustrated in the accompanying drawings. Wherever possible, the same numerals will be used to refer to the same or like parts. Embodiments of the disclosure are described in the following paragraphs with reference to Figures 1 to 7. In Figures 1 to 7, the same element or elements which have the same functions are indicated by the same reference signs.

[0042] Referring to Figure 1, illustrating a microfluidic device (100), may also be called as a microfluidic chip. The microfluidic device (100) includes a base member (110), a magnet holder (130), a glass panel (120) disposed between the base member (110) and the magnet holder (130). The base member (110) is act as a platform for flow of Bovine sperm cells or other biological samples. Hereinafter referred as the biological sample. The biological sample may be a semen sample, blood, saliva, urine, bone marrow, tissue, cerebrospinal fluid, feces, or cell lines. In a preferred embodiment, the biological sample is the semen sample of animal or human being. The biological sample is pre-treated with the silica-coated magnetite nanoparticles (800) before being received in the inlet conduit (112) of the device (100) (described below in greater detail). The biological sample contains the X chromosomes and the Y chromosomes. The base member (110) is made up of polydimethylsiloxane [PDMS] polymer material. The shape of the base member (110) is rectangular. In an embodiment, the shape of the base member (110) may be but not limited to circular, oval, square, triangular, pentagonal, hexagonal, heptagonal, trapezoidal, or rhombus etc. The base member (110) having an upper surface (110a). The base member (110) is defined with an inlet (112a) and an outlet (114b) (shown in Figure 2). Both the inlet (112a) and the outlet (114b) are positioned at opposite side ends of the base member (110). Specifically, the inlet (112a) and the outlet (114b) are arranged along a width of the base member (110). In an embodiment, the inlet (112a) and the outlet (114b) may be arranged along a length of the base member (110). Further, the inlet (112a) and the outlet (114b) are positioned at a middle portion of both side ends of the base member (110). In an embodiment, the inlet (112a) and the outlet (114b) may be positioned anywhere along the length of the base member (110). The inlet (112a) is fluidly connected to an inlet conduit (112) and the outlet (114b) is fluidly connected to an outlet conduit (114). The inlet (112a) is configured for an entrance of the biological sample and the outlet (114b) is configured to eject a processed biological sample. The base member (110) having an upper surface (110a) provided with a working portion (110b).

[0043] The device (100) further includes a magnet (140) which is positioned below the working portion (110b) of the base member (110). The magnet holder (130) is adapted to removably receive the magnet (140). In an embodiment, the magnet (140) may be integrated into the magnet holder (130). The magnet (140) may be a permanent magnet, a temporary magnet, or an electromagnet. In a preferred embodiment, the magnet (140) is the permanent magnet. The magnet holder (130) with the magnet (140) is attached to a bottom of the base member (110).

[0042]

[0044] Referring now to Figure 2, depicts a top side view of the device (100). The base member (110) includes a plurality of microchannels (116) is defined in an array on the upper surface (110a). The plurality of microchannels (116) is configured to trap a biological sample bounded with the silica-coated magnetite nanoparticles (800) under the influence of magnetic field of the magnet (140). The plurality of microchannels (116) is arranged in a direction orthogonal to a longitudinal direction of the device (100). In an embodiment, the plurality of microchannels (116) may be provided along the longitudinal direction of the device (100). The plurality of microchannels (116) provides an area for the biological sample to flow. Further, the base member (110) includes a plurality of partition walls (118) separating the plurality of microchannels (116). The plurality of partition walls (118) extending from the upper surface (110a) of the base member (110). Each partition wall is defined with a plurality of microgrooves (118a) fluidly interconnecting the adjacent microchannels. The plurality of microgrooves (118a) is positioned between each of the plurality of microchannels (116). The plurality of microgrooves (118a) is defined in a non-parallel configuration and are configured to provide a passage between the adjacent microchannels. Each microgroove of the plurality of microgrooves (118a) is defined having a cross sectional shape of circular, semi-circular, rectangular, square, pentagonal, hexagonal, heptagonal or combinations thereof. The plurality of microgrooves (118a) is configured to selectively allow flow of the processed biological sample to the outlet conduit (114) of the device (100). The biological sample (200) containing the X perm cells and the Y sperm cells is inserted into the inlet conduit (112) for flowing through the working portion (110b) of the base member (110) of the device (100).

[0043]

[0045] Referring now to Figure 3, illustrates flow of the biological sample containing the X sperms cells and the Y sperms cells over the working portion (110b) of the base member (110). The biological sample (200) which is incubated with the silica-coated magnetite nanoparticles (800) is passed through the inlet (112a) to flow over the working portion (110b) of the base member (110). The magnet holder (130) along with the magnet (140) is positioned below the working portion (110b) of the base member (110). The magnet (140) is configured to attract a biological sample bounded with silica-coated magnetite nanoparticles (800) flowing over the working portion (110b). Specifically, the silica-coated magnetite nanoparticles (800) are attracted under the influence of magnetic field of the magnet (140) and gets trap in the plurality of microchannels (116) and the plurality of microgrooves (118a), thereby trapping the biological sample bounded with silica- coated magnetite nanoparticles (800). Furthermore, a processed biological sample unbounded with the silica-coated magnetite nanoparticles (800) is collected at the outlet conduit (114), when the magnet (140) is attached to the base member (110) of the device (100). The processed biological sample bounded with the silica-coated magnetite nanoparticles (800) are retrieved from the outlet conduit (114), when the magnet (140) is removed from the base member (110) of the device (100). The processed biological sample bounded with the silica-coated magnetite nanoparticles (800) and collected at the outlet conduit (114) is Y sperm cells (200 Y). The processed biological sample unbounded with the silica-coated magnetite nanoparticles (800) and collected at the outlet conduit (114) is X sperm cells (200X). Accordingly, the X sperm cells (200X) are getting segregated from the Y sperm cells (200Y).

[0044]

[0046] Referring now to Figure 4, illustrates the microfluidic device (100) having different size of the plurality of microgrooves (118a). The device (100) including the plurality of microchannels (116) are created in the array and interconnected with the plurality of microgrooves (118a). A length of the plurality of microchannels (116) is in the range of 8 mm to 32 mm. In a preferred embodiment, the length of the plurality of microchannels (116) is in the range of 10 mm to 30 mm. A width of the plurality of microchannels (116) is in the range of 0.3 mm to 1.5 mm. In a preferred embodiment, the width of the plurality of microchannels (116) is in the range of 0.5 mm to 1.5 mm. Further, a length of the plurality of microgrooves (118a) is in the range of 0.2 mm and 0.8 mm. In a preferred embodiment, the length the plurality of microgrooves (118a) is 0.5 mm. In an embodiment, the length of the plurality of microgrooves (118a) may be different from each other. In a preferred embodiment, the length of the plurality of microgrooves (118a) is equal. A width of the plurality of microgrooves (118a) is in the range of 12 pm to 55 pm. In a preferred embodiment, the width of the plurality of microgrooves (118a) is in the range of 15 pm to 55 pm In an embodiment, the width of the plurality of microgrooves (118a) may be different from each other. In a preferred embodiment, the width of the plurality of microgrooves (118a) may be gradually decreased from the inlet (112a) towards the outlet (114b) of the device (100).

[0045]

[0047] Referring now to Figure 5, illustrates the different configuration of the microfluidic devices (100) each having different size (width) of the plurality of microgrooves (118a). The size of the plurality of microgrooves (118a) is varied from with 15 pm, 20 pm, 40 pm and 50 pm. The microfluidic device (100) is used depending on the biological sample. A method of fabrication of the microfluidic device (100) is described below in greater detail.

[0046]

[0048] Referring now to Figures 6(a) to 6(k), illustrates the method for fabrication of the microfluidic device (100). The method for the fabrication of the microfluidic device (100) includes sequential steps shown in Figure 6(a) to 6(k) followed one after another sequentially. Figure 6(a) showcases a silicon wafer (300) that is polished. The silicon wafer (300) having a thickness of 540 pm. The silicon wafer (300) is cleaned with piranha solution and exposed for plasma ashing at 150 W. Further, the silicon wafer (300) is set for the dehydration bake at 200°C for 10 mins and then cooled to room temperature. The silicon wafer (300) is then coated with a hexamethyldisilazane [HMDS] primer agent prior to an adhesion of a photoresist agent (310) to enhance the adhesion of the photoresist agent (310). The silicon wafer (300) is further spin coated with the photoresist agent (SU8-3050) (310) having the thickness of 100 pm and then soft baked at a gradual heating cycle (as shown in Figure 6(b)). Figure 6(c) showcases a photomask (320) as a plate with dark (opaque) and clear (transparent) patterns that is placed over the photoresist agent (310) adhered to the silicon wafer (300). The photomask (320) is then exposed to ultraviolet light (400) or UV light with the exposure dosage of 250 mJ / cm2. The photomask (320) allows the ultraviolet light (400) to pass through the clear patterns. The ultraviolet light (400) cures the photoresist agent (310) underneath the clear patterns engraved on the photomask (320), thereby creating the desired pattern on the silicon wafer (300). After the development of the desired pattern, the uncured areas of the photoresist agent (310) get washed away. Further, the silicon wafer (300) is then hard baked at 150°C for 20 mins to anneal any surface cracks that may be evident after the development (as shown in Figure 6(d)). Further, a curing agent such as polydimethylsiloxane [PDMS], also known as dimethylpolysiloxane or dimethicone is poured on the silicon wafer (300) to form the base member (110). The curing agent is prepared by mixing the silicone elastomer base 10 parts and curing agent 1 part. In an embodiment, the curing agent may also be prepared by flexdym, poly (methyl methacrylate), polycarbonate, etc. The pattern of the the photoresist agent (310) is transferred to the base member (110) after curing at 60°C for 12 hours (shown in Figure 6(e)). Then the base member is peeled off from the silicon wafer (300) and the desired patterns (herein plurality of microchannels (116) are created on the base member (110) (as shown Figure 6(f)). Further, the base member (110) is punched to create the inlet (112a) and the outlet (114b) and treated with oxygen plasma (500) (shown in Figure 6(g)). Figure 6(h) shows a glass panel (120) which is treated with the oxygen plasma (500). Further, as shown on Figure 6(i), the glass panel (120) is bonded to a bottom of the base member (110). Furthermore, the inlet conduit (112) and the outlet conduit (114) are fluidly connected to the inlet (112a) and the outlet (114b) of the base member (110), respectively. Figure 6(j) showcases the magnet holder (130) to hold the magnet (140). The magnet holder (130) is prepared by a 3D printing process. In an embodiment, the magnet holder (130) may be prepared using any other suitable method. The magnet holder (130) having a cavity to accommodate the magnet (140), for example neodymium magnet. The magnet holder (130) including the magnet (140) is attached to the base member (110), and the glass panel (120) is sandwiched between the magnet (140) and the base member (110) forming the microfluidic device (100).

[0047]

[0049] Referring now to Figure 7, illustrates a process of synthesizing the silica- coated magnetite nanoparticles (800). The silica-coated magnetite nanoparticles (800) is synthesized in a two-step process, a first process (600) and a second process (700). The first step (600) is a polyol mediated synthetic route, and the second step (700) is the silica coating to the nanoparticles through reverse microemulsion. The first step (600) includes a first step (602) in which iron (III) chloride hexahydrate and sodium acetate trihydrate is dissolved in polyethylene glycol [PEG] 200 under constant stirring @500 rpm for 30 mins at 50°C under dark. Further, in a second step (604) a rection dish is purged with nitrogen gas for 5 mins and the flask is tightly sealed and heated up to 300°C for 1 hours. Then, in the third step (606), the precipitated nanoparticles are washed with ethanol and sequentially with deionized water [DI] water and dried overnight. The second step (700) includes a fourth step (702) in which the magnetite nanoparticles are dispersed in ethanol and added to Cyclohexane and Polyoxyethylene nonylphenyl ether or IGEPAL CO-520 and mixed for 30 mins, in a fifth step (704). The nanoparticles mixture is then added to (3 -Aminopropyl)ti ethoxy silane [APTES], tetraethoxysilane [TEOS] and Ammonium hydroxide mixture and mixed until the particles precipitate, in a sixth step (706). Further, in a seventh step (708), the particles are washed with ethanol and Deionized water [DI] water.

[0048]

[0050] The experimental test is performed to calculate a sorting efficiency by using the microfluidic device (100). In the experimental test gel free semen sample is collected and diluted to a concentration of 50 million cell / ml. The magnetic nanoparticles are added to the semen sample in a concentration of 3 Magnetic nanoparticle per sperm and mixed for the homogenization of the nanoparticles through the semen sample. The difference in the Z potential of the spermatozoa carrying the X sperm cells or X chromosomes (-20m V) and those carrying the Y sperm cells or Y chromosomes (-16mV) makes the Y chromosomes to preferentially attach to the magnetic nanoparticle forming a complex. The biological sample incubated with the silica-coated magnetite nanoparticles passed through the plurality of microchannels (116), the Y sperm cells (200Y) bearing spermatozoa remained trap at the plurality of microchannels (116) while the X sperm cells (200X) bearing spermatozoa passes through the plurality of microchannels (116) and the plurality of microgrooves (118a) of the microfluidic device (100) and get collected at the outlet (114b). The sperm cells which are passed through the microfluidic device (100) at a controlled flow rate and collected at the outlet (114b) is stained and observed for the nucleus head morphology and tried to distinguish between the X sperm cells (200X) and the Y sperm cells (200Y) bearing chromosome. The cells are collected at outlet (114b) when the magnet is attached to the microfluidic device (100) and also without attaching the magnet to the microfluidic device (100).

[0049]

[0051] Further, an image annotation is performed on the sperm cells collected at the outlet (114b) when the magnet is attached to the microfluidic device (100) and without the magnet. The image annotation of the collected cells is performed to quantify the sorting efficiency by using Miniconda docking images. The total sixty images from each of the outlet (114b) is analyzed for nucleus shape of the cells. When the magnet is attached to the microfluidic device (100) and the sperm cells collected at the outlet conduit (114), the efficiency of the X sperm cells (200X) is 81% and the Y sperm cells (200 Y) is 19%. Further, when the magnet is removed from the microfluidic device (100) and the sperm cells collected at the outlet conduit (114), the efficiency of the X sperm cells (200X) is 7% and the Y sperm cells (200Y) is 93%. The efficiency of the X sperm cells (200X) is grater than the efficiency of the Y sperm cells (200Y) under optimal flow conditions, when the magnet is attached to the microfluidic device (100). The experimental results are shown in below table 1.

[0052] The sperm cells collected at the outlet (114b) exhibit a sorting efficiency of 81% for the X sperm cells (200X) and 19% for the Y sperm cells (200 Y), when the magnet (140) is attached to the base member (110) of the microfluidic device (100). The sperm cells collected at the outlet (114b) exhibit the sorting efficiency of 7% for the X sperm cells (200X) and 93% for the Y sperm cells (200Y), when the magnet (140) is removed from the base member (110) of the microfluidic device (100).

[0050] Table 1: Percentage of the sorting efficiency from the image analysis

[0051]

[0053] The configuration of the microfluidic device (100) uses the difference in the zeta potential of the sperm cells as key separating factor and the magnetic nanoparticles of similar surface charges are synthesized. The charged specific magnetic nanoparticles have a specific affinity to Y chromosome cells and not to the X chromosome cells. This magnetic specificity is employed in sorting the cells by placing a strong permanent magnet below the microfluidic device which has a close contact to the cells through the magnet holder.

[0052]

[0054] The microfluidic device (100) in which the premixed biological sample with the magnetic nanoparticles are passed at the inlet (112a) of the microfluidic device (100) and the configuration of the microfluidic device (100) ensures the maximum time of interaction of the magnetic nanoparticle bound sperm cell to the magnet (140) attached to the device (100). The magnet (140) covers the working portion (110b) of the plurality of microchannels (116) and the plurality of microgrooves (118a) to ensure that the 100% of the sperm cells are interacting with the magnet (140) and the time of interaction is also uniform. This increases the sorting efficiency and enhance the consistency of the multiple results.

[0053]

[0055] The configuration of the microfluidic device allows accurate sex sorting of the sperm cell with minimal cell damage and high viability by the Z electrical potential difference between the sperm cell plasma membrane and the ambient environment. This property can be used to segregate the X and Y spermatozoa according to their differential capability.

[0054]

[0056] The inventors have developed the invention, so that advantage can be achieved in an economical, practical, and facile manner. While preferred aspects and example configurations have been shown and described, it is to be understood that various further modifications and additional configurations will be apparent to those skilled in the art. It is intended that the specific embodiments and configurations herein disclosed are illustrative of the preferred nature of the invention and should not be interpreted as limitations on the scope of the invention.

[0055]

[0057] The various embodiments of the present disclosure have been described above with reference to the accompanying drawings. The present disclosure is not limited to the illustrated embodiments; rather, these embodiments are intended to fully and completely disclose the subject matter of the disclosure to those skilled in this art. In the drawings, like numbers refer to like elements throughout. The thicknesses and dimensions of some components may be exaggerated for clarity.

[0056]

[0058] Herein, the terms “attached,” “connected,” “interconnected,” “contacting,” “mounted,” “coupled” and the like can mean either direct or indirect attachment or contact between elements, unless stated otherwise.

[0057]

[0059] Well-known functions or constructions may not be described in detail for brevity and / or clarity. As used herein the expression “and / or” includes any and all combinations of one or more of the associated listed items.

[0060] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises”, “comprising”, “includes” and / or “including” when used in this specification, specify the presence of stated features, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, operations, elements, components, and / or groups thereof.

[0058]

[0061] While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

[0059]

[0062] EQUIVALENTS:

[0060]

[0063] The embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0061]

[0064] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

[0062]

[0065] Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[0063]

[0066] The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher / lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.

[0064]

[0067] Reference numerals:

Claims

I / We claim:

1. A microfluidic device (100) for processing a biological sample (200), the microfluidic device (100) comprising: a base member (110) defined with an inlet conduit (112) and an outlet conduit (114) positioned opposite to the inlet conduit (112), the inlet conduit (112) is adapted to receive the biological sample (200), and the outlet conduit (114) is configured to selectively eject the processed biological sample; a magnet (140) positioned below the base member (110), and is configured to attract the biological sample bounded with silica-coated magnetite nanoparticles (800) flowing over an upper surface (110a) of the base member (110); the base member (110) of the microfluidic device (100) comprises: a plurality of microchannels (116) defined in an array on the upper surface (110a) of the base member (110), the plurality of microchannels (116) is configured to trap the biological sample bounded with the silica-coated magnetite nanoparticles (800) under the influence of magnetic field of the magnet (140); and a plurality of partition walls (118) separating the plurality of microchannels (116), wherein each partition wall is defined with a plurality of microgrooves (118a) fluidly interconnecting the adjacent microchannels (116); the plurality of microgrooves (118a) is configured to selectively allow flow of the processed biological sample to the outlet conduit (114) of the device (100).

2. The microfluidic device (100) as claimed in claim 1, wherein the processed biological sample bounded with the silica-coated magnetite nanoparticles (800) and collected at the outlet conduit (114) is Y sperm cells (200Y).

3. The microfluidic device (100) as claimed in claim 2, wherein the Y sperm cells (200Y) are retrieved from the outlet conduit (114), when the magnet (140) is removed from the base member (110) of the device (100).

4. The microfluidic device (100) as claimed in claim 1, wherein the processed biological sample unbounded with the silica-coated magnetite nanoparticles (800) and collected at the outlet conduit (114) is X sperm cells (200X).

5. The microfluidic device (100) as claimed in claim 1, wherein the device comprises a magnet holder (130) disposed below the base member (110) and the magnet holder (130) is adapted to removably receive the magnet (140).

6. The microfluidic device (100) as claimed in claim 1, the device comprises a glass panel (120) disposed between the base member (110) and the magnet holder (130).

7. The microfluidic device (100) as claimed in claim 1, wherein the plurality of microchannels (116) is arranged in a direction orthogonal to a longitudinal direction of the device (100).

8. The microfluidic device (100) as claimed in claim 1, wherein the plurality of microgrooves (118a) is defined in a non-parallel configuration and are configured to provide a passage between the adjacent microchannels.

9. The microfluidic device (100) as claimed in claim 1, wherein a length of the plurality of microchannels (116) is in the range of 8 mm to 32 mm, and a width of the plurality of microchannels (116) is in the range of 0.3 mm to 1.8 mm.

10. The microfluidic device (100) as claimed in claim 1, wherein the length of the plurality of microgrooves (118a) is in the range of 0.1 mm to 0.9 mm,and the width of the plurality of microgrooves (118a) is in the range of 12 pm to 55 pm.

11. The microfluidic device (100) as claimed in claim 1, wherein the biological sample (200) is pre-treated with the silica-coated magnetite nanoparticles (800) before being received in the inlet conduit (112) of the device (100).

12. The microfluidic device (100) as claimed in claim 1, wherein each microgroove of the plurality of microgrooves (118a) is defined having a cross sectional shape of circular, semi-circular, rectangular, square, pentagonal, hexagonal, heptagonal or combinations thereof.

13. A method for processing a biological sample (200), the method comprising steps of: a) synthesizing silica-coated magnetite nanoparticles (800); b) collecting gel-free biological samples; c) diluting the collected gel-free semen samples to a concentration of 40-60 million cells / ml; d) adding the silica-coated magnetite nanoparticles (800) to the biological sample (200) in a concentration of 3 to 5 magnetic nanoparticles per sperm and mixing for the homogenization of the nanoparticles through the biological sample (200) to get the mixture of the biological sample (200) containing the silica-coated magnetite nanoparticles (800); e) subjecting the biological sample (200) containing the magnetite nanoparticle to a microfluidic device (100) at a controlled flow rate; f) trapping of the biological sample (200) bounded with the silica- coated magnetite nanoparticles (800) in a plurality of microgrooves (118a) using a magnet (140); and g) collecting a biological sample containing unbounded sperm cells from an outlet (114b)14. A method of synthesizing silica-coated magnetite nanoparticles (800) as claimed in claim 13, the method comprising steps of: a) dissolving iron (III) chloride hexahydrate and sodium acetate trihydrate in Polyethylene glycol (PEG) under constant stirring @500 rpm for 30 mins at 50°C under dark in a reaction dish; b) purging of a reaction dish with Nitrogen gas for 5 minutes and tightly sealed and heated up to 300°C for Ihour; c) washing precipitated nanoparticles with ethanol and deionised water DI water and subjecting to dry overnight; d) collecting the precipitated nanoparticles; e) coating of silica to the nanoparticles through reverse microemulsion; f) dispersing of magnetic nanoparticles in ethanol; g) adding of the magnetic nanoparticles to cyclohexane and IGEPAL CO-520 and mixing for 30 minutes; h) adding the above nanoparticle mixture to (3- Aminopropyl)ti ethoxy silane (APTES), Tetraethyl orthosilicate (TEOS) and Ammonium hydroxide mixture and mixing until the particles precipitate; and i) washing the particles precipitate with ethanol and the deionised water DI water.

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