Antibody-Conjugated Magnetic Beads For Sperm Sexing By A Multi-Wall Carbon Nanotube Microfluidic Device


 Bull production industries demand specific sexual bulls in response to different business productivity requirements. Therefore, sexual separation of sperm is essential for the livestock industry, increasing the demand for assisted reproductive technologies (ARTs). A popular sexual separation method in ART is monoclonal antibody enhancement, which does not affect the quality of the sperm and has specificity with high sexual separation accuracy. Moreover, microfluidic technique is an innovative tool for sperm separation, with increased installation efficiency and convenience but low accuracy and speed of detection. In this study, particles were separated using magnetic beads, magnetic beads on monoclonal antibodies, and sperm with the function of magnetic beads on monoclonal antibodies by controlling the microfluidic system fluid flow rate at 2 µL/min and releasing a 2.5-V electric field. This microfluidic device separated 100% and 95.42% of the magnetic beads and magnetic beads on monoclonal antibodies, respectively. The sperm separation test results showed that positively charged sperm Y moved to the negative electrical fluid channel with a specially designed multi-walled carbon nanotube microfluidic device that showed an X- and Y-sperm separation with a success rate of 80.12%. Compared with natural pregnancy (X- and Y-sperm ration 50:50, 50%), our proposed method is approximately 30.12% better.


Introduction
Bovine sperm separation technology has a signi cant impact on both the food and livestock industry, which leads to an increasing demand for assisted reproductive technologies (ARTs). For example, female cattle are required for the dairy industry, while males are preferred in the beef cattle industry [1] because male cattle have a higher daily growth rate and can be fattened more easier than female cattle.
Conventional sperm sexing techniques rely on centrifugation, which is known to cause oxidative stress and consequently damage cells. Moreover, the sexing of X-and Y-spermatozoa based on differences in their chromosomal content analyzed by ow cytometry is considered to be the most reliable and effective method, in which spermatozoa of the desired sex can be obtained with greater than 90% purity [2,3] . Sperm X has 3.8% more DNA weight than Y-sperm, thus providing an opportunity to use the properties of sperm with different body weights. Sexing sperm with this technique can increase the proportion of sperm with the X chromosome by 90% [4] . The disadvantages of using ow cytometry for sexing sperm are as follows: (1) effect of the sexually transmitted sperm during fertilization with the egg due to the pregnancy rate for 2 x 10 6 sexed sperm per inseminate was 56%, whereas routine operation of a ow cytometer/cell sorter for sexing sperm results in sexing about 10x10 6 sperm/h of each sex and the typically a dose of frozen bull sperm for arti cial insemination contains 20 x 10 6 sperm; (2) ow cytometers are expensive (250,000 $ USD/device); (3) the conception rate has reduced from 50-42%; and (4) ow cytometry requires a highly skilled technician [5] .
Recently, lab-on-a-chip (LOC) technology, in combination with micro uidics, has emerged as an innovative tool for handling small volumes of samples within a small device. Micro uidics is a eld that involves the ow of liquid inside channels of micrometer size. Micro uidics can be considered both as a science, involving the study of the behavior of uids in microchannels [6,7] , and technology, including manufacturing of micro uidic devices for applications, especially clinical applications [8][9][10] . Currently, micro uidics is utilized in many clinical applications, including biological and chemical analyses, pointof-care testing, clinical and forensic analyses, and molecular and medical diagnostics. Moreover, micro uidics has been successfully incorporated into various elds of ART, including 1) infertility diagnosis, 2) sperm selection, 3) sperm guidance, 4) oocyte analysis, 5) insemination, 6) embryo culture, 7) embryo selection, and 8) cryopreservation and veri cation [11] . Successful applications of micro uidic systems for sperm sorting and analysis have been discussed by Samuel et al. [12] . In addition, sperm separation methods such as uorescence-activated cell sorting (FACS) [13,14] , dielectrophoretic sorting [15] , electrophoretic sorting [16][17][18] , magnetic activated sperm cell sorting [19][20][21] , sperm sorting to utilize the difference in motility or nonmotility [22][23][24][25][26][27] , immunological markers [28,29] , electrokinetic isolation [30] , inertial separation [31] , and controlled pressure sorting [32] are used to isolate homogeneous and concentrated cell populations from heterogeneous cell mixtures. Each sorting method has its own advantages and disadvantages. For example, the swim up method provides higher yield [33] but low DNA integrity [34] compared to the micro uidic technique. In terms of sorting sperm for sexual selection in fertilization, variation in electrophoretic mobility between sperm carrying X or Y chromosomes (X and Y sperms hereafter) due to a slight difference in the electrical charge of these sperms has been observed [35] . Moreover, the difference in the zeta potential of human X and Y sperms was measured using electrophoretic light scattering spectrophotometry and a laser-rotating prism. The X and Y sperms were then separated by free-ow electrophoresis, and their purities were determined by staining the F-body. The X-sperm was found to have a greater net negative charge (− 20 mV) than Y-sperm (− 16 mV) [36] . Male and female mammalian sperm cells were also found to differ in sialic acid content. It was shown that motility of X and Y chromosome-containing sperm cells differs under a magnetic eld, where the female sperm cell is faster than the male. It was shown that the electrophoretic motility differs, with mobilities equal to 1.07 and 0.41 µm s − 1 V − 1 for X and Y chromosomes containing spermatozoa, respectively. The difference in sialylation was further con rmed when the motility of the male female sperm cells became similar were treated with sialidase, an enzyme that hydrolyzes the negatively charged sialic acid. The changes were visible after 15 and 28 min for Y and X chromosome-containing sperm cells, respectively [37] . It was also observed that X and Y sperm cells have different zeta potentials [36] . The rst evidence of this difference was seen when the X chromosome-containing sperm cell had better motility than the Y sperm cell under the presence of an electric eld [38] . The difference in motility was explained by the fact that male and female sperm cells differ in surface charge, thus showing variable zeta potential in solution. Therefore, the difference in the surface charge was used for sexing separation.
Currently, the cattle production industry demands an increasing number of sex-speci c calves. Therefore, the fabrication of micro uidic devices capable of sorting sperm for sexual selection has become a signi cant topic in assisted reproductive technology based on electric elds [39] . Our proposed micro uidic device design can be separated into X-/Y-sperm separation, which uses monoclonal antibody-conjugated magnetic beads. The monoclonal antibody used in this study was a monoclonal antibody speci c to Y-sperm. The proposed method did not affect the quality of the desired sex sperm and showed speci city and accuracy in terms of gender separation. The Y sperm captured by the monoclonal antibody is destroyed and will not affect the quality of sperm X, along with the use of magnetic particles and electric elds, which aids in the separation of the sex of the sperm. To study the properties of sperm X and Y for use in the design and construction of a micro uid system device for gender separation of cattle sperm, real-time polymerase chain reaction (PCR) was used for sperm counting. Moreover, the monoclonalantibody conjugated magnetic beads were separated on electric elds via evaluations with ow cytometry, which was found to be up to 95.42% effective. After sorting with an in-built device to test the sorting accuracy for the development of technology, the equipment can lead to future applications in the livestock industry.

Materials And Methods
A. Preparation of monoclonal antibodies conjugated with magnetic particles and sperm

Control Sample
The monoclonal antibody reaction test with ow cytometer used in the screening of sperm sex was divided into two groups: the control sample test and its negative result (control and negative control, respectively) and a monoclonal antibody 1F9, which is an IgG antibody that binds to the H-Y antigen located on the surface of Y sperm-speci c cells. The control samples were A01 and A02; A01 was the conjugate control (CC), which consisted of 50 µL of FACS buffer and 50 µL of bull sperm; A02 was isotype control (IC), which consisted of 1 µL of ST28A antibody, 50 µL of FACS buffer, and 50 µL of bull sperm. However, there is no speci city for the sperm that it should not bind to the sperm. The control samples A01 and A02 are collectively referred to as negative controls (NC). They probability of the population (P1) to analyze the results of the control sample (Control), which shows the probability of the relationship between forward scatter height (FSC-H) and side scatter height (SSC-H) is represented as a dot plot format with the probability of correlation between uorescent light intensity (FL1-H) and number. The cell (Count) in the form of a histogram, is shown in Fig. 1.

Monoclonal Antibody 1F9
Comparison with the negative control sample revealed that the monoclonal antibody 1F9 affected the bindable sperm, as mentioned above. Negative controls are unable to bind to sperm. However, the positive results obtained with the 1F9 monoclonal antibody revealed that the 1F9 monoclonal antibody had the ability to bind to sperm, as shown in Fig. 2. A03 is a 1F9 monoclonal antibody at a concentration of 50 µg/mL, consisting of 100 µg/mL monoclonal antibody (50 µL). A04 is a 1F9 monoclonal antibody at a concentration of 25 µg/mL, consisting of 50 µL of 50 µg/mL monoclonal antibody and 50 µL of cow sperm. And A06 is a monoclonal antibody 1F9 at a concentration of 6.25 µg/ml, consisting of a monoclonal antibody at a concentration of 12.5 µg/ml of 50 µl and cow sperm of 50 µl.
Sperm preparation with the 1F9 monoclonal antibody at various concentrations, it yielded different results with the sperm test and the sperm test with the ST28A antibody yielding a negative (Negative) test showing that the sperm with the 1F9 monoclonal antibody bind together and at reduced concentrations.
There is a tendency to catch each other down as well. The optimum concentration for sperm binding was A04 at a concentration of 25 µg/mL.

Preparation of monoclonal antibodies conjugated with magnetic particles
Magnetic beads (Pierce™ NHS-Activated Magnetic Beads; Thermo Scienti c™) were used for the preparation of monoclonal antibodies conjugated with magnetic particles. Magnetic particles were prepared by balancing protein and magnetic particles at room temperature, and then 300 µL of magnetic particles were inserted into a 1.5 mL microcentrifuge tube. Next, the tubes were placed in a magnetic stand, the magnetic particles were collected, and the suspended solids were disposed. Ice-cold 1 mM hydrochloric acid (1 ml) was added to the tube and blended for 15 s. The tubes were placed in a magnetic stand, the magnetic particles were collected, and suspended solids were discarded. Then, 300 µL of protein solution (1 mg/mL in coupling buffer) was added to the tube and blended for 300 s. The tube was incubated for 2 h at room temperature on a rotary agitator. During the rst 30 min of incubation, the mixture was mixed for 15 s every 5 min and then every 15 s every 15 min, until the curing was complete.
The tube was then placed in the magnetic stand, the magnetic particles were collected, and the suspended solids were discarded. Then, 1 mL of 0.1 M glycine (pH 2) was added to the magnetic particles and mixed for 15 s, and the tubes were placed in the magnetic stand. The magnetic particles were collected, and the solution of suspended solids was discarded; this process was repeated once. Thereafter, 1 mL of ultrapure water was added to the magnetic particles and mixed for 15 s in the magnetic stand. The magnetic particles were collected, while the suspended solids were discarded. A 1 mL of 3 M ethanolamine (pH 9) was added to the magnetic particles, blended for 30 s, and incubated for 2 h at room temperature on a rotary agitator, and the tubes were placed in a magnetic stand. The magnetic particles were collected and the suspensions were discarded again. Subsequently, 1 mL of puri ed water was added and mixed well, the magnetic particles were collected with a magnetic stand and suspended solids were discarded before adding 1 mL of coupling buffer (50 M borate, pH 8.5) with 0.05% sodium azide and mixed well. The magnetic particles were collected with a magnetic stand, discarding the suspension, and the process was repeated two more times, with 300 µL of coupling buffer and 0.05% sodium azide, mixed well, and stored at 4°C until use. Finally, monoclonal antibodies conjugated with magnetic particles were rechecked by ow cytometry, as shown in Fig. 3.
From the ow cytometry test of the A07 sample, the test result was negative. The result of the A07 sample was used as a negative control sample for comparison with the A08 sample. The A08 sample is a GαM-FITC-dyed magnetic bead on a monoclonal antibody, as shown in Fig. 4. From the ow cytometry test of the A08 sample, the test was positive when comparing the negative result from the A07 sample, the magnetic bead and the monoclonal antibody matched each other. The compatibility was 94.54%, as shown in Table 1. was added to remove the egg yolk extender from the spermatozoa. Subsequently, a tube of sperm sample was centrifuged at 12,000 rpm for 10 s thrice, and the supernatant was discarded to collect the sperm pellet. Turk's solution was mixed with sperm at a ratio of 1:10. Then, the sperm was blocked with AB serum (FC Receptor) for 30 min. Finally, a total of six sperm tubes were prepared: Tube 1, monoclonal antibody 100 µg/mL, 50 µL volume and 50 µL sperm. Tube 2, 50 µg/mL monoclonal antibody, 50 µL volume and 50 µL sperm. Tube 3, 25 µg/mL monoclonal antibody, 50 µL, and 50 µL sperm. Tube 4, 12.5 µg/mL monoclonal antibody, 50 µL volume, and 50 µL sperm. Tube 5, 1 µL ST28A antibody, 50 µL FACS buffer, and 50 µL sperm. Tube 6, 50 µL FACS Buffer, and 50 µL sperm.

B. Micro uidic Device Fabrication
The design of the micro uidic chip, which mainly consists of a Y channel cell micro uidic channel (Fig.  5a) with a pair of electrodes positioned at the separation regions is shown in Fig. 5b. The micro uidic chip was designed using a layout editor program. The dimensions of the micro uidic chip were 6,000 µm × 150 µm, and it was 50 µm in thickness. A pair of Ti/Au electrodes was deposited with dimensions of 5,000 × 500 µm and a thickness of 100 nm.
The micro uidic chip was placed on a glass wafer using UV and soft lithography processes. Figure 6 illustrates the microfabrication process. To create a microelectrode, the UV patterned electrode deposition was fabricated by spin-coating the AZ P1512 photoresist on the substrate and exposed to UV to develop the patterned electrode. Thin les of Ti/Au layers (50/50 nm) were deposited on the glass substrate by thermal evaporation. Then, left off by acetone was employed to achieve the nal electrodes (Fig. 6a). To create a microchannel, the SU-8 photoresist (SU-8 drysheet 50 µm) for the microstructure was laminatecoated and patterned. UV exposure was performed on SU-8 at 60 s to achieve a thickness of 50 µm of SU-8. The entrapping structure was designed to be wider than the space between the two sidewall electrodes to create the detection signal in the chamber. To provide the inlet and outlet ow of uidic interconnections, a replicated polydimethylsiloxane sylgard 184 (PDMS-184) with multi-wall carbon nanotubes was used as the master mold replication (Fig. 6b). Finally, PDMS with multi-wall carbon nanotubes, which have a microchannel-patterned underneath, were bonded by the plasma O 2 method on a pair of Ti/Au electrodes (Fig. 6c).
To set up the electrical base on the microelectrode connection, the microdevice was attached to the print circuit board and connected by using the wire bonding method in the experimentation.

Results
Testing the magnetic particle beads The magnetic particle beads were separated using a micro uidic chip. The properties of the magnetic beads were attributed to the positively charged N-hydroxysuccinimide (NHS) functional groups on the blocked magnetic bead surface. Using electrophoresis, the magnetic beads were tested by separating the beads on a micro uidic device using the principle of the charge on the surface of magnetic beads. Electrophoresis is a laboratory technique used extensively to separate DNA, RNA, or protein molecules based on their size and electrical charge. Therefore, the optimized condition for the separation of charged beads, as understood from Phiphattanaphiphop's research, included a micro uidic device with a 150 µm microchannel and 100 µm electrode distance and showed the highest performance of 87.07% validation (95% con dence level) for the separation of negatively charged TiO 2 -coated PS beads [18] . An optimally designed structure used previously for the separation of the micro uidic beads was used to test the magnetic particle beads. The magnetic particle beads were prepared by controlling the sample ow rate at 2 µL/min. This micro uidic system device was able to separate magnetic particle beads (Fig. 7).
Moreover, magnetic particle beads were tested on the MWCNT-micro uidic chip. It was processed into a pelletized form for use in the microelectrode area, as shown in Fig. 8. The magnetic particle beads were controlled using a BS-8000 syringe pump (Braintree Scienti c, Inc., Braintree, MA, USA) at a ow rate at 2 µl/min. It was found that this MWCNT-micro uidic system device was able to separate magnetic particle beads at 100% success as shows in Table 2. MCNT-Micro uidic chip Magnetic particle 100 100 Testing the monoclonal antibodies conjugated with magnetic particles In additional the monoclonal antibody conjugated with magnetic particles was added to achieve a better effect of separating in a micro uidic chip. From the experiment test for separating various particles, including magnetic particles on a monoclonal antibody with the function of monoclonal antibodies and magnetic particles by controlling the sample ow rate at 2 µl/min. It was found that this micro uidic system device was able to separate magnetic particles on a monoclonal antibody. The results were achieved at 95.42% as shown in the Fig. 9. And when it was used to sort the monoclonal antibody conjugated with magnetic particles in MWCNT-micro uidic chip, it was found that the results were in line with the theory, that is, the results of separating the monoclonal antibody conjugated with magnetic particles with higher positive charge at 98.84% as shows in Table 3. Testing Sperm and sperm with monoclonal antibodies conjugated magnetic particle beads in micro uidic chip Sperm and sperm with monoclonal antibody-conjugated magnetic particle beads were added to achieve better separation on a micro uidic chip compared with that on a MCNT-micro uidic chip. The sperm with monoclonal antibodies conjugated with magnetic particles in a microchannel in a micro uidic system device and microelectrode was tested. The particle ow was observed in the microscopic ow channel. The particle in ow rate was controlled at 2 µL/min, and the electric elds were discharged at 2.5 V for the micro uidic chip. The results were achieved at 61.11% and 80.12% for sperm conjugated monoclonal antibodies and magnetic particle beads, respectively, as shown in the micro uidic chip and MCNTmicro uidic chip, respectively in Fig. 10. The results of the sperm in combination with monoclonal antibodies conjugated with magnetic particles show better e cacy compared with those by non-antibody coadministration with beads, as shown in Table 4. The micro-uid system and micro-electrodes were created from the experiments for separating particles with the device. The particle ow was observed in the microscopic ow channel. By controlling the in ow rate of particles and emitting electric elds of various sizes, the following results were obtained.
The magnetic particles beads in Micro uidic system The magnetic particle separation experiment by controlling the in ow rate of magnetic particles at 2 µL/min and emitting a 1 V electric eld found that the magnetic particles were released into the microstructure ow channel in the micro uidic chip. It ows through the main ow channel and into the negative-ion ow channel. The magnetic particles released into the microstructure ow channel were positively charged at the north poles. Therefore, the magnetic particles ow out of the negative-ion ow channel rather than the positive charge ow channel. According to the theory of electricity and magnetism, the same polarity or charge repel each other. This shows that the generated micro uid system device can separate magnetic particles with a success rate of 100% on both the micro uidic chip and MCNT-micro uidic chip, as understood from using ow cytometry.
The monoclonal antibodies conjugated with magnetic particle beads The monoclonal antibody, 1F9, was conjugated with magnetic particle beads. Monoclonal antibody 1F9 will only react with Y sperm; in other words, monoclonal antibody1F9 will bind to sperm Y. Positively charged magnetic particle beads conjugated with monoclonal antibodies 1F9 are attracted by the generated micro uidic device structure. By generating a magnetization electric eld at 2.5 V, the device was found to be able to selectively screen antibody-coated magnetic beads with a success rate of 95.42% in micro uidic chip and 98.84% in the MCNT-Micro uidic chip. The analyzes were conducted using ow cytometry machines. Therefore, the magnetic particles ow out of the negative ion ow channel rather than the positive charge ow channel. The monoclonal antibodies with magnetic particles had little effect on magnetization, which could adjust the size of the electric eld from 1 to 2.5 V. The magnitude of the electric eld at 2.5 V was the maximum value of the electric eld. It can be used without forming air bubbles or adversely affecting the micro uidic systems. This shows that the generated micro uidic system device can isolate magnetic particle beads on a monoclonal antibody.
Sperm and sperm with monoclonal antibodies conjugated magnetic particle beads Sperm sexing experiments combined with magnetic particle beads function on a monoclonal antibody by controlling the in ow rate of the sample at 2 µL/min and releasing a 2.5-V electric eld found that the sperms working with magnetic particles on the monoclonal antibody were released into the micromicrostructure ow channel. It ows through the main ow channel and separately to both exits-the positive charge ow channel and the negative charge ow channel. In order to check for clear results, the accuracy was checked using real-time PCR.
The analysis of the sperm screened with the micro uidic system using real-time PCR technique was used to determine the DNA content of the Y-sperm in the sample. To investigate the accuracy of sperm gender separation, samples that owed through the anion channel containing the Y-sperm DNA load were collected from the outlet of the anion ow channel, which was 53.99% non-antibody coadministration with beads in the micro uidic chip. The results of the test for sperm without monoclonal antibodies at the magnetic bead conjugate in the MCNT-micro uidic chip were 74.62% [18] . The test results of sperm with monoclonal antibodies at the magnetic bead conjugate were isolated at 61.11% of the micro uidic chip and 80.12% of the MCNT-micro uidic chip. It can be seen that the monoclonal antibodies with the magnetic bead conjugate will result in superior sperm separation in both the micro uidic and MCNTmicro uidic chip. Based on the principle of separation of using a monoclonal antibody and magnetic beads to increase the screening capacity of sperm using an electric eld, the result supports the hypothesis that an improved non-monoclonal antibody at the magnetic bead conjugate is achieved with a success rate of up to 80%. However, the magnetic bead conjugate monoclonal antibody method is used for speci c sample selection. Speci cally tailored to speci c sex needs, samples using conjugated antibodies were not available, and the sample used for this nding was sperm. X, which is 20% of the Y sperm in size, with the ratio of X-sperms being greater than Y-sperms, resulting in an increased success rate in obtaining sex X-sperm.

Conclusions
In this study, magnetic particle beads were designed to conjugate monoclonal antibody-speci c Y-sperm. The separation was performed using a micro uidic system, designed using multi-walled carbon nanotubes for separation by sexing X-and Y-sperms. Separation was performed based on the assumption that the X and Y sperms had different electrical charges on the surface. The micro uidic devices used in this research were designed based on the optimal values from previous research in which a micro uidic device with a 150-µm microchannel and 100-µm electrode distance were used, and the structure was constructed from MCNT-micro uidic chip [18] . Based on the sorting test results in which monoclonal antibodies were conjugatedwith the magnetic particle beads, the device could sort the magnetic particle beads, monoclonal antibody-conjugated magnetic particle beads, and spermconjugated monoclonal antibodies with success rates of 100%, 95.42%, and 61.11%, respectively, in the micro uidic structure. The MCNT-micro uidic chip successfully sorted magnetic particle beads, monoclonal antibody-conjugated magnetic particle beads, and sperm-conjugated monoclonal antibodies with magnetic particles, with success rates of 100%, 98.84%, and 80.12%, respectively. The original device sorted the antibody-free sperm in the micro uidic device at only 53.99% success rute and in the MCNTmicro uidic chip at 74.62% success rate; therefore, success in sperm sorting when the monoclonal antibody is applied to the magnetic particle beads has a better effect than without it, but there are limitations in the speci city of the monoclonal antibody application because it can result in only one gender segregation. Moreover, it can be seen that the best sperm-sexing structures are those created using multi-wall carbon nanotubes in micro uidic devices. Sperm separation was performed using an electric eld separation principle and a micro uidic structure design using multi-wall carbon nanotubes to optimize the separation and magnetic bead conductivity and achieve better rejection. This separation result supports the hypothesis that X-and Y-sperms have different electrical charges on their surfaces.      The ow of the magnetic particle in the microstructure ow channel with micro uidic chip.

Figure 8
Page 20/21 The structure of MWCNT-micro uidic chip Figure 9 Magnetic particles ow on a monoclonal antibody in microstructure ow channel with micro uidic chip. Figure 10