Biosensor for male sterility

Abstract

The present invention relates to a biosensor and its use in the quantification of sperm function. Methods and tools for the diagnosis of male infertility are also disclosed.

Description

TECHNICAL FIELD

[0001] The present invention relates to a biosensor for quantifying sperm function and assessing male fertility and its use. BACKGROUND

[0002] Approximately 15% of the global population is affected by infertility, of which male infertility is known to account for 20-70% of all cases (Reprod Biol Endocrinol. 2015, 13:37). There are also estimates that 2.5-12% of all men are infertile. This is a cause for concern with the declining birth rates and other male infertility related factors in Europe. Since treatment of male infertility relies on accurate diagnosis, analysis and detection of underlying causes is of utmost importance. Apart from anatomical and endocrine analysis, current trends in laboratory diagnosis of male infertility encompass the characterization of sperm and semen (Mayo Clinic). 30-40% of cases of male infertility are associated with unknown male infertility related factors (European Association of Urology, 2015). This is called idiopathic male infertility, the treatment of which requires an in-depth functional analysis.

[0003] In addition, assisted reproductive technology (ART) as a standard treatment option also encounters this obstacle. In 2015, 1.6% of babies in the United States were born through ART. Although there were 231,936 rounds of ART in the United States in 2015 alone, they resulted in 60,778 live births (Centers for Disease Control and Prevention, 2016). In 2017, the European Society of Human Reproduction and Embryology identified the post-IVF failure fertilization rate (FFR) of oocytes as a key performance indicator for ART laboratories, with a very low competency level of only 5% (Human Reprod Open. 2017, 2). It is worth noting that although sperm morphology cannot be linked to IVF success / failure, fertilization failure is caused by sperm function problems (Human Reprod. 2000, 15(3):702; Fertil Steril. 2003 Jan; 79(1):74). Therefore, there is a need to develop a robust method to diagnose the fertilization capacity of sperm cells to predict the feasibility of IVF and better select suitable ART methods.

[0004] Prior to ART, men with infertility are usually tested for factors associated with male infertility, including sperm quality analysis, sperm count, concentration, morphology and motility, identification of atypical cell types in the semen and presence of autoantibodies. Other analyses can include studies of the interaction of sperm cells with cervical mucus, acrosome reaction, biochemical assays of accessory organ function and estimation of reactive oxygen species and DNA damage (World Health Organization, 2010). In the case of idiopathic male infertility and IVF fertilization failure, these values do not provide much information about the underlying cause of infertility. In most cases, intracytoplasmic sperm injection (ICSI) is the preferred method, but the unnecessary IVF step leads to waste of healthy oocytes and financial burden.

[0005] While the morphological and kinetic properties of sperm cells are essential for in vivo fertilization or IVF fertilization, the final step of fertilization is the fusion of the sperm cell with the oocyte. Research methods such as Hemizona assay, human sperm-oocyte interaction test and human zona pellucida binding test are able to mimic certain parts of this step. However, these methods cannot be commercialized since they rely on human oocytes or parts thereof which are not easily available. To use hamster oocytes instead of human oocytes, a zona-free hamster oocyte penetration test was developed. Although this test eliminates the need for human oocytes, it has a very low predictive value for the success of fertilization at any concentration of sperm in IVF treatment, so the use of this test is significantly limited.

[0006] The primary binding between the two gametes is mediated by the extracellular layer of zona pellucida (ZP) glycoproteins around the oocyte (Cell. 2017, 169(7): 1315; Reprod Biomed Online. 2003, 7(6): 641). This interaction is responsible for triggering the acrosome reaction in the sperm cell. Moreover, sperm cells that have not started the acrosome reaction before encountering the ZP cannot fertilize the oocyte. Next, hydrolytic enzymes released from the acrosome are needed to digest the ZP, thus allowing the sperm to enter the oocyte membrane.

[0007] A key step in this binding was discovered in 2014. The sperm surface antigen IZUMOl binds to the female counterpart JUNO protein, formerly known as folate receptor 4 (Nature. 508:483-487; Nature. 2016, 534(7608): 566). This biochemical event has been found to be essential for the fusion of the two gametes. Any biochemical mismatch can lead to fertilization failure.

[0008] Therefore, it is important to be able to quantify the number of sperm cells in a semen sample that are able to bind to the JUNO protein for the assessment of fertility.

[0009] A biosensor is a sensor that utilizes the molecular recognition function of biological materials such as microorganisms, enzymes, antibodies, DNA, and RNA, and uses these biological materials as molecular recognition elements. In other words, a biosensor utilizes reactions that occur when a fixed biological material recognizes a target substrate, oxygen consumed by microorganism respiration, enzyme reactions, luminescence, etc. In a biosensor, practical applications of enzyme sensors are being developed. For example, enzyme sensors for glucose, lactic acid, uric acid, and amino acids can be used in medical devices and food processing industries.

[0010] Different techniques can be used to track the interaction between, for example, proteins bound to electrodes and target substances such as sperm. One of these techniques relies on surface plasmon resonance (SPR). In SPR, one molecular partner (e.g., a protein) is immobilized on a metal (chip). Light excites surface plasmons in the metal; when the binding partner binds to the immobilized molecule, this causes a detectable change in the surface plasmon signal. Another of these techniques relies on electrochemical transduction, in which the content of a biological sample is analyzed by directly converting a biological event into an electrical signal. The most common techniques in electrochemical biosensing include methods based on cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, and field effect transistors, as well as biosensing technologies based on nanowires or magnetic nanoparticles. SUMMARY

[0011] The inventors have used the biochemical reaction between the JUNO protein and the sperm surface antigen IZUMOl to determine the fertilization potential of sperm cells. In addition to revealing potential causes of infertility, this can be particularly useful for selecting the appropriate ART technique, i.e., between conventional IVF and ICSI, while reducing the waste of oocytes. The inventors have also proposed the development and clinical validation of an electrochemical and / or optical sensing platform for detecting the fertilization potential of sperm cells for the diagnosis of male infertility. Existing commercial methods for diagnosing male infertility and checking sperm quality focus on the physical aspects of sperm cells, ignoring the biochemical interactions necessary for the fertilization event. The method proposed by the inventors is more advantageous than existing diagnostic methods because it is the first biomimetic assay for male fertility that uses the biological receptors of sperm cells, and it mimics the key steps of sperm cell fertilization, thus directly revealing their fertilization potential. Detection methods for sperm- oocyte interactions have been developed before, but they either require the use of human oocytes and zonapellucida, which are not readily available, or are unreliable due to the low correlation between the results of the detection and various semen parameters. The present invention overcomes the problem of oocyte utilization by creating conditions that mimic oocytes, and in particular by using one or more key protein receptors involved in sperm-oocyte fusion.

[0012] One aspect of the present disclosure provides a biosensor for quantifying sperm function, the biosensor comprising a substrate and a JUNO protein or fragment thereof, wherein the JUNO protein or fragment thereof is immobilized on the substrate.

[0013] A further aspect of the present disclosure provides a biosensor for detecting sperm function, the biosensor comprising a substrate and a JUNO protein or fragment thereof, wherein the JUNO protein or fragment thereof is immobilized on the substrate.

[0014] Another aspect of the present disclosure provides a method for detecting and / or quantifying sperm function, wherein the method comprises the steps of:

[0015] a. providing a semen sample from a subject, wherein the semen sample comprises one or more sperm cells,

[0016] b. contacting the semen sample with a biosensor according to any one of the preceding claims,

[0017] c. determining the binding of the sperm cells to the protein immobilized on the sensor,

[0018] thereby detecting and / or quantifying sperm function of the sample.

[0019] Another aspect of the present disclosure provides a method of diagnosing male infertility, wherein the method comprises the steps of:

[0020] a. providing a semen sample from a subject,

[0021] b. contacting the semen sample with a biosensor according to the present disclosure,

[0022] c. quantifying sperm function of the sample according to the method of the present disclosure,

[0023] d. using the sperm function to diagnose whether the subject is infertile.

[0024] Another aspect of the present disclosure provides a method of diagnosing male infertility, wherein the method comprises the steps of:

[0025] a. providing a sperm sample from a subject,

[0026] b. contacting the sperm sample with a biosensor according to the present disclosure,

[0027] c. quantifying sperm function of the sample according to the method of the present disclosure,

[0028] d. using the sperm function to diagnose whether the subject is infertile.

[0029] Another aspect of the disclosure provides a method for manufacturing a biosensor comprising a JUNO protein, the method comprising:

[0030] a. providing a substrate,

[0031] b. providing a JUNO protein,

[0032] c. immobilizing the JUNO protein on the substrate,

[0033] thereby manufacturing a biosensor comprising a JUNO protein.

[0034] A further aspect of the disclosure provides a method of selecting a sperm, the method comprising:

[0035] a. providing a semen sample from a subject, wherein the semen sample comprises one or more sperm cells,

[0036] b. contacting the semen sample with a biosensor of the disclosure,

[0037] c. visualizing the sperm bound to the biosensor by microscopy,

[0038] thereby selecting the sperm.

[0039] A further aspect of the disclosure provides a method of selecting a sperm, the method comprising:

[0040] a. providing a sperm sample from a subject,

[0041] b. contacting the semen sample with a biosensor of the disclosure,

[0042] c. visualizing the sperm bound to the biosensor by microscopy,

[0043] thereby selecting the sperm.

[0044] Another aspect of the disclosure provides a hand-held device for detecting and / or quantifying sperm function, the device comprising:

[0045] a. a sample inlet;

[0046] b. a biosensor comprising a JUNO protein or fragment thereof, wherein the JUNO protein is immobilized on the biosensor, and wherein the inlet is configured to contact the sample with the sensor;

[0047] c. a detector configured to receive a signal from the sensor and convert it into a format readable by a user; and

[0048] d. optionally, means for isolating cellular components from the sample. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 : a) after addition of buffer only (solid line) and after addition of 1.67 x 10 -2 X seminal sample diluted in buffer (dotted line), b) electrode-gold nanoparticle-JUNO-BSA blocked cyclic voltammetry response (dotted-dashed line), and c) after addition of 1.67 x 10 -2 X seminal sample diluted in buffer (dotted line), b) electrode-gold nanoparticle-JUNO-BSA blocked cyclic voltammetry response (dotted-dashed line), and c) after addition of 1.67 x 10

[0050] Figure 2 : after addition of 3 different dilutions (1.56 x 10 -5 X (dotted line), 1.67 x 10 -2 X (dotted-dashed line) and 2.5 x 10 - 2 X (dashed line) of 6 different seminal samples (S1, S2, S3, S4, S5 and S6, respectively Figure 2A -F). The solid line shows the electrode response using buffer only (no seminal sample).

[0051] Figure 3 : after addition of 3 different dilutions (1.56 x 10 -5 X (dotted line), 1.67 x 10 -2 X (dotted-dashed line) and 2.5 x 10 -2 X (dashed line) of seminal samples S1, S2, S3, S4, S5 and S6 (respectively Figure 3A -F). The solid line shows the electrode response using buffer only (no seminal sample). DETAILED DESCRIPTION

[0053] Disclosed herein are biosensors for quantifying sperm function to assess male infertility and applications thereof. Further, the present disclosure relates to methods for diagnosing infertility in a subject, comprising determining sperm function of sperm cells in a sample obtained from the subject.

[0054] Sperm function

[0055] The main function of sperm is to reach the egg cell and deliver two subcellular structures: (i) the male pronucleus containing the genetic material and (ii) the centriole as a structure that helps to organize the microtubular cytoskeleton to induce fertilization. Thus, the function of sperm can be understood as the ability of sperm to reach the egg cell and induce fertilization.

[0056] The primary binding between the two gametes is mediated by the zona pellucida (ZP) glycoprotein extracellular layer surrounding the egg cell (Cell. 2017, 169(7): 1315; Reprod Biomed Online. 2003, 7(6): 641). This interaction is responsible for triggering the acrosome reaction of the sperm cell. Moreover, sperm cells that have not started the acrosome reaction before encountering the ZP cannot fertilize the egg cell. Next, hydrolytic enzymes released from the acrosome are required to digest the ZP, thus allowing the sperm to enter the egg cell membrane.

[0057] A key step in this binding was discovered in 2014. The sperm surface antigen IZUMOl binds to the female counterpart JUNO protein, formerly known as folate receptor 4 (Nature. 508:483-487; Nature. 2016, 534(7608): 566). This biochemical event has been found to be crucial for the fusion of the two gametes.

[0058] The present disclosure relates to a biosensor for quantifying sperm function, said biosensor comprising a substrate and a JUNO protein or fragment thereof, wherein said JUNO protein or fragment thereof is immobilized on the substrate. Indeed, the detection of sperm function by binding sperm cells to a biosensor comprising a JUNO protein can be a viable strategy and can ultimately enable the diagnosis of male infertility, thus overcoming the need for human or animal oocytes or parts thereof.

[0059] In some embodiments, there is provided a biosensor of the present disclosure, wherein said sperm function is determined by the binding of at least a portion of a sperm to a protein or fragment thereof that has been immobilized on the sensor, wherein said protein is a JUNO protein, ZPl, ZP2, ZP3 and / or an anti-IZUMO antibody, or fragment thereof. In some embodiments, said at least a portion of a sperm comprises the IZUMOl surface antigen.

[0060] An aspect of the present disclosure provides a method for detecting and / or quantifying sperm function, wherein said method comprises the steps of:

[0061] a. providing a semen sample from a subject, wherein said semen sample comprises one or more sperm cells,

[0062] b. contacting the semen sample with a biosensor according to any one of the preceding claims,

[0063] c. determining the binding of the sperm cells to the protein immobilized on the sensor,

[0064] thereby detecting and / or quantifying sperm function of said sample.

[0065] A further aspect of the disclosure provides a method of diagnosing male infertility, wherein the method comprises the steps of:

[0066] a. providing a semen sample from a subject,

[0067] b. contacting the semen sample with a biosensor of the disclosure,

[0068] c. quantifying sperm function of the sample according to the methods of the disclosure,

[0069] d. using the sperm function to diagnose whether the subject is infertile.

[0070] A further aspect of the disclosure provides a method of diagnosing male infertility, wherein the method comprises the steps of:

[0071] a. providing a sperm sample from a subject,

[0072] b. contacting the sperm sample with a biosensor of the disclosure,

[0073] c. quantifying sperm function of the sample according to the methods of the disclosure,

[0074] d. using the sperm function to diagnose whether the subject is infertile.

[0075] As used herein, the term“sperm function” refers to the health of sperm, which is the ability of sperm to capacitate and fertilize an egg. Sperm function assays are methods of diagnosing or investigating biochemical or molecular characteristics of sperm cells. Some examples of sperm function assays are found in the following references: Talwar and Hayatnagarkar 2015. J Hum Reprod Sci. 8(2): 61-69. The present disclosure relates to sperm function assays, which are also known as sperm- oocyte interaction assays, wherein the assays do not use oocytes, but rather mimic the conditions of sperm-oocyte interaction by using one or more key protein receptors involved in sperm-oocyte fusion.

[0076] In one embodiment of the methods of the disclosure, the semen sample comprises or is suspected of comprising one or more sperm cells.

[0077] In one embodiment of the methods of the disclosure, sperm function of the sample is detected by assaying binding of sperm cells to a protein immobilized on the sensor, wherein the protein is selected from the group consisting of: JUNO protein, ZPl, ZP2, ZP3, and / or anti-IZUMO antibody, or fragments thereof, and wherein the binding is detected by microscopy analysis, electrochemical detection, and / or surface plasmon resonance.

[0078] In one embodiment of the method of the disclosure, sperm function of the sample is quantified by assaying the binding of sperm cells to a protein immobilized on a sensor, wherein the protein is selected from the group consisting of JUNO protein, ZPl, ZP2, ZP3 and / or anti-IZUMO antibody, or fragments thereof, and wherein the binding is detected by microscopy analysis, electrochemical detection and / or surface plasmon resonance.

[0079] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the percentage of bound sperm cells over unbound sperm cells by microscopy analysis.

[0080] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the percentage of bound sperm cells over unbound sperm cells by electrochemical detection.

[0081] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the percentage of bound sperm cells over unbound sperm cells by surface plasmon resonance.

[0082] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the acrosome status of sperm cells in the semen sample and / or sperm sample by microscopy analysis.

[0083] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the acrosome status of sperm cells in the semen sample and / or sperm sample by electrochemical detection.

[0084] In one embodiment of the method of the disclosure, sperm function is quantified in step c) by determining the acrosome status of sperm cells in the semen sample and / or sperm sample by surface plasmon resonance.

[0085] In one embodiment of the method of the disclosure, the method further comprises comparing the percentage of bound sperm cells over unbound sperm cells and / or the acrosome status of sperm cells to respective reference values, wherein the reference values can be positive reference values (representing functional sperm) and / or negative reference values (representing non-functional sperm). The reference values can be obtained by detecting control semen samples and / or control sperm samples. The reference values can also be obtained from data available in scientific literature.

[0086] In one embodiment of the method of the disclosure, the sample is treated prior to step b. For example, semen sample treatment can comprise liquefaction of sperm. Semen sample treatment can optionally comprise capacitation. Sperm sample treatment can comprise liquefaction of sperm. Sperm sample treatment can optionally comprise capacitation.

[0087] The biosensors disclosed herein can be used to determine various levels of sperm function.

[0088] For example, the disclosed biosensors can be used to determine the ability of sperm cells in a semen sample and / or a sperm sample to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3.

[0089] The disclosed biosensors can be used to determine the ability of sperm cells in a semen sample and / or a sperm sample to undergo acrosome reaction.

[0090] Binding to the zona pellucida proteins ZP1, ZP2 and / or ZP3 is a necessary step for sperm cells to undergo acrosome reaction. Therefore, sperm cells that are not able to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3 will not undergo acrosome reaction.

[0091] Determining whether sperm cells are able to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3 and undergo acrosome reaction is important to determine which assisted reproductive technologies can be used. Specifically, sperm cells that are not able to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3 and / or do not undergo acrosome reaction can be suitable for IVF, provided that the zona pellucida coat has been removed from the egg.

[0092] The disclosed biosensors can be used to determine the ability of sperm cells in a semen sample and / or a sperm sample to bind to the JUNO protein.

[0093] Even if sperm cells are not able to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3 and / or do not undergo acrosome reaction, the sperm cells are able to bind to the JUNO protein. This is because sperm cells can be induced to capacitate and expose surface antigens necessary to bind to the oocyte cell membrane.

[0094] In some embodiments, sperm function is determined by the ability of sperm to bind to immobilized JUNO protein or fragments thereof. The binding can occur through, for example, IZUMOl protein or fragments thereof located on the sperm. The binding can occur through, for example, IZUMOl protein or fragments thereof expressed by the sperm.

[0095] In fact, sperm that have been capacitated can present IZUMOl protein or fragments thereof on their surface and the IZUMOl protein or fragments thereof are able to bind to their oocyte counterpart, for example, JUNO protein or fragments thereof or anti-IZUMO antibodies or fragments thereof.

[0096] In some embodiments, sperm function is determined by the binding of IZUMOl protein or fragments thereof to immobilized JUNO protein or fragments thereof.

[0097] In one particular embodiment, the method of the present disclosure further comprises the step of treating the male infertility.

[0098] In one particular embodiment of the method of the disclosure, the treatment comprises administering a therapeutically effective amount of a drug and / or by assisted reproductive technology (ART). For example, a subject diagnosed with reduced sperm function can reproduce by means of ART, such as intrauterine insemination (IUI), in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (ICSI).

[0099] For example, a subject diagnosed with reduced sperm function, in particular whose sperm cells are characterized by:

[0100] - a reduced ability to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3,

[0101] - a reduced ability to perform acrosome reaction;

[0102] - a normal ability to bind to the JUNO protein,

[0103] may reproduce by means of IVF.

[0104] For example, a subject diagnosed with reduced sperm function, in particular whose sperm cells are characterized by:

[0105] - a reduced ability to bind to the zona pellucida proteins ZP1, ZP2 and / or ZP3,

[0106] - a reduced ability to perform acrosome reaction;

[0107] - a reduced ability to bind to the JUNO protein,

[0108] may reproduce by means of IVF with ICSI.

[0109] As used herein, the terms "reduced ability" and "normal ability" are relative to a reference value, which can be a positive control. The reference value can be obtained by calculating the average of the seminal fluid samples of fertile subjects. The reference value can be obtained by calculating the average of the sperm samples of fertile subjects. The reference value can also be obtained from scientific reports. Thus, in order to diagnose a subject with reduced sperm function and / or male sterility, the ability of the sperm cells of a seminal fluid sample and / or a sperm sample taken from said subject to bind to the zona pellucida proteins ZP1, ZP2 and ZP3 and / or to perform acrosome reaction is compared to at least one reference value, such as a positive control and / or a negative control.

[0110] Any treatment for male sterility known to the person skilled in the art can be used.

[0111] In some embodiments, the biosensor of the disclosure is configured for detecting and / or quantifying sperm function.

[0112] In one embodiment of the method of the disclosure, in step c) sperm function is quantified by determining the acrosome status of sperm cells in the semen sample and / or sperm sample, thereby converting the degree of acrosome reactivity into a measure of sperm function. In a particular embodiment, a fluorophore is added to the sample, which is then analyzed by SPR or microscopy to determine the acrosome status of the sperm cells (i.e. whether the sperm cells are “acrosomally reactive”). In some embodiments, the fluorophore is added to the sample at the biosensor inlet only after the sample is brought into contact with the substrate. In some embodiments, the fluorophore is added to the sample at the biosensor inlet only after acrosome reaction has occurred. The presence of a fluorophore can aid the step of determining the acrosome status of sperm cells. For example, a fluorescently labeled lectin, such as Pisum sativum (pea lectin) or Arachis hypogaea (peanut lectin), or a monoclonal antibody against the acrosomal antigen CD46, can be used to assess the acrosome status of sperm cells. Methods of detecting acrosome reaction can be found in the WHO Laboratory Manual Examination and processing of human semen, WHO, 5th edition, 2010, ISBN 978 92 4 154778 9 (see in particular chapter 4).

[0113] In some embodiments of the method of the disclosure, in step c) sperm function is quantified by determining the acrosome status of sperm cells in the semen sample and / or sperm sample, thereby converting the degree of acrosome reactivity into a measure of sperm function, wherein the acrosome reactivity in the semen sample and / or sperm sample is compared to the average acrosome reactivity of sperm collected from a fertile male individual, and wherein the acrosome reactivity of a functional sperm can be equal to or higher than the average acrosome reactivity of sperm collected from a fertile male individual. Data on the average acrosome reactivity of sperm collected from a fertile male individual can be found in the scientific literature and in clinical reports.

[0114] In some embodiments of the method of the disclosure, in step c) sperm function is quantified by converting the degree of acrosome reactivity into a measure of sperm function, wherein an acrosome reactivity of 15% or more can indicate a functional sperm.

[0115] In some embodiments of the method of the disclosure, in step c) sperm function is quantified by converting the degree of acrosome reactivity into a measure of sperm function, wherein an acrosome reactivity of 10% or less can indicate a non-functional sperm.

[0116] In some embodiments of the method of the disclosure, in step c) sperm function is quantified by converting the degree of acrosome reactivity into a measure of sperm function, wherein an acrosome reactivity of between 10% and 15% can indicate an abnormal sperm function.

[0117] Acrosome reaction

[0118] During fertilization, sperm must first fuse with the plasma membrane and then penetrate the female egg to fertilize it. Before penetrating the egg's hard shell or extracellular matrix, the sperm cell undergoes a process called the acrosome reaction. The acrosome reaction is an exocytosis process that occurs after the sperm binds to the zona pellucida and must occur before the sperm can penetrate the oocytevestment and fuse with the oocyte. The acrosome is a cap-like structure on the front half of the sperm head. When the sperm approaches the zona pellucida of the egg, which is necessary to initiate the acrosome reaction, the membrane around the acrosome fuses with the sperm head's plasma membrane, exposing the contents of the acrosome. The contents include surface antigens necessary for binding to the egg's cell membrane and a number of enzymes responsible for penetrating the egg's tough outer coating and allowing fertilization to occur. For example, the contents of the acrosome can include an IZUMOl protein or fragment thereof.

[0119] Biosensor

[0120] One aspect of the present disclosure provides a biosensor for quantifying sperm function, the biosensor comprising a substrate and a JUNO protein or fragment thereof, wherein the JUNO protein or fragment thereof is immobilized on the substrate.

[0121] Another aspect of the present disclosure provides a biosensor for detecting sperm function, the biosensor comprising a substrate and a JUNO protein or fragment thereof, wherein the JUNO protein or fragment thereof is immobilized on the substrate.

[0122] In one embodiment, the biosensor of the present disclosure is a sensor. The sensor or biosensor of the present disclosure is configured for detecting and / or quantifying sperm function.

[0123] As used herein, a "biosensor" is sometimes referred to as a "sensor". There are multiple devices known for detecting ligand / receptor interactions. The most basic of these are pure chemical / enzymatic assays, in which the presence or amount of an analyte is detected by measuring or quantifying a detectable reaction product. Ligand / receptor interactions can also be detected and quantified by radiolabeled assays.

[0124] This type of quantitative binding assay comprises two separate components: a reaction substrate, such as a solid phase test strip, dish, chip, or electrode, and a separate reader or detector device, such as a scintillation counter, spectrophotometer, microscope, or any other detector known in the art. The substrate is typically not amenable to multiple assays or miniaturization for multiple analyte assays on small amounts of body fluid samples.

[0125] In contrast, in a biosensor, the assay substrate and detector surface can be integrated into a single device. One general type of biosensor employs an electrode surface and is combined with a current or impedance measurement element to detect changes in current or impedance that occur in response to the presence of a ligand-receptor binding event. Another type of biosensor can employ a chip (e.g., a glass chip) and is combined with an optical detector (e.g., combined with surface plasmon resonance). Another type of biosensor can employ a dish and is combined with a microscope. Another type of biosensor can employ a microbead and is combined with an optical or electrical detector, e.g., suitable for latex agglutination detection. Another type of biosensor can employ a polymeric substrate (e.g., cellulose or nitrocellulose paper) and is combined with, e.g., an optical or electrical detector, e.g., suitable for lateral flow detection.

[0126] As used herein, the term "dish" can refer to a container or slide made of glass, ceramic, plastic, cellulose, nitrocellulose, or any other material, and can be used as a substrate for microscopic or macroscopic optical detection and selection. Examples include glass slides, microtiter plates, multiwell plates, Petri dishes, surface dishes, and the like. The dish can be made of a material that can be modified (e.g., coated with a layer of gold).

[0127] As used herein, the term "microbead" refers to a particle that is 1 mm or less in diameter. Microbeads can be made of natural or synthetic polymeric materials. Microbeads can be made of materials that feature a surface that can be modified, e.g., they can have a surface that can be bound to nanoparticles (e.g., gold nanoparticles) and / or molecules (e.g., peptides).

[0128] A biosensor refers to a sensor that incorporates a biological element. Biosensors are in effect a replacement for traditional analytical techniques that can be lengthy, expensive, complex, and not suitable for in situ monitoring. A biosensor can be a chemical analysis device that integrates a biological element with a transducer. It has a biological element immobilized within or in close contact with the transducer, resulting in a signal that is proportional to a single analyte that is further transmitted to a detector. In some embodiments, the signal output from the binding of the analyte can be visualized by microscopy. In some embodiments, the signal output from the binding of the analyte can be visualized by an optical detector.

[0129] A biosensor contains three basic components, a biological receptor (biological element), a transducer, and an electronic circuit. The biological receptor or biological element is a biomolecule, such as an enzyme, DNA, protein, whole cell, antibody, etc., embedded with a transducer. In the present application, the biological receptor can be a JUNO protein. In some embodiments of the present disclosure, the biosensor includes more than one type of biological receptor, for example, one type of biological receptor can be a JUNO protein or fragment thereof, and another type of biological receptor can be selected from the group consisting of ZP1, ZP2, ZP3, and an anti-IZUMO antibody.

[0130] The detector encompassed by the methods of the present disclosure is an optical detector, such as a surface plasmon resonance detector, an electrochemical detector, and a measurement circuit. The electronic circuit contains signal processing biosensors that convert the electrical signal into a processable signal. In some embodiments, the detector can be a microscope.

[0131] Biosensors based on the surface plasmon resonance (SPR) effect utilize changes in the angle of reflection of the SPR surface due to perturbations at the SPR interface, such as binding events. Finally, biosensors can also utilize changes in the optical properties of the biosensor surface.

[0132] Electrochemical biosensors are generally based on enzyme catalysis of reactions that produce or consume electrons (oxidoreductases). The sensor substrate typically contains three electrodes: a reference electrode, a working electrode, and a counter electrode. The target analyte participates in a reaction that occurs at the active electrode surface, and this reaction can result in electron transfer across the double layer (producing a current) or can contribute to the potential of the double layer (producing a voltage). The current can be measured, where the flow rate of electrons is proportional to the analyte concentration at a fixed potential, or the potential can be measured at zero current, which has a logarithmic response. Additionally, small peptides and proteins can be directly and label-free electrically detected using a biologically functionalized ion-sensitive field effect transistor through its inherent charge.

[0133] Potentiometric biosensors that generate a potential under zero current, producing a logarithmic response with a high dynamic range. Such biosensors are typically made by screen-printing an electrode pattern onto a plastic substrate coated with a conducting polymer, and then attaching some protein (enzyme or antibody) on top. They have only two electrodes, and are extremely sensitive and robust. They are able to detect analytes at levels previously only achievable by HPLC and LC / MS, and without the need for rigorous sample preparation. Since the biosensing component is highly selective for the relevant analyte, all biosensors typically require only minimal sample preparation. The signal is generated due to electrochemical and physical changes in the conducting polymer layer as a result of changes at the sensor surface. These changes can be attributed to ionic strength, pH, hydration, and redox reactions. Field effect transistors (FETs) with a gate region modified with an enzyme or antibody can also detect very low concentrations of various analytes, since binding of the analyte to the FET gate region causes a change in the drain-source current.

[0134] Biosensors have many potential advantages over traditional binding assays. One important advantage is the ability to use microchip manufacturing methods to make small-scale but highly reproducible biosensor units.

[0135] Various types of biosensors have many potential applications. The main conditions under which biosensor methods become valuable in research and commercial applications are the recognition of the target molecule, the presence of a suitable biological recognition element, and the potential for a disposable, portable detection biosensor to be preferred over a sensitive, laboratory-based technique in some cases.

[0136] In one embodiment, the biosensor of the present disclosure further comprises Zona Pellucida 1 (ZP1), Zona Pellucida 2 (ZP2), Zona Pellucida 3 (ZP3), and / or an anti-IZUMO antibody or fragment thereof, wherein the ZP1, ZP2, ZP3, and / or anti-IZUMO antibody or fragment thereof is immobilized on the substrate.

[0137] In one embodiment, the biosensor of the present disclosure comprises the proteins JUNO and ZP3, wherein the proteins are immobilized on the substrate.

[0138] In one embodiment, the biosensor of the present disclosure comprises the proteins JUNO and ZP2, wherein the proteins are immobilized on the substrate.

[0139] In one embodiment, the biosensor of the present disclosure comprises the proteins JUNO and ZP1, wherein the proteins are immobilized on the substrate.

[0140] In one embodiment, the biosensor of the present disclosure comprises the protein JUNO and the anti-IZUMO antibody, wherein the proteins are immobilized on a substrate.

[0141] In one embodiment, the biosensor of the present disclosure comprises the proteins JUNO, ZP2 and ZP3, wherein the proteins are immobilized on a substrate.

[0142] The presence of a combination of JUNO and one or more of ZP1, ZP2, ZP3 and anti-IZUMO antibody can improve the specificity of the biosensor and can more accurately determine sperm function of the analyzed semen sample and / or the analyzed sperm sample. For example, the presence of a combination of JUNO and one or more of ZP1, ZP2, ZP3 and anti-IZUMO antibody can more accurately determine the ability of sperm to bind to zona pellucida proteins, to perform acrosome reaction and to bind to oocytes.

[0143] In one embodiment, the proteins JUNO, ZP1, ZP2 and ZP3 that can be immobilized on a substrate are mammalian proteins. For example, the proteins JUNO, ZP1, ZP2 and ZP3 can be human proteins or fragments thereof. For example, the proteins JUNO, ZP1, ZP2 and ZP3 can be equine proteins or fragments thereof. For example, the proteins JUNO, ZP1, ZP2 and ZP3 can be canine proteins or fragments thereof. For example, the proteins JUNO, ZP1, ZP2 and ZP3 can be bovine proteins or fragments thereof.

[0144] In a further embodiment, the biosensor of the present disclosure comprises a JUNO protein comprising or consisting of a polypeptide having at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity identity, such as about 100% sequence identity to SEQ ID NO: 1 or an ortholog thereof or a fragment of said protein.

[0145] In one embodiment, the biosensor of the present disclosure is provided, wherein the ZP1 comprises or consists of a polypeptide having at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity identity, such as about 100% sequence identity to SEQ ID NO: 2 or an ortholog thereof or a fragment of said protein.

[0146] In a further embodiment, there is provided a biosensor of the disclosure, wherein the ZP2 comprises or consists of a polypeptide having at least 95% sequence identity, for example at least 96% sequence identity, for example at least 97% sequence identity, for example at least 98% sequence identity, for example at least 99% sequence identity identity, for example about 100% sequence identity to SEQ ID NO: 3 or an ortholog thereof or a fragment of the protein.

[0147] In a further embodiment, there is provided a biosensor of the disclosure, wherein the ZP3 comprises or consists of a polypeptide having at least 95% sequence identity, for example at least 96% sequence identity, for example at least 97% sequence identity, for example at least 98% sequence identity, for example at least 99% sequence identity identity, for example about 100% sequence identity to SEQ ID NO: 4 or an ortholog thereof or a fragment of the protein.

[0148] In some embodiments, at least one of the JUNO protein, ZP1, ZP2, ZP3 and anti-IZUMO antibody is bound to a further moiety. For example, the further moiety can be a peptide or a label. For example, at least one of the JUNO protein, ZP1, ZP2, ZP3 and anti-IZUMO antibody is bound to a polyhistidine tag.

[0149] In some embodiments, there is provided a biosensor of the disclosure, wherein the substrate is a microbead, a dish, a chip or an electrode.

[0150] In one embodiment, the substrate is a polymeric microbead. For example, the substrate can be an agarose, cellulose, nitrocellulose or latex microbead. In some embodiments, the microbead is configured such that it can be coupled to a microscope, optical transducer or measurement circuitry.

[0151] In one embodiment, the substrate is a dish, for example a plastic dish, a ceramic dish or a glass dish. In a further embodiment, the dish is configured such that it can be coupled to a microscope or optical transducer.

[0152] In some embodiments, the chip is a glass chip.

[0153] As used herein, the term "glass" is equivalent to quartz or silicon dioxide, which comprises silicon and oxygen atoms in a continuous framework, with the overall chemical formula Si02.

[0154] In particular embodiments, the electrode is a carbon, gold or platinum electrode. In one embodiment, the electrode is a screen printed electrode.

[0155] In some embodiments, the substrate has a modified surface. In one embodiment, at least one surface of the substrate is coated with a layer of gold. In some embodiments, at least one surface of the substrate is modified with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.

[0156] In specific embodiments, the biosensor of the present disclosure is configured such that the substrate can be coupled to a microscope, an electrochemical workstation, a surface plasmon resonance detector, a measurement circuit, or an optical transducer.

[0157] In one embodiment, the substrate is an electrode and is configured such that it can be coupled to an electrochemical workstation or a measurement circuit.

[0158] In one embodiment, the substrate is a chip and is configured such that it can be coupled to a surface plasmon resonance detector.

[0159] Protein immobilization

[0160] The present disclosure relates to a biosensor.

[0161] For the detection and quantification of sperm function, the biosensor comprises a substrate and a protein JUNO or a fragment thereof immobilized on the substrate.

[0162] Immobilization of a biological element, such as a protein of interest, on a sensor surface (metal, polymer or glass) is an essential and critical step in the design of a sensor. Different immobilization techniques exist depending on the substrate employed, which are known to the person skilled in the art.

[0163] In some embodiments, the biosensor of the present disclosure is provided, wherein at least one of the JUNO protein, ZP1, ZP2, ZP3 and anti-IZUMO antibody is bound to an additional moiety. In some embodiments, the additional moiety is a peptide. In one embodiment, the additional moiety is a label.

[0164] In some embodiments, the additional moiety is a peptide, such as a polyhistidine tag (His-tag).

[0165] In some embodiments, the additional moiety is a label, also known as a fluorescent tag or probe.

[0166] A polyhistidine tag can be successfully used to immobilize a protein on a surface, such as a metal surface, for example a gold, nickel or cobalt coated microtiter plate or protein array.

[0167] In some embodiments, the biosensor of the present disclosure is provided, wherein the JUNO protein, ZP1, ZP2, ZP3 and / or anti-IZUMO antibody is immobilized on the substrate.

[0168] In some embodiments, there is provided a biosensor of the disclosure, wherein the JUNO protein, ZP1, ZP2, ZP3 and / or anti-IZUMO antibody is immobilized on the substrate by nanoparticles, for example by gold nanoparticles. The presence of nanoparticles between the substrate and the protein is beneficial in that it prevents the unfolding of the protein and helps the protein to maintain the correct conformation.

[0169] Method

[0170] An aspect of the disclosure provides a method for manufacturing a biosensor comprising a JUNO protein, for example a biosensor disclosed herein, the method comprising:

[0171] a. providing a substrate,

[0172] b. providing a JUNO protein,

[0173] c. immobilizing the JUNO protein on the substrate,

[0174] thereby manufacturing a biosensor comprising a JUNO protein.

[0175] In some embodiments, the method for manufacturing a biosensor further comprises immobilizing one or more of ZP1, ZP2, ZP3 and anti-IZUMO antibody on the substrate.

[0176] A further aspect of the disclosure provides a method of selecting sperm, the method comprising:

[0177] a. providing a semen sample from a subject,

[0178] b. contacting the semen sample with a biosensor of the disclosure,

[0179] c. visualizing sperm bound to the biosensor by microscopy, and

[0180] d. selecting sperm bound to the biosensor.

[0181] A further aspect of the disclosure provides a method of selecting sperm, the method comprising:

[0182] a. providing a sperm sample from a subject,

[0183] b. contacting the sperm sample with a biosensor of the disclosure,

[0184] c. visualizing sperm bound to the biosensor by microscopy, and

[0185] d. selecting sperm bound to the biosensor.

[0186] In one embodiment of the methods of the disclosure, the semen sample comprises or is suspected of comprising one or more sperm cells.

[0187] Household device

[0188] Another aspect of the disclosure provides a hand-held device for detecting and / or quantifying sperm function, the device comprising:

[0189] a. a sample inlet;

[0190] b. a biosensor comprising a JUNO protein or fragment thereof, wherein the JUNO protein is immobilized on the biosensor, and wherein the inlet is configured to bring the sample into contact with the sensor;

[0191] c. a detector configured to receive a signal from the sensor and convert it into a format readable by a user;

[0192] d. optionally, means for isolating cellular components from the sample.

[0193] In particular embodiments, the hand-held device of the disclosure comprises a biosensor as defined in any embodiment of the disclosure.

[0194] Subject

[0195] An aspect of the disclosure provides the methods of the disclosure, wherein the subject is a human subject. In particular embodiments, the human subject is a child or an adult.

[0196] In further embodiments of the methods of the disclosure, the subject is a mammal. In further embodiments of the methods of the disclosure, the subject is a horse, a cow, a buffalo, a sheep, a pig, a goat, a cat, or a dog.

[0197] In some embodiments of the disclosure, the subject is a horse, and the biosensor comprises a JUNO protein or fragment thereof immobilized on a substrate, wherein the JUNO protein has a sequence having at least 95% sequence identity to the horse JUNO protein of SEQ ID NO: 6, for example at least 96% sequence identity, for example at least 97% sequence identity, for example at least 98% sequence identity, for example at least 98% sequence identity, for example at least 99% sequence identity, for example about 100% sequence identity to the horse JUNO protein of SEQ ID NO: 6.

[0198] In some embodiments of the disclosure, the subject is a dog, and the biosensor comprises a JUNO protein or fragment thereof immobilized on a substrate, wherein the JUNO protein is a canine JUNO protein.

[0199] In some embodiments of the present disclosure, the subject is a bovine, and the biosensor comprises a JUNO protein or fragment thereof immobilized on a substrate, wherein the JUNO protein is a bovine JUNO protein.

[0200] Sample

[0201] In specific embodiments of the methods of the present disclosure, the sample is a semen sample, optionally wherein the sample has been treated prior to analysis.

[0202] In specific embodiments of the methods of the present disclosure, the sample is a sperm sample, optionally wherein the sample has been treated prior to analysis.

[0203] In one embodiment of the methods of the present disclosure, the semen sample and / or sperm sample comprises or is suspected of comprising one or more sperm cells.

[0204] In some embodiments, the semen sample is treated prior to analysis, and the treatment comprises liquefaction of the sperm.

[0205] Semen sample can be used interchangeably with sperm sample and semen herein. Semen sample and / or sperm sample is defined herein as a semen sample that can contain sperm. In specific embodiments of the present disclosure, the semen sample can be further treated (e.g., diluted) by using solvents, media, buffers, and / or fluids suitable for treating (e.g., diluting) the semen / sperm sample.

[0206] Semen is secreted by the male or hermaphroditic gonads and other sex organs and enables the fertilization of the female ovum. In humans, semen contains several components in addition to sperm: proteolytic enzymes and other enzymes and fructose as a component of semen, which promote the survival of sperm and provide a medium for sperm to move or "swim" in. Semen is produced and derived from the seminal vesicles located in the pelvis.

[0207] The term "sperm" as used herein refers to a male reproductive cell and is synonymous with "sperm cell". A motile, single flagellated sperm cell is referred to as a sperm, while a non-motile sperm cell is referred to as a spermatium. In the present disclosure, the term "sperm", i.e., both sperm and spermatozoa are used interchangeably.

[0208] Detection technique

[0209] In some embodiments of the methods of the present disclosure, surface plasmon resonance (SPR) is used to detect sperm function. In specific embodiments, surface plasmon resonance readout is used to determine the concentration of one or more thyroid hormones.

[0210] Surface plasmon resonance is the resonant oscillation of conduction electrons at the interface between a negative dielectric constant material and a positive dielectric constant material under excitation by incident light. SPR is the basis of many standard tools for measuring adsorption of materials onto planar metal (e.g. gold or silver) surfaces or onto metal nanoparticle surfaces. This is the underlying principle behind many color-based biosensor applications, different sensors, and diatom photosynthesis. SPR can be used to detect biomolecular binding interactions. In SPR, one molecular partner, e.g. a protein, is immobilized on a metal film. Light excites surface plasmons in the metal; when the binding partner binds to the immobilized molecule, this causes a detectable change in the surface plasmon signal.

[0211] In some embodiments of the methods of the present disclosure, sperm function is detected or quantified by electrochemical transduction.

[0212] Electrochemical biosensors, also known as biosensors that utilize electrochemical transduction, offer an attractive means of analyzing the content of a biological sample due to the direct conversion of a biological event into an electronic signal. The most common techniques in electrochemical biosensing include cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, and field effect transistor based methods, as well as nanowire or magnetic nanoparticle based biosensing techniques. Other measurement techniques that can be used in combination with electrochemical detection can also include surface plasmon resonance, optical waveguide light mode spectroscopy, ellipsometry, quartz crystal microbalance, and electrochemical forms of scanning probe microscopy.

[0213] Male sterility

[0214] Male infertility refers to the inability of a male to impregnate a female. In humans, it accounts for 40-50% of infertility. It affects about 7% of all men. Male infertility is usually due to a defect in the semen and semen quality is used as a surrogate measure of male fecundity.

[0215] Male infertility is defined as the inability of a male individual to impregnate a fertile female individual. One of the causes of male infertility can be the production of non-functional spermatozoa.

[0216] The diagnosis of male infertility can be based on a series of functional tests, such as any one of the sperm function tests disclosed herein. The tests can be evaluated according to guidelines known to the person skilled in the art, such as described in the WHO Laboratory Manual for the Examination and processing of human semen, WHO, 5th edition, 2010, ISBN 978 92 4 154778 9 (see in particular chapter 4).

[0217] Sequence

[0218] SEQ ID NO: 1: JUNO Human sperm-egg fusion protein Juno

[0219] MACWWPLLLELWTVMPTWAGDELLNICMNAKHHKRVPSPEDKLYEECIPWKDNACCTLTTSWEAHLDVSPLYNFSLFHCGLLMPGCRKHFIQAICFYECSPNLGPWIQPVGSLGWEVAPSGQGERVVNVPLCQEDCEEWWEDCRMSYTCKSNWRGGWDWSQGKNRCPKGAQCLPFSHYFPTPADLCEKTWSNSFKASPERRNSGRCLQKWFEPAQGNPNVAVARLFASSAPSWELSYTIMVCSLFLPFLS

[0220] SEQ ID NO: 2: ZP1 Human zona pellucida sperm-binding protein 1

[0221] MAGGSATTWGYPVALLLLVATLGLGRWLQPDPGLPGLRHSYDCGIKGMQLLVFPRPGQTLRFKVVDEFGNRFDVNNCSICYHWVTSRPQEPAVFSADYRGCHVLEKDGRFHLRVFMEAVLPNGRVDVAQDATLICPKPDPSRTLDSQLAPPAMFSVSTPQTLSFLPTSGHTSQGSGHAFPSPLDPGHSSVHPTPALPSPGPGPTLATLAQPHWGTLEHWDVNKRDYIGTHLSQEQCQVASGHLPCIVRRTSKEACQQAGCCYDNTREVPCYYGNTATVQCFRDGYFVLVVSQEMALTHRITLANIHLAYAPTSCSPTQHTEAFVVFYFPLTHCGTTMQVAGDQLIYENWLVSGIHIQKGPQGSITRDSTFQLHVRCVFNASDFLPIQASIFPPPSPAPMTQPGPLRLELRIAKDETFSSYYGEDDYPIVRLLREPVHVEVRLLQRTDPNLVLLLHQCWGAPSANPFQQPQWPILSDGCPFKGDSYRTQMVALDGATPFQSHYQRFTVATFALLDSGSQRALRGLVYLFCSTSACHTSGLETCSTACSTGTTRQRRSSGHRNDTARPQDIVSSPGPVGFEDSYGQEPTLGPTDSNGNSSLRPLLWAVLLLPAVALVLGFGVFVGLSQTWAQKLWESNRQ

[0222] SEQ ID NO: 3: ZP2 Human zona pellucida sperm-binding protein 2

[0223] MACRQRGGSWSPSGWFNAGWSTYRSISLFFALVTSGNSIDVSQLVNPAFPGTVTCDEREITVEFPSSPGTKKWHASVVDPLGLDMPNCTYILDPEKLTLRATYDNCTRRVHGGHQMTIRVMNNSAALRHGAVMYQFFCPAMQVEETQGLSASTICQKDFMSFSLPRVFSGLADDSKGTKVQMGWSIEVGDGARAKTLTLPEAMKEGFSLLIDNHRMTFHVPFNATGVTHYVQGNSHLYMVSLKLTFISPGQKVIFSSQAICAPDPVTCNATHMTLTIPEFPGKLKSVSFENQNIDVSQLHDNGIDLEATNGMKLHFSKTLLKTKLSEKCLLHQFYLASLKLTFLLRPETVSMVIYPECLCESPVSIVTGELCTQDGFMDVEVYSYQTQPALDLGTLRVGNSSCQPVFEAQSQGLVRFHIPLNGCGTRYKFEDDKVVYENEIHALWTDFPPSKISRDSEFRMTVKCSYSRNDMLLNINVESLTPPVASVKLGPFTLILQSYPDNSYQQPYGENEYPLVRFLRQPIYMEVRVLNRDDPNIKLVLDDCWATSTMDPDSFPQWNVVVDGCAYDLDNYQTTFHPVGSSVTHPDHYQRFDMKAFAFVSEAHVLSSLVYFHCSALICNRLSPDSPLCSVTCPVSSRHRRATGATEAEKMTVSLPGPILLLSDDSSFRGVGSSDLKASGSSGEKSRSETGEEVGSRGAMDTKGHKTAGDVGSKAVAAVAAFAGVVATLGFIYYLYEKRTVSNH

[0224] SEQ ID NO:4: ZP3 Human zona pellucida sperm-binding protein 3

[0225] MELSYRLFICLLLWGSTELCYPQPLWLLQGGASHPETSVQPVLVECQEATLMVMVSKDLFGTG KLIRAADLTLGPEACEPLVSMDTEDVVRFEVGLHECGNSMQVTDDALVYSTFLLHDPRPVGNLS IVRTNRAEIPIECRYPRQGNVSSQAILPTWLPFRTTVFSEEKLTFSLRLMEENWNAEKRSPTF HLGDAAHLQAEIHTGSHVPLRLFVDHCVATPTPDQNASPYHTIVDFHGCLVDGLTDASSAFKV PRPGPDTLQFTVDVFHFANDSRNMIYITCHLKVTLAEQDPDELNKACSFSKPSNSWFPVEGSAD ICQCCNKGDCGTPSHSRRQPHVMSQWSRSASRNRRHVTEEADVTVGPLIFLDRRGDHEVEQWA LPSDTSVVLLGVGLAVVVSLTLTAVILVLTRRCRTASHPVSASE

[0226] SEQ ID NO: 5: IZUMOl Human Izumo sperm-egg fusion protein 1

[0227] MGPHFTLLCAALAGCLLPAEGCVICDPSVVLALKSLEKDYLPGHLDAKHHKAMMERVENAVKDF QELSLNEDAYMGVVDEATLQKGSWSLLKDLKRITDSDVKGDLFVKELFWMLHLQKETFATYVAR FQKEAYCPNKCGVMLQTLIWCKNCKKEVHACRKSYDCGERNVEVPQMEDMILDCELNWHQASE GLTDYSFYRVWGNNTETLVSKGKEATLTKPMVGPEDAGSYRCELGSVNSSPATIINFHVTVLPK MIKEEKPSPNIVTPGEATTESSISLQPLQPEKMLASRLLGLLICGSLALITGLTFAIFRRRKVIDF IKSSLFGLGSGAAEQTQVPKEKATDSRQQ Example

[0228] Example 1. Electrochemical biosensor for sperm binding using cyclic voltammetry

[0229] A biosensor was fabricated on a screen-printed gold electrode. Briefly, 10 μL of 2 mg / mL cysteine ​​hydrochloride was added to the gold electrode and dried in the dark at room temperature. The electrode was washed with deionized water and then modified with 10 μL of citrate-capped gold nanoparticles. After drying and washing with deionized water, 10 μL of 50 mM hypotriacetic acid was added and incubated overnight at room temperature. Subsequently, it was blocked with 0.01 M phosphate buffer (pH 7.4) containing 1% bovine serum albumin. 10 μL of 10 mM nickel sulfate was added and incubated at room temperature for 2 hours. After washing the electrode with 0.01 M phosphate buffer (pH 7.4), 0.5 μg of JUNO or ZP3 containing a multihistidine tag was immobilized on the biosensor.

[0230] Semen samples were liquefied according to standard procedures and diluted to different dilutions in semen preparation media (#1070 / 1069, OrigioA / SDenmark, https: / / origio.marketport.net / MarketingZone / MZDirect / Source / 510e96f4-075b-4684-9a99-d472c1e3d31b). A biosensor was connected to an electrochemical workstation, and the diluted semen samples were added to the biosensor. Cyclic voltammetric responses of the interactions between the immobilized proteins and the sperm present in the semen samples were recorded.

[0231] The results are as follows Figure 1 As shown in Figure 3, Figure 1 The diagram shows a) after adding buffer only (solid line) and after adding 1.67x10. -2 After diluting the semen sample in buffer solution (dotted line), Electrode - gold nanoparticles - BSA blocking The cyclic volt-ampere response, b) Electrode - gold nanoparticles - JUNO - BSA blocking The cyclic voltammetric response (dotted line), and c) adding 1.67x10 -2 After diluting the semen sample in buffer solution, X Electrode - gold nanoparticles - ZP3 - BSA blocking The cyclic voltammetric response (underlined), where the dilution factor is determined by the initial volume / final volume.

[0232] Figure 2 shows the addition of 3 different dilutions (1.56x10). -5 X (dashed line), 1.67 x 10 -2 X (dotted line) and 2.5x10 -2 Six different semen samples (X ( dashed)) Figure 2A After -F), Electrode - gold nanoparticles - ZP3 - BSA blocking The cyclic voltammetric response is shown, while the solid line shows the electrode response using only buffer (without semen sample).

[0233] Figure 3 shows the cyclic voltammetry response of the electrodes after the addition of semen samples S1, S2, S3, S4, S5 and S6 (respectively -5 X (dotted line), 1.67 x 10 -2 X (dotted-dashed line) and 2.5 x 10 -2 X (short dashed line)) at three different dilutions (1.56 x 10 Figure 3A -F) after, Electrode - gold nanoparticles - JUNO - BSA blocking Conclusion: the solid line shows the electrode response using only the buffer (no semen sample).

[0234] Figure 1

[0235] As shown in Figure 1 , sperm do not bind to the biosensor without immobilization of JUNO or ZP3. The interaction of sperm with the biosensor when JUNO or ZP3 is present can be seen from the reduction and / or oxidation peaks that are apparent in the cyclic voltammograms. In addition, the binding can be quantified using cyclic voltammetry. As shown in Figures 2 and 3 respectively for ZP3-sperm and JUNO-sperm interactions, the shift in the reduction and oxidation peaks is consistent with the number of sperm that can bind to ZP3 and JUNO. Furthermore, the interaction detected is protein specific since the signal for the negative control (i.e. absence of binding protein Example 2. Electrochemical biosensor for sperm binding using electrochemical impedance spectroscopy ) remains constant between zero and non-zero sperm concentration.

[0236] Conclusion:

[0237] The screen-printed gold electrodes were functionalized with 10 mΐ of 2 mg / ml cysteamine hydrochloride (Cy HCI) and incubated for 2-4 h at room temperature in the dark. After washing the electrodes with deionized water, 5 mΐ of Asn-AuNPs were added to the functionalized electrodes. The electrodes were incubated for 5 h at room temperature and then washed with deionized water. A further 5 mΐ of 5 mΐ PB (10 mM, pH 7.4) containing 160 pg / ml JUNO and 160 pg / ml ZP3 were added and incubated overnight at 4 °C. Finally, the electrodes were blocked with 10 mΐ PBS containing 1 % BSA for 5 h at 4 °C.

[0238] The modified electrodes were connected to an electrochemical workstation.

[0239] 5-50 mΐ of pre-treated semen sample was added to the working electrode or the electrode was immersed in the sample vial. The sperm cells were allowed to bind to the modified electrode for 1-10 minutes and then washed with deionized water. The binding was analyzed by electrochemical impedance spectroscopy in the presence of a suitable concentration of redox mediator.

[0240] Similar experiments were performed using JUNO only, ZP2 only and a combination of ZP2, ZP3, JUNO.

[0241] For this example and all the following examples, prior to analysis, the semen sample was pre-treated (liquefied) following the following steps:

[0242] - The semen sample collected in the laboratory or clinic was liquefied at room temperature or in an incubator at 37°C for 15-60 minutes. Gentle rotation or agitation can be used to prevent heterogeneous liquefaction of the semen sample.

[0243] - This liquefied semen sample can be washed, reconstituted or diluted with a suitable semen preparation medium.

[0244] Example 3. Electrochemical biosensor for acrosome reaction

[0245] The sensor comprising a screen-printed gold electrode functionalized and modified with JUNO or a combination of JUNO with any one of ZP2 and ZP3 or a combination thereof can be used to detect and quantify sperm function by means of an electrochemical workstation.

[0246] Conclusion

[0247] The screen-printed gold electrode was functionalized by 10 μΐ of 2 mg / ml cysteamine hydrochloride (CyHCl) and incubated for 2-4 h at room temperature in the dark. After washing the electrode with deionized water, 5 μΐ of Asn-AuNPs were added to the functionalized electrode. The electrode was then incubated for 5 hours at room temperature and then washed with deionized water. Further, 5 μΐ of 5 μΐ PB (10 mM, pH 7.4) containing 160 μg / ml JUNO and 160 μg / ml ZP3 were added and incubated overnight at 4°C. Finally, the electrode was blocked with 10 μΐ PBS containing 1% BSA for 5 h at 4°C.

[0248] The modified electrode was connected to an electrochemical workstation.

[0249] 5-50 μΐ of the pre-treated semen sample was added to the working electrode or the electrode was immersed in the sample vial. The sperm cells were allowed to bind to the modified electrode while the binding was electrochemically analyzed by electrochemical impedance spectroscopy or amperometry.

[0250] Similar experiments were performed using ZP2 and a combination of JUNO, ZP3, ZP2.

[0251] Example 4. Detection / quantification of sperm function by surface plasmon resonance (SPR)

[0252] Sensors comprising screen printed gold electrodes functionalized and modified with JUNO or a combination of JUNO with any one of ZP2 and ZP3 or combinations thereof can be used to detect and quantify sperm function by electrochemical workstations.

[0253] Conclusion:

[0254] SPR chips are chemically functionalized and modified with any one of ZP2, ZP3 and JUNO or combinations thereof. The chips are then washed with deionized water and uncoated areas are blocked with bovine serum albumin (BSA). These chips are used in conjunction with an SPR detector. Pre-treated semen samples are flowed over the chip surface. Interactions between the coated proteins and sperm cells are analyzed from changes in binding kinetics and affinity. Sperm function is thereby detected and quantified.

[0255] Example 5. Detection / quantification of sperm binding by optical transducer

[0256] Sensors comprising SPR chips chemically functionalized and modified with any one of ZP2, ZP3 and JUNO or combinations thereof can be used to detect and quantify sperm function by an SPR detector.

[0257] Conclusion:

[0258] Entire or partial glass or plastic dishes are first chemically functionalized and modified with AuNPs and then with any one of ZP2, ZP3 and JUNO or combinations thereof. The dishes are washed with deionized water and uncoated areas are blocked with BSA. Pre-treated semen samples are added as drops to the coated areas and binding is observed under an optical sensor such as a bright field or dark field microscope. Sperm function is thereby detected and quantified.

[0259] Example 6. Detection / quantification of acrosome reaction by optical transducer

[0260] Sensors comprising any one of ZP2 and ZP3 or combinations thereof bound to AuNPs or AuNP-modified glass or plastic dishes can be used to detect and quantify sperm function by optical sensors.

[0261] Conclusion:

[0262] The pre-treated semen sample is incubated with any one or a combination of ZP2 and ZP3 previously bound to AuNPs. The sperm are allowed to interact with the proteins for 5-60 minutes, then washed by centrifugation. The sperm are then stained for acrosome reaction with fluorescein isothiocyanate (FITC)-labelled Pisum sativum agglutinin (PSA) (PSA-FITC) and then observed under an optical transducer such as a fluorescence microscope or a flow cytometer (see section 4.4.1 in http: / / apps.who.int / iris / bitstream / handle / 10665 / 44261 / 9789241547789_eng.pdf;jsessionid=2EF9F9030760BB60B84C83708999AF64?sequence=1). Sperm function is thereby detected and quantified.

[0263] Alternatively, the pre-treated semen sample is incubated with any one or a combination of ZP2 and ZP3 previously bound to AuNP-modified glass or plastic dishes for 5-60 minutes. The sperm are removed from the surface and then stained for acrosome reaction with PSA-FITC and then observed under an optical transducer such as a fluorescence microscope or a flow cytometer. Sperm function is thereby detected and quantified.

[0264] Alternatively, the pre-treated semen sample is incubated with any one or a combination of ZP2 and ZP3 previously bound to AuNP-modified glass or plastic dishes for 5-60 minutes. The sperm are then labelled in situ with anti-CD46 antibodies or labelled after removal from the substrate to detect sperm that have undergone acrosome reaction. Detection is performed by an optical transducer such as a fluorescence microscope or a flow cytometer. Sperm function is thereby detected and quantified.

[0265] ​

[0266] A sensor comprising any one or a combination of ZP2, ZP3 and JUNO bound to AuNPs or AuNP-modified glass or plastic dishes can be used to detect and quantify sperm function by an optical transducer. SEQUENCE LISTING <110> Spermosens AB <120> Biosensor for male sterility <130> P4957PC00 <150> EP 18190720.5 <151> 2018-08-24 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 250 <212> PRT <213> Human (Homo sapiens) <400> 1 Met Ala Cys Trp Trp Pro Leu Leu Leu Glu Leu Trp Thr Val Met Pro 1 5 10 15 Thr Trp Ala Gly Asp Glu Leu Leu Asn Ile Cys Met Asn Ala Lys His 20 25 30 His Lys Arg Val Pro Ser Pro Glu Asp Lys Leu Tyr Glu Glu Cys Ile 35 40 45 Pro Trp Lys Asp Asn Ala Cys Cys Thr Leu Thr Thr Ser Trp Glu Ala 50 55 60 His Leu Asp Val Ser Pro Leu Tyr Asn Phe Ser Leu Phe His Cys Gly 65 70 75 80 Leu Leu Met Pro Gly Cys Arg Lys His Phe Ile Gln Ala Ile Cys Phe 85 90 95 Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Pro Val Gly Ser 100 105 110 Leu Gly Trp Glu Val Ala Pro Ser Gly Gln Gly Glu Arg Val Val Asn 115 120 125 Val Pro Leu Cys Gin Glu Asp Cys Glu Glu Trp Trp Glu Asp Cys Arg 130 135 140 Met Ser Tyr Thr Cys Lys Ser Asn Trp Arg Gly Gly Trp Asp Trp Ser 145 150 155 160 Gln Gly Lys Asn Arg Cys Pro Lys Gly Ala Gin Cys Leu Pro Phe Ser 165 170 175 His Tyr Phe Pro Thr Pro Ala Asp Leu Cys Glu Lys Thr Trp Ser Asn 180 185 190 Ser Phe Lys Ala Ser Pro Glu Arg Arg Asn Ser Gly Arg Cys Leu Gin 195 200 205 Lys Trp Phe Glu Pro Ala Gin Gly Asn Pro Asn Val Ala Val Ala Arg 210 215 220 Leu Phe Ala Ser Ser Ala Pro Ser Trp Glu Leu Ser Tyr Thr Ile Met 225 230 235 240 Val Cys Ser Leu Phe Leu Pro Phe Leu Ser 245 250 <210> 2 <211> 638 <212> PRT <213> Homo sapiens <400> 2 Met Ala Gly Gly Ser Ala Thr Thr Trp Gly Tyr Pro Val Ala Leu Leu 1 5 10 15 Leu Leu Val Ala Thr Leu Gly Leu Gly Arg Trp Leu Gin Pro Asp Pro 20 25 30 Gly Leu Pro Gly Leu Arg His Ser Tyr Asp Cys Gly Ile Lys Gly Met 35 40 45 Gln Leu Leu Val Phe Pro Arg Pro Gly Gin Thr Leu Arg Phe Lys Val 50 55 60 Val Asp Glu Phe Gly Asn Arg Phe Asp Val Asn Asn Cys Ser Ile Cys 65 70 75 80 Tyr His Trp Val Thr Ser Arg Pro Gin Gin Pro Ala Val Phe Ser Ala 85 90 95 Asp Tyr Arg Gly Cys His Val Leu Glu Lys Asp Gly Arg Phe His Leu 100 105 110 Arg Val Phe Met Glu Ala Val Leu Pro Asn Gly Arg Val Asp Val Ala 115 120 125 Gln Asp Ala Thr Leu Ile Cys Pro Lys Pro Asp Pro Ser Arg Thr Leu 130 135 140 Asp Ser Gin Leu Ala Pro Pro Ala Met Phe Ser Val Ser Thr Pro Gin 145 150 155 160 Thr Leu Ser Phe Leu Pro Thr Ser Gly His Thr Ser Gin Gly Ser Gly 165 170 175 His Ala Phe Pro Ser Pro Leu Asp Pro Gly His Ser Ser Val His Pro 180 185 190 Thr Pro Ala Leu Pro Ser Pro Gly Pro Gly Pro Thr Leu Ala Thr Leu 195 200 205 Ala Gln Pro His Trp Gly Thr Leu Glu His Trp Asp Val Asn Lys Arg 210 215 220 Asp Tyr Ile Gly Thr His Leu Ser Gln Glu Cys Cys Gln Val Ala Ser 225 230 235 240 Gly His Leu Pro Cys Ile Val Arg Arg Thr Ser Lys Glu Ala Cys Gln 245 250 255 Gln Ala Gly Cys Cys Tyr Asp Asn Thr Arg Glu Val Pro Cys Tyr Tyr 260 265 270 Gly Asn Thr Ala Thr Val Gln Cys Phe Arg Asp Gly Tyr Phe Val Leu 275 280 285 Val Val Ser Gln Glu Met Ala Leu Thr His Arg Ile Thr Leu Ala Asn 290 295 300 Ile His Leu Ala Tyr Ala Pro Thr Ser Cys Ser Pro Thr Gln His Thr 305 310 315 320 Glu Ala Phe Val Val Phe Tyr Phe Pro Leu Thr His Cys Gly Thr Thr 325 330 335 Met Gin Val Ala Gly Asp Gin Leu lie Tyr Gin Asn Trp Leu Val Ser 340 345 350 Gly lie His lie Gin Lys Gly Pro Gin Gly Ser lie Thr Arg Asp Ser 355 360 365 Thr Phe Gin Leu His Val Arg Cys Val Phe Asn Ala Ser Asp Phe Leu 370 375 380 Pro lie Gin Ala Ser lie Phe Pro Pro Pro Ser Pro Ala Pro Met Thr 385 390 395 400 Gln Pro Gly Pro Leu Arg Leu Gin Leu Arg lie Ala Lys Asp Gin Thr 405 410 415 Phe Ser Ser Tyr Tyr Gin Glu Asp Asp Tyr Pro lie Val Arg Leu Leu 420 425 430 Arg Gin Pro Val His Val Gin Val Arg Leu Leu Gin Arg Thr Asp Pro 435 440 445 Asn Leu Val Leu Leu Leu His Gin Cys Trp Gly Ala Pro Ser Ala Asn 450 455 460 Pro Phe Gin Gin Pro Gin Trp Pro lie Leu Ser Asp Gly Cys Pro Phe 465 470 475 480 Lys Gly Asp Ser Tyr Arg Thr Gin Met Val Ala Leu Asp Gly Ala Thr 485 490 495 Pro Phe Gin Ser His Tyr Gin Arg Phe Thr Val Ala Thr Phe Ala Leu 500 505 510 Leu Asp Ser Gly Ser Gin Arg Ala Leu Arg Gly Leu Val Tyr Leu Phe 515 520 525 Cys Ser Thr Ser Ala Cys His Thr Ser Gly Leu Glu Thr Cys Ser Thr 530 535 540 Ala Cys Ser Thr Gly Thr Thr Arg Gin Arg Arg Ser Ser Gly His Arg 545 550 555 560 Asn Asp Thr Ala Arg Pro Gin Asp He Val Ser Ser Pro Gly Pro Val 565 570 575 Gly Phe Glu Asp Ser Tyr Gly Gin Glu Pro Thr Leu Gly Pro Thr Asp 580 585 590 Ser Asn Gly Asn Ser Ser Leu Arg Pro Leu Leu Trp Ala Val Leu Leu 595 600 605 Leu Pro Ala Val Ala Leu Val Leu Gly Phe Gly Val Phe Val Gly Leu 610 615 620 Ser Gin Thr Trp Ala Gin Lys Leu Trp Glu Ser Asn Arg Gin 625 630 635 <210> 3 <211> 745 <212> PRT <213> Homo sapiens <400> 3 Met Ala Cys Arg Gin Arg Gly Gly Ser Trp Ser Pro Ser Gly Trp Phe 1 5 10 15 Asn Ala Gly Trp Ser Thr Tyr Arg Ser Ile Ser Leu Phe Phe Ala Leu 20 25 30 Val Thr Ser Gly Asn Ser Ile Asp Val Ser Gin Leu Val Asn Pro Ala 35 40 45 Phe Pro Gly Thr Val Thr Cys Asp Glu Arg Glu Ile Thr Val Glu Phe 50 55 60 Pro Ser Ser Pro Gly Thr Lys Lys Trp His Ala Ser Val Val Asp Pro 65 70 75 80 Leu Gly Leu Asp Met Pro Asn Cys Thr Tyr Ile Leu Asp Pro Glu Lys 85 90 95 Leu Thr Leu Arg Ala Thr Tyr Asp Asn Cys Thr Arg Arg Val His Gly 100 105 110 Gly His Gin Met Thr Ile Arg Val Met Asn Asn Ser Ala Ala Leu Arg 115 120 125 His Gly Ala Val Met Tyr Gin Phe Phe Cys Pro Ala Met Gin Val Glu 130 135 140 Glu Thr Gin Gly Leu Ser Ala Ser Thr lie Cys Gin Lys Asp Phe Met 145 150 155 160 Ser Phe Ser Leu Pro Arg Val Phe Ser Gly Leu Ala Asp Asp Ser Lys 165 170 175 Gly Thr Lys Val Gin Met Gly Trp Ser lie Glu Val Gly Asp Gly Ala 180 185 190 Arg Ala Lys Thr Leu Thr Leu Pro Glu Ala Met Lys Glu Gly Phe Ser 195 200 205 Leu Leu lie Asp Asn His Arg Met Thr Phe His Val Pro Phe Asn Ala 210 215 220 Thr Gly Val Thr His Tyr Val Gin Gly Asn Ser His Leu Tyr Met Val 225 230 235 240 Ser Leu Lys Leu Thr Phe lie Ser Pro Gly Gin Lys Val lie Phe Ser 245 250 255 Ser Gin Ala lie Cys Ala Pro Asp Pro Val Thr Cys Asn Ala Thr His 260 265 270 Met Thr Leu Thr lie Pro Glu Phe Pro Gly Lys Leu Lys Ser Val Ser 275 280 285 Phe Glu Asn Gin Asn lie Asp Val Ser Gin Leu His Asp Asn Gly lie 290 295 300 Asp Leu Glu Ala Thr Asn Gly Met Lys Leu His Phe Ser Lys Thr Leu 305 310 315 320 Leu Lys Thr Lys Leu Ser Glu Lys Cys Leu Leu His Gln Phe Tyr Leu 325 330 335 Ala Ser Leu Lys Leu Thr Phe Leu Leu Arg Pro Glu Thr Val Ser Met 340 345 350 Val Ile Tyr Pro Glu Cys Leu Cys Glu Ser Pro Val Ser Ile Val Thr 355 360 365 Gly Glu Leu Cys Thr Gln Asp Gly Phe Met Asp Val Glu Val Tyr Ser 370 375 380 Tyr Gln Thr Gln Pro Ala Leu Asp Leu Gly Thr Leu Arg Val Gly Asn 385 390 395 400 Ser Ser Cys Gln Pro Val Phe Glu Ala Gln Ser Gln Gly Leu Val Arg 405 410 415 Phe His Ile Pro Leu Asn Gly Cys Gly Thr Arg Tyr Lys Phe Glu Asp 420 425 430 Asp Lys Val Val Tyr Glu Asn Glu Ile His Ala Leu Trp Thr Asp Phe 435 440 445 Pro Pro Ser Lys Ile Ser Arg Asp Ser Glu Phe Arg Met Thr Val Lys 450 455 460 Cys Ser Tyr Ser Arg Asn Asp Met Leu Leu Asn Ile Asn Val Glu Ser 465 470 475 480 Leu Thr Pro Pro Val Ala Ser Val Lys Leu Gly Pro Phe Thr Leu Ile 485 490 495 Leu Gln Ser Tyr Pro Asp Asn Ser Tyr Gln Gln Pro Tyr Gly Glu Asn 500 505 510 Glu Tyr Pro Leu Val Arg Phe Leu Arg Gln Pro Ile Tyr Met Glu Val 515 520 525 Arg Val Leu Asn Arg Asp Asp Pro Asn Ile Lys Leu Val Leu Asp Asp 530 535 540 Cys Trp Ala Thr Ser Thr Met Asp Pro Asp Ser Phe Pro Gln Trp Asn 545 550 555 560 Val Val Val Asp Gly Cys Ala Tyr Asp Leu Asp Asn Tyr Gln Thr Thr 565 570 575 Phe His Pro Val Gly Ser Ser Val Thr His Pro Asp His Tyr Gln Arg 580 585 590 Phe Asp Met Lys Ala Phe Ala Phe Val Ser Glu Ala His Val Leu Ser 595 600 605 Ser Leu Val Tyr Phe His Cys Ser Ala Leu Ile Cys Asn Arg Leu Ser 610 615 620 Pro Asp Ser Pro Leu Cys Ser Val Thr Cys Pro Val Ser Ser Arg His 625 630 635 640 Arg Arg Ala Thr Gly Ala Thr Glu Ala Glu Lys Met Thr Val Ser Leu 645 650 655 Pro Gly Pro Ile Leu Leu Leu Ser Asp Asp Ser Ser Phe Arg Gly Val 660 665 670 Gly Ser Ser Asp Leu Lys Ala Ser Gly Ser Ser Gly Glu Lys Ser Arg 675 680 685 Ser Glu Thr Gly Glu Glu Val Gly Ser Arg Gly Ala Met Asp Thr Lys 690 695 700 Gly His Lys Thr Ala Gly Asp Val Gly Ser Lys Ala Val Ala Ala Val 705 710 715 720 Ala Ala Phe Ala Gly Val Val Ala Thr Leu Gly Phe Ile Tyr Tyr Leu 725 730 735 Tyr Glu Lys Arg Thr Val Ser Asn His 740 745 <210> 4 <211> 424 <212> PRT <213> Human (Homo sapiens) <400> 4 Met Glu Leu Ser Tyr Arg Leu Phe Ile Cys Leu Leu Leu Trp Gly Ser 1 5 10 15 Thr Glu Leu Cys Tyr Pro Gln Pro Leu Trp Leu Leu Gln Gly Gly Ala 20 25 30 Ser His Pro Glu Thr Ser Val Gln Pro Val Leu Val Glu Cys Gln Glu 35 40 45 Ala Thr Leu Met Val Met Val Ser Lys Asp Leu Phe Gly Thr Gly Lys 50 55 60 Leu Ile Arg Ala Ala Asp Leu Thr Leu Gly Pro Glu Ala Cys Glu Pro 65 70 75 80 Leu Val Ser Met Asp Thr Glu Asp Val Val Arg Phe Glu Val Gly Leu 85 90 95 His Glu Cys Gly Asn Ser Met Gln Val Thr Asp Asp Ala Leu Val Tyr 100 105 110 Ser Thr Phe Leu Leu His Asp Pro Arg Pro Val Gly Asn Leu Ser Ile 115 120 125 Val Arg Thr Asn Arg Ala Glu Ile Pro Ile Glu Cys Arg Tyr Pro Arg 130 135 140 Gln Gly Asn Val Ser Ser Gln Ala Ile Leu Pro Thr Trp Leu Pro Phe 145 150 155 160 Arg Thr Thr Val Phe Ser Glu Glu Lys Leu Thr Phe Ser Leu Arg Leu 165 170 175 Met Glu Glu Asn Trp Asn Ala Glu Lys Arg Ser Pro Thr Phe His Leu 180 185 190 Gly Asp Ala Ala His Leu Gln Ala Glu Ile His Thr Gly Ser His Val 195 200 205 Pro Leu Arg Leu Phe Val Asp His Cys Val Ala Thr Pro Thr Pro Asp 210 215 220 Gln Asn Ala Ser Pro Tyr His Thr Ile Val Asp Phe His Gly Cys Leu 225 230 235 240 Val Asp Gly Leu Thr Asp Ala Ser Ser Ala Phe Lys Val Pro Arg Pro 245 250 255 Gly Pro Asp Thr Leu Gln Phe Thr Val Asp Val Phe His Phe Ala Asn 260 265 270 Asp Ser Arg Asn Met Ile Tyr Ile Thr Cys His Leu Lys Val Thr Leu 275 280 285 Ala Glu Gln Asp Pro Asp Glu Leu Asn Lys Ala Cys Ser Phe Ser Lys 290 295 300 Pro Ser Asn Ser Trp Phe Pro Val Glu Gly Ser Ala Asp Ile Cys Gln 305 310 315 320 Cys Cys Asn Lys Gly Asp Cys Gly Thr Pro Ser His Ser Arg Arg Gln 325 330 335 Pro His Val Met Ser Gin Trp Ser Arg Ser Ala Ser Arg Asn Arg Arg 340 345 350 His Val Thr Glu Glu Ala Asp Val Thr Val Gly Pro Leu Ile Phe Leu 355 360 365 Asp Arg Arg Gly Asp His Glu Val Glu Gin Trp Ala Leu Pro Ser Asp 370 375 380 Thr Ser Val Val Leu Leu Gly Val Gly Leu Ala Val Val Val Ser Leu 385 390 395 400 Thr Leu Thr Ala Val Ile Leu Val Leu Thr Arg Arg Cys Arg Thr Ala 405 410 415 Ser His Pro Val Ser Ala Ser Glu 420 <210> 5 <211> 350 <212> PRT <213> Human (Homo sapiens) <400> 5 Met Gly Pro His Phe Thr Leu Leu Cys Ala Ala Leu Ala Gly Cys Leu 1 5 10 15 Leu Pro Ala Glu Gly Cys Val Ile Cys Asp Pro Ser Val Val Leu Ala 20 25 30 Leu Lys Ser Leu Glu Lys Asp Tyr Leu Pro Gly His Leu Asp Ala Lys 35 40 45 His His Lys Ala Met Met Glu Arg Val Glu Asn Ala Val Lys Asp Phe 50 55 60 Gln Glu Leu Ser Leu Asn Glu Asp Ala Tyr Met Gly Val Val Asp Glu 65 70 75 80 Ala Thr Leu Gln Lys Gly Ser Trp Ser Leu Leu Lys Asp Leu Lys Arg 85 90 95 Ile Thr Asp Ser Asp Val Lys Gly Asp Leu Phe Val Lys Glu Leu Phe 100 105 110 Trp Met Leu His Leu Gln Lys Glu Thr Phe Ala Thr Tyr Val Ala Arg 115 120 125 Phe Gln Lys Glu Ala Tyr Cys Pro Asn Lys Cys Gly Val Met Leu Gln 130 135 140 Thr Leu Ile Trp Cys Lys Asn Cys Lys Lys Glu Val His Ala Cys Arg 145 150 155 160 Lys Ser Tyr Asp Cys Gly Glu Arg Asn Val Glu Val Pro Gln Met Glu 165 170 175 Asp Met Ile Leu Asp Cys Glu Leu Asn Trp His Gln Ala Ser Glu Gly 180 185 190 Leu Thr Asp Tyr Ser Phe Tyr Arg Val Trp Gly Asn Asn Thr Glu Thr 195 200 205 Leu Val Ser Lys Gly Lys Glu Ala Thr Leu Thr Lys Pro Met Val Gly 210 215 220 Pro Glu Asp Ala Gly Ser Tyr Arg Cys Glu Leu Gly Ser Val Asn Ser 225 230 235 240 Ser Pro Ala Thr Ile Ile Asn Phe His Val Thr Val Leu Pro Lys Met 245 250 255 Ile Lys Glu Glu Lys Pro Ser Pro Asn Ile Val Thr Pro Gly Glu Ala 260 265 270 Thr Thr Glu Ser Ser Ile Ser Leu Gln Pro Leu Gln Pro Glu Lys Met 275 280 285 Leu Ala Ser Arg Leu Leu Gly Leu Leu Ile Cys Gly Ser Leu Ala Leu 290 295 300 Ile Thr Gly Leu Thr Phe Ala Ile Phe Arg Arg Arg Lys Val Ile Asp 305 310 315 320 Phe Ile Lys Ser Ser Leu Phe Gly Leu Gly Ser Gly Ala Ala Glu Gln 325 330 335 Thr Gln Val Pro Lys Glu Lys Ala Thr Asp Ser Arg Gln Gln 340 345 350

Claims

1. A biosensor for detecting and / or quantifying sperm binding function, said biosensor comprising a substrate and a JUNO protein, wherein said JUNO protein is a polypeptide of SEQ ID NO: 1, wherein said JUNO protein is immobilized on said substrate, and wherein said biosensor is configured for determining binding of at least a portion of a sperm cell to a protein immobilized on a substrate.

2. The biosensor of claim 1, further comprising a zona pellucida protein 1 (ZP1), a zona pellucida protein 2 (ZP2), a zona pellucida protein 3 (ZP3), and / or an anti-IZUMO antibody, and wherein said ZP1, ZP2, ZP3, and / or anti-IZUMO antibody is immobilized on said substrate.

3. The biosensor of claim 2, wherein said proteins JUNO, ZP1, ZP2, and ZP3 are mammalian proteins.

4. The biosensor of claim 2, wherein said proteins JUNO, ZP1, ZP2, and ZP3 are human, equine, canine, or bovine proteins.

5. The biosensor of any one of claims 2-4, wherein said ZP1 is a polypeptide of SEQ ID NO:

2.

6. The biosensor of any one of claims 2-4, wherein said ZP2 is a polypeptide of SEQ ID NO:

3.

7. The biosensor of any one of claims 2-4, wherein said ZP3 is a polypeptide of SEQ ID NO:

4.

8. The biosensor of any one of the preceding claims 1-4, wherein at least one of JUNO, ZP1, ZP2, ZP3, and anti-IZUMO antibody is bound to an additional moiety.

9. The biosensor of claim 8, wherein said additional moiety is a peptide.

10. The biosensor of claim 8, wherein said additional moiety is a label.

11. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said sperm binding function is determined by binding of at least a portion of a sperm cell to a protein immobilized on the sensor, wherein said protein comprises JUNO and a protein selected from the group consisting of ZP1, ZP2, ZP3, and / or an anti-IZUMO antibody.

12. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said at least a portion of a sperm cell comprises an IZUMOl surface antigen.

13. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said substrate is a microbead, a dish, a chip, or an electrode.

14. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said substrate is an electrode.

15. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said substrate is a polymeric microbead.

16. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein said substrate is an agarose, cellulose, nitrocellulose, or latex microbead.

17. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein the substrate is a plastic dish, a ceramic dish or a glass dish.

18. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein the substrate is a dish, and wherein the dish is configured such that it can be coupled to a microscope or an optical transducer.

19. The biosensor of any one of the preceding claims 1-4 and 9-10, wherein the substrate is a microbead, wherein the microbead is configured such that it can be coupled to a microscope, an optical transducer or a measurement circuit.

20. The biosensor of the preceding claim 13, wherein the chip is a glass chip.

21. The biosensor of the preceding claim 13, wherein the electrode is a carbon, gold or platinum electrode.

22. The biosensor of the preceding claim 13, wherein the electrode is a screen printed electrode.

23. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein the substrate has a modified surface.

24. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein at least a portion of the substrate is coated with a gold layer.

25. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein at least a portion of the substrate is modified with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide and combinations thereof.

26. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein JUNO protein and proteins ZP1, ZP2, ZP3 and / or anti-IZUMO antibody are immobilized on the substrate.

27. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, configured such that the substrate can be coupled to a microscope, an electrochemical workstation, a surface plasmon resonance detector, a measurement circuit or an optical transducer.

28. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein the substrate is a dish, and wherein the dish is configured such that it can be coupled to a microscope or an optical transducer.

29. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein the substrate is an electrode, and wherein the electrode is configured such that it can be coupled to an electrochemical workstation or a measurement circuit.

30. The biosensor of any one of the preceding claims 1-4, 9-10 and 20-22, wherein the substrate is a chip, and wherein the chip is configured such that it can be coupled to a surface plasmon resonance detector.

31. The biosensor of any one of claims 1-4, 9-10 and 20-22, wherein the biosensor is configured for detecting and / or quantifying sperm binding function, wherein the sperm are in a semen sample.

32. A method of selecting sperm, the method comprising: a. providing a semen sample from a subject, wherein the semen sample comprises one or more sperm cells, b. contacting the semen sample with a biosensor according to any of the preceding claims, c. visualizing the sperm cells bound to the biosensor by microscopy, and d. selecting at least one sperm cell bound to the biosensor.

33. A method for manufacturing a biosensor according to claim 1, the method comprising: a. providing a substrate, b. providing a JUNO protein, c. immobilizing the JUNO protein to the substrate, and d. providing means for determining the binding of a sperm cell to the protein immobilized on the substrate, thereby manufacturing a biosensor comprising a JUNO protein.

34. The method according to claim 33, wherein the substrate is as defined in any of the preceding claims.

35. The method according to any of claims 33 to 34, wherein the method further comprises immobilizing one or more of ZP1, ZP2, ZP3 and an anti-IZUMO antibody on the substrate.

36. The method according to any of claims 33 to 34, wherein the proteins JUNO, ZP1, ZP2, ZP3 and an anti-IZUMO antibody are as defined in any of the preceding claims.

37. A hand-held device for detecting and / or quantifying sperm binding function, the device comprising: a. a sample inlet; b. a biosensor according to any of claims 1-31, wherein the inlet is configured to bring the sample into contact with the substrate; c. a detector configured to receive a signal from the substrate and convert it into a format readable by a user; and d. optionally, means for separating cellular components from the sample.

Images