Biosensor for Male Infertility

Abstract

The present invention relates to a biosensor and applications thereof for the quantification of sperm function. Methods and tools for diagnosis of male infertility are also disclosed herein.

1 . A biosensor for detection and/or quantification of sperm function, the biosensor comprising a substrate and a JUNO protein or a fragment thereof, wherein the JUNO protein or fragment thereof is immobilized on the substrate. 
     
     
         2 . The biosensor according to any one of the preceding claims, further comprising Zona pellucida 1 (ZP1), Zona pellucida 2 (ZP2), Zona pellucida 3 (ZP3) and/or an anti-IZUMO antibody or fragments thereof, wherein the ZP1, ZP2, ZP3, and/or the anti-IZUMO antibody or fragments thereof are immobilized on the substrate. 
     
     
         3 . The biosensor according to any one of the preceding claims, wherein the proteins JUNO, ZP1, ZP2 and ZP3 are mammalian proteins.
 The biosensor according to any one of the preceding claims, wherein the proteins JUNO, ZP1, ZP2 and ZP3 are human, equine, canine or bovine proteins.   
     
     
         4 . The biosensor according to any one of the preceding claims, wherein the JUNO protein 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 entity, such as about 100% sequence identity to a protein of SEQ ID NO: 1 or an orthologue thereof, or a fragment of said protein. 
     
     
         5 . The biosensor according to any one of the preceding claims, 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 entity, such as about 100% sequence identity to SEQ ID NO: 2 or an orthologue thereof, or a fragment of said protein. 
     
     
         6 . The biosensor according to any one of the preceding claims, wherein the ZP2 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 entity, such as about 100% sequence identity to SEQ ID NO: 3 or an orthologue thereof, or a fragment of said protein. 
     
     
         7 . The biosensor according to any one of the preceding claims, wherein the ZP3 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 entity, such as about 100% sequence identity to SEQ ID NO: 4 or an orthologue thereof, or a fragment of said protein. 
     
     
         8 . The biosensor according to any one of the preceding claims, wherein at least one of the JUNO protein, ZP1, ZP2, ZP3, and anti-IZUMO antibody is conjugated to an additional moiety. 
     
     
         9 . The biosensor according to any one of the preceding claims, wherein said additional moiety is a peptide. 
     
     
         10 . The biosensor according to any one of the preceding claims, wherein said additional moiety is a label. 
     
     
         11 . The biosensor according to any one of the preceding claims, wherein the sperm function is determined from the binding of at least a portion of sperm to a protein immobilized on the sensor, wherein said protein is selected from the groups consisting of JUNO protein, ZP1, ZP2, ZP3 and/or the anti-IZUMO antibody, or fragments thereof. 
     
     
         12 . The biosensor according to any one of the preceding claims, wherein at least a portion of sperm comprises an IZUMO1 surface antigen. 
     
     
         13 . The biosensor according to any one of the preceding claims, wherein the substrate is a microbead, a dish, a chip or an electrode. 
     
     
         14 . The biosensor according to any one of the preceding claims, wherein the substrate is a polymeric microbead. 
     
     
         15 . The biosensor according to any one of the preceding claims, wherein the substrate is agarose, cellulose, nitrocellulose or latex microbeads. 
     
     
         16 . The biosensor according to any one of the preceding claims, wherein the substrate is a dish, such as a plastic dish, a ceramic dish or a glass dish. 
     
     
         17 . The biosensor according to any one of the preceding claims, wherein the substrate is a dish and wherein said dish is configured such that it can be coupled to a microscope or an optical transducer. 
     
     
         18 . The biosensor according to any one of the preceding claims, wherein the substrate is microbeads, wherein said microbeads are configured such that they can be coupled to a microscope, an optical transducer or a measurement circuit. 
     
     
         19 . The biosensor according to any one of the preceding claims, wherein chip is a glass chip. 
     
     
         20 . The biosensor according to any one of the preceding claims, wherein the electrode is a carbon, gold or platinum electrode. 
     
     
         21 . The biosensor according to any one of the preceding claims, wherein the electrode is a screen printed electrode. 
     
     
         22 . The biosensor according to any one of the preceding claims, wherein the substrate has a modified surface. 
     
     
         23 . The biosensor according to any one of the preceding claims, wherein at least a portion of the substrate is coated with a layer of gold. 
     
     
         24 . The biosensor according to any one of the preceding claims, 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. 
     
     
         25 . The biosensor according to any one of the preceding claims, wherein the JUNO protein, ZP1, ZP2, ZP3 and/or the anti-IZUMO antibody is immobilized on the substrate. 
     
     
         26 . The biosensor according to any one of the preceding claims, 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. 
     
     
         27 . The biosensor according to any one of the preceding claims, wherein the substrate is a dish and wherein said dish is configured such that it can be coupled to a microscope or an optical transducer. 
     
     
         28 . The biosensor according to any one of the preceding claims, wherein the substrate is an electrode and wherein said electrode is configured such that it can be coupled to an electrochemical workstation or a measurement circuit. 
     
     
         29 . The biosensor according to any one of the preceding claims, wherein the substrate is a chip and wherein said chip is configured such that it can be coupled to a surface plasmon resonance detector. 
     
     
         30 . The biosensor according to any one of the preceding claims, wherein the biosensor is configured for detection and/or quantification of sperm function, wherein said sperm is in the semen sample. 
     
     
         31 . A method for detecting and/or quantifying sperm function, wherein the method comprises the steps of:
 a. Providing a semen sample from a subject, wherein said semen sample comprises one or more sperm cells,   b. Contacting the semen sample with the biosensor according to any one of the preceding claims,   c. Determining binding of the sperm cells to a protein immobilized on the sensor,   
       thereby detecting and/or quantifying the sperm function of said sample. 
     
     
         32 . A method for diagnosis of male infertility, wherein the method comprises the steps of:
 a. Providing a semen sample from a subject,   b. Contacting the semen sample with the biosensor according to any one of the preceding claims,   c. Quantifying the sperm function of said sample according to the method of  claim 31 ,   d. Using the sperm function to diagnose if the subject is infertile.   
     
     
         33 . The method according to any one of  claim 32 , wherein the semen sample comprises one or more sperm cells. 
     
     
         34 . The method according to any one of  claims 31  to  33 , wherein the protein immobilized on the sensor is selected from the group consisting of JUNO protein, ZP1, ZP2, ZP3 and/or the anti-IZUMO antibody, or fragments thereof, and wherein said binding is detected by microscopic analysis, electrochemical detection and/or surface plasmon resonance. 
     
     
         35 . The method according to any one of  claims 31  and  34 , wherein the sperm function of said sample is quantified by determining the percentage of bound versus unbound sperm cells by microscopic analysis, electrochemical detection and/or surface plasmon resonance. 
     
     
         36 . The method according to any one of  claims 31  to  35 , wherein the sperm function of said sample is quantified by determining the acrosomal status of the sperm cells in the semen sample by microscopic analysis, electrochemical detection and/or surface plasmon resonance. 
     
     
         37 . The method according to any one of  claims 31  to  36 , wherein said method further comprises comparing the percentage of bound versus unbound sperm cells and/or the acrosomal status of the sperm cells with respective reference values. 
     
     
         38 . The method according to any one of  claims 31  to  37 , wherein the sample is treated prior to step b., and wherein said treatment comprises liquefaction of the sperm and optionally capacitation. 
     
     
         39 . The method according to any one of  claims 31  to  38 , wherein the method further comprises a step of adding a fluorophore to the sample prior to quantifying the sperm function of said sample. 
     
     
         40 . The method according to any one of  claims 31  to  39 , wherein sperm function is determined by the ability of a sperm to bind to the immobilized JUNO protein. 
     
     
         41 . The method according to any one of  claims 31  to  40 , wherein said binding of a sperm to the immobilized JUNO proteins occurs via an IZUMO1 protein expressed by the sperm. 
     
     
         42 . The method according to any one of  claims 31  to  41 , further comprising a step of treating said male infertility. 
     
     
         43 . The method according to  claim 42 , wherein the treatment comprises administration of a medicament in a therapeutically effective amount and/or an artificial reproductive technology (ART). 
     
     
         44 . The method according to any one of  claims 31  to  43 , wherein the subject is a human subject. 
     
     
         45 . The method according to any one of  claims 31  to  44 , wherein the human subject is a child or an adult. 
     
     
         46 . The method according to any one of  claims 31  to  45 , wherein the subject is a mammal, such as a horse, cow, buffalo, sheep, pig, goat, cat or dog. 
     
     
         47 . A method of selecting sperm, said method comprising:
 a. Providing a semen sample from a subject, wherein said semen sample comprises one or more sperm cells,   b. Contacting the semen sample with the biosensor according to any one of the preceding claims,   c. Visualizing a sperm bound to the biosensor by microscopy, and   d. Selecting sperm bound to the biosensor.   
     
     
         48 . A method for manufacturing a biosensor comprising a JUNO protein according to  claim 1 , the method comprising:
 a. Providing a substrate,   b. providing the JUNO protein, and   c. immobilizing the JUNO protein on the substrate,   
       thereby manufacturing a biosensor comprising the JUNO protein. 
     
     
         49 . The method according to  claim 48 , wherein the substrate is as defined in any one of the preceding claims. 
     
     
         50 . The method according to any one of  claims 48  to  49 , wherein the method further comprises immobilizing on the substrate one or more of ZP1, ZP2, ZP3 and an anti-IZUMO antibody. 
     
     
         51 . The method according to any one  claims 48  to  50 , wherein the proteins JUNO, ZP1, ZP2, ZP3 and the anti-IZUMO antibody are as defined in any one of the preceding claims. 
     
     
         52 . A hand-held device for detection and/or quantification of sperm function, the device comprising:
 a. An inlet for a sample;   b. A biosensor comprising a JUNO protein or a fragment thereof, wherein the JUNO protein is immobilized on the biosensor, and wherein the inlet is configured to place the sample in contact with the sensor;   c. A detector configured to receive a signal from the sensor and transform it into a format readable by a user; and   d. Optionally, means for separating cellular components from the sample.   
     
     
         53 . The hand-held device according to  claim 52 , wherein the biosensor is as defined in any one of the preceding claims.