Biosensors and methods for detection of lysophosphatidic acid for signaling of ovarian cancer

Abstract

The present disclosure relates to biosensors, kits and methods for detecting and/or quantifying lysophosphatidic acid (LPA) in a liquid sample such as a serum sample from a subject. The present disclosure also relates to linker compounds that are useful, for example, in the biosensors, kits and methods of the present disclosure and to methods for preparing a biosensor for detecting and/or quantifying lysophosphatidic acid (LPA) in a liquid sample.

1 . A biosensor for detecting and/or quantifying lysophosphatidic acid (LPA) in a liquid sample, the biosensor comprising:
 (a) a solid support; and   (b) a dual protein system comprising gelsolin and dye-modified actin,   
       wherein
 the actin is bound to an actin-binding site of the gelsolin; and 
 the gelsolin is bound to the solid support. 
 
     
     
         2 . (canceled) 
     
     
         3 . The biosensor of  claim 1 , wherein the gelsolin is bound to the solid support via a linker having the following structure: 
       
         
           
           
               
               
           
         
       
       wherein
 R S  is a functional group covalently bonded to the solid support; 
 R G  is a functional group covalently bonded to the gelsolin; and 
 L is C 2-20 alkylene, wherein one or more available carbon atoms in the alkylene are optionally replaced by —O— or —S—. 
 
     
     
         4 . The biosensor of  claim 3 , wherein L is —(CH 2 ) 2 —O—(CH 2 ) 2 —. 
     
     
         5 . The biosensor of  claim 3 , wherein the gelsolin comprises a histidine tag and R G  is bonded to the histidine tag of the gelsolin. 
     
     
         6 . (canceled) 
     
     
         7 . The biosensor of  claim 5 , wherein R G  comprises a Ni-NTA moiety that is bonded to the histidine tag of the gelsolin, the Ni-NTA moiety having the following structure that is coordinated to a Ni 2+  ion: 
       
         
           
           
               
               
           
         
       
       wherein
 Z is C 2-14 alkylene or —(CH 2 CH 2 —O—) m CH 2 CH 2 ; and 
 m is 1, 2 or 3. 
 
     
     
         8 . The biosensor of  claim 3 , wherein R S  comprises a silicon atom and the linker is bound to the solid support via a silicon-oxygen bond. 
     
     
         9 . (canceled) 
     
     
         10 . The biosensor of  claim 9 , wherein the linker is comprised in a self-assembled monolayer (SAM) that is on a surface of the solid support and the SAM comprises a siloxane network comprising linkers of the following structure: 
       
         
           
           
               
               
           
         
       
       wherein
    represents the surface of the solid support; and 
 each   represents an oxygen atom in the siloxane network. 
 
     
     
         11 . (canceled) 
     
     
         12 . The biosensor of  claim 10 , wherein the SAM is a mixed SAM and the siloxane network further comprises diluents of the following structure: 
       
         
           
           
               
               
           
         
       
       wherein
 R C  is selected from: 
 C 1-24 alkyl, wherein one or more available carbon atoms in the alkyl are optionally replaced by —O— or —S—; 
 (ii) C 1-24 alkylene-O—C(O)CF 3 , wherein one or more available carbon atoms in the alkylene are optionally replaced by —O— or —S—; and 
 (iii) C 1-24 alkylene-OH, wherein one or more available carbon atoms in the alkylene are optionally replaced by —O— or —S—; 
    represents the surface of the solid support; and 
 each   represents an oxygen atom in the siloxane network. 
 
     
     
         13 . The biosensor of  claim 12 , wherein R C  is hexyl, octadecyl or 3-(2-(2,2,2-trifluoroacetoxy)ethoxy)propyl, optionally wherein R C  is hexyl. 
     
     
         14 . The biosensor of  claim 1 , wherein the dye-modified actin is rhodamine-modified actin, optionally wherein the dye-modified actin is N-hydroxysuccinimide(NHS)-rhodamine-modified actin. 
     
     
         15 . (canceled) 
     
     
         16 . The biosensor of  claim 1 , wherein
 (a) the solid support comprises plastic, glass, metal, metal oxide or crystal, and the solid support is in the form of a testing strip, a well in a microwell plate or a microcentrifuge tube, optionally wherein the solid support is in the form of a microcentrifuge tube; or   (b) the solid support comprises or consists essentially of silica gel or comprises magnetic nanoparticles, and the silica gel or magnetic nanoparticles are housed in a column, vial or tube.   
     
     
         17 . (canceled) 
     
     
         18 . A kit for detecting and/or quantifying lysophosphatidic acid (LPA) in a liquid sample, the kit comprising:
 (a) a biosensor according to  claim 1 ; and   (b) optionally instructions for use.   
     
     
         19 . The kit of  claim 18 , wherein the solid support of the biosensor is in the form of a testing strip, a well in a microwell plate or a microcentrifuge tube or wherein the solid support consists essentially of silica gel or magnetic nanoparticles housed in a column, vial or tube, and wherein:
 (a) when the solid support is in the form of a testing strip, the kit further comprises one control testing strip per each 2 or 3 biosensors in the kit, the control testing strip made of the same solid support as the biosensors but not the dual protein system;   (b) when the solid support is in the form of a well in a microwell plate, the microwell plate comprises one control well per each 2 or 3 biosensors in the microwell plate, the control well made of the same solid support as the biosensors but not the dual protein system;   (c) when the solid support is in the form of a microcentrifuge tube, the kit further comprises one control tube per each 2 or 3 biosensors in the kit, the control tube made of the same solid support as the biosensors but not the dual protein system; and   (d) when the solid support consists essentially of silica gel or magnetic nanoparticles housed in a column, vial or tube, the kit further comprises one control column, vial or tube per each 2 or 3 biosensors in the kit, the control column, vial or tube containing the same solid support as the biosensors but not the dual protein system.   
     
     
         20 - 21 . (canceled) 
     
     
         22 . A method for detecting lysophosphatidic acid (LPA) in a liquid sample, the method comprising:
 exposing the sample to a biosensor according to  claim 1  under conditions to bind the LPA to the gelsolin and thereby release the dye-modified actin into the liquid; and   analyzing the sample after exposure to the biosensor to determine if LPA was present in the liquid sample,   
       wherein the step of analyzing comprises spectroscopically measuring a signal associated with the dye of the dye-modified actin. 
     
     
         23 . The method of  claim 22 , wherein the method quantifies the amount of LPA in a liquid sample, and comprises:
 exposing a first portion of the sample to a first biosensor;   analyzing the first portion of the sample after exposure to the first biosensor to obtain a first signal value;   adding a known amount of LPA to a second portion of the sample;   exposing the second portion of the sample to a second biosensor;   analyzing the second portion of the sample after exposure to the second biosensor to obtain a second signal value;   exposing a third portion of the sample to a control made of the same solid support as the biosensors but not the dual protein system;   analyzing the third portion of the sample after exposure to the control to obtain a control signal value; and   calculating the ratio of the first signal value minus the control signal value to the second signal value minus the control signal value to determine the concentration of LPA that was present in the sample,   
       wherein the first biosensor and the second biosensor are each made of the same solid support, and dual protein system; and wherein the steps of analyzing comprise spectroscopically measuring a signal associated with the dye of the dye-modified actin. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The method of  claim 22 , wherein the method quantifies the amount of LPA in a liquid sample, and comprises:
 exposing a first portion of the sample to a first biosensor;   analyzing the first portion of the sample after exposure to the first biosensor to obtain a first signal value;   adding different known amounts of LPA to each of a plurality of additional portions of the sample;   exposing each of the plurality of additional portions of the sample to a respective plurality of additional biosensors;   analyzing each of the plurality of additional portions of the sample after exposure to the respective plurality of additional biosensors to obtain a plurality of additional signal values;   plotting the signal value obtained for each portion of the sample against the corresponding concentration of LPA added to the portion of the sample; and   obtaining the concentration of LPA that was present in the sample from the y-intercept of the line of best fit for the plot,   
       wherein the steps of analyzing comprise spectroscopically measuring a signal associated with the dye of the dye-modified actin. 
     
     
         27 - 30 . (canceled) 
     
     
         31 . The method of  claim 22 , wherein the actin is modified with a dye suitable for detection by UV-visible absorbance spectroscopy and the step(s) of analyzing comprise(s) measuring, by ultraviolet-visible absorbance spectroscopy, a signal associated with said dye or wherein the actin is modified with a dye suitable for detection by fluorescence spectroscopy and the step(s) of analyzing comprise(s) measuring, by fluorescence spectroscopy, a signal associated with said dye. 
     
     
         32 - 34 . (canceled) 
     
     
         35 . The method of  claim 22 , wherein the liquid sample is a serum sample from a subject, optionally wherein the subject is a woman over the age of fifty and/or wherein the woman has a family history of ovarian cancer, wherein the concentration of LPA in the serum sample is determined to be above about 1.5 μM and the method further comprises determining whether or not the subject has ovarian cancer by a method which comprises using an imaging technique, optionally wherein the imaging technique comprises transvaginal ultrasound (TVU). 
     
     
         36 . (canceled) 
     
     
         37 . A compound of Formula I: 
       
         
           
           
               
               
           
         
       
       wherein
 R 1 , R 2  and R 3  are each independently a hydrolysable group that does not react with an acid chloride; 
 j is 0, 1, 2 or 3; 
 k is 2, 3, 4 or 5; and 
 n is 1, 2, 3, 4 or 5. 
 
     
     
         38 - 40 . (canceled) 
     
     
         41 . A method for preparing a biosensor for detecting and/or quantifying lysophosphatidic acid (LPA) in a liquid sample, the method comprising:
 (a) reacting a solid support having hydroxyl groups on its surface with a compound of Formula I according to  claim 37  under conditions to hydrolyze the hydrolysable groups and thereby form a self-assembled monolayer (SAM) comprising a siloxane network bound to the surface of the solid support;   (b) reacting the product obtained from (a) with an NTA moiety of the Formula II under conditions so that the acid chloride from the compound of Formula I and the amine from the compound of Formula II react to form an amide linkage:   
       
         
           
           
               
               
           
         
         
           wherein
 Z is C 2-14 alkylene or —(CH 2 CH 2 —O—) m CH 2 CH 2 ; and 
 m is 1, 2 or 3; 
 
         
         (c) reacting the product obtained from (b) with a nickel (II) compound under conditions to form a complex between the Ni 2+  and the carboxylate groups from the compound of Formula II; 
         (d) reacting the product obtained from (c) with his-tagged gelsolin under conditions to bind the Ni-NTA moiety to the his-tag; and 
         (e) reacting the product obtained from (d) with dye-modified actin under conditions to bind the actin to the gelsolin. 
       
     
     
         42 - 48 . (canceled)