O-ring systems and methods for quantification of multiplex biomarkers in multiple samples

Abstract

Kits and methods of using O-rings having apertures are provided herein. O-rings are useful for incubating with a sample fluid potentially having one or more biomarkers, in order to detect the presence of the biomarkers. O-rings can be readily organized in a trackable manner prior to and during incubation with the sample fluid. O-rings can also be readily transferred and organized into one or more trackable arrays for detecting the presence of bound biomarkers and measuring the signaling product generated by bound detect molecule-linked enzymes present in a homogeneous solution with a spectrophotometer.

1 . A method of detecting the presence of one or more biomarkers in a fluid, comprising:
 a) providing a fluid that potentially contains one or more biomarkers;   b) providing one or more O-rings, each individually comprising an aperture;   c) coating the one or more O-rings with a coupling reagent to enhance binding to one or more capture molecules;   d) covalently coupling said one or more capture molecules with the one or more O-rings, to form one or more O-ring/capture molecule complexes, wherein each capture molecule is selected to bind to the one or more biomarkers potentially present in the fluid;   e) incubating the one or more O-ring/capture molecule complexes with the fluid to sufficiently allow binding between the one or more capture molecules and their respective biomarkers so as to form one or more O-ring/capture molecule/biomarker complexes; and   f) detecting the presence of the one or more biomarkers in the one or more O-ring/capture molecule/biomarker complexes.   
     
     
         2 . The method of  claim 1 , wherein a plurality O-rings of substantially the same size and shape are provided and arranged in a trackable order after covalently coupling with a plurality of capture molecules and prior to incubating with the fluid. 
     
     
         3 . The method of  claim 2 , wherein each O-ring is individually covalently coupled to a plurality of capture molecules such that no O-ring contains more than one particular type of capture molecule. 
     
     
         4 . The method of  claim 2 , wherein the trackable order comprises a column of stacked O-ring/capture molecule complexes. 
     
     
         5 . The method of  claim 4 , wherein the column of stacked O-ring/capture molecule complexes are positioned inside a sealable container via a probe that traverses through the apertures of the O-rings, such that the positional order of the O-rings is maintained during incubation with the fluid. 
     
     
         6 . The method of  claim 4 , wherein the column of stacked O-ring/capture molecule complexes are positioned on a pin in a pin plate, such that the pin traverses through the apertures of the O-rings and maintains the positional order of the O-rings during incubation with the fluid. 
     
     
         7 . The method of  claim 4 , wherein after incubation with the fluid, the plurality of O-ring/capture molecule/biomarker complexes are transferred in a trackable order and formed into an array on a microplate, wherein only one O-ring/capture molecule/biomarker complex is placed in a single well on the microplate. 
     
     
         8 . A method of detecting the presence of one or more biomarkers in a fluid, comprising:
 a) providing a fluid that potentially contains one or more biomarkers;   b) providing one or more O-rings, each individually comprising an aperture;   c) coating said one or more O-rings with a coupling reagent to enhance binding to said one or more biomarkers;   d) incubating the one or more O-rings with the fluid to sufficiently allow binding with the one or more biomarkers so as to form one or more O-ring/biomarker complexes; and   e) detecting the presence of the one or more biomarkers in the one or more O-ring/biomarker complexes.   
     
     
         9 . The method of  claim 8 , wherein a plurality O-rings of substantially the same size and shape are provided and arranged in a trackable order prior to incubating with the fluid to bind to a plurality of biomarkers. 
     
     
         10 . The method of  claim 9 , wherein prior to incubation, the plurality of O-rings are coated with a coupling reagent to enhance binding to the plurality of biomarkers potentially present in the sample fluid. 
     
     
         11 . The method of  claim 9 , wherein the plurality of O-rings are arranged in a trackable order on a pin plate having a plurality of pins configured to traverse through the apertures of the plurality of O-rings. 
     
     
         12 . The method of  claim 9 , wherein after incubation with the fluid, the plurality of O-ring/biomarker complexes are transferred in a trackable order and formed into an array on a welled microplate, wherein only one O-ring/biomarker complex is placed in a single well on the microplate. 
     
     
         13 . A method of measuring the concentration of a signaling product comprising:
 a) providing a plurality of biomarker complexes, that individually comprise an O-ring and a plurality of biomarkers;   b) arranging said biomarker complexes individually into single wells on a microplate;   c) providing a plurality of detect molecule-linked enzyme complexes that are selected to bind to the plurality of biomarkers and are capable of producing a measurable signaling product when contacted with a substrate specific for said linked enzyme in a reactive solution;   d) adding said detect molecule-linked enzyme complexes to the wells on the microplate such that the detect molecules bind to the biomarkers in the biomarker complexes;   e) adding said substrate in the reactive solution to the wells on the microplates such that it comes into contact with bound detect molecule-linked enzyme complexes; and   f) measuring the signaling product produced by the bound detect molecule-linked enzyme complexes and the reactive solution.