Affinity based system for detecting particulates in a fluid

Abstract

This invention provides methods and apparatus for detecting and quantifying particulate matter suspended in a fluid. Specifically, the invention provides an integrated, affinity-binding based, analytical system comprising a platform for performing an affinity-binding based assay for specifically binding particulates including microbial cells, and a detection means for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. Methods for using the analytical systems of the invention are also provided.

1 . An apparatus for detecting a particulate in a fluid, the apparatus comprising 
 a platform comprising a substrate having a surface, wherein said surface defines a detection chamber comprising an area that is coated with a specific binding reagent that specifically binds to the particulate to be detected;    a fluid sample input means in fluid communication with the detection chamber that is coated with the specific binding reagent;    a wash buffer reservoir containing a wash buffer in fluid communication with the detection chamber that is coated with the specific binding reagent;    a fluid waste receptacle in fluid communication with the detection chamber that is coated with the specific binding reagent;    wherein an amount of a fluid sample comprising a particulate is moved from the fluid sample input means to the detection chamber and incubated thereon for a time sufficient to result in specific binding between the particulate in the fluid sample and the specific binding reagent in the detection chamber; and    wherein the fluid sample is replaced with the wash buffer and displaced into the fluid waste receptacle; and    wherein the wash buffer is further displaced into the fluid waste receptacle; and    wherein a particulate specifically bound to the detection chamber is specifically bound to the specific binding reagent and is detected thereupon.    
     
     
         2 . An apparatus according to  claim 1 , wherein the surface of the detection chamber is a porous surface.  
     
     
         3 . The apparatus of  claim 1 , wherein the sample input means comprises an area on said first surface, wherein a metered amount of an unmetered fluid sample applied to the surface is retained in the fluid sample input means, the surface further comprising an overflow reservoir in fluid communication with the fluid sample input means, wherein fluid comprising the unmetered fluid sample in excess of the metered amount of the fluid sample that is retained in the fluid sample input means is retained in the overflow reservoir on the surface of the platform.  
     
     
         4 . An apparatus according to  claim 1 , wherein the particulate is a cell.  
     
     
         5 . An apparatus according to  claim 1  wherein each of the components of the apparatus in fluid communication with one another are connected by a plurality of microchannels that enable fluid communication between the components of the apparatus.  
     
     
         6 . An apparatus according to  claim 4 , further comprising: 
 a label reservoir comprising an amount of a detectable labeling moiety that is specific for the particulate to be detected and is in fluid communication with the detection chamber, wherein the wash buffer is displaced from the detection chamber by the solution comprising the detectable labeling moiety.    
     
     
         7 . An apparatus according to  claim 6 , wherein the detectable labeling moiety is a histochemical stain.  
     
     
         8 . An apparatus according to  claim 6  wherein the detectable labeling moiety is a immunochemical reagent.  
     
     
         9 . An apparatus according to  claim 8  wherein the immunochemical reagent is a detectably labeled antibody.  
     
     
         10 . An apparatus according to  claim 9  wherein the detectable label is a fluorescent label.  
     
     
         11 . An apparatus according to  claim 9  wherein the detectable label is produced by an enzymatic moiety, wherein the apparatus further comprises a substrate for the enzymatic moiety wherein the substrate is converted to a detectable label by enzymatic action.  
     
     
         12 . An apparatus according to  claim 11  wherein the substrate is a component of the wash buffer.  
     
     
         13 . An apparatus according to  claim 1  wherein the wash buffer further comprises a detectable label.  
     
     
         14 . An apparatus according to  claim 11  wherein the substrate comprises a component of the reservoir containing the detectable labeling moiety.  
     
     
         15 . An apparatus according to  claim 1  wherein a portion of the detection chamber is optically transparent.  
     
     
         16 . An apparatus according to  claim 3  wherein the metered amount of the fluid sample is from about 10 μL to about 500 μL.  
     
     
         17 . An apparatus according to  claim 1  wherein the detection chamber comprises a volume from about 5 μL to about 1000 μL.  
     
     
         18 . An apparatus according to  claim 1  wherein the specific binding reagent is an antibody, a ligand, a lectin, an integrin, an antigen, a receptor, a carbohydrate or an adhesion molecule.  
     
     
         19 . An apparatus according to  claim 1  further comprising a device, wherein the device has a surface or cavity that accommodates the platform, and a light source positioned to illuminate the platform.  
     
     
         20 . An apparatus according to  claim 19  further comprising a photodetector, wherein the photodetector is positioned to detect light from the light source transmitted through or reflected from the detection chamber on the platform surface.  
     
     
         21 . An apparatus according to  claim 20  wherein a portion of the detection chamber is optically transparent, and the photodetector and the light source are positioned so that light from the light source illuminates the detection chamber and is detected by the photodetector through the optically transparent portion of the platform.  
     
     
         22 . An apparatus according to  claim 20  wherein a portion of the detection chamber comprises a reflective surface, and the photodetector and the light source are positioned so that light from the light source illuminates the detection chamber is reflected therefrom and the reflected light is detected by the photodetector.  
     
     
         23 . An apparatus according to  claim 1  wherein the detection chamber that is coated with a specific binding reagent is further treated with a blocking agent that prevents non-specific binding to the surface of the platform.  
     
     
         24 . An method for detecting a particulate in a fluid using the apparatus of  claim 1 , the method comprising the steps of: 
 applying an amount of a sample to the fluid sample input means of the apparatus;    moving the fluid sample from the fluid sample input means to the detection chamber;    incubating the fluid sample in the detection chamber for a time sufficient to result in specific binding between the particulate in the fluid sample and the specific binding reagent in the detection chamber;    replacing the fluid of the fluid sample in the detection chamber with the wash buffer;    displacing the wash buffer into the fluid waste receptacle; and    detecting the particulate specifically bound to the detection chamber.