Structured substrates for optical surface profiling

Abstract

This disclosure provides methods and devices for the label-free detection of target molecules of interest. The principles of the disclosure are particularly applicable to the detection of biological molecules (e.g., DNA, RNA, and protein) using standard SiO2-based microarray technology.

1 - 15 . (canceled) 
     
     
         16 . An optical detection apparatus comprising:
 (i) a light source, wherein said light source produces an illumination beam comprising one or more wavelength(s);   (ii) a reflective substrate comprising capture molecules bound to an uppermost surface, wherein the illumination beam is directed onto the reflective substrate;   (iii) a flow cell into which the reflective substrate is incorporated to allow delivery of target molecules to the capture molecules in a fluid environment; and   (iv) a photodetector array operably linked to a central processor capable of measuring an intensity of light from the illumination beam reflected from the reflective substrate.   
     
     
         17 - 56 . (canceled) 
     
     
         57 . The optical detection apparatus of  claim 16 , wherein the light source is a laser. 
     
     
         58 . The optical detection apparatus of  claim 57 , wherein the laser is a tunable laser. 
     
     
         59 . The optical detection apparatus of  claim 16 , wherein the illumination beam comprises substantially a single wavelength. 
     
     
         60 . The optical detection apparatus of  claim 16 , wherein the reflective substrate comprises silicon dioxide (SiO 2 ). 
     
     
         61 . The optical detection apparatus of  claim 16 , wherein the reflective substrate comprises a plurality of spatially distinct binding locations, each location comprising a plurality of substantially identical capture molecules that specifically bind a single type of target molecule. 
     
     
         62 . The optical detection apparatus of  claim 16 , comprising one or more objectives to focus the illumination beam and/or imaged light. 
     
     
         63 . The optical detection apparatus of  claim 16 , wherein the photodetector array and central processor operably linked thereto are capable of recording an image comprising pixels, each measuring an intensity of the light reflected at a corresponding physical location on the substrate surface. 
     
     
         64 . The optical detection apparatus of  claim 63 , wherein the central processor is capable of determining a height change and/or mass of target molecule bound to locations on the substrate surface. 
     
     
         65 . A method for measuring target molecule binding to a microarray, the method comprising:
 (a) illuminating the microarray with an illumination beam, the illumination beam directed onto the microarray in a direction substantially perpendicular to the plane of the microarray comprising capture molecules on a surface of the microarray, wherein:   the microarray is incorporated within a flow cell, allowing for delivery of target molecules to the microarray;   (b) delivering, via the flow cell, target molecules to the microarray in a fluid environment;   (c) imaging light from the illumination beam reflected from the surface of the microarray onto a photodetector array thereby recording one or more image(s) of the microarray surface; and   (d) using the recorded image(s) of the microarray surface to assess binding of the target molecules to the capture molecules on the microarray surface.   
     
     
         66 . The method of  claim 65 , comprising performing steps (c) and (d) repeatedly to assess binding at various times during a pre-equilibrium state. 
     
     
         67 . The method of  claim 66 , comprising using the assessment of binding at various times to calculate binding kinetics between the target and capture molecules. 
     
     
         68 . The method of  claim 65 , comprising using a change in intensity between pixels of the one or more images to determine binding of the target molecules at each point of the microarray surface. 
     
     
         69 . The method of  claim 65 , comprising determining a corresponding mass of bound target molecules. 
     
     
         70 . The method of  claim 65 , comprising repeating steps (c) and (d) to assess the binding between the target molecules and the capture molecules at least 3 times. 
     
     
         71 . The method of  claim 70 , comprising assessing the binding between the target molecules and the capture molecules at least one time prior to binding equilibrium between the target molecules and the capture molecules. 
     
     
         72 . The method of  claim 65 , comprising illuminating the microarray at varying wavelengths and capturing an image at each wavelength. 
     
     
         73 . The method of  claim 72 , wherein the light source is a tunable laser. 
     
     
         74 . The method of  claim 65 , wherein the microarray surface comprises silicon dioxide (SiO 2 ). 
     
     
         75 . The method of  claim 65 , wherein the microarray surface comprises a plurality of spatially distinct binding locations, each location comprising a plurality of substantially identical capture molecules that specifically bind a single type of target molecule.