Nucleic acid hybridization assay

Abstract

Provided herein is a method and device for performing a homogeneous nucleic acid detection assay. The device can contain a pair of plates where one of the plates comprises (i) surface amplification surface; and (ii) target-specific nucleic acid probes that are immobilized on said amplification surface and that specifically binds to a part of the target nucleic acid; and the second plate comprises a sample contact area comprising a reagent storage site that comprises target-specific nucleic acid detection agents that specifically binds to another part of the target nucleic acid. In some embodiments, the device can be read without a washing unbound label from the surface of the device.

We claim: 
     
         1 . A device for performing a homogeneous nucleic acid detection assay, comprising a first plate, a second plate, a hinge, spacers, and an imager, wherein
 (i) the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration,   (ii) the first plate and the second plate each comprises a sample contact area for contacting a sample that contains or is suspected of containing a target analyte comprising one or more target nucleic acids;   (iii) the sample contact area of the first plate comprises a binding site comprising a capture agent or target-specific nucleic acid probe that is immobilized and that specifically binds to a part of the target nucleic acid;   (iv) one of the sample contact areas comprises a reagent storage site comprising a labeled detection agent or target-specific nucleic acid detection agent that binds to another part of the target nucleic acid,   (v) the spacers have a pillar shape, a predetermined substantially uniform height, and a predetermined inter-spacer distance, wherein at least one of the spacers is inside the sample contact area of one or both of the plates;   (vi) one or both of the plates is flexible, and the thickness of the flexible plate times the Young's modulus of the flexible plate is in the range 60 to 750 GPa-μm;   (vii) the fourth power of the inter-spacer-distance (ISD) divided by the thickness of the flexible plate (h) and the Young's modulus (E) of the flexible plate, ISD 4 /(hE), is equal to or less than 5×10 6  um 3 /GPa;   (viii) the plates are coupled by a hinge that is configured to transition the plates between an open configuration and a closed configuration; and   (ix) the imager detects a label of the labeled detection agent.   
     
     
         2 . The device of  claim 1 , wherein the sample contact area of the first plate further comprises a proximity-dependent signal amplification layer, wherein the capture agent is immobilized on the surface of the proximity-dependent signal amplification layer. 
     
     
         3 . The device of  claim 1  further comprising a thermal cycler. 
     
     
         4 . The device of  claim 1 , wherein, in the closed configuration, the majority of the target analyte binds to the target-specific nucleic acid probe in 300 seconds or less. 
     
     
         5 . The device of  claim 1 , wherein, in the closed configuration, the majority of the target analyte binds to the target-specific nucleic acid probe in 60 seconds or less. 
     
     
         6 . The device of  claim 1 , wherein the target nucleic acid is a DNA or RNA comprising one or more selected from the group consisting of genomic DNA, cfDNA, cDNA ctDNA, mRNA, and miRNA. 
     
     
         7 . The device of  claim 1 , wherein the thickness of the sample in the closed configuration, the concentration of labels dissolved in the sample in the closed configuration, and the amplification factor of the surface amplification layer are configured such that any of the labels that are bound directly or indirectly to the probe are visible in the closed configuration without washing away of the unbound labels. 
     
     
         8 . The device of  claim 2 , wherein the labeled detection agent that is bound indirectly to the target-specific nucleic acid probe via a target nucleic acid is visible in less than 60 seconds. 
     
     
         9 . The device of  claim 2 , wherein the storage site is approximately above the binding site on the first plate in the closed configuration. 
     
     
         10 . The device of  claim 1 , wherein the signal amplification layer comprises a disk-coupled dots-on-pillar antenna-array (D2PA). 
     
     
         11 . The device of  claim 1 , wherein the signal amplification layer comprises a layer of metallic material. 
     
     
         12 . The device of  claim 2 , wherein the proximity-dependent signal amplification layer comprises a continuous metallic film comprising a material selected from the group consisting of gold, silver, copper, aluminum, alloys thereof, and combinations thereof. 
     
     
         13 . The device of  claim 1 , wherein the sample contact area of the first plate further comprises a metallic material layer that either locally enhances or acts as a reflector, or both, to enhance an optical signal. 
     
     
         14 . The device of  claim 2 , wherein the proximity-dependent signal amplification layer comprises a layer of metallic material and a dielectric material on top of the metallic material layer, wherein the target-specific nucleic acid probe is on the dielectric material. 
     
     
         15 . The device of  claim 1 , wherein the sample contact area of the first plate further comprises a metallic material layer that is a uniform metallic layer, nanostructured metallic layer, or a combination thereof. 
     
     
         16 . The device of  claim 2 , wherein the sample contact area of the first plate further comprises a site that comprises the proximity-dependent signal amplification layer but not the target-specific nucleic acid probe. 
     
     
         17 . The device of  claim 1 , wherein the spacers are fixed on one of the plates, wherein the spacers regulate the spacing between the first plate and the second plate in the closed configuration.