Binding interactions in dipstick assays

Abstract

Use of dipsticks to test for the presence of target nucleic acid in a sample solution is described. The dipsticks comprise a contact end for contacting the sample solution and a capture zone, remote from the contact end, to which a capture probe is immobilised. The capture probe is capable of hybridising to the target nucleic acid. The sample solution is contacted with the contact end of the dipstick and travels by capillary action to the capture zone. If target nucleic acid is present in the sample solution it is captured and can be detected at the capture zone. The capture probe is immobilised to the capture zone by a spacer. Use of the spacer increases the stability of the interaction between the capture probe and the target nucleic acid and thus improves the sensitivity of target nucleic acid detection. Detection probes with spacers are also described.

1 .- 66 . (canceled) 
     
     
         67 . A method for testing for the presence of a target nucleic acid in a sample solution, which comprises:
 providing a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution, and a capture probe that is immobilized at the capture zone of the chromatographic strip remote from the contact end, wherein the capture probe comprises an oligonucleotide capable of hybridizing to the target nucleic acid or to a hook capture probe bound to the target nucleic acid, and wherein the capture probe is linked to a capture probe spacer and the capture probe spacer is linked to the capture zone, thereby immobilizing the capture probe at the capture zone and spacing the capture probe from the capture zone; and   contacting the contact end of the dipstick with the sample solution to cause sample solution to move up the dipstick by capillary action to the capture zone of the dipstick, thereby allowing target nucleic acid in the sample solution to be captured and detected at the capture zone, wherein:
 i) the capture probe spacer is linked to one end of the capture probe, and the end of the capture probe that is not linked to the capture probe spacer is coupled to one or more nucleotides, which do not hybridize to the target nucleic acid when the capture probe is hybridized to the target nucleic acid or do not hybridize to the hook capture probe when the capture probe is hybridized to the hook capture probe; 
 ii) the capture probe spacer is linked to a part of the capture probe between the ends of the capture probe, and one or both ends of the capture probe are coupled to one or more nucleotides, which do not hybridize to the target nucleic acid when the capture probe is hybridized to the target nucleic acid or do not hybridize to the hook capture probe when the capture probe is hybridized to the hook capture probe; or 
 iii) the capture probe spacer comprises a protein adsorbed directly to the capture zone, wherein the capture probe is covalently linked to the protein by a linker. 
   
     
     
         68 . The method of  claim 67 , which further comprises contacting the sample solution with a detection probe that comprises an oligonucleotide capable of hybridizing to the target nucleic acid, and detecting for the detection probe at the capture zone. 
     
     
         69 . The method of  claim 68 , wherein the detection probe is incubated with the sample solution under conditions for hybridization of the detection probe to the target nucleic acid prior to contacting the sample solution with the contact end of the dipstick, thereby allowing target nucleic acid and the detection probe to move with the sample solution to the capture zone once the contact end of the dipstick is contacted with the sample solution. 
     
     
         70 . The method of  claim 68 , wherein the detection probe is releasably immobilized at a probe zone located between the contact end and the capture zone of the chromatographic strip, such that sample solution passing the probe zone mobilizes the detection probe allowing the detection probe to hybridize to target nucleic acid in the sample solution and move with the target nucleic acid to the capture zone. 
     
     
         71 . The method of  claim 68 , wherein the detection probe is coupled to a label, which allows direct detection of the target nucleic acid when the detection probe is hybridized to the target nucleic acid, or to a detection ligand, which can be bound by a detection ligand binding moiety to allow indirect detection of the target nucleic acid when the detection probe is hybridized to the target nucleic acid. 
     
     
         72 . The method of  claim 71 , wherein the label or detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe, thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe. 
     
     
         73 . The method of  claim 71 , wherein the label is a visible label that can be detected by direct visual observation of the dipstick when the detection probe has been captured at the capture zone. 
     
     
         74 . The method of  claim 73 , wherein the label is selected from the group consisting of a metal sol, a textile dye, and coloured particles.