Sensor constructs and detection methods

Abstract

Sensor constructs for detecting the presence of an analyte in a sample comprising a molecular scaffold, a pair of labels which interact via Förster resonance energy transfer, and at least one molecular recognition domain for binding the analyte. A changed optical signal is produced when the analyte is bound by the molecular recognition domain.

1 . A sensor construct for determining the presence of an analyte in a sample, comprising in a single molecule: 
 (a) a molecular scaffold;    (b) a first label comprising an RET donor bound to the molecular scaffold;    (c) a second label comprising an RET acceptor bound to the molecular scaffold, wherein the RET acceptor is separated from the RET donor by a distance that allows a FRET interaction to occur between the RET donor and the RET acceptor; and    (d) a molecular recognition domain which specifically binds the analyte without producing a conformational change in the molecular scaffold, wherein the molecular recognition domain is bound to a component of the sensor construct selected from the group consisting of the molecular scaffold, the first label, and the second label;    wherein the first label, the second label, and the molecular recognition domain remain bound to the molecular scaffold as a single molecule when the molecular recognition domain binds the analyte.    
   
   
       2 . The construct of  claim 1 , wherein the RET donor is selected from the group consisting of a fluorescent protein, a luminescent protein, a non-protein fluorophore, a non-protein chemiluminescent compound, and a fluorescent nanocrystal.  
   
   
       3 . The construct of  claim 2 , wherein the RET donor is a fluorescent protein selected from the group consisting of GFP, EGFP, RFP, BFP, CFP, ECFP, YFP, EYFP, and derivatives of one of the foregoing.  
   
   
       4 . The construct of  claim 1 , wherein the RET acceptor is selected from the group consisting of a fluorescent protein, a non-protein fluorophore, a fluorescent nanocrystal, and a quencher.  
   
   
       5 . The construct of  claim 4 , wherein the RET acceptor is a fluorescent protein selected from the group consisting of GFP, EGFP, RFP, BFP, CFP, ECFP, YFP, EYFP, and derivatives of one of the foregoing.  
   
   
       6 . The construct of  claim 1 , further comprising a plurality of RET donors and RET acceptors.  
   
   
       7 . The construct of  claim 1 , wherein the molecular scaffold is bound to the RET donor with a flexible linker.  
   
   
       8 . The construct of  claim 1 , wherein the molecular scaffold is bound to the RET donor with a rigid linker.  
   
   
       9 . The construct of  claim 1 , wherein the molecular recognition domain comprises a polypeptide.  
   
   
       10 . The construct of  claim 9  wherein a further component of the sensor construct comprises a polypeptide, the further component being selected from the group consisting of the molecular scaffold, the first label, and the second label, and wherein the molecular recognition domain is recombinantly expressed within the further component of the sensor construct, the molecular recognition domain and further component thereby comprising a recombinantly expressed fusion protein.  
   
   
       11 . The construct of  claim 1 , wherein the molecular recognition domain comprises a chelated metal.  
   
   
       12 . The construct of  claim 1 , wherein the molecular recognition domain comprises an oligonucleotide.  
   
   
       13 . The construct of  claim 1 , wherein the molecular scaffold of the sensor construct comprises a polypeptide.  
   
   
       14 . The construct of  claim 13 , wherein the sensor construct comprises an antibody, the molecular recognition domain comprises a CDR region of the antibody, and the molecular scaffold comprises a remaining portion of the antibody.  
   
   
       15 . The construct of  claim 14 , wherein the first label comprises the E domain of Protein A and the second label comprises a binding domain of Protein L.  
   
   
       16 . The construct of  claim 13 , wherein the sensor construct comprises an antibody fragment, the molecular recognition domain comprises a CDR region of the antibody fragment, and the molecular scaffold comprises a remaining portion of the antibody fragment.  
   
   
       17 . The construct of  claim 16 , wherein the antibody fragment is a single-chain Fv.  
   
   
       18 . The construct of  claim 16 , wherein the CDR region of the antibody fragment is a portion of a CDR region of an antibody.  
   
   
       19 . The construct of  claim 16 , wherein the antibody fragment comprises an Fv portion of an antibody.  
   
   
       20 . The construct of  claim 13 , wherein the polypeptide is derived from a periplasmic binding protein.  
   
   
       21 . The construct of  claim 13 , wherein the first label is a fluorescent protein produced together with the molecular scaffold as a single polypeptide molecule.  
   
   
       22 . The construct of  claim 21 , wherein the second label is a fluorescent protein produced together with the molecular scaffold as a single polypeptide molecule.  
   
   
       23 . The construct of  claim 13 , wherein the molecular recognition domain is chemically coupled to a component of the sensor construct selected from the group consisting of the molecular scaffold, the first label, and the second label.  
   
   
       24 . The construct of  claim 13 , wherein the molecular scaffold comprises a pair of heterodimeric coiled coil polypeptides.  
   
   
       25 . The construct of  claim 24 , wherein the pair of heterodimeric coiled coil polypeptides is selected from the group consisting of a WinZip-A2/WinZip-B1 coil pair and an E/K coil pair.  
   
   
       26 . The construct of  claim 1 , wherein the first label comprises a plurality of RET donors.  
   
   
       27 . The construct of  claim 1 , wherein the second label comprises a plurality of RET acceptors.  
   
   
       28 . The construct of  claim 1 , wherein the molecular recognition domain of the sensor construct is bound to the molecular scaffold of the sensor construct.  
   
   
       29 . The construct of  claim 1 , wherein the molecular recognition domain of the sensor construct is bound to a component of the sensor construct selected from the group consisting of the first label and the second label.  
   
   
       30 . The construct of  claim 1 , wherein the sensor construct is derived from a maltose binding protein.