Molecular biosensors for use in competition assays

1. A molecular biosensor, the molecular biosensor comprising two epitope binding agent constructs, which together have formula (VI):

R 47 —R 48 —R 49 —R 50 ; and

R 51 —R 52 —R 53 —R 54 ;

wherein:

R 47 is an epitope-binding agent that binds to a first epitope on a target molecule and is selected from the group consisting of a peptide, a small molecule, and a protein;

R 48 is a flexible linker attaching R 47 to R 49 ;

R 49 and R 53 are a pair of complementary nucleotide sequences having a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. and at a salt concentration from about 1 mM to about 100 mM;

R 50 and R 54 together comprise a detection means such that when R 49 and R 53 associate a detectable signal is produced;

R 51 is an epitope binding agent selected from the group consisting of an antibody, an antibody fragment, peptide, protein, and an aptamer that binds to R 47 such that in the absence of a target molecule:

(i) R 51 binds to R 47 ,

(ii) R 49 and R 53 associate, and

(iii) R 50 and R 54 produce a detectable signal; and

R 52 is a flexible linker attaching R 51 to R 53 .

2. The molecular biosensor of claim 1 , wherein R 51 is an antibody or antibody fragment selected from the group consisting of polyclonal antibodies, ascites, Fab fragments, Fab′ fragments, monoclonal antibodies and humanized antibodies.

3. The molecular biosensor of claim 1 , wherein R 48 and R 52 are from about 50 to about 250 angstroms in length and are selected independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.

4. The molecular biosensor of claim 1 , wherein R 49 and R 53 are from about 2 to about 20 nucleotides in length.

5. The molecular biosensor of claim 1 , wherein R 50 and R 54 are independently selected from the group consisting of fluorescence resonance electron transfer (FRET), lanthamide resonance electron transfer (LRET), fluorescence cross-correlation spectroscopy, fluorescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescence resonance energy transfer, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence resonance energy transfer, excimer formation, colorimetric substrates detection, phosphorescence, electro-chemical changes, and redox potential changes.

6. A molecular biosensor, the molecular biosensor comprising two epitope binding agent constructs, which together have formula (VI):

R 47 —R 48 —R 49 —R 50 ; and

R51—R52—R53—R54;

wherein:

R 47 is an antibody or antibody fragment epitope-binding agent that binds to a first epitope on a target molecule;

R 48 is a flexible linker attaching R 47 to R 49 ;

R 49 and R 53 are a pair of complementary nucleotide sequences having a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. and at a salt concentration from about 1 mM to about 100 mM;

R 50 and R 54 together comprise a detection means such that when R 49 and R 53 associate a detectable signal is produced;

R 51 is a peptide or protein epitope binding agent that binds to R 47 such that in the absence of a target molecule:

(i) R 51 binds to R 47 ,

(ii) R 49 and R 53 associate, and

(iii) R 50 and R 54 produce a detectable signal, and;

R 52 is a flexible linker attaching R 51 to R 53 .

7. The molecular biosensor of claim 6 , wherein R 51 is an antibody or antibody fragment selected from the group consisting of polyclonal antibodies, ascites, Fab fragments, Fab′ fragments, monoclonal antibodies and humanized antibodies.

8. The molecular biosensor of claim 7 , wherein R 48 and R 52 are from about 50 to about 250 angstroms in length and are selected independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.

9. The molecular biosensor of claim 8 , wherein R 49 and R 53 are from about 2 to about 20 nucleotides in length.

10. The molecular biosensor of claim 9 , wherein R 50 and R 54 are independently selected from the group consisting of fluorescence resonance electron transfer (FRET), lanthamide resonance electron transfer (LRET), fluorescence cross-correlation spectroscopy, fluorescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescence resonance energy transfer, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence resonance energy transfer, excimer formation, colorimetric substrates detection, phosphorescence, electro-chemical changes, and redox potential changes.

11. A method for determining the presence of a target molecule in a sample, the method comprising:

a) measuring the signal of a molecular biosensor without the target molecule being present, the molecular biosensor comprising two epitope binding agent constructs, which together have formula (VI)

R 47 —R 48 —R 49 —R 50 ; and

R 51 —R 52 —R 53 —R 54 ;

wherein:

R 47 is an epitope-binding agent that binds to a first epitope on a target molecule and is selected from the group consisting of a peptide, a small molecule, and a protein;

R 48 is a flexible linker attaching R 47 to R 49 ;

R 49 and R 53 are a pair of complementary nucleotide sequences having a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. and at a salt concentration from about 1 mM to about 100 mM;

R 50 and R 54 together comprise a detection means such that when R 49 and R 53 associate a detectable signal is produced;

R 51 is an epitope binding agent selected from the croup consisting of an antibody, an antibody fragment, and an aptamer that binds to R 47 such that in the absence of a target molecule:

(i) R 51 binds to R 47 ,

(ii) R 49 and R 53 associate, and

(iii) R 50 and R 54 produce a detectable signal; and

R 52 is a flexible linker attaching R 51 to R 53 .

b) combining the molecular biosensor with the sample; and

c) measuring the signal of the biosensor, wherein a decrease in signal indicates the presence of a target molecule.

12. The method of claim 10 , wherein R 51 is an antibody or antibody fragment selected from the group consisting of polyclonal antibodies, ascites, Fab fragments, Fab′ fragments, monoclonal antibodies and humanized antibodies.

13. The method of claim 12 , wherein R 48 and R 52 are from about 50 to about 250 angstroms in length and are selected independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.

14. The method of claim 13 , wherein R 49 and R 53 are from about 2 to about 20 nucleotides in length.

15. The method of claim 14 , wherein R 50 and R 54 are independently selected from the group consisting of fluorescence resonance electron transfer (FRET), lanthamide resonance electron transfer (LRET), fluorescence cross-correlation spectroscopy, fluorescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescence resonance energy transfer, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence resonance energy transfer, excimer formation, colorimetric substrates detection, phosphorescence, electro-chemical changes, and redox potential changes.

16. The method of claim 11 , wherein the concentration of the target molecule is determined.

17. The method of claim 11 , wherein the target molecule is a macromolecule selected from the group consisting of a protein, a polypeptide, a prion, a nucleic acid, a lipid, and a carbohydrate.

18. The method of claim 11 , wherein the target molecule is selected from a kinase, a binding protein, and an antigen.