Biosensor for detecting multiple epitopes on a target

1. A method for detecting a target comprising at least one repeating epitope, the method comprising contacting a sample comprising the target with a molecular biosensor, the biosensor comprising:

R 24 —R 25 —R 26 —R 27 ,

R 28 —R 29 —R 30 —R 31 , and

O

wherein:

R 24 is an epitope binding agent that binds to a repeating epitope on a target;

R 25 is a flexible linker attaching R 24 to R 26 ;

R 26 and R 30 are a pair of nucleotide sequences that are not complementary to each other, but are complementary to two distinct regions on O;

R 27 and R 31 together comprise a detection means such that when R 26 and R 30 associate with O a detectable signal is produced;

R 28 is an epitope-binding agent that binds to the same repeating epitope on the target as R 24 ;

R 29 is a flexible linker attaching R 28 to R 30 ; and

O is a nucleotide sequence comprising a first region that is complementary to R 26 , and a second region that is complementary to R 30 , and detecting the signal produced by the association of R 26 with O and R 30 with O wherein the signal indicates the presence of the target.

2. The method of claim 1 , wherein the target is selected from the group consisting of a protein, a polypeptide, a nucleic acid, a lipid, a carbohydrate, and a microbial organism.

3. The method of claim 1 , wherein the target is a microbial organism selected from the group consisting of a bacterium, a virus, and a fungus.

4. The method of claim 3 , wherein R 25 and R 29 are from 50 to 250 angstroms in length and are independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.

5. The method of claim 1 , wherein the detection means is selected from the group consisting of FRET, fluorescence cross-correlation spectroscopy, fluorescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescense 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. The method of claim 1 , wherein 80% of the maximum signal may be detected at 5 min.

7. The method of claim 1 , wherein O comprises formula (IV):

R 32 —R 33 —R 34 —R 35 —R 36   (IV)

wherein:

R 32 , R 34 , and R 36 are nucleotide sequences not complementary to any of R 26 , R 30 , R 33 , or R 35 ;

R 33 is a nucleotide sequence complementary to R 26 ;

R 35 is a nucleotide sequence that is complementary to R 30 ;

R 33 is not adjacent to R 35 ; and

wherein R 32 , R 34 , and R 36 are from 2 to 20 nucleotides in length;

and R 33 and R 35 comprise a length such that the free energy of association between R 33 and R 26 and R 35 and R 30 is from −5 to −12 kcal/mole at a temperature from 21° C. to 40° C. and at a salt concentration from 1 mM to 100 mM.

8. The method of claim 1 , wherein R 24 and R 28 are independently selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, modified nucleic acids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.

9. The method of claim 1 , wherein R 26 and R 30 are from 2 to 20 nucleotides in length.

10. A method for detecting a target comprising at least one repeating epitope, the method comprising contacting a sample comprising the target with a molecular biosensor comprising two epitope binding agent constructs and an oligonucleotide construct, which together have formula (III):

R 24 —R 25 —R 26 —R 27 ;

R 28 —R 29 —R 30 —R 31 ; and

R 32 —R 33 —R 34 —R 35 —R 36   (III)

wherein:

R 24 is an epitope binding agent that binds to a repeating epitope on a target molecule;

R 25 is a flexible linker attaching R 24 to R 26 ;

R 26 and R 30 are a pair of nucleotide sequences that are not complementary to each other, but are complementary to R 33 and R 35 respectively;

R 27 and R 31 are labels that together comprise a detection means such that when R 26 and R 30 associate with R 33 and R 35 a detectable signal is produced;

R 28 is an epitope binding agent that binds to the same repeating epitope on the target molecule as R 24 ;

R 29 is a flexible linker attaching R 28 to R 30 ;

R 32 , R 34 , and R 36 are nucleotide sequences not complementary to any of R 25 , R 30 , R 33 , or R 35 ;

R 33 is a nucleotide sequence complementary to R 26 ;

R 35 is a nucleotide sequence that is complementary to R 30 ; and R 33 is not adjacent to R 35 ;

wherein R 32 , R 34 , and R 36 are from 2 to 20 nucleotides in length; and R 33 and R 35 comprise a length such that the free energy of association between R 33 and R 26 and R 35 and R 30 is from −5 to −12 kcal/mole at a temperature from 21° C. to 40° C. and at a salt concentration from 1 mM to 100 mM; and

detecting the signal produced by the association of R 26 with O and R 30 with O wherein the signal indicates the presence of the target.

11. A method for detecting a target in a sample, the method comprising:

(a) contacting a surface comprising an immobilized oligonucleotide construct O, with two epitope binding agents constructs, and a sample, the epitope binding agents constructs comprising:

R 24 —R 25 —R 26 —R 27 ;

R 28 —R 29 —R 30 —R 31 ;

wherein:

R 24 is an epitope binding agent that binds to a repeating epitope on a target molecule;

R 25 is a flexible linker attaching R 24 to R 26 ;

R 26 and R 30 are a pair of nucleotide sequences that are not complementary to each other, but are complementary to two distinct regions on O;

R 27 and R 31 are labels that together comprise a detection means such that when R 26 and R 3 ° associate with O a detectable signal is produced;

R 28 is an epitope binding agent that binds to the same repeating epitope on the target molecule as R 24 ;

R 29 is a flexible linker attaching R 28 to R 30 ; and

O is a nucleotide sequence comprising a first region that is complementary to R 26 , and a second region that is complementary to R 30 , wherein O is immobilized to the surface irrespective of the association between R 26 and O or R 30 and O; and

(b) detecting whether R 26 and R 30 bind to O, wherein the binding indicates that the target is present in the sample.