Methods, compositions, and kits for assay signal amplification

1 . A method of detecting an analyte of interest in a sample, comprising:

(I):

a. contacting a first complex that comprises (A) a first detectable label, wherein the first detectable label is an electrochemiluminescent (ECL) label, and (B) the analyte of interest, with a signal amplification reagent that specifically binds to the first detectable label, wherein the signal amplification reagent comprises a nucleic acid probe, thereby forming a second complex comprising the first complex and the signal amplification reagent;

b. extending the nucleic acid probe to form an extended sequence; and

c. measuring the amount of extended sequence, thereby detecting the analyte of interest;

or

(II):

d. forming a first complex on a surface comprising the analyte of interest, a capture reagent that specifically binds to the analyte, wherein the capture reagent is immobilized on the surface or wherein the capture reagent is capable of being immobilized to the surface; and a detection reagent that specifically binds to the analyte and that comprises a first nucleic acid probe;

e. extending the first nucleic acid probe to form a first extended sequence comprising a first anchoring region, wherein the first anchoring region binds a first anchoring reagent that is immobilized on the surface;

f. binding the first extended sequence to a first labeled probe comprising a first detectable label, wherein the first detectable label is an electrochemiluminescent (ECL) label;

g. contacting the first labeled probe bound to the first extended sequence with a signal amplification reagent that specifically binds to the first detectable label, wherein the signal amplification reagent comprises a second nucleic acid probe, thereby forming a second complex comprising the signal amplification reagent and the first labeled probe;

h. extending the second nucleic acid probe to form a second extended sequence comprising a second anchoring region, wherein the second anchoring region binds a second anchoring reagent that is immobilized on the surface; and;

i. measuring the amount of (I) the second extended sequence or (II) the first extended sequence and the second extended sequence bound to the surface, thereby detecting the analyte of interest.

2 . The method of claim 1 , wherein the first complex of (I) is on a surface, optionally wherein the surface comprises a particle or a well of a multi-well plate.

3 . The method of claim 2 , wherein the first complex of (I) comprises:

a capture reagent that specifically binds to the analyte, wherein the capture reagent is immobilized on the surface prior to the contacting of (a); and

a detection reagent that specifically binds to the analyte and that comprises the first detectable label.

4 . The method of claim 3 , wherein the capture reagent, the detection reagent, and the signal amplification reagent each comprises an antibody or antigen-binding fragment thereof, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer.

5 . The method of claim 3 , wherein the first detectable label is linked to the detection reagent via a conjugation linker.

6 . The method of claim 2 , wherein the first complex of (I) comprises:

a capture reagent that specifically binds to the analyte, wherein the capture reagent is immobilized on the surface prior to the contacting of (a);

a first detection reagent that specifically binds to the analyte; and

a second detection reagent that specifically binds to the analyte,

wherein the first complex comprises at least two first detectable labels, and wherein each of the first detection reagent and the second detection reagent comprises one of the at least two first detectable labels.

7 . The method of claim 6 , wherein the first detectable label is linked to the second detection reagent via a conjugation linker, and wherein the signal amplification reagent is capable of binding simultaneously to the first detectable label on the first detection reagent and to the first detectable label on the second detection reagent.

8 . The method of claim 2 , wherein the surface comprises an electrode, and the measuring further comprises applying a voltage waveform to the electrode to generate an electrochemiluminescence signal; or

wherein the surface comprises a particle, and the method further comprises collecting the particle on an electrode, and the measuring further comprises applying a voltage waveform to the electrode to generate an electrochemiluminescence signal.

9 . The method of claim 1 ,

wherein the extending of (b), (e), or (h) comprises polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), self-sustained synthetic reaction (3SR), isothermal amplification, or combination thereof; and

when the extending of (b), (e), or (h) comprises PCR, the extending comprises binding the nucleic acid probe to a template oligonucleotide and extending the nucleic acid probe by PCR;

when the extending of (b), (e), or (h) comprises isothermal amplification, the extending comprises binding the nucleic acid probe to a template oligonucleotide, forming a circular template, and extending the nucleic acid probe by rolling circle amplification (RCA).

10 . The method of claim 9 , wherein the first complex of (I) is on a surface, and wherein the surface comprises an anchoring reagent that is immobilized on the surface prior to or during the extending of (b), wherein the anchoring reagent binds to an anchoring region of the extended sequence.

11 . The method of claim 10 , wherein the anchoring reagent and the anchoring region comprise complementary oligonucleotides, and

wherein the measuring of (c) comprises measuring the amount of extended sequence bound to the surface via the anchoring reagent.

12 . The method of claim 1 , wherein the first complex of (I) comprises at least two first detectable labels;

wherein the signal amplification reagent is a first signal amplification reagent, and the contacting of (a) further comprises contacting the first complex with a second signal amplification reagent that comprises a nucleic acid probe, wherein the first and second signal amplification reagents each binds to a distinct first detectable label, and the second complex comprises the first complex and the first and second signal amplification reagents.

13 . The method of claim 12 , wherein the extending of (b) comprises:

binding one or both of the nucleic acid probes of the first and second signal amplification reagents to a template oligonucleotide, forming a circular template, and extending one or both nucleic acid probes by RCA, wherein:

each nucleic acid probe is bound to a distinct template oligonucleotide; a circular template is formed from each template oligonucleotide; and each nucleic acid probe is extended by RCA; or

both nucleic acid probes are contacted with two template oligonucleotides, wherein each template oligonucleotide binds to a portion of each nucleic acid probe; the two template oligonucleotides are ligated to form a circular template; and one or both of the nucleic acid probes are extended by RCA.

14 . The method of claim 13 , wherein the nucleic acid probes of the first and second signal amplification reagents comprise or consist of a same sequence.

15 . The method of claim 1 , wherein the method further comprises, prior to the contacting of (a) or (g) detecting the first complex, wherein detecting the first complex comprises measuring the amount of the first detectable label.

16 . The method of claim 1 , wherein the signal amplification reagent is an antibody or antigen-binding fragment thereof comprising an antigen binding domain specific to an electrochemiluminescent (ECL) label or to an ECL label and a conjugation linker.

17 . The method of claim 1 , wherein the measuring comprises binding the extended sequence of (I) to a labeled probe or binding the second extended sequence of (II) to a second labeled probe, wherein the labeled probe or the second labeled probe comprises a second detectable label, and measuring the amount of (i) the second detectable label or (ii) the first and second detectable labels on the surface.

18 . The method of claim 17 , wherein the second detectable label is capable of being measured using light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or combinations thereof.

19 . The method of claim 18 , wherein each of the first detectable label and the second detectable label is an ECL label, and wherein the ECL label of the first and/or the second detectable label comprises an organometallic complex comprising at least one substituted bipyridine ligand, wherein the substituted bipyridine ligand comprises at least one sulfonate group, and optionally wherein:

the organometallic complex comprises at least two substituted bipyridine ligands, wherein each substituted bipyridine ligand comprises at least one sulfonate group;

the organometallic complex comprises ruthenium, osmium, or rhenium; and/or

the substituted bipyridine ligand is a compound of Formula I:

20 . The method of claim 18 , wherein each of the first detectable label and the second detectable label is an ECL label, and wherein the ECL label of the first and the second detectable label is a compound of Formula II:

or

wherein the first detectable label comprises a compound of Formula II, and the second detectable label comprises a compound of any one of Formulas III, IV, V, or VI:

wherein X is a phosphate, a carbonate, a borate, or combination thereof;

or

wherein the first detectable label comprises a compound of Formula III, and the second detectable label comprises a compound of any one of Formulas II, IV, V, or VI; or

wherein the first detectable label comprises a compound of Formula IV, and the second detectable label comprises a compound of any one of Formulas II, III, V, or VI; or

wherein the first detectable label comprises a compound of Formula V, and the second detectable label comprises a compound of any one of Formulas II, III, IV, or VI; or

wherein the first detectable label comprises a compound of Formula VI, and the second detectable label comprises a compound of any one of Formulas II, III, IV, or V.

21 . The method of claim 1 , wherein the surface of (II) comprises an electrode, and the measuring further comprises applying a voltage waveform to the electrode to generate an electrochemiluminescence signal; or

wherein the surface of (II) comprises a particle, and the method further comprises collecting the particle on an electrode, and the measuring further comprises applying a voltage waveform to the electrode to generate an electrochemiluminescence signal.

22 . The method of claim 1 , wherein, in the first complex of (II), the capture reagent, the detection reagent, and the signal amplification reagent each comprises an antibody or antigen-binding fragment thereof, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer.

23 . The method of claim 1 , wherein the first extended sequence of (II) binds to at least two first labeled probes that each comprise a first detectable label;

wherein the signal amplification reagent of (II) is a first signal amplification reagent, and the contacting of (g) further comprises contacting the first extended sequence with a second signal amplification reagent that comprises a third nucleic acid probe, wherein the first and second signal amplification reagents each binds to a distinct first detectable label, and the second complex comprises the first and second signal amplification reagents each bound to a first labeled probe.

24 . The method of claim 23 , wherein the extending of (h) comprises:

binding one or both of the second and third nucleic acid probes of the first and second signal amplification reagents to a template oligonucleotide, forming a circular template, and extending one or both second nucleic acid probes by RCA, wherein:

each second nucleic acid probe is bound to a distinct template oligonucleotide; a circular template is formed from each template oligonucleotide; and each second nucleic acid probe is extended by RCA; or

both second nucleic acid probes are contacted with two template oligonucleotides, wherein each template oligonucleotide binds to a portion of each second nucleic acid probe; the two template oligonucleotides are ligated to form a circular template; and one or both of the second nucleic acid probes are extended by RCA.