Detection units and methods for detecting a target analyte
The present application relates to detection units and methods for detecting one or more target analytes in a sample using a complex formed by a target and first and second probes, wherein the first probe is coupled to a detectable piece, the target is coupled to the first probe and the second probe, and the second probe is coupled to a solid support. Specific binding of the detectable piece to the target analyte can be distinguished from non-specific binding of the detectable piece by measuring the number of detectable pieces that leave their initial location after exposure to a disruptor that uncouples the detectable piece from the solid support.
1 . A method of detecting two or more different target analytes in a sample, the method comprising:
a) providing at least one detectable piece coupled to a solid support via a complex formed by a target analyte and a first probe and a second probe, wherein:
i) the first probe is coupled to the detectable piece and bound to the target analyte if present, and
ii) the second probe is coupled to the solid support and bound to the target analyte if present, so that only if the target analyte is present in the sample, the detectable piece is directly or indirectly coupled to the solid support at an initial location via the complex, wherein the complex comprises an elongated region that is at least 100 nanometers in length;
b) either applying a force to the detectable piece and measuring the displacement of the detectable piece or measuring the amount of Brownian motion of the detectable piece;
c) exposing the complex to a disruptor that is capable of uncoupling the detectable piece from the solid support;
d) optionally applying a further force to the detectable piece; and
e) detecting if the detectable piece was uncoupled from the solid support by the disruptor; wherein presence of the target analyte in the sample is indicated by the detectable pieces:
i) suffering the displacement or the amount of Brownian motion within a pre-determined range and
ii) uncoupling from the solid support by the disruptor,
wherein a different target analyte is detected by using a first probe specific to the different target analyte and a second probe specific to the different target analyte, wherein the different target analyte, the first probe specific to the different target analyte, and the second probe specific to the different target analyte form a further complex, and wherein the disruptor optionally disrupts the further complex.
2 . The method of claim 1 , wherein step e) is conducted by determining if the detectable piece left the initial location.
3 . The method of claim 1 , further comprising the step of estimating the concentration of the target analyte and/or the different target analyte based on the number of detectable pieces that are uncoupled from the solid support by the disruptor.
4 . The method of claim 1 , wherein the first probe comprises a first nucleic acid that binds to a first region of the target analyte and the second probe comprises a second nucleic acid that binds to a second region of the target analyte; and/or wherein the first probe specific to the different target analyte comprises a first nucleic acid that binds to a first region of the different target analyte and the second probe specific to the different target analyte comprises a second nucleic acid that binds to a second region of the different target analyte.
5 . The method of claim 1 , wherein the first probe comprises a first antibody or a first aptamer that binds to a first region of the target analyte and the second probe comprises a second antibody or a second aptamer that binds to a second region of the target analyte; and/or wherein the first probe specific to the different target analyte comprises a first antibody or a first aptamer that binds to a first region of the different target analyte and the second probe specific to the different target analyte comprises a second antibody or a second aptamer that binds to a second region of the different target analyte.
6 . The method of claim 1 , wherein the target analyte comprises a protein, the disruptor comprises an enzyme capable of degrading the target analyte, and the first and second probes do not comprise proteins; and/or wherein the different target analyte comprises a protein, the disruptor comprises an enzyme capable of degrading the different target analyte, and the first probe specific to the different target analyte and the second probe specific to the different target analyte do not comprise proteins.
7 . The method of claim 6 , wherein the disruptor comprises proteinase K.
8 . The method of claim 1 , wherein the target analyte comprises RNA, the disruptor comprises an enzyme capable of degrading the target analyte, and the first and second probes are not degradable by the disruptor; and/or wherein the different target analyte comprises RNA, the disruptor comprises an enzyme capable of degrading the different target analyte, and the first probe specific to the different target analyte and the second probe specific to the different target analyte are not degradable by the disruptor.
9 . The method of claim 8 , wherein the disruptor comprises RNase H, RNase A, RNase III, RNase L, RNase P, RNase PhyM, RNase TI, RNase T2, RNase U2, or RNase V.
10 . The method of claim 1 , wherein the target analyte and/or the different target analyte comprise a nucleic acid and the disruptor comprises an endonuclease enzyme or an exonuclease enzyme.
11 . The method of claim 10 , wherein the disruptor comprises a restriction enzyme.
12 . The method of claim 1 , wherein the disruptor comprises a nucleic acid that can hybridize to the target analyte in a region where the target analyte hybridizes to the first probe or the second probe; and/or wherein the disruptor comprises a nucleic acid that can hybridize to the different target analyte in a region where the different target analyte hybridizes to the first probe specific to the different target analyte or the second probe specific to the different target analyte.
13 . The method of claim 1 , wherein the disruptor comprises a nucleic acid that can hybridize to the first probe or to the second probe; and/or wherein the disruptor comprises a nucleic acid that can hybridize to the first probe specific to the different target analyte or the second probe specific to the different target analyte.
14 . The method of claim 1 , wherein the disruptor comprises two different disruptors that are sequentially contacted with the complex and the further complex and the detectable pieces that are uncoupled from the solid support by the two different disruptors are quantified after each different disruptor is contacted with the complex and the further complex.
15 . The method of claim 1 , which further comprises the step of coupling the first probe and/or the first probe specific to the different target analyte to the detectable piece.
16 . The method of claim 1 , which further comprises the step of coupling the second probe and/or the second probe specific to the different target analyte to the solid support.
17 . The method of claim 1 , which further comprises the steps of exposing the sample to the first probe and exposing the sample to the second probe and/or the steps of exposing the sample to the first probe specific to the different target analyte and exposing the sample to the second probe specific to the different target analyte.
18 . The method of claim 17 , wherein the further steps are performed in either order, or simultaneously.
19 . The method of claim 18 , which further comprises a step of exposing the sample to at least one detectable piece.
20 . The method of claim 19 , further comprising a washing step after one or more of the steps.
21 . The method of claim 20 , further comprising a lysing step.
22 . The method of claim 1 , wherein the force applied to the detectable piece comprises a magnetic force, a fluid drag force, an electrical force, or a centrifugal force.
23 . The method of claim 1 , wherein the detectable piece comprises a particle.
24 . The method of claim 23 , wherein the particle is magnetic.
25 . The method of claim 24 , wherein the particle is superparamagnetic.
26 . The method of claim 1 , wherein the detectable piece is fluorescent.
27 . The method of claim 1 , wherein the detectable piece is detected using an imaging system with a lens, with a lens-free microscope, or with a coherent imaging technique.
28 . The method of claim 3 , which further comprises controlling the temperature of the sample to produce denaturation of double stranded nucleic acids in the sample and/or specific hybridization of nucleic acids in the sample to the first and second probes and/or the first probe specific to the different target analyte and the second probe specific to the different target analyte.
29 . The method of claim 3 , wherein the sample is initially treated with an exonuclease enzyme to convert double stranded nucleic acids into single stranded nucleic acids.
30 . The method of claim 1 , wherein the target analyte and/or the different target analyte comprises a protein, carbohydrate, lipid, hormone, steroid, toxin, vitamin, hapten, metabolite, drug, or a combination thereof.
31 . A method of detecting two or more different target analytes in a sample, the method comprising:
a) providing at least one detectable piece coupled to a solid support via a complex formed by a target analyte and a first probe and a second probe, wherein:
i) the first probe is coupled to the detectable piece and bound to the target analyte if present, and
ii) the second probe is coupled to the solid support and bound to the target analyte if present, so that only if the target analyte is present in the sample, the detectable piece is directly or indirectly coupled to the solid support via the complex,
b) optionally detecting presence of the detectable piece;
c) exposing the complex to a disruptor that is capable of uncoupling the detectable piece from the solid support, wherein the disruptor comprises a strand-displacement molecule capable of dissociating one or more nucleic acid duplexes formed between the target analyte and the first probe or the second probe, or the disruptor comprises a degradation molecule capable of breaking one or more covalent bonds of the target analyte;
d) optionally applying a force to the detectable piece; and
e) detecting if the detectable piece was uncoupled from the solid support by the disruptor;
wherein the detectable piece if indirectly bound to the target analyte is likely to be uncoupled from the solid support by the disruptor, whereas the detectable piece if not indirectly bound to the target analyte is unlikely to be uncoupled from the solid support by the disruptor;
wherein a different target analyte is detected by using a first probe specific to the different target analyte and a second probe specific to the different target analyte, wherein the different target analyte, the first probe specific to the different target analyte, and the second probe specific to the different target analyte form a further complex, and wherein the disruptor optionally comprises a degradation molecule capable of breaking one or more covalent bonds of the different target analyte or comprises a strand-displacement molecule capable of dissociating one or more nucleic acid duplexes formed between the different target analyte and the first probe specific to the different target analyte or the second probe specific to the different target analyte.
32 . A method of detecting a target analyte in a sample, the method comprising:
a) providing at least one detectable piece coupled to a solid support via a complex formed by the target analyte and a first probe and a second probe, wherein:
i) the first probe is coupled to the detectable piece and bound to the target analyte if present, and
ii) the second probe is coupled to the solid support and bound to the target analyte if present, so that only if the target analyte is present in the sample, the detectable piece is directly or indirectly coupled to the solid support at an initial location via the complex, wherein the complex comprises an elongated region that is at least 100 nanometers in length;
b) either applying a force to the detectable piece and measuring the displacement of the detectable piece or measuring the amount of Brownian motion of the detectable piece;
c) exposing the complex to a disruptor that is capable of uncoupling the detectable piece from the solid support;
d) optionally applying a further force to the detectable piece; and
e) detecting if the detectable piece was uncoupled from the solid support by the disruptor; wherein presence of the target analyte in the sample is indicated by the detectable piece:
i) suffering the displacement or the amount of Brownian motion within a pre-determined range and
ii) uncoupling from the solid support by the disruptor; and
wherein the disruptor comprises a nucleic acid that can hybridize to the first probe or to the second probe.