Methods and kits for detecting target substances
Provided is a method for detecting target substances. The method includes a) introducing a sample containing target substances onto a substrate, b) allowing detection probes conjugated with docking strands to specifically bind to the target substances, c) introducing one or more separate strands capable of complementary binding to the docking strands into the docking strands, either the docking strands or the separate strands, or both, are labeled with at least one donor fluorescent substance and at least one acceptor fluorescent substance, and d) measuring fluorescence signals generated by the FRET between the donor fluorescent substance and the acceptor fluorescent substance to identify the target substances.
1 . A method for detecting target substances, comprising:
a) introducing a sample containing target substances onto a substrate;
b) allowing detection probes conjugated with docking strands to specifically bind to the target substances;
c) introducing separate strands capable of complementary binding to the docking strand,
wherein the separate strands comprise one or more of donor strand labeled with a first donor fluorescent substance and one or more of acceptor strand labeled with a second donor fluorescent substance and an acceptor fluorescent substance that forms a FRET pair with the second donor fluorescent substance; and
d) measuring fluorescence signals generated by the FRET between the second donor fluorescent substance and the acceptor fluorescent substance to identify the target substances.
2 . The method according to claim 1 , wherein the peak absorption wavelength of the second donor fluorescent substance is shorter than that of the acceptor fluorescent substance and the peak emission wavelength of the second donor fluorescent substance is shorter than that of the acceptor fluorescent substance.
3 . The method according to claim 1 , wherein the extinction coefficient of the second donor fluorescent substance at a wavelength of light exciting the second donor fluorescent substance is at least 3 times higher than that of the acceptor fluorescent substance.
4 . The method according to claim 1 , wherein the Förster radius between the second donor fluorescent substance and the acceptor fluorescent substance is at least 0.208 nm.
5 . The method according to claim 1 , wherein the second donor fluorescent substance and the acceptor fluorescent substance are attached to the acceptor strand via linkers.
6 . The method according to claim 5 , wherein the linkers are 10 nm or less in length.
7 . The method according to claim 1 , wherein the signal-to-noise ratio of the fluorescence signals is at least 2.
8 . The method according to claim 1 , wherein the target substances are of two or more types and one or more of the donor strands or the acceptor strands have different sequences depending on the types of the target substances or are labeled with fluorescent substances of different types or at different sites.
9 . The method according to claim 8 , further comprising removing the donor strands and/or the acceptor strands after identification of the target substances and repeating step d) using donor strands and/or acceptor strands different from the removed donor strands and/or acceptor strands to identify the other target substances.
10 . The method according to claim 1 , wherein when the donor strands and the acceptor strands bind simultaneously or sequentially to the docking strands, any gaps—between the donor strands and the acceptor strands are smaller than the persistence length of the docking strands.
11 . The method according to claim 1 , further comprising controlling the concentration of the donor strands or the acceptor strands such that the time it takes for the donor strands or the acceptor strands to bind to the docking strands is 10 minutes or less while maintaining the signal-to-noise ratio of the fluorescence signals at 2 or more.
12 . The method according to claim 11 , wherein the concentration of the donor strands or the acceptor strands is from 10 nM to 10 μM.
13 . The method according to claim 1 , wherein when the docking strands or the acceptor strands are nucleic acids, the number of the bases of the acceptor strands complementary to the docking strands is at least 8.
14 . The method according to claim 13 , wherein the number of the bases of the donor strands complementary to the docking strands is from 6 to 12.
15 . The method according to claim 1 , wherein when the docking strands or the acceptor strands are nucleic acid analogues, the number of the bases of the acceptor strands complementary to the docking strands is at least 5.
16 . The method according to claim 15 , wherein the number of the bases of the donor strands complementary to the docking strands is from 3 to 9.
17 . The method according to claim 1 , wherein the following equation:
FRET efficiency=(Intensity of light from acceptor)/(Sum of intensity of light from donor and intensity of light from acceptor)
is used to measure FRET efficiency of the fluorescence signals.
18 . The method according to claim 1 , wherein two or more target substances are simultaneously identified by determining the difference between the FRET efficiencies distinguished depending on the distances between the second donor fluorescent substance and the acceptor fluorescent substance.
19 . The method according to claim 1 , wherein two or more target substances are simultaneously identified by determining the difference between the emission spectra by the FRET for different types of the second donor fluorescent substance or the acceptor fluorescent substance.
20 . The method according to claim 1 , further comprising determining the concentrations of the target substances in the sample from the numbers of the target substances per unit area of a region where the fluorescence signals are imaged.