Targeted probes of cellular physiology
Biosensor comprising an activatable acceptor fluorogen linked via a linker to a donor which transfers energy to the fluorogen on detecting an analyte wherein the fluorogen component reacts and a 100 fold increase in intensity results when the fluorogen interacts non-covalently with an activator e.g. fluorogen activator peptide.
1. A complex comprising:
a biosensor comprising malachite green covalently linked by a linker to a single ion-sensitive cyanine dye or an ion-sensitive rhodamine dye that interacts with an analyte ion; and
an activator of malachite green bound to the malachite green, wherein the activator comprises one of SEQ ID NOS: 3-9,
wherein the malachite green produces a fluorescence signal increase of at least 100-fold when it is bound to the activator as compared to when no activator is bound to the malachite green,
wherein the ion-sensitive cyanine dye or ion-sensitive rhodamine dye transfers excitation energy to the malachite green such that, when activated by binding to the activator, the malachite green produces a detectable fluorescent signal when the ion-sensitive cyanine dye or ion-sensitive rhodamine dye is excited and the ion-sensitive cyanine dye or ion-sensitive rhodamine dye transfers different amounts of excitation energy to the malachite green, and the malachite green fluoresces at a different intensity at different analyte ion concentrations, and
wherein the ion-sensitive cyanine dye is a trimethine cyanine dye.
2. The complex of claim 1 , in which the increase in fluorescence is at least 1000-fold.
3. The complex of claim 1 , in which the activator of malachite green is attached to a selectivity component.
4. The complex of claim 3 , in which the selectivity component and the activator of malachite green form a fusion protein.
5. The complex of claim 3 , in which the selectivity component is crosslinked to the activator of malachite green.
6. The complex of claim 1 , in which the activator of malachite green comprises one of SEQ ID NOS: 3-6.
7. The complex of claim 6 , in which the activator of malachite green further comprises an amino acid sequence of a selectivity component.
8. The complex of claim 1 , wherein the activator of malachite green is linked via a peptide bond or a linker to a targeting group that interacts with a target.
9. The complex of claim 8 , wherein the target is one of an epitope, a protein, a modified protein, a nucleic acid, a nucleotide sequence, a small molecule, an active agent, an antibody, a cell, a cell-surface marker, a tissue, a site in an array or a particle.
10. The complex of claim 1 , wherein the ion-sensitive trimethine cyanine dye comprises a protonated indole.
11. The complex of claim 1 , wherein the fluorescent intensity of the malachite green of the complex is sensitive to pH.
12. The complex of claim 1 , wherein the malachite green of the complex fluoresces at different intensities when the ion-sensitive rhodamine dye interacts with different concentrations of calcium or zinc ions.
13. The complex of claim 1 , wherein the biosensor is selected from the group consisting of:
14. An environmental sensing method for detecting the presence of an analyte ion or for quantifying the analyte ion, comprising:
a) contacting a biosensor with an activator of malachite green, wherein the activator comprises one of SEQ ID NOS: 3-9, the biosensor comprising: malachite green covalently linked by a linker to a single ion-sensitive trimethine cyanine dye or an ion-sensitive rhodamine dye that interacts with the analyte ion,
wherein the malachite green produces a fluorescence signal increase of at least 100-fold when it is bound to the activator of malachite green as compared to when no activator of malachite green is bound to the malachite green, and wherein the ion-sensitive trimethine cyanine dye or ion-sensitive rhodamine dye transfers excitation energy to the malachite green such that, when activated, the malachite green produces a detectable fluorescent signal when the ion-sensitive trimethine cyanine dye or ion-sensitive rhodamine dye is excited and the ion-sensitive trimethine cyanine dye or ion-sensitive rhodamine dye transfers different amounts of excitation energy to the malachite green when it interacts with the analyte ion as compared to when no analyte ion is present; and
b) illuminating the biosensor with light of a wavelength overlapping an excitation spectrum of the ion-sensitive trimethine cyanine dye or ion-sensitive rhodamine dye, but not overlapping the excitation spectrum of the malachite green and measuring emissions from the malachite green.
15. The method of claim 14 , further comprising, c) after contacting the biosensor with the activator of malachite green, illuminating the biosensor with light of a wavelength overlapping an excitation spectrum of the malachite green, but not overlapping an excitation spectrum of the ion-sensitive trimethine cyanine dye or ion-sensitive rhodamine dye and measuring emissions from the malachite green.
16. The method of claim 15 , further comprising determining a ratio between the emissions obtained from steps b) and c).
17. The method of claim 14 , wherein the activator of malachite green comprises a selectivity component.
18. The method of claim 17 , wherein the selectivity component binds to an epitope, a protein, a modified protein, a nucleic acid, a nucleotide sequence, a small molecule, an active agent, an antibody, a cell, a cell-surface marker, a tissue, a site in an array or a particle by the selectivity component.
19. The method of claim 14 , further comprising comparing the measured emissions from the malachite green to a control sample or to control sample data to determine the presence of or to quantify amounts of the analyte ion.