Luminogenic and nonluminogenic multiplex assay
View Patent ↗A method to detect the presence or amount of at least one molecule for an enzyme-mediated reaction in a multiplex luminogenic/nonluminogenic assay is provided.
1. A method to detect live and/or dead cells in a sample, comprising:
a) contacting a sample comprising cells suspected of having one or more proteases with a peptide substrate for a first nonsecreted protease and a peptide substrate for a second nonsecreted protease, wherein a reaction with one of the substrates mediated by one of the proteases yields a fluorogenic product and a reaction with the other substrate mediated by the other protease yields a luminogenic or a fluorogenic product, wherein one of the substrates is substantially cell membrane permeant and the other substrate is substantially cell membrane impermeant, wherein if both reactions yield fluorogenic products, the fluorophores on the two substrates are spectrally distinct, and wherein the substantially cell membrane permeant substrate is capable of detecting or determining the number or presence of live cells in the sample and the substantially cell membrane impermeant substrate is capable of detecting or determining the number or presence of dead cells in the sample as a result of protease activity released into the extracellular environment due to loss of membrane integrity; and
b) detecting or determining fluorescence and/or luminescence in the sample, thereby detecting or determining the number or presence of live and/or dead cells in the sample.
2. The method of claim 1 wherein a reaction mediated by each protease yields a fluorogenic product.
3. The method of claim 1 wherein a reaction mediated by one protease yields a luminogenic product and a reaction mediated by the other protease yields a fluorogenic product.
4. The method of claim 1 wherein the substantially cell permeant substrate is a substrate for a protease associated with a proteasome, an aminopeptidase or a cathepsin.
5. The method of claim 1 wherein the substantially cell impermeant substrate is a substrate for a tripeptidyl peptidase or calpain.
6. The method of claim 1 wherein the sample comprises mammalian cells.
7. The method of claim 1 wherein the two substrates are combined before contact with the sample.
8. The method of claim 3 wherein fluorescence and luminescence are detected or determined sequentially.
9. The method of claim 2 wherein the fluorescence of each fluorogenic product is detected or determined simultaneously.
10. The method of claim 1 further comprising contacting the sample with one or more agents suspected of altering the number of live and/or dead cells prior to contact with the substrates.
11. A method to detect live and/or dead cells and at least one other molecule in a sample comprising cells, comprising:
a) contacting a sample comprising cells suspected of having one or more proteases with a peptide substrate for a first nonsecreted protease and a peptide substrate for a second nonsecreted protease, wherein a reaction with one of the substrates mediated by one of the proteases yields a fluorogenic product and a reaction with the other substrate mediated by the other protease yields a luminogenic or a fluorogenic product, wherein one of the substrates is substantially cell membrane permeant and the other substrate is substantially cell membrane impermeant, wherein if both reactions yield fluorogenic products, the fluorophores on the two substrates are spectrally distinct, and wherein the substantially cell membrane permeant substrate is capable of detecting or determining the number or presence of live cells in the sample and the substantially cell membrane impermeant substrate is capable of detecting or determining the number or presence of dead cells in the sample as a result of protease activity released into the extracellular environment due to loss of membrane integrity; and
b) detecting or determining fluorescence and/or luminescence, thereby detecting or determining the number or presence of live and/or dead cells, and the presence or amount of at least one molecule, in the sample.
12. The method of claim 11 wherein the molecule is DNA.
13. The method of claim 11 wherein the molecule is an enzyme.
14. The method of claim 11 wherein the molecule is ATP.
15. The method of claim 11 wherein the sample is subjected to conditions that lyse cells prior to detecting or determining the presence or amount of the molecule.
16. The method of claim 1 or 11 wherein one of the substrates comprises Gly-Phe-AFC.
17. The method of claim 1 or 11 wherein one of the substrates comprises (Ala-Ala-Phe) 2 -R110.
18. The method of claim 1 or 11 wherein one of the substrates comprises Gly-Phe-AMC.
19. The method of claim 1 or 11 wherein one of the substrates comprises Ala-Ala-Phe-AMC or Ala-Ala-Phe-aminoluciferin.