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. 2007 Aug 15;367(2):201-9.
doi: 10.1016/j.ab.2007.04.023. Epub 2007 Apr 25.

A multienzyme bioluminescent time-resolved pyrophosphate assay

Ye Sun  1 , K Bruce Jacobson, Val Golovlev
Affiliations

A multienzyme bioluminescent time-resolved pyrophosphate assay

Ye Sun et al. Anal Biochem. .

Abstract

We have developed a high-sensitivity assay for measurement of inorganic pyrophosphate (PPi) in adenosine 5'-triphosphate (ATP)-contaminated samples. The assay is based on time-resolved measurements of the luminescence kinetics and implements multiple enzymes to convert PPi to ATP that is, in turn, utilized to produce light and to hydrolyze PPi for measurement of the steady state background luminescence. A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented. We found that the model is in excellent agreement with the experimental results. We have developed and evaluated two algorithms for PPi measurement from luminescence kinetics acquired from ATP-contaminated samples. The first algorithm is considered to be the method of choice for analysis of long, i.e., 3-5 min, kinetics. The activity of enzymes is controlled during the experiment; the sensitivity of PPi detection is about 7 pg/ml or 15 pM of PPi in ATP-contaminated samples. The second algorithm is designed for analysis of short, i.e., less than 1-min, luminescence kinetics. It has about 20 pM PPi detection sensitivity and may be the better choice for assays in microplate format, where a short measurement time is required. The PPi assay is primarily developed for RNA expression analysis, but it also can be used in various applications that require high-sensitivity PPi detection in ATP-contaminated samples.

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Figures

Figure 1
Figure 1
Luminescence kinetics detected from three samples, containing: (a) 1.8 fmol ATP; (b) 11.2 fmol PPi; and (c) a mixture of ATP and PPi.
Figure 2
Figure 2
Example of fitting the luminescence kinetics, IExper(t), by sum of the fast, A(t), the slow, B(t), and the steady-state background, IBackgr, components.
Figure 3
Figure 3
Detection sensitivity of PPi assay vs. the assay time and concentration of the luciferin-luciferase in the assay solution: (a) :2-diluted ATP-detection solution, γL-L= 0.012 s-1; (b) x1 ATP-detection solution, γL-L= 0.024 s-1; and (c) x2 ATP-detection solution, γL-L= 0.048 s-1.
Figure 4
Figure 4
Luminescence kinetics from samples prepared by mixing known amount of ATP and PPi. Shown are kinetics replicates acquired from samples with amount of PPi in the range of 0.6 pg – 60 pg and ATP amount of 0, 1 pg, and 2 pg in 5-μl sample : (a) 60 pg PPi; (b) 20 pg PPi and 2 pg ATP; (c) 20 pg PPi and 1 pg ATP; (d) 20 pg PPi, etc. The set of kinetics have been used to evaluate different algorithms for measuring PPi in ATP-contaminated samples.
Figure 5
Figure 5
Selection of time intervals for application of the algorithm given by Eqs. (38-42). The number of photons emitted during corresponding time interval is Ni. The smooth solid line is the fit of the experimental data by Eq. (7) with kinetic parameters determined by algorithm described in the text.
Figure 6
Figure 6
Measurement of PPi in three samples with simulated contamination by ATP. The amount of ATP is varied between samples and is 0, 1 pg and 2 pg of ATP.
Figure 7
Figure 7
Two reference kinetics A and B are used to calculate the fit curve for sample kinetics C. The fit curve D is given by a linear combination of the kinetics A and B (see the Eq.(36). The coefficients in the Eq.(36) are calculated using data acquired during the first 45 sec, as indicated by dashed line.
Figure 8
Figure 8
Measurement of PPi by 45-sec assay vs. the measurements by 300-sec assay. The dashed line shows the least square linear fit.
Figure 9
Figure 9
Detection of RNA spike in complex RNA sample by time-resolved bioluminescence assay.
See this image and copyright information in PMC

References

    1. Hassibi A, Contag C, Vlad MO, Hafezi M, Lee TH, Davis RW, Pourmad N. Bioluminescence regenerative cycle (BRC) system: Theoretical consideration for nucleic acid quantification assays. Biophys Chem. 2005;116:175–185. - PMC - PubMed
    1. Sun Y, Golovlev V. Bioluminescence assay for RNA quantitation. Luminescence. 2006;21:293.
    1. Hassibi A, Lee TH, Davis RW, Pourmand N. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays, Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis. Poceedings of SPIE. 2003;4966:65–75.
    1. Sun Y, Jacobson KB, Golovlev V. Quantitative bioluminescent RNA assay. Proceedings of 14th International Symposium on Bioluminescence and Chemiluminescence; San Diego CA. 2006. in press.
    1. Brovko L, Gandel'man O, Polenova T, Ugarova N. Kinetics of bioluminescence in the firefly luciferin-luciferase system. Biochem (Moscow) 1994;59:195–201.

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