Fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes

Abstract

Ratiometric fluorescent assays have a built-in correction factor which enhances assay accuracy and reliability. We have developed fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes using a mixture of three molecular components. One of the molecules is a tetra-cationic fluorescence quencher called CalixPyr which can bind and quench the polyanionic pyrene fluorophore, CMP, that emits at 430 nm. Polyphosphates can disrupt the CMP/CalixPyr complex and alter the fluorescence intensity (responsive signal). CalixPyr has no effect on the fluorescence emission of cationic pentamethine cyanine fluorophore, cCy5, which emits at 665 nm and acts as a non-responsive reference signal. The continuous ratiometric fluorescent assay for alkaline phosphatase monitored hydrolytic consumption of adenosine triphosphate (ATP). The continuous ratiometric fluorescent assay for phytase activity monitored hydrolytic consumption of phytate. With further development this latter assay may be useful for high throughput assessment of phytase activity in individual batches of fortified animal feed. It is likely that the three-molecule mixture (CMP, CalixPyr, cCy5) can become a general assay platform for other enzymes that catalyse addition/removal of phosphate groups from appropriate molecular substrates.

Scheme 1. (a) Chemical structures of the STA molecules used in this study. (b) Polyphosphates used in this study. (c) Selective binding and quenching of CMP fluorescence by CalixPyr is modulated by the presence of polyphosphate. (d) Ratiometric assay reports time-dependent change in fluorescence signals for non-responsive reference cCy5 and responsive CMP which can be plotted as the cCy5/CalixPyr fluorescence ratio.

Fig. 1. Fluorescence spectra for a single solution containing a binary mixture of CMP (5 μM) and cCy5 (5 μM) in water (pH 6.8) and titrated with CalixPyr. λ_ex = 401 nm (CMP) and 630 nm (cCy5), slit width = 1 nm.

Fig. 2. (a) Partial ¹H NMR spectrum (500 MHz, D₂O, pD 6.63, 25 °C) of cCy5 [1 mM] (top), CalixPyr [1 mM] + cCy5 [1 mM] (middle), and CalixPyr [1 mM] (bottom). (b) Partial ¹H NMR spectrum (500 MHz, D₂O, pD 6.63, 25 °C) of CMP [1 mM] (top), CalixPyr [0.5 mM] + CMP [1 mM] (middle), and CalixPyr [1 mM] (bottom).

Fig. 3. Change in fluorescence intensity (F/F₀) for CMP (λ_ex = 401 nm, λ_em = 430 nm) upon incremental addition of ATP, ADP, or AMP to separate solutions containing CMP (1 μM), cCy5 (1 μM) and CalixPyr (4 μM) in 10 mM NaOAc solution, pH 7.2, room temperature.

Fig. 4. (a) Summary of continuous ratiometric fluorescent assay that monitors consumption of ATP catalysed by AP. (b) Typical data set showing change in normalized CMP and cCy5 fluorescence after addition of AP (1.5 U mL⁻¹) at 1 min, also in the assay mixture was CalixPyr (4 μM), CMP (1 μM), cCy5 (1 μM), ATP (60 μM), 10 mM NaOAc, pH 7.2, 37 °C. (c) Same assay data set plotted as the ratio of fluorescence intensities, F_cCy5/F_CMP, in the presence and absence of AP, (CMP, λ_ex = 401 nm, λ_em 430 nm; cCy5, λ_ex = 630 nm, λ_em 665 nm).

Fig. 5. (a) Summary of continuous ratiometric fluorescent assay that monitors consumption of Phyt catalysed by phytase. (b) Typical data set showing change in normalized CMP and cCy5 fluorescence after addition of phytase (0.01 U mL⁻¹) at time zero the assay mixture contained CalixPyr (4 μM), CMP (1 μM), cCy5 (1 μM), and phytate (100 μM) in 5 mM HEPES buffer, pH 5.1 at 45 °C. (c) Ratio of fluorescence intensities, F_cCy5/F_CMP, over time for samples containing different of phytase levels, (CMP, λ_ex = 401 nm, λ_em 430 nm; cCy5, λ_ex = 630 nm, λ_em 665 nm).