Simple and ultrastable all-inclusive pullulan tablets for challenging bioassays

Abstract

Many biodetection systems employ labile enzymes and substrates that need special care, making it hard to routinely use them for point-of-care or field applications. In this work we provide a simple solution to this challenging problem through the creation of all-inclusive pullulan assay tablets. The proposed tablet system not only enhances the long-term stability of both enzymes and organic substrates, but also simplifies the assay procedure. The enhanced stability is attributed to two factors: the restriction of the molecular motion of proteins and impermeability to molecular oxygen afforded by the tables. These tablets dissolve rapidly upon addition to testing samples, making the test very easy to perform. Using the ATP-detecting luciferase-luciferin system as an example, we show that the tablet-based assay can achieve highly sensitive detection of ATP in biological samples and that the activity of the assay tablets remains unchanged for over a month at room temperature.

Fig. 1. Method to produce all-inclusive pullulan assay tablets for ATP detection. A photograph of an all-inclusive pullulan tablet is shown on the right, where the tablet is a disc-shaped film with a diameter of 6 mm.

Fig. 2. (A) Thermal stability of all-inclusive tablet vs. solution. (B) Steady-state anisotropy of Trp within HSA in pullulan tablet, pullulan solution and buffer solution. (C) Comparison of luminescence of all-inclusive pullulan vs. dextran tablets cast under ambient conditions (refer to Fig. S5 for the data obtained when luciferase and luciferin were included in separate pullulan tablets). (D) Long-term stability of pullulan tablet produced under nitrogen vs. solution.

Fig. 3. (A) Effect of CoA concentration on assay performance (values are final concentrations after tablet dissolution). 54 mM represents the optimal concentration of CoA in our all-inclusive assay system. (B) Comparison of signal evolution from a tablet-based assay and a solution assay.

Fig. 4. (A) ATP detection using all-inclusive pullulan tablets. (B) Detection of lysed E. coli cells using the all-inclusive luminescent tablets.