Immobilization of Firefly Bioluminescent System: Development and Application of Reagents

Abstract

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL−1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination.

Enzymes Immobilized into Starch- and Gelatin-Based Hydrogels: Properties and Application in Inhibition Assay

“…The final step may include pipetting the mixture onto the support and drying (Figure 2). This is the way to produce single-use reagents containing the preset amount of enzyme (Figure 3) [57][58][59][60]. In this form, they can be used in cuvette-based instruments (spectrophotometer, luminometer).…”

“…However, the advantage of this method is that it saves on expensive enzyme preparations. The above-mentioned method of immobilizing enzymes by entrapping them in starch and gelatin hydrogels without any previous modifications was successfully used to produce stable preparations of butyrylcholinesterase (BChE), trypsin (Try), firefly luciferase (FLuc), and a bioluminescent bi-enzyme system of luminous bacteria: NAD(P)H:FMNoxidoreductase and luciferase (Red + Luc) [58][59][60].…”

“…In the gelatin gel, though, the function of the firefly luciferin-luciferase system did not change substantially even when polymer content reached 5% [86]. By immobilizing FLuc in the 1% gelatin gel, we produced high-activity single-use preparations for assessing microbial contamination based on ATP content [59,87]. Although other authors also reported successful immobilization of In contrast, higher BChE activity (comparable to the activity of the free enzyme) was achieved by the entrapment of the enzyme in the gelatin gel (Table 1).…”

“…Although other authors also reported successful immobilization of In contrast, higher BChE activity (comparable to the activity of the free enzyme) was achieved by the entrapment of the enzyme in the gelatin gel (Table 1). The starch gel was completely unsuitable for immobilizing FLuc [59]. A possible reason for that was that components of the buffer solution used for this reaction interacted with starch gel, preventing the disks from drying completely even in the long term.…”

Water-Soluble Polymer Polyethylene Glycol: Effect on the Bioluminescent Reaction of the Marine Coelenterate Obelia and Coelenteramide-Containing Fluorescent Protein

“…It is known that water-soluble polymers can change the rates of cellular processes via stabilizing biological structures. It is shown [ 1 , 2 , 3 ] that enzymes included in the gelatin or starch gel matrix become more stable and lead to an increase in the activity. Moreover, water-soluble polymers such as polyethylene glycol (PEG) may cause toxic effects on living organisms [ 4 , 5 ].…”