CLARIOstar Plus simplifies enzymatic reaction monitoring with its novel Enhanced Dynamic Range (EDR) technology

Introduction

How active are enzymes? This is an often addressed question in biochemical laboratories. As enzymes are the catalyzers of most chemical reactions taking place in an organism, they and their regulators are often characterized in detail.

Enzymatic assays typically employ a substrate that is converted to a chromophore, fluorophore or a luciferase substrate in course of the enzymatic reaction. This means in turn that the signal increases with increasing reaction time until all the substrate is converted by the enzyme. As it is hard to foresee at which range of signal intensity an enzymatic assay ends their detection is challenging and is often associated with various rounds of finding the right amplifi cation for fluorescent and luminescent signals. A virtually unlimited dynamic range as found with the enhanced dynamic range (EDR) feature drastically simplifies fluorescent and luminescent enzyme assays and saves time to set up the measurement.

Assay Principle

The enzyme β-galactosidase (β-gal) catalyzes the hydrolysis of β-galactosides by breaking up glycosidic bonds. It is used in biological research to report on cellular senescence which is indicated by senescence-associated β-gal. A second and very popular use of β-gal is as a reporter gene: a DNA construct contains the genetic information for β-gal and a promoter that regulates the expression of the enzyme. Mostly, β-gal serves as control gene. This means it is inserted into the cells of interest during transient transfection along with a reporter gene of interest. In this case β-gal reports on the transfection efficiency. A high transfection efficiency results in high enzyme production; low transfection efficiency results in low enzyme expression. The amount of expressed β-gal can be tested with a non-fluorescent synthetic β-gal substrate: Fluorescein di (β-D-galactopyranoside) (FDG). It is cleaved by the enzyme to galactose and the fluorescent molecule fluorescein (FITC) (Fig. 1). Here, we measured various enzyme concentrations on the CLARIOstar Plus with and without use of the novel EDR feature.

The enhanced dynamic range (EDR) refers to a technology that measures very dim and very intense signals in one plate measurement. This way, a dynamic range of 8 decades can be covered. In contrast, traditional measurements require an amplification of the signal that is typically set by the gain. This limits the range in which signals can be detected (Fig. 2).

Materials & Methods

• Black 96-well plate with flat bottom (Greiner bio-one # 655076)
• CLARIOstar Plus
• β-Galactosidase from Escherichia coli (G6008-1KU, Sigma-Aldrich / Merck)
• Fluorescein Di-β-D-Galactopyranoside (F1179, Invitrogen / Thermo Fisher Scientific)

Experimental procedure
The assay buffer was prepared by weighing 0.8370 g KH₂PO₄, 0.6855 g Na₂HPO₄·2H₂O, 0.0625 TCEP and 0.0203 g MgCl₂·6H₂O, dissolving in distilled water and adjusting the volume to 50 ml (fill up to 50 ml).

The β-Galactosidase was dissolved in 1 ml of distilled water (enzyme stock solution) and the substrate fluorescein Di-β-D-Galactopyranoside was dissolved in 10 ml of water (0.5 mg/ml substrate buffer). Standards of 0.5 U/ml; 0.25 U/ml; 0.1 U/ml; 0.05 U/ml; 0.025 U/ml; 0.01 U/ml were prepared by preparing double of the end concentration in distilled water and subsequently diluting it 1:2 in substrate buffer. The substrate concentration was kept constant at 0.25 mg/ml. Total reaction volume was 200 µl and four replicates were measured. Fluorescence intensity measurements were performed by the CLARIOstar Plus multi-mode microplate reader with the settings indicated below.

Instrument Settings

Results & Discussion

In order to test the suitability of the CLARIOstar Plus for measuring β-gal reporter assays, different enzyme concentrations were incubated with the synthetic substrate FDG and its conversion was monitored by measuring FITC fluorescence every 10 minutes. Traditionally, a signal range needed to be chosen up-front in order to detect such a kinetic measurement. For demonstration purpose, we selected a low gain (800) which translates to low signal amplification and should prevent excessive amplification of the increasing fluorescent signal.

However, detection in this wisely preselected range resulted in overflow measurements for all of the enzyme concentrations as the signal increased further than expected (Fig. 3). The lowest enzyme concentration showed an increase in fluorescence until 1 h 10 min after reaction start (orange line Fig. 3), but flattened out the following timepoints as the signal exceeded the preselected intensity range. A quantification of the enzyme is not possible with the acquired data.