Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Abstract

Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.

Influence of the experimental setup on the determination of enzyme kinetic parameters

“…Although the solutions and pipette tips were preheated, the liquid temperatures increase for 30–40 min. During this heating phase the higher temperatures in the outer wells than in the inner wells agree well with described edge effects . Different temperature profiles result in different reaction rates, and the corresponding reaction progresses in the wells clearly split after 5 min (Figure ).…”

“…The cresol red assay was adapted for temperature measurement in MTP . 10 mg mL −1 cresol red was dissolved in ethanol and diluted to 20 µg mL −1 with 100 mmol L −1 TRIS buffer.…”

“…Mainly in microbial cultivations, but also occasionally for biocatalytic applications, strong influence of physicochemical properties were observed. For instance, changes of sample volumes in MTP and the corresponding change in surface/volume ratios affect possible protein‐surface/liquid interactions.…”

“…12 Accordingly, biocatalytic assays and kinetic measurements are increasingly transferred to MTPs, although experiments in microwell scale are often accompanied by reduced accuracy and reproducibility. [13][14][15][16] Mainly in microbial cultivations, [17][18][19][20] but also occasionally for biocatalytic applications, [21][22][23] strong influence of physicochemical properties were observed. For instance, changes of sample volumes in MTP and the corresponding change in surface/volume ratios affect possible protein-surface/liquid interactions.…”

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

“…The cofactor solution (6 mmol L − NADPH) was freshly prepared. The assay solution (8:1 parts AcPh and NADPH) was preheated for 5 min before measurement . First, 20 µL Lb ADH solution was given into the reaction wells followed by 180 µL of the preheated assay solution (final concentrations 0.6 mmol L − NADPH and 25 mmol L − AcPh).…”