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Optimization of Instrument Settings and Measurement Parameters for Kinetic Fluorometric and Luminometric Assays

Abstract

The speed of reaction kinetics of fluorometric and luminometric assays sets certain requirements for the detection instrument. Fluorometric peroxidase assay, luminometric ATP assay and luminescence reaction of Aequorin and calcium were used as examples for optimizing measurements of different types of kinetic reactions in microplate format. The results present many ways of selecting optimal instrument settings according to the reaction speed.

Introduction

When fluorometric and luminometric assays are performed in microplate format, a high number of samples can be analyzed at a time. The speed of the reaction kinetics determines the requirements for the detection instrument and measurement parameters for achieving the best possible performance. If the reaction is started by adding the starting reagent to microplate wells, the speed of the reaction determines if it’s possible to add the reagent manually or if an automatic instrument dispenser must be used. Some assays include several reagent addition steps, therefore instrument with more than one dispenser may be required.

In some measurements the baseline signal is first measured before starting the reaction with reagent addition. For those assays the user must be able to select from the software settings at which kinetic reading the reagent is dispensed. It must also be possible to choose in which order the kinetic readings of different wells are measured dispensing. If the reaction is really fast, all kinetic readings of the first well must be measured instantly after reagent addition before proceeding to dispense and measure the next well. Otherwise the peak signal may be lost while the other wells are measured. So the consecutive kinetic readings following the dispensing can either be measured by one well, the whole plate or by certain group of wells according to the rate of the reaction.

The dispensing speed should also be selectable. The more viscose the solution is, the faster the dispensing speed should be. The optimal dispensing speed is also important for reagent mixing. If the speed is too low, the reagents may not get mixed well and fast enough. And if the speed is too high, the reagents may splash outside the well.

Methods

All the fluorometric and luminometric measurements were performed with Thermo Scientific Varioskan^® Flash, a spectral scanning multitechnology microplate reader. The instrument was controlled via Thermo Scientific SkanIt^® Software.

Amplex^® Red hydrogen peroxide / peroxidase assay kit (Invitrogen, USA) was used for kinetic fluorometric measurements. In the presence of peroxidase Amplex Red reagent reacts with H2O2 to produce resorufin, which can be detected fluorometrically.

Two ATP detection kits were used for comparison of luminescence kinetics. One was the CheckLiteTM HS Set (Kikkoman Corp., Japan) producing fast flash type luminescence and the other was the ATP Biomass HS kit (BioThema AB, Sweden) producing more stable glow type luminescence. The ATP detection of both of the kits is based on the light producing reaction between D-luciferin and ATP catalyzed by firefly luciferase.

One example of an extremely fast luminescent reaction is the reaction between Aequorin and calcium. Aequorin is a photoprotein complex which emits blue light upon binding calcium. When loaded inside cells it can be used as an intracellular Ca2+ indicator. The luminescence of AquaLite^® Recombinant Aequorin (Invitrogen, USA) was detected by adding Aequorin to microplate wells and the reaction was started by dispensing CaCl2 solution with maximum dispensing speed.

Results

Picture 1 presents two different ways of combining reagent dispensing and signal detection in kinetic measurement of AmplexRed fluorescence. If the starting reagent is dispensed at the first kinetic reading, the detected signal represents only the fluorescence after reagent addition. In case the reagent is dispensed e.g at the fifth reading, the baseline can be detected separately. The software creates the kinetic curves automatically and the baseline signal of each sample can be subtracted from the curve.

Picture 1. Fluorescence kinetics of of Amplex Red hydrogen peroxide / peroxidase assay. The blue curve presents the measurement with dispensing at the first kinetic reading and the red curve presents the measurement with dispensing at the fifth kinetic reading.

The reaction speed of flash and glow type luminescence ATP reactions were compared (picture 2). The flash type luminescence of the ATP reaction reaches the peak maximum in about 4 seconds after reagent addition. After that the light decays rapidly with a signal half-life of about two minutes, therefore the flash type luminescence reaction can not be measured without instrument dispensers. Starting the reaction by manual pipetting decreases the assay sensitivity significantly as the real peak signal is never detected. Also the signal instability indicates that the signal of each well must be detected with constant time difference between dispensing and measurement.

The glow type luminescence of the ATP reaction on the other hand is quite stable. The peak maximum is reached in about a minute after reagent addition. With this type of reaction kinetics it’s possible to add the reagents manually to the whole plate at a time. But reagent addition with instrument dispensers helps to achieve reliable and reproducible assay results also in glow type assays.

Picture 2. Kinetics of flash (A) and glow (B) type luminescence reactions of two ATP detection kits.

Picture 3 presents the luminescence kinetics of Aequorin reaction. As can be seen, the peak maximum is reached instantly in 0.5 second after addition of calcium solution and the signal decays in half in about two seconds. After 10 seconds there’s less than 10 % of the signal left. The measurement of the kinetic readings must be started simultaneously with automatic reagent dispensing or otherwise the peak maximum will never be detected. So the kinetic measurements must obviously be done by one well. The reagent must be dispensed at high speed so that the reagents will get mixed fast and thoroughly. Also the measurement integration time of the kinetic readings must be very short, like 10 ms. With too long integration time the real peak of the curve may be lost.

Picture 3. Luminesecence kinetics of Aequorin reaction.

Conclusions

The reaction kinetics of fluorometric and luminometric reactions affects the way the measurements must be performed to get the best possible results. Optimization of the instrument settings and measurement parameters can improve the assay sensitivity considerably. Instrument dispensers help to achieve reliable and reproducible results and decrease the inaccuracy caused by manual pipetting. Several microplate assays are based on such fast reactions, which can not be measured without automatic instrument dispensers without losing the assay sensitivity at the same time.

With Thermo Scientific Varioskan Flash it’s possible to have up to three dispensers in the instrument for multiple reagent addition. The flexible SkanIt Software allows the user to create kinetic measurement protocols according to the reaction speed. The measurement can be started simultaneously with dispensing, which is essential in really fast kinetic reactions. Also the time of dispensing inside the measurement protocol can be selected. Fast reactions must be measured by one well at a time and the more stable kind of reactions allow the measurements to be performed by the whole plate or in a groups of wells. The dispensing speed can be optimised based on the liquid type and reaction requirements. All these choices help the user to achieve maximal efficiency and high result quality of all types of kinetic microplate assays.

Thermo Fisher Scientific

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