Using Spark® Te-Cool™ Module for Optimization of Temperature-Sensitive Assays

A wide range of factors can easily affect scientific results and therefore, it becomes all the more important to ensure that steady experimental conditions, such as temperature and other environmental factors, are defined and created to achieve reproducible and reliable results.

Elucidated by the Van’t Hoff equation, the speed of a chemical reaction largely depends on temperature, but the temperature in global laboratories will definitely vary based on the season, local climatic conditions, and also the time of day (Figure 1).

Example of temperature fluctuations in a laboratory. The temperature fluctuates by around 4 °C, and is higher during the day than in the night.

Figure 1. Example of temperature fluctuations in a laboratory. The temperature fluctuates by around 4 °C, and is higher during the day than in the night.

Multimode readers are extensively employed for both kinetic and endpoint measurements; however, the reader’s measurement chamber temperature is not always the same as room temperature. The system is integrated with many electronics that can cause the temperature in the measurement chamber to increase up to +4 °C over ambient, particularly if the reader has been running for some time.

Traditionally, multimode readers can only be heated using temperature control and cannot be set at ambient temperature or lower. However, this method can create problems for enzymatic assays and other experiments that are sensitive to temperatures, and thus can affect the reproducibility of results over a period of time.

Additionally, during measurements, when the plate heats up even for a few minutes it will affect the precision of the experiment. Now, this issue can be resolved by using Tecan’s Te-Cool temperature control module, the first-ever air conditioning system developed for Spark multimode readers.

The device is very unique and can easily cool the measurement chamber, which means it can be conveniently set at any user-defined temperature between 18 and 42 °C and will ensure steady and continuous conditions in laboratories across the globe and across any season.

Materials and methods

Temperature-dependent signal intensities in an enzymatic assay

In this study, enzymatic temperature dependency was evaluated with a luminescent kinase assay. A reaction buffer containing the luciferase enzyme was taken to which Ultra-pure ADP was added. Next, 20 μl of this reaction mixture was gradually pipetted into the center and boundary wells of a 384-well plate, as shown in Figure 2 for the plate layout. Water was subsequently used to fill the remaining wells.

Pipetting scheme for glow luminescence measurements in a 384-well plate. Wells filled with luciferase and reaction buffer are colored yellow, wells filled with water are blue.

Figure 2. Pipetting scheme for glow luminescence measurements in a 384-well plate. Wells filled with luciferase and reaction buffer are colored yellow, wells filled with water are blue.

Kinetic and endpoint measurements were carried out (Table 1) by equilibrating the plate at 22 °C for 10 minutes, and using a Spark 20M reader, the luminescent signal at various temperatures – 18, 22 (ambient), 26 and 30 °C – was measured.

Table 1. Measurement parameters used for luminescence measurements

Plate type

Greiner® Bio-One 384 Flat White [GRE384sw]

Temperature

18, 22 (ambient), 26 or 30 °C

Measurement mode

Luminescence

Attenuation

Automatic

Settle time

0 ms

Integration time

200 ms

Output

Counts/second

Temperature-dependent enzyme stability

Enzyme stability is another important point in enzymatic assays. In order to show the decreased enzyme stability at increasing temperature, a sequential luciferase assay was carried out, and the purified luciferases from Photinus pyralis (firefly beetle) and Renilla reniformis (sea pansy) were subsequently measured.

After pipetting the luciferases into the wells of a 96-well plate, the plate was shifted into the Spark 20M reader, which was set to various temperatures (18, 22 (ambient), 26 and 30 °C). Each luciferase emitted a luminescent signal, which was sequentially identified for 10 seconds after administering 100 μl of the related substrate (Table 2).

Table 2. Measurement parameters for a double luciferase assay consisting of two sequential luminescence measurements

Plate type

Greiner Bio-One 96 Flat White [GRE96fw_chimney]

Temperature

18, 22 (ambient), 26 or 30 °C

Well-wise measurement

Measurement mode

Luminescence – well wise

Label 1 – Luciferase 1 (firefly luciferase)

Inject

Injector A; 100 μl substrate 1; 200 μl/sec; refill after injection

Wait

3 seconds

Measure

Luminescence

Attenuation

Automatic

Integration time

10,000 ms

Settle time

0 ms

Output

Counts/sec

Label 2 – Luciferase 2 (Renilla luciferase)

Inject

Injector B; 100 μl substrate 2; 200 μl/sec; refill after injection

Wait

3 seconds

Measure

Luminescence

Attenuation

Automatic

Integration time

10,000 ms

Settle time

0 ms

Output

Counts/second

Results

Temperature-dependent signal intensities in an enzymatic assay

The rate of conversion in enzymatic reactions usually depends on the temperature, with increased rates of reaction at elevated temperatures. Such a behavior was seen in an ideal luminescent kinase assay, where a glow luciferase activity was determined at various temperatures, as shown in Figure 3. With each 1 °C increase in temperature, the luminescent signal was found to increase by 10%.

The luminescent signal of an exemplary glow luciferase is temperature dependent. Increasing temperature results in a higher luminescent signal.

Figure 3. The luminescent signal of an exemplary glow luciferase is temperature dependent. Increasing temperature results in a higher luminescent signal.

During kinetic measurements, when the Spark 20M reader was equilibrated to room temperature, the luminescent signal existing in the plates, which were equilibrated to room temperature, was found to be stable (Figure 4A).

When the reader’s temperature was increased, it led to a higher luminescent signal as opposed to the initial values. This is attributed to the heating of the plate. Over a 6 minute-measurement time, the luminescent signal was increased by 78% and 34% at 8 °C and 4 °C above ambient temperature, as opposed to a 2% reduction in the intensity of signal when using the Spark 20M reader at ambient temperature. In addition, it was observed that at higher measurement temperatures, the coefficient of variation (CV) of the results increased considerably (Figure 4B).

Luminescent signal over time in a kinetic measurement. During a measurement time of 6 minutes, the luminescent signal increases due to heating of the liquid in the wells (A). This results in elevated signal variations and higher CVs for the measurement results (B).

Figure 4. Luminescent signal over time in a kinetic measurement. During a measurement time of 6 minutes, the luminescent signal increases due to heating of the liquid in the wells (A). This results in elevated signal variations and higher CVs for the measurement results (B).

Temperature-dependent enzyme stability

Enzyme activity is not always affected by assay temperature. There are certain enzymes that may show modified rates of conversion at various temperatures, while other enzymes will have decreased stability. This phenomenon was shown in a luminescence assay, where both Renilla luciferase and firefly luciferase were sequentially identified during a well-wise measurement at various temperatures of the reader.

Relatively extended integration times were required for this type of assay, which led to a measurement time of nearly 30 seconds for each well and took about 45 minutes for the entire 96-well plate. In Figure 5, it is seen how the luminescent signal for the firefly luciferase reduced over time owing to the instability of proteins at a higher temperature, particularly at 30 °C (A).

Sequential luminescence assay employing firefly luciferase and Renilla luciferase.

Figure 5. Sequential luminescence assay employing firefly luciferase and Renilla luciferase. While the signal of Renilla luciferase is stable with increasing temperature (B), elevated temperature decreases the activity of firefly luciferase (A).

However, it was observed that Renilla luciferase remained unaffected by altered temperatures within the Spark 20M reader (B). On comparing the CV over different plate wells, the impact of temperature on enzyme activity became very clear (Figure 6).

Temperature-dependent decrease in firefly luciferase activity is shown by the higher CV at 30 °C.

Figure 6. Temperature-dependent decrease in firefly luciferase activity is shown by the higher CV at 30 °C.

Conclusion

The above data shows that temperature is a critical parameter, especially for enzymatic assays and generally for chemical reactions. In this analysis, three types of enzymes were used to demonstrate the possible enzymatic temperature dependency.

Temperature change considerably affected two of the three enzymes, leading to reduced stability or modified activity. Therefore, during an experiment, this parameter should be controlled to ensure optimal data quality.

Tecan’s Te-Cool temperature control module for the Spark multimode readers enables both heating and cooling of the reader, even below ambient temperature. This makes it possible to precisely define experimental conditions to meet assay requirements and also improve the experiments’ quality through improved precision and reproducibility for individual measurements. Therefore, the Te-Cool module ensures optimized results all the time, in laboratories all over the world, across any season.

References

  1. Atkins, Peter; De Paula, Julio (10 March 2006). Physical Chemistry (8th ed.). W.H. Freeman and Company. p. 212. ISBN 0-7167-8759-8

Tecan

About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 1, 2019 at 4:47 AM

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