Analytical Enzymatic Reactions in Microfluidic Chips

Abstract

A number of approaches have been proposed and tested to transfer enzymatic reactions into the functional elements of microfluidic chips on the example of the bienzyme bioluminescent reaction involving NAD(P)H:FMN-oxidoreductase and luciferase. Measurement of the catalytic activity of these enzymes (under the influence of pollutants) is the basis of enzymatic bioassay of various liquids. It was found that all of the components of the reaction must be placed in the same cell of the chip to improve the reproducibility of the measurements. The use of starch gel as a carrier for immobilization and gelatin as a scaffold in the reactor of the chip enables the preservation of enzyme activity in the course of sealing the chip at room temperature. It is shown that the components of the reaction should be vigorously stirred in a microfluidic chip reactor to improve the efficiency of the analysis. As a result of the studies, a prototype of microfluidic chip based on the enzymatic bioluminescent reaction is proposed. It is characterized by a detection limit of copper sulfate of 3 μM that corresponds to the sensitivity of traditional lux-biosensors based on living cells. The analysis time is reduced to 1 min, and the analysis can be performed by individuals without special laboratory skills.

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Abbreviations

PMMA:

polymethyl methacrylate

ЕС 50 :

effective concentration of the active substance, causing reduction of the luminescent activity of the bienzyme system by 50%

EDC:

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

FMNH2 :

reduced flavin mononucleotide

NAD(P)H:

reduced nicotinamide adenine dinucleotide phosphate

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Correspondence to I. A. Denisov.

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Original Russian Text © K.A. Lukyanenko, I.A. Denisov, A.S. Yakimov, E.N. Esimbekova, K.I. Belousov, A.S. Bukatin, I.V. Kukhtevich, V.V. Sorokin, A.A. Evstrapov, P.I. Belobrov, 2016, published in Biotekhnologiya, 2016, Vol. 32, No. 5, pp. 69–76.

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Lukyanenko, K.A., Denisov, I.A., Yakimov, A.S. et al. Analytical Enzymatic Reactions in Microfluidic Chips. Appl Biochem Microbiol 53, 775–780 (2017). https://doi.org/10.1134/S0003683817070043

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Keywords

  • bioluminescence
  • luciferase
  • microfluidics
  • microfluidic chip
  • enzymatic bioassay