Development of enzymatic bioluminescent bioassay for soil, air and body fluids

Rimatscaya N.V. 1, Ivanova G.V. 1, Chernyaev V.A. 1, Kratasyuk V.A.1,2

1 Siberian Federal University, 79 Svobodny Prospect, Krasnoyarsk, 660041, Russia2 Institute of Biophysics SB RAS, 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia

e-mail: Shmanko_Nadya@mail.ru

Comparative characteristics of bioluminescent assays used to monitor pollution

Type of bioassay Soluble 

coupled 

system 

Luminescent bacteria

«Enzymolum»

Number of components

(simplicity)5 2 3

Duration of assay, min 10 5–30 7

Sensitivity

На примере воздуха

Water 2000 / 3 700 / 3 16000 / 3

Ethanol 700 / 1 1 / 1 250 / 3

Acetone 2000 / 3 1 / 1 >2000 / 3

Storage conditions

2 months at +15C, 3 years

at –18C

6 months at +5C, 1 year

at –18C

2 years at +4 - +25C

The project is directed to development and design of new biophysical methods for contact monitoring of water ecosystems. The results allow expanding the application scopes of bioluminescent enzymatic methods for toxicity detection developed in the REC "Yenisei". In REC “Enisey” there has been developed a platform technology of enzyme-based bioluminescent enzyme system technology (BESTTM ) for ecological monitoring of water media. “Method to measure intensity of bioluminescence with the help of the reagent “Enzymolum” for detection of toxicity of drinking, waste, and treated waste water samples” was certified and the in-lab production of “Enzymolum” reagent was established.

Technology is based on application of highly sensitive enzymes from bioluminescent bacteria. The enzyme reaction instantly signals about presence or absence any toxic compounds in the medium. The main advantages of developed technology are the possibility of express analysis (less than two minutes), high precision and sensitivity, coupling of the components in disposal sensor, and easy handling. This advantages confirm the possibility of successful commercialization of new techniques based on bioluminescent enzyme system technology (BESTTM ).

The aim of the presented project is to expand the scopes of bioluminescent enzymatic bioassay technology for environmental monitoring toxicity in soil, air, and toxicity testing in sports medicine on the basis of bioluminescent enzyme system technology BESTTM. This technology brings together the chain of education-science-technology-market.

To achieve this aim we need to adapt bioassay method to other media, develop methods for sample preparation providing high sensitivity of bioluminescent method, determine connection between the results of bioluminescent enzymatic bioassays and physico-chemical composition of the samples, develop recommendations how to use bioluminescent biotests for the analysis of the new media.

Test-systems

“Enzymolum” Immobilized couple enzyme system

NADH:FMN-oxidoreductase-luciferase

soluble couple enzyme system NADH:FMN-

oxidoreductase-luciferase

Lyophilized Luminescent

bacteria

Т= Iexp/Iс*100%80% >Т > 120% → dilute в 3, 9, 27…times

The results indicate that water is the better than ethanol or acetone medium for air sample preparation because of its sufficient capacity to absorb organic compounds, absence of interfering effects on bioluminescent. The sensitivity of soluble and immobilized enzymes is 3-24 times higher than sensitivity of bacterial-based test. The immobilized reagent provides the reduction of the time required to complete the analysis (down to 7 minutes), easy-to-use, higher sensitivity (allowed dilutions is up to 16000), possibility to increase the volume of the sample up to 97% of the total one. Thus we showed the possibility to apply the bioluminescent bioassays based on immobilized reagent "Enzimolyum" for air pollution monitoring.

This work was supported by the 1) Minigrant CRDF (US Civilian R&D Foundation) # RUX0-002-KR-06/BG2102 «Development of enzymatic bioluminescent bioassay for soil, air and body fluids»; 2) Federal agency of science and innovations (contract No 02.740.11.0766), the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058).

NADH:FMN-oxidoreductase (R)NADH:FMN-oxidoreductase (R)

NADH (NADPH) + H+ + FMN NAD(NADP)+ + FMNH2

Luciferase (L)Luciferase (L)

FMNH2 + RCHO + O2 FMN + RCOOH + H2О + h

TriStar LB 941 (Berthold)

Light

t, s

I, RU

tmax

100

200

Control sample

Analyzed sample

400

200

0300

Iexp

Ic

P

The method of assessment of the toxicity

H2О

C2H5OH

C3H6O

saliva samplesair samples

soil samples