Yudinà N.Yu., Arlyapov V.A.
Tula State University, Russia, Tula
Development of mediator
amperometric biosensor for rapid analysis of biochemical oxygen demand
To
assess the degree of
contamination of the water is
often used such a parameter
- biochemical oxygen demand (BOD).
Currently, all companies and water treatment plants using for daily analysis of water samples the standard method, the duration of which is
5 days. Alternatives are express
methods for determining the BOD -
biosensors using analyzers
based on the use of microorganisms,
capable of oxidizing a wide range
of organic compounds [1]. An important distinction between this method and the standard technique is to reduce the analysis time from 5
days to 10 - 20 min. Significant progress in the development
of amperometric biosensors has become possible by the use of compounds capable for transferring electrons from the active sites of enzymes to the electrode - electron transport mediators. It is known that the surface localization of enzymes in the membranes of bacterial cells facilitates their interaction with the electron transport mediators. Thus, for the determination of organic compounds in the sample (in particular the definition of the BOD index) can be
used mediator biosensors, based on bacteria Gluconobacier oxydans, contating membrane localized enzymes. Thus, the aim is to develop a mediator amperometric biosensor
for rapid analysis of BOD based on
bacteria Gluconobacier oxydans.
Materials and
methods. Electrochemical
measurements were performed using
galvanic potentiostat
«IPC-micro» («Kronas», Moscow, Russia),
integrated with the PC. Range of recording amperage is 5 nA to 20 uA. To recording the
sensor response using the two electrode system. The working electrode was a carbon paste electrode
with the immobilized bacterial cells G. oxydans, the reference electrode - silver chloride. As electron
transport mediator used ferrocene
at a constant potential 250 mV. Measurements were
performed in phosphate buffer pH 6.0.
As biomaterial were chosen acetic acid bacterial cells Gluconobacter oxydans.
When introducing the substrate into the measurement cell microorganisms oxidize it,
in consequence of
which a change in the current
flowing in the system over time [2].
Results and discussion. Bacteria G.oxydans have wide substrate specificity and are able to oxidize
many alcohols (ethanol, propanol, butanol, amyl alcohol, glycerol, sorbitol),
carbohydrate (D-glucose, D-xylose, D-galactose, D-mannose, D-maltose
L-arabinose, D-fructose, D-ribose), amino acids and other organic substances,
which is a promising for determining BOD index.
A mixture of
glucose and glutamic acid (GGA) at a ratio of 1:1 (w/w) was taken as a model
mixture; it is used as the standard in BOD5 detection in the Russian
Federation and in international practice (BOD5 = 0.68 × CGGA)
[3].
Important
characteristics of the
biosensor are operational and
long-term stability. Operating stability shows resistance
sensor response on
the same substrate concentration during a large number of successive measurements. Answers sensor remained
stable over the 15 measurements, the relative standard deviation was 4.8%.
Long-term
stability characterized the sensor resistance
over a long period of time. Long-term stability of the
sensors, based on bacteria
G.oxydans was 13 days.
To obtain quantitative information
on the content of the analytes in the sample need to know characteristics of biosensor calibration, i.e. the
dependence of the concentration of the analytical signal. The dependence of the
response of the
sensor BOD has a hyperbolic shape and was
approximated by the Michaelis-Menten equation.
To
reduce the error of analysis
is often limited to the use of the linear portion of
the calibration curve. The
numerical value of the upper limit of detectable concentrations of BOD is constant Mihaelesa, K'm
and was 440 ±
40 mg/dm3. The bottom line of the area is the
lower limit of detectable concentrations of BOD, which was 5.1 mg/dm3.
The high value of the lower limit
of detectable concentrations of BOD
(5 mg/dm3) does not
allow to analyze samples of tap water, where the BOD value is in the range 0.5 - 1.9 mg/dm3. But perhaps
express BOD polluted
water, as well as semi-fermented, where BOD value is
greater than 5 mg/dm3. Thus, the range of detectable concentrations
of BOD is 5.1 -
440 mg/dm3.
An important characteristic of this method become its rapidity.
Time of analysis using amperometric sensors BOD
based on G.oxydans bacteria does
not exceed 15 minutes. On the
last step has been made the measurement of
the BOD5 of real
samples of water and semi-fermented
using the developed biosensor bacteria G.oxydans.
It is shown that the use of the mediator biosensor bacteria G.oxydans
for BOD water
samples and semi-fermentation
products are allows data to be highly
correlated with the reference
method (R = 0,9875). Thus, the
mediator BOD biosensor
can be effectively used to determine
the BOD5 in water samples
with BOD values
greater than 6 mg/dm3.
References
1.
Bourgeois W., Burgess J.E., Stuetz
R.M. On-line monitoring of wastewater quality: a review // Journal of Chemical
Technology and Biotechnology. 2001. V 76. P. 337-348.
2.
Deppenmeier U., Hoffmeister M.,
Prust C. Biochemistry and biotechnological applications of Gluconobacter
strains Appl Microbiol. Biotechnol. 2002. V. 60. P. 233–242.
3.
Environmental Normative Documents,
Federative 14. 1:2:3:4. 123-97. Quantitative chemical water analysis. The
method of measurements of biochemical oxygen demand after n days of incubation (BODcompl) in the surface fresh,
subsurface (ground), drinking, waste, and purified wastewaters. Moscow, 1997,
25 pp.
Acknowledgements
The work was supported by the Federal Goal-oriented
Program “Scientific and Scientific-Pedagogical Cadres of Innovative Russia” for
2009–2013, State Contract No 16.740.11.0766 and agreement 14.B37.21.0561