Development of amperometric biosensor for choline determination

D. Yu. Kucherenko, D. V. Siediuko, D. V. Knyzhnykova © 2016 D. Yu. Kucherenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 543.06 + 577.15 + 543.553


Introduction
Choline (2-oxyethyltrimethyl ammonium hydroxide) belongs to the vitamin-like substances [1].It is an important ingredient for the nervous system due to the synthesis of neurotransmitter acetylcholine.The m onitoring of choline in blood serum is helpful to identify neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, myasthenia gravis and disruption of cholinergic neurotransmission [2].
The choline deficiency leads to delays in the development and growth of organism, an increase in blood cholesterol level, fatty infiltration of the liver, varicose veins, high blood pressure and excess body weight [1,3,4].
In clinical analysis, the choline quantification in biological samples is of great importance.Spectrophotometry, gas chromatography [5], mass spectrometry and chromatography-mass spectrometry [6] are known as mostly used physical and chemical methods for the choline identification.However, they are also known to have significant drawbacks: complicated and expensive equipment, requirement for qualified personnel, and laboriousness.
The way to overcome these challenges is the usage of new bioanalytical devices -biosensors.Many enzyme-based sensors have been developed for the choline detection, such as the amperometric enzyme sensor, electrochemiluminescent biosensor, amperometric aqueous sol-gel biosensor and chemilumines-

Molecular and Cell Biotechnologies
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32. N 3. P 229-234 doi: http://dx.doi.org/10.7124/bc.000924cent flow sensor [7].The amperometric biosensors are considered the most promising and successful among the electrochemical biosensors.Thus, this work was aimed at the development of an amperometric biosensor for the quantitative analysis of choline in aqueous solutions.Moreover, this biosensor could be a part of the biosensor array for the simultaneous detection of several substances in future investigation.

Materials
In the work we used the enzyme choline oxidase (ChOx), activity 15 unites/mg, from Sigma-Aldrich (Japan).Bovine serum albumin (BSA, fraction V), glycerol, HEPES, 50 % aqueous solution of glutaraldehyde (GA), choline chloride and m-phenylenediamine were obtained from Sigma-Aldrich Chemie (Germany).KH 2 PO 4 and other compounds were of Ukrainian production and of analytical purity grade.

Preparation of bioselective elements
In the work, the polyphenylenediamine (PPD) membrane was prepared by the method described in [8].The procedure was as follows.The bare working electrodes were immersed in 5 mM m-phenylenediamine solution, afterwards 10-12 cyclic voltammograms were obtained.Next, the enzyme membrane was deposited onto the PPD membrane.
The bioselective elements of biosensors were obtained by immobilization of the enzyme and additives on the surface of amperometric transducer with the PPD membrane .
The gel stock solution for preparation of the bioselective membrane contained 8 % (hereinafterw/w) of ChOx, 4 % of BSA, 10 % of glycerol in 100 mM phosphate buffer, pH 6.5.Glycerol was added to stabilize the enzymes during immobilization to prevent early drop drying and to improve the membrane adhesion to the transducer surface.Before deposition onto the transducer surface, the enzyme gel was mixed with 1.6 % aqueous solution of glutaraldehyde (crosslinking agent) in a 1:1 ratio.
Immediately thereafter, the mixture was deposited onto the working surfaces of transducers and kept for 10 min in air at room temperature.After immobilization, the biosensors were washed in the working buffer from unbound components of a bioselective membrane and excess glutaraldehyde.

Measuring procedure
Three-electrode scheme of amperometric analysis was used.Platinum disc electrodes of our own fabrication [9] served as amperometric transducers.The working electrodes, auxiliary platinum electrode and Ag/AgCl reference electrode were connected to the potentiostat PalmSens (Palm Instruments BV, Netherlands).The 8-channel device (CH-8 multiplexer from the same manufacturer) connected to the potentiostat allowed simultaneous registration of the signals from eight working electrodes, but actually only two or three working electrodes were attached.
The measurements were carried out at room temperature in an open measuring cell, 3.5 ml in volume, with constant stirring at a constant potential of +0.6 V vs Ag/AgCl reference electrode.The working buffer was 25 mM HEPES, pH 7.4.All experiments were performed in triplicate.The substrate concentrations in the working cell were set by adding aliquots of the substrate stock solution.

Principle of biosensor functioning
The functioning of amperometric biosensor for the choline determination is based on the enzymatic reaction (1), which takes place in a bioselective membrane resulting in choline oxidation and formation of electrochemically active hydrogen peroxide.While applying a positive potential, the hydrogen peroxide decomposion occurs on the electrode (2), which causes the current changes directly recorded by the amperometric transducer.

Choline oxidase
(1) H 2 O 2 2H + + O 2 + 2e - First, the biosensor was submerged in the 3.5 ml measuring cell filled with 25 mM HEPES buffer (pH 7.4) and kept there for several minutes to obtain a stable baseline.Then aliquots of the stock choline solution were added and the signals were obtained.The data were automatically processed and graphically displayed on the computer monitor.

Choice of optimal conditions of choline oxidase immobilization
The procedure of bioselective element immobilization is an important factor, which affects analytical characteristics and operation of the biosensor.The functioning of enzyme biosensors depends on the composition of bioselective element (the enzyme and GA concentrations and their ratio) and time of the immobilization.
As the first step, an optimal GA concentration was determined.The bioselective elements were prepared with the GA mass fraction in the membrane of 0.1 %, 0.2 %, 0.4 %, 0.8 %, 1 % and 2.5 %.At low GA concentrations, the biosensor responses were low (Fig. 1), and they quickly decreased during repeated use.This was due to the weak binding of the enzyme and its rapid leaching out the bioselective membrane.At high GA concentrations, the responses to choline were also low, this time because the cross-linking was too strong and the enzyme could not normally interact with the substrate.
In the further work, the membrane contains 0.8 % of GA since at this concentration the biosensor responses to choline were the highest and most stable during the measurement.
Another important parameter, which influences the biosensor characteristics, is the enzyme concentration in biomembranes.To determine the optimal enzyme concentration, the bioselective elements with ChOx mass fractions of 1 %, 2 %, 4 % and 6 % were used.As seen (Fig. 2), the highest responses to choline were observed at concentrations of 4 % and 6 %.Since these response values were about identical, in the further experiments the ChOx concentration in the bioselective element was 4 %.
The biosensors were also tested depending on the duration of bioselective element formation ranging from 5 to 40 min.The highest responses to choline were observed when immobilization lasted 10 min, at longer procedure the responses were significantly lower (Fig. 3).

Reproducibility of biosensor responses
Reproducibility of the biosensor response is one of the main characteristics of the efficiency of its operation.Therefore, the next step was to test the reproducibility of biosensor response during continuous operation.For the purpose, the responses to choline concentrations of 10 µM, 100 µM and 1000 µM were measured over one working day (Fig. 4).One measurement lasted about 5 min, interval between measurements was about 10 min (during this time the biosensor was washed from the substrates changing the working buffer several times).
No notable decrease in the response value was observed during 4 h continuous operation (15 measurements); the relative standard deviation was 5 -7 %.

Selectivity of biosensor
The mediatorless biosensor with relatively high operating potential (+0.6 V vs Ag/AgCl reference electrode) was used, which made possible oxidation of a number of electroactive compounds (for example, ascorbic acid, uric acid and other) on the electrode surface.Therefore, the selectivity of amperometric electrode was improved by deposition of an additional polymer membrane, which limits the diffusion of interfering substances to the electrode surface.In the work, the poly-m-phenylenediamine (PPD) membrane was used.
To confirm the improvement of selectivity of the modified transducer, the electrodes sensitivity towards interfering substances was tested.The bare transducer reacted to certain substances quite strongly, which could be a problem while analyzing the real samples.The deposition of PPD membrane resulted in essential decrease or even complete absence of the biosensor responses to interferents whereas the sensitivity to hydrogen peroxide remained almost the same (Table 1).It was also found that the reproducibility of the biosensor signals to choline is significantly improved with the PPD membrane deposition due to better adhesion of biomembrane to the electrode.

Analytical characteristics of biosensor for choline determination
The linear working range of the biosensor and minimum limit of choline detection were determined.
The minimum limit of choline detection was defined as the choline concentration, the biosensor response to which was three times higher than the