ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2007, том 62, № 9, с. 979-984
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УДК 543
A SOLID-STATE pH TRANSDUCER FABRICATED FROM SELF-PLASTICISED METHACRYLIC-ACRYLIC MEMBRANE FOR POTENTIOMETRIC ACETYLCHOLINE CHLORIDE BIOSENSOR
© 2007 r. Lim Boon Peng, Lee Yook Heng, Siti Aishah Hasbullah, Musa Ahmad
School of Chemical Sciences & Food Technology, Faculty Science & Technology, University Kebangsaan Malaysia
43600 Bangi, Selangor, Malaysia Received 11.01.2006
A solid-state pH sensor based on self-plasticising film of methacrylic-acrylic copolymer was developed. The sensor was able to detect changes in pH after tridodecylamine ionophore was immobilized together with a lipophilic anionic salt. The pH sensor exhibited almost Nernstian response (57.6 mV pH1) with a linear pH response range of 6-10. It demonstrated a fast response (<2 min) to changes in pH and good selectivity against other common cations such as sodium, potassium, magnesium, lithium and calcium. The sensor has a shelf life of at least 30 days without obvious deterioration in response. By depositing a layer of poly(hydroxylethyl-methacrylate) immobilized with enzyme acetylcholinesterase on top of the pH selective methacrylic-acrylic film, the pH sensor was able to detect acitylcholine chloride (AChCl). The linear response range of the poten-tiometric biosensor to AChCl was dependent on the buffer concentrations used, and for buffer concentration less than 1 mM, the linear response range obtained was 3.98-31.62 |M.
The measurement of pH is commonly carried out using a glass electrode. The popularity of the glass electrode is mainly due to its high selectivity, reliability and a large dynamic pH response range. However, the glass electrode has a major draw back, i.e. it is fragile and easily breakable. This makes the glass pH electrode unsuitable for in vivo analysis especially in terms of biomedical applications. For the same reason, glass pH electrode is less robust in terms of design and cannot be constructed into solid-state sensing devices that do not need any internal liquid. It is due to these many factors that development of non-glass pH sensitive electrodes has become recently an important task.
The intention to find a substitute for the glass pH electrode has led to the use of polymer membranes as a material for the fabrication of pH sensitive electrodes. Polymer based electrode is actually an extension from the liquid membrane electrode. The electrode fabricated from polymer membrane can be designed in various shapes and sizes. The use of polymer has also open up a new dimension in the construction of potentiometry ion-selective electrode as it is now possible for the ele-crode to be miniaturized. Poly(vinylchloride) (PVC) has been the main matrix in the development of pH sensitive electrodes [1, 2] for many years. However, PVC membrane requires a large amount of plasticiser to be incorporated and this made it unsuitable to be used in clinical analysis because of the possible leakage of plasticiser, which is often toxic and harmful to the human body. Thus, plasticiser-free membrane materials are essential for the application of ion-selective sensor in may fields [3].
Recently, several self-plasticising polymer matrices which do not require a plasticiser, have been developed based on methacrylate and acrylate types of co-polymers [4, 5]. These polymers are verstile materials especially in term of solid-state ion sensor fabrication. Not only they can be prepared directly and rapidly by simple photocuring procedures, but more importantly these materials have good adhesion property and are suitable for the design of solid-state sensing devices [6-8].
One application of solid-state pH sensor is as a transducer for potentiometric biosensor in the analysis of acetylcholine. Acetylcholine is a neurotransmitter found in the brain and peripheral nervous system, it has an important function involving learning and memory [9]. Upon stimulation, acetylcholine is released into the synaptic cleft. Acetylcholine has important biomedical implication to human health. A shortage of acetylcho-line can lead to Alzheimer's disease. The enzyme acetylcholinesterase (AChE) plays a role in the hydrolysis of acetylcholine at the synaptic cleft so that the function of a neurotransmitter can be achieved. However, the action of AChE can be irreversibly inhibited by pesticides. This results in acute pesticide toxicity.
AChCl is used in autonomic testing where it acts as an agent to evoke sudomotor reflex axon response or QSART [10]. The concentration of AChCl and its stability is important for such purpose and therefore a rapid analytical technique for the determination of AChCl is required. The determination of AChCl is also important in the rapid screening of pesticides, especially the organophosphates and carbamates group where level AChCl is used as a substrate to evaluate the degree of
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inhibition of the enzyme AChE after exposure to these compounds. The normal method of AChCl determination is reverse-phase HPLC with electrochemical detection from an acetylcholine/choline assay kit [10]. Therefore, such method can not satisfy the requirement of rapid analysis and simple instrumentation setup.
In this work, we have designed an acetylcholine biosensor, which is based on two membranes, i.e. a non-plasticised hydrogen ion-selective methacrylic-acrylic membrane to detect pH changes and a poly(hydroxyl-ethylmethacrylate) hydrogen membrane to immoblise the enzyme AChE. The enzyme AChE layer was deposited on top of the pH transducer to create an all-solid-state potentiometric biosensor. The hydrolysis of AChCl by the enzyme AChE leads to production of acetic acid, which results in a change of pH of the sample. This solid-state potentiometric biosensor can be used for rapid and quantitative determination of AChCl.
EXPERIMENTAL
Materials. Methylmethacrylate (MMA), «-butyl acrylate (nBA), lithium acetate, sodium chloride and potassium chloride were all obtained from Aldrich. Tri-
dodecylamine (hydrogen ionophore 1), sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), agar, sodium hydroxide and calcium chloride were purchased from Fluka. Benzyol peroxide and benzene were supplied by BDH-chemical. Petroleum ether (80-100°C) was from UNIVAR. Acetylcholineterase enzyme (EC 7.1.3) (AChE), acetylcholine chloride (AChCl), poly(2-hydroxylethylmethacrylate) (pHEMA), magnesium chloride, hydrochloric acid, silver chloride and dichlo-romethane were purchased from Sigma. Tris(hydroxym-ethyl) animomethane was from Duchefa. All the chemicals above were of analytical grade and were used as supplied. Doubly deionised water was use throughout the experiments.
Instrumentation. The potentiometric measurements were carried out using an ion meter 420 A plus from Orion. The reference electrode was a double junction Ag/AgCl electrode with 1 M lithium acetate as the bridge solution [11]. The working electrode is a Ag/AgCl electrode (Warner, USA) with a tip diameter of 2 mm. The schematic potentiometric cell used to for the biosensor evaluation is:
Ag/Aga/Tris-HCl(01.M)/Lithium(OAc)(1M)/sample/pHEMA/H+ selective membrane/Ag/AgCl (Reference electrode) (Working electrode)
Synthesis of polymer for a self-plasticizing membrane of the pH transducer. The copolymer was synthesized using solution polymerization initiated by free radicals. This method was adopted with some minor modifications [4]. 2 g of MMA and 8 g of nBA were mixed together with 5 mL of benzene. 0.05 g of benzy-ol peroxide was then added to the mixture as initiator. The mixture was reflux in a fume hood for 8 h at 80-100°C. As the polymerization occurred, the mixture turned viscous. After 8 h, the polymer solution was cooled to room temperature before petroleum ether was added to in. The polymer was precipitated as a sticky solid and all the non-reacted monomers were rinsed from the polymer mass by several portion of petroleum ether. The purified polymer was left to dry for 36-48 h before it is used to cast sensor film.
Preparation of the pH sensor from self-plasticis-ing membrane. A pH selective film was prepared by dissolving 50 mg of the co-polymer together with 7 wt % of hydrogen ionophore and 14 mol % of NaTFPB (relative to the ionophore) in 250 ^L of dichlo-romethane. 10 ^L of the cocktail was drop-coated onto the top of a polished solid Ag/AgCl electrode. The mixture was then dried at 4°C for 24 h to allow a thin film to be formed slowly.
Construction of AChCl biosensor. The biosensor for AChCl was constructed by depositing 5 ^L of pHEMA
solution, which contained 0.3 mg pHEMA in water : diox-ane (4 : 1) mixture with 20U of AChE enzyme added, on the top of the pH transducer membrane and left to dry for 24 h at 4°C. After a thin film of the enzyme layer was formed, the biosensor was rinsed with TrisHCl buffer of pH 7 before use.
Evaluation of solid-state pH transducer response. The pH sensor was characterized by using Tris-HCl buffers of different pH from pH 2-10. This is to establish the linear range of the sensor to pH changes and the Nernstian response behaviour of the sensor. The response was measured as cell electromotive force (emf) or mV in a potentiometric cell described above. The potential of the ion meter was recorded when the reading became stable for more than 2 min. A graph of mV against pH was plotted according to the Nernst Equation to determine the dynamic response range.
The selectivity of the pH sensors was tested using the fixed-interference method (FIM) as recommended by the IUPAC [12] where pH response of the sensor was determined in the presence of 0.1 M of sodium, potassium, lithium, magnesium or calcium ions. The selectivity coefficients of each interference cation were then determined from the plot of mV against pH values. The response of the pH sensor towards different pH solutions was recorded for a period of 30 days to determine the long-term performance of th
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