Biosensors based on conductometric detection

The present paper is a self-review on the development of about 20 conductometric biosensors based on planar electrodes and containing different biological material (enzymes, cells, antibodies), bio-mimics or synthetic membranes, including Imprinting polymers, as a sensitive element. Highly specific, sensitive, simple, fast and cheap determination of different analytes makes them promising for needs of medicine, biotechnology, environmental control, agriculture and food industry. Non-specific interference of back­ ground ions may be overcome by the differential mode of measurement, the usage of rather concentrated sample buffer and additional negatively or positively charged membranes, which decrease buffer capacity influence and extend a dynamic range of sensors response. For development of easy-to-use small conductometric immunosensors several approaches seem to be promising: i) the usage of polyaniline as electroconductive label for antibodies detection in competitive electroimmunoassay; ii) the elaboration of multilayer structures with phtalocyanine films; Hi) the usage of acrylic copolymeric membranes. The advantages and disadvantages of conductometric biosensors created are discussed. For future commer­ cialisation our effort are aimed to unite a thin-film technology with membranes deposition and to find the ways of membrane stabilisation, including bio-mimics creation, utilisation of bioaffinity polymeric membranes, imprinting polymers etc.


Introduction. The last decade has seen unprecedented interest in the development of analytical devices for the detection, quantification and monitoring of dif ferent biological and chemical compounds. The dyna mic field of biosensors is covered by extensive number of reviews [1-5].
As a rule a biosensor is a device containing two functional parts: a bios elective membrane in dired contact with a physical transducer, which transforms the biochemical signal into the electrical or optical signal.The amplitude of this signal depends on the concentration of the analysed compound (analyte) in the sample.Biologically active materials used for construction of biosensor systems can be divided into two main groups: catalytic (enzymes, cells, tissues) and noncatalytic or affinity (antibodies, receptors, nucleic acids).Electrochemical (ampero-, potentio-, and conductometric), optical, calorimetric, and acou stic transducers are currently used in measuring systems.
The main efforts in biosensor development are focused on the exploration of the various combi nations of biological components (or their synthetic mimics) with measuring principles, i. e. different transducers.
The conductometric sensors are based on the fact that many biochemical reactions in solution produce changes in the electrical resistance (reciprocal con ductance) due to changes of ionic compounds.Con ductance measurements usually involve the resistance determination of a sample solution between two parallel electrodes.
The biosensors based on conductometric principle seem to be the most advantageous in several aspects: i) thin-film electrodes are suitable for miniaturisation and large scale production using inexpensive tech nology; moreover, noble metals can be changed for cheaper ones, e. g.Ni; ii) they do not require reference electrode, have no light sensitivity, the driving voltage can be small to decrease power consumption substantially; iii) large spectrum of ana lytes of different nature can be determined on the basis of various reactions and mechanisms.
Conductometry is a unique method for direct assay of many enzymes and their substrates.Ac cording to Lawrence [7] five factors allow the appli cation of conductometric methods for enzymatic ana lysis: 1) generation of ionic groups (e. g. amidases):; 2) separation of unlike charges (e. g. decarboxylases):; 3) proton migration (e. g. esterases); 4) changes in the degree of association of ionic groups resulting from chelation (e. g. kinases); and 5) changes in the sizes of charge-carrying groups (e. g. phosphatases), Factors 1 and 2 give rise to very large coefficients and make conductometry the assay method of choice.
Conductometric transducers for biosensing devi ces have been introduced by Watson et al. [12].The device consisted of an oxidised silicon wafer with interdigitated gold electrodes pairs on one surface in a planar configuration.
The transducer was tested for urea detection in human serum samples [12] and for the enzymes penicillinase and acyl amilamidohydrolase activity study [13].
However, there was no sufficient progress in the development of the conductometric biosensors due to some disadvantages, mainly a very strong response dependence on a sample buffer capacity and a pos sible influence of non-specific ions.
Taking into consideration rather attractive featu res of conductometric principle and a simplicity of corresponding device, our efforts were aimed at the elaboration of different conductometric biosensors with improved working characteristics.
In this article we have reviewed our achievements: in the development of conductometric biosensors ba sed on planar electrodes and containing different biological material (enzymes, cells, antibodies), biomimics or synthetic membranes, including imprinting polymers, as a sensitive element.
Enzyme-based conductometric biosensors.The enzyme-based conductometric biosensors can be used for both direct and inhibitory analysis.
In case of direct analysis, the conductometric biosensors for determination of glucose, urea, acetyl choline chloride, butyrylcholine chloride and penicil lin have been developed.The resulting conductivity changes are produced by enzymatically catalysed conversion of different substrates according to the general scheme of reaction: The conductometric enzyme biosensors, emp loying the inhibitory analysis, have been created for organophosphorous pesticides and heavy metal ions determination.The scheme for pesticide detection is based on their ability to inhibit acetylcholinesterase and butyrylcholinesterase activity by phosphorylating the serine OH group in enzymes active sites.In the second case, immobilised urease can be inactivated by heavy metal ions through their direct interaction with the thiol group of the enzyme active site.
The assay protocol included measurement of the biosensor response to a fixed concentration of the specific substrate before and after the incubation of the biosensor for a definite time in a solution con taining inhibitor.

The main analytical characteristics of created biosensors are presented in Table L
The biosensors demonstrated reproducible and stable response to the addition of substrates with a measurement time within 0.5-2 min.The influence of pH, buffer capacity and ionic strength of the samples on the biosensors response has been studied earlier and will be discussed further [14][15][16].

It is worth noting that the urease and cholinesterase-based conductometric biosensors may also serve as reliable tool for the estimation of the overall toxin level in liquid samples [17, 18].
However, all the enzyme sensors presented in Table 1 have been shown to possess strong depend ence of their response on buffer capacity and ionic strength.The possibility to overcome this difficulty has been investigated on the glucose biosensor exam ple [19].The dynamic range of its response is very small and does not exceed 2 mM, the response value decreases 20-fold when the concentration of buffer is changed from 1 to 10 mM.Further increase of the buffer concentration up to 20-40 mM makes the measurement of glucose concentration practically im possible [16,20].The reason for the limited dynamic range of the glucose sensor is a lack of oxygen for biocataiytic oxidation of glucose [21 ].The response dependence on buffer concentration may be explained by some kind of «саггіег-mediated» transport of protons («facilitated diffusion») out of the enzyme membrane in the presence of mobile buffer species [19].
We have studied different additional membranes which may control diffusion of substrates and pro ducts of the biochemical reaction, thus optimising the  The microbial sensor can be used with the same biological membrane several times after 30 min inte rval needed for washing and system stabilising Relative standard deviation of the measurements is 10-12 % for 15 repealed assays.A decrease of the sensor output In slightly buffered sample solutions has been found, The biosensor signal is stable for 12 days, when the system is operated 2 hours daily and then stored at 4 °С.
The results of ethanol determination in different diluted alcoholic beverages by the cell-biosensor crea ted as compared to those obtained by method of gas chromatography are presented in Fig. 5.It is shown, that each value coincided relatively well with that determined by the reference method with a corre lation coefficient 0.9988.
Conductometric immunosensors.Despite the ex cellent sensitivity of E^LISA and fluorescent immu noassay, their application is limited sometimes by short lifetime of labels, necessity of complex and expensive equipment, relatively long time of analysis.So alternative procedures may be of special interest for express immunological testing.
For the development of easy-to-use small conduc tometric immunosensors several approaches seem to be promising: i) the usage of polyaniline as electroconductive label for antibodies detection in competi tive electroimmunoassay; ii) the elaboration of multi layer structures with phthalocyanine films; iii) the usage of acrylic copolymeric membranes.
In the first case the conductometric scheme of a sensor is the same as described above.A non competitive mode of antibodies detection has been employed with the usage of polyaniline (PA) as an electrically conductive label.Various water-soluble forms of PA with different molecular weights and oxidation levels have been compared.The maximum specific response has been observed for the PA- fraction of 45 kDa, synthesised in the presence of 0.2 mM ammonium peroxidisulfate.The sensor res ponse for the specific Ab-PA conjugate has been revealed to be three times larger than for non-specific one.Minimum quantity of labelled antibodies easily detected is 50 ng/ml (Fig. 6).At the competitive mode of antibodies detection the sensor sensitivity is lower, which needs further improvement.
The second approach to conductometric immunosensor creation utilises iodine-sensitive phthalocyanine thin -films [25 ].Excellent sensitivity of the tetra-tert-butyl CuPc (ttb-CuPc) to free iodine is used in an aqueous medium to detect the peroxidase reaction.To minimise interfering effect of aqueous electrolytes Au/Cr inter digitated planar electrodes bearing ttb-CuPc thin-films were overcoated with a hydrophobic gas-permeable membrane.After optimi sing the sensor working parameters the detection of peroxidase-labelled antibodies in the range of 0.1 -2 /ig/ml has been demonstrated.Therefore, for the first time, the chemoresistor based on organic semi conductor, phthalocyanine, has been used as a con ductometric transducer for biosensors operating in aqueous media.
However, phthalocyanines main shortcomings are evident: low conductivity and rather slow response, and this stimulates a search for alternative chemoresistive compounds exhibiting good chemical sensi tivity, high conductivity and faster response.
A new way to detect different antibodies or antigens is based on the usage of copolymers of acrylonitrile with acrylic acid [26,27 ].The films cased from such polymer are stable and flexible, Electroconductivity (resistance) of such membranes is determined by two factors: properties of the polymer surface and material porosity.Immunochemical reac tions between one of the immunocomponents being immobilised within the porous membrane and the other in analysed sample causes changes in the membrane charge distribution and porous structure which results in alteration of the membrane resistance and can be used for development of immunosensors.The response cf such sensor system depends on the polymer composition and conditions of membrane formation.
It has been shown that during Ab-Ag binding the membrane conductivity increases with the increase of analysed Ag concentration.However, the porous stru cture must be optimised to ensure: i) maximum binding of free antigen in solution by biomembrane with immobilised antibody; ii) maximum influence o:f the immunological reaction on the ion transfer, res ponsible for the membrane electroconductivity; iii) occurring of Immunological reaction mainly inside of Using molecular imprinting approach new types of polymeric membranes containing molecular recog nition sites for L-phenylalanine (L~Phe), 6-amino-lpropyluracil (APU), atrazine and sialic acid have been prepared [28 J.The membrane synthesis inclu des radical polymerisation of ethylene glycol dimethacrylate and functional monomer in the presence of template.Several substances-diethyl aminoethyl raethacrylate, methacrylic acid, allylamine, or vinylphenylboronic acid have been tested as functional monomers, able to form covalent, ionic or hydrogen bonds with corresponding templates.After splitting off the template molecules, these polymers have been used as materials for conductometric sensors, specific for a corresponding template (Fig. 9).The sensors created detect the analytes in solution in the range of 1-50 /tiM concentrations (Table 2).
Besides high sensitivity, analytical devices contai ning imprinted polymers are expected to be extremely stable and operate in such conditions, where usual biosensors are ineligible, which is especially important for health and environmental control and protection.