Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2001-05-29
2004-08-03
Noguerola, Alex (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
Reexamination Certificate
active
06770190
ABSTRACT:
The invention relates to methods of electrochemical detection of analytes and to sensing electrodes for use in methods of electrochemical detection.
Electrochemical analysis of analytes, such as various antigens, antibodies, DNA molecules etc, in biological fluids using biosensors is one of the most promising and attractive methods of instrument analysis. The sustained interest and large number of publications in this field are explained by a number of basic advantages of the method, namely high sensitivity, simplicity and the use of relatively simple and inexpensive equipment.
It is known in the art to construct biosensor devices based on the use of electroconductive polymer films, such as polypyrrole or polythiophene, which transduce a chemical signal associated with the presence of an analyte into a measurable electrical signal (see [1] and [2]).
EP-A-0 193 154 describes an electrode for use in electrochemical detection, the electrode being coated with a polypyrrole or polythiophene film. Bioreceptors complementary to the analyte to be tested are adsorbed onto the surface of the electroconductive polymer film after polymerisation. WO 89/11649 describes an alternative process for the production of polymeric electrodes for use in electrochemical assays. In this process bioreceptor molecules having the desired binding specificity are incorporated into a film of electroconductive polymer during polymerisation. Using the processes described in EP-A-0 193 154 and WO 89/11649 for each given assay it is necessary to synthesise a different sensing electrode having immobilized bioreceptors capable of specifically binding to the analyte for which one wants to test.
The applicants' published application PCT/GB98/00548 describes a potentiometric method of electrochemical analysis using an electrochemical sensing electrode comprising a metallic potentiometric electrode again coated with a layer of electroconductive polymer containing immobilised bioreceptor molecules which bind specifically to the analyte under test. The presence of analyte is indicated by a change in surface charge of the sensing electrode upon binding of analyte to the immobilised bioreceptors. The analyte detection procedure is carried out by first assembling an electrochemical cell comprising the sensing electrode and a reference electrode connected together by means of a measuring device immersed in a working buffer solution of fixed pH. A base value of potential difference between the sensing electrode and the reference electrode is recorded, the sensing and reference electrodes are then brought into contact with a solution of higher ionic strength suspected of containing the analyte but with pH the same as the working buffer and potential difference is again recorded. The sensing and reference electrodes are finally transferred to clean working buffer and potential is again recorded. The change in potential difference between the sensing electrode and the reference electrode resulting from a change in ionic strength of the buffer at constant pH in the presence of analyte is proportional to the concentration of analyte.
As shown in references [13, 14, 15, 16] and in [3], the response (amount and rate of potential change) of the sensing electrode with polymer film containing bioreceptors to a step-change in ionic strength of the ambient solution (so-called “ion-step” procedure) is to a large extent determined by the charge on the polymer film. Apart from the material from which it is made, the polymer film charge is determined by the charge of the receptor molecules bound in it. If the receptor charge changes as a result of an affinity reaction with a specific analyte, the response of the sensing electrode will also change as a result of the ion-step procedure carried out after contact between the sensing electrode and the test fluid. It should be noted that, because of the amphoteric nature of the majority of analytes, the receptor charge depends on the pH of the solution and it is therefore very important to maintain a constant pH of the solution during the ion-step procedure.
Thus, in the previously described methods, based on measurement of the change in response of the sensing electrode to the ion-step procedure carried out before and after contact between the sensing electrode and test fluid, it is possible to make a determination as to the presence in the test fluid of analyte specific to the receptors bound on the sensing electrode. In the ideal case, the variation in the charge of the receptors in the membrane and, hence, the change in the sensing electrode response is directly proportional to the concentration in the test fluid of analyte specific to the receptors bound on the sensing electrode. However, in real conditions, the charge of the same analyte can vary considerably, which produces inconsistent quantitative results. Moreover, the affinity reactions are not always accompanied by a change in receptor charge. This normally occurs when testing small or non-charged antigens [14].
In summary, the shortcomings of the prior art methods of electrochemical detection based on the use of electroconductive polymer electrodes include complexity and limited amenability to industrialisation of the sensing electrode manufacturing process, inconsistency of the characteristics of the sensing electrodes obtained, limited ability to store the sensing electrodes without loss of performance. In addition, the previously described protocols for electrochemical detection, particularly the method described in PCT/GB98/00548, are of limited use for the detection of small and non charged molecules or molecules whose isoelectric point is close to the isoelectric point of the receptors immobilised on the surface of the sensing electrode.
The present invention provides a method of electrochemical analysis of an analyte in a sample which is to a large extent free of the shortcomings inherent in the methods described above in that it widens the scope of application by virtue of the ability to analyse small and non-charged molecules, provides strictly quantitative results, makes the electrode manufacturing process more amenable to industrial production methods, increases the productivity of the analysis, improves reproducibility and therefore enhances reliability of the results obtained.
Thus, in a first aspect the invention provides a sensing electrode for use in methods of electrochemical detection of an analyte, the sensing electrode comprising an electrically conductive electrode coated with an electroconductive polymer with adaptor molecules selected from the group consisting of avidin, streptavidin, anti-FITC antibodies and a molecule capable of binding to at least one class of receptor molecules immobilised therein or adsorbed thereto.
One of the principal problems inherent in electrochemical analysis methods using sensing electrodes is the problem of retention over time of the native properties of the receptors fixed on the sensing electrodes. Relative progress has been achieved in this field only for a limited number of enzyme sensing electrodes [7]. For the majority of electrochemical sensing electrodes using refined receptors known in the literature [8, 9, 10], their useful storage life is simply not stated. The retention of the native properties of immobilised receptors is particularly critical where antibodies are used as the receptors, which is attributable to their inherent high degree of conformational variability.
In contrast, it is known that antibodies and other biomolecules retain their useful properties over very long periods of time when stored in the form of concentrated solutions; therefore the problem of prolonged storage of the sensing electrodes without loss of working characteristics may be overcome by rapid immobilisation of receptors before use or even during the electrochemical detection procedure.
This problem is solved in the declared invention by use of so-called adaptor molecules which are immobilized in or adsorbed to th
Biryukov Nadejda
Biryukov Yuri Sergeyevich
Cherkasov Vladimir Rurikovich
Farmakovski Dmitri Alexandrovich
Leonardova Olga
Biryukov Nadejda
Noguerola Alex
Sensor-Tech Limited
Wolf Greenfield & Sacks P.C.
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