Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-12-31
2002-01-22
Graser, Jennifer (Department: 1645)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C422S082010, C422S082020, C435S004000, C435S006120, C435S007100, C435S025000, C435S026000, C435S820000, C436S518000, C436S532000, C204S157970, C204S402000, C204S403060, C204S435000
Reexamination Certificate
active
06340597
ABSTRACT:
The present application is the national stage filing of and claims priority to International Application No. PCT/EP96/02919, filed Jul. 3, 1996 and Italian Application Serial No. MI95A001441 filed Jul. 5, 1995.
FIELD OF THE INVENTION
The present invention concerns new electrochemical biosensors based on composite transducers containing solid binding makers, prepared by incorporating a biocatalyst into the bulk of said transducers or by applying a biocatalytic layer onto the surface of said transducers. The biosensors of the invention are useful in the determination of the concentration of specific analytes in sample solutions or suspensions.
PRIOR ART DISCLOSURE
A biosensor is a device incorporating a biological sensing element either intimately connected to or integrated within a transducer. Its usual aim is to produce electronic signals which are proportional to the concentration of the specific substance which has to be determined.
Analytical advantages of biosensors consist in their specificity, sensitivity, simple manipulation, rapid response and consequent low costs of analysis. These specific and sensitive devices have been used in medical diagnostics, in monitoring food quality and freshness, in environmental monitoring, in fermentation and analytical control, and so on.
Electrochemical biosensors, especially amperometric ones, play a significant role in the applications of these devices. Amperometric biosensors of the second generation, based on redox reactions, are characterized by the use of chemical mediators instead of molecular oxygen. During the transformation of a substrate by a bioactive material, chemical mediators shuttle electrons from the redox centre of the biocatalyst itself to the indicating electrode and the corresponding amperometric signal is measured. One of the most promising groups of chemical mediators consists of metallocenes, and in particular ferrocenes. The first successful enzyme electrode based on ferrocene was prepared by Cass et al. (
Anal. Chem.
56, 667-671, 1984); in this glucose sensitive sensor, the electrode was prepared by soaking a spongy carbon foil with 1,1′-dimethyl-ferrocene and the enzyme glucose oxidase was chemically immobilized on the surface of this electrode.
Later on, Dicks et al. (
Ann. Biol. Clin.
47, 607-619, 1989) prepared a gold microelectrode covered with a polypyrrol film, on which glucose oxidase and ferrocene were adsorbed. However, these electrodes, having chemical mediators adsorbed on the surface of the device, show poor stability due to the leaking of the mediators out of the transducer.
Since the late 1980s, intensive research activities were devoted to the development of biosensors based on Carbon Paste Electrodes (CPE); a carbon paste electrode is a mixture of electrically conducting graphite or carbon with a pasting liquid, e.g. paraffin oil, silicon oil, Nujol etc.
This kind of electrodes has the advantage of allowing bulk modification of the eletrode material with biocatalysts, as well as with other advantageous components, essential for the effective functioning of the device. Furthermore, bulk modification allows to create sensors with renewable surfaces, so that each measurement can be performed on a new surface of the electrode, thus avoiding the drawbacks due to previuos measurement residuals.
A CPE is commonly prepared by mixing carbon or graphite powder with a biocatalyst, a chemical mediator and optionally a co-factor, and by finally adding one of the above mentioned pasting liquids. The thus obtained paste is packed in a suitable tube, so to obtain a disc electrode (L. Gorton,
Electroanalysis,
7, 23-45, 1995). These biosensors can be used for the detection of different analytes, such as glucose, fructose, galactose, ethanol, glycerol, aminoacids, lactate, xanthines etc., and are based on the corresponding oxidases and co-factor dependent dehydrogenases.
Nevertheless, CPE based biosensors show the following practical drawbacks and limitations:
i) poor mechanical properties, due to their paste creamy character imparted by the pasting liquid, often leading to easy disintegration of the system;
ii) poor compatibility of the paste with the biocatalytic enzyme, due to their opposite chemical properties (an enzyme is usually hydrophilic, while paste is strongly hydrophobic), often leading to phase separation (biocatalyst/transducer);
iii) leaking of the mediator out of the CPE, due to poor compatibility of CPE with the mediator.
Finally, the above mentioned features of CPE hinder or significantly reduce the electron transfer from the biocatalytic site to the electrode.
In alternative to the use of paste matrices for the fabrication of composite enzyme electrodes, direct modification of enzymes with chemical mediators was carried out by A. E. G. Cass and M. H. Smit (Trends in Electrochemical Biosensors, G. Costa e S. Miertus Ed.,
Word Sci. Publ.,
25-42, 1992); in order to prevent the mediator leaking out of the transducer, peroxidase was covalently modified with ferrocenyl groups, by derivatizing the enzyme chains near the active redox centre.
Furthermore, Karube et al. recently described enzyme-immobilized electrodes based on electrically conducting enzymes (EPA 0 563 795), i.e. enzymes to which electrical conductivity is imparted by covalently linking a chemical mediator. The electrical conductivity of such enzymes varies depending upon the amount of presence of the substrate to be detected in a specimen sample. Chemical mediators can be attached through a covalent bond to enzymes, such as oxidases or dehydrogenases, either to the proteic body (for example, to an aminic group of a lysine residue) or to a side chain of the enzyme (for example, to an oligosaccharide chain, to an alkyl chain, to a peptidic chain linked to the main chain, etc.).
Nevertheless, the covalent attachment of a mediator to the enzyme has the great disadvantage of changing the electrochemical properties of the mediator itself, consequently reducing its mobility and affecting its reaction rate with the enzyme. Moreover, the procedures and operative conditions used in the modification of enzymes are rather drastic for biocatalysts; chemical modification can significantly reduce enzyme activity and stability, leading to a consequent decrease or total loss of activity, when naturally low-stable enzymes are used. Furthermore, said chemical modification leads to an increase of production costs.
Therefore, it is felt the need of devising alternative electrochemical biosensors, endowed with higher stability and efficacy with respect to the known devices.
SUMMARY OF THE INVENTION
Now, the Applicant has unexpectedly found new electrochemical biosensors based on composite transducers, comprising at least a solid binding maker, as defined hereunder, able to overcome the drawbacks of the prior art.
The biosensors of the invention comprise:
a) at least an electro-conducting material, in the form of powder or grains;
b) at least a chemical mediator;
c) optionally, a substance capable of sorption of said chemical mediator;
d) at least a solid binding maker, which is a compound in solid state at room temperature, selected from the group consisting of: linear or branched, saturated or unsaturated hydrocarbons, containing from 12 to 60 carbon atoms, preferably from 12 to 30 carbon atoms, optionally substituted with at least a group selected from —OH, —SH, —NH2, —CO—, —CHO, —SO3H, —COOH, —OR1, —SR1, —NR1R2 and —COOR1, wherein R1 and R2 are independently hydrocarbon groups C
1
-C
30
, optionally containing one or more heteroatoms; esters of fatty acids with glycerol; and esters of fatty acids with cholesterol; and
e) at least a biocatalyst, selected from the group consisting of enzymes, cells, cellular components, tissues, immunoproteins and DNA.
In the biosensors of the invention, said biocatalyst (e) can be either incorporated into the body of the composite transducer, made up of components (a)-(d), or applied onto the surface of said composite transducer, in the form of a film layer.
The biosensor of the invention can be optionally
Miertus Stanislav
Stredansky Miroslav
Svorc Josef
Graser Jennifer
Hedman, Gibson & Costigan, P.C.
Hines Ja'Na
Saicom S.r.l.
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