Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-31
2002-11-05
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C204S403060, C204S412000, C324S071500, C435S034000, C435S035000
Reexamination Certificate
active
06475728
ABSTRACT:
This is a 371 of PCT/FR98/01213 filed on Jun. 11, 1999.
TECHNICAL FIELD
The field of the invention is that of the detection of products, preferably “affinity” biological products, such as nucleic acids, or else biopolymers of a protein nature.
More specially, present invention relates, on the one hand, to a method for the qualitative and/or quantitative analysis of biological substances CBS, preferably polynucleotides PN present in a conductive liquid (solution or gel) medium CL, by means of optoelectrochemical measurements and, on the other hand, to the devices and the affinity sensors intended for implementing this method.
The biological substances more particularly, but not exclusively, for which the invention is targeted are polynucleotides PN. This general term encompasses, according to the invention, molecules composed of at least two nucleotides (oligonucleotides and polynucleotides stricto sensu), including especially the nucleic acids RNA or DNA and any genetic structure comprising them. The invention also relates to the compounds that can be involved in immunological coupling [antigen Ag/antibody Ab] reactions or even Enzyme/Substrate E/S recognition reactions.
PRIOR ART
In order to detect, identify or quantitatively determine these molecules, their bioaffinity properties. have been exploited, namely their particular ability to specifically pair with their complementary species, according to Ag/Ab immunological coupling or E/S recognition PN/cPN genetic hybridization mechanisms. Thus, in the immunological field, methods are known which are based on antigen/antibody coupling and which involve a step of revealing the pairs formed using radioactive, enzyme, fluorescent, colored or similar labels. Such methods are lengthy and complex to implement. In addition, the reactants used are not commonly available and are expensive. Finally, these methods do not allow continuous measurements and even less in vivo measurements. These techniques have been transposed with their shortcomings to the field of the detection of nucleic sequences, labeled in order to be able to be identified and/or quantitatively determined.
In another context, it has been proposed to detect physical signals of any kind that are capable of being induced by nucleotide-hybridization or immunological coupling biochemical reactions. To do this, it is appropriate, first of all, to isolate one type of particular and characteristic signal and then to use a transducer capable of converting said signals into a measurable physical quantity. These signals, may, for example, be produced by a chemical species, a variation in thickness, in optical index or in mass, or even a variation in electrical properties. The transducers may therefore be electrochemical, piezoelectric, optical or electrical sensors. The entire difficulty resides in how to bring out the specific signals from the pairing and how to develop a corresponding transducer which is reliable, sensitive and reproducible.
The invention described in French Patent Application No. 94/08688 falls perfectly within this prior art based on the detection of electrical signals induced by PN/cPN hybridizations or by antigen-antibody reactions, in an electrically conductive liquid medium.
That patent application describes a method for qualitatively and/or quantitatively analyzing biological substances, particularly polynucleotides, antigens, antibodies, enzymes and substrates, in which a multilayer structure comprising a semiconductor wafer is used, said wafer being covered with an insulator whose surface is functionalized by one of the species of the pairs of specifically pairable biological substances mentioned above. According to that method, the semiconductor containing polynucleotides PN to be quantitatively determined or to be identified are biased and the variations in the electrical signals induced by a charge effect phenomenon, directly and essentially related to the pairings of the intended biological substances with their complementary ligands immobilized on the insulator, possibly by means of a sensitive membrane, are collected.
This electrical transduction measurement technique does not require a reaction intermediate, a specific label, or an enzyme reaction. It gives satisfactory results but it nevertheless remains to be improved with regard to simplicity of implementation and capability of taking a series of rapid measurements on various substrates, without using as many sensors as there are different kinds of substrates to be analyzed. According to that technique, the charge-effect phenomenon may be apprehended by measuring the electrochemical impedance of a semiconductor/-insulator/sensitive membrane/conductive liquid structure. According to one embodiment, the field effect due to the variation in the surface charge induced by the pairing may be characterized by means of a biased field-effect transistor on which the variations in the gate/source potential induced by the field effect are measured.
Moreover, technical proposals have been made for the identification and/or quantitative determination of biological molecules, which combine
on the one hand, means for revealing the substances to be quantitatively determined, using radioactive, enzyme, fluorescent or colored labels, redox-potential or pH modifiers, or similar means, and
on the other hand, electrochemical or optoelectrochemical means of transduction.
Unfortunately, such a combination is not of the type to eliminate the drawbacks associated with the revelation-based techniques, nor to improve the electrochemical analysis techniques.
Thus, U.S. Pat. Nos. 4,591,550 and 5,500,188 disclose a method and a device for the determination and quantitative analysis of one or more substances which are contained in a gaseous or solid liquid medium and are capable of modifying the photoresponse characteristics of a photosensitive element, comprising means for recognizing said substances. These means involve a mechanism of revelation by one or more tracer substances capable of modifying the physicochemical characteristics of the analysis medium (pH or redox potential) and/or capable of being distinguished by a radioactive, colored or fluorescent label. It is therefore a hybrid technique which combines optoelectrochemical means of transduction with physicochemical means of revelation.
The device employed in that known method comprises one or more sensors each consisting of a semiconductor wafer (silicon covered with an SiO
2
or silicon nitride insulator layer), the surface of the latter possibly being functionalized by means for recognizing the substances to be identified and/or quantitatively determined. That device is also provided with means for biasing the semiconductor/insulator structure (e.g. a circuit for applying a biasing voltage, said circuit comprising, on the one hand, a counterelectrode and a reference electrode and being connected, on the other hand, to the semiconductor/insulator structure) The device furthermore includes means for irradiating the photosensitive element or elements as well as means for measuring the resulting electrical signals, for detecting and/or for identifying the substances in question.
It should be noted that each photosensitive element comprising the semiconductor/insulator structure is necessarily associated with illumination means, biasing means and measurement means. It follows that the device is extremely complex in its alternative forms aimed at the multiple detection of different substances, both with regard to the structure as it is and to the taking and processing of the measurements and of the resulting signals.
According to this prior art, the insulator layer of the photosensitive elements is functionalized if the substances to be analyzed are PN/cPN, E/S or Ag/Ab affinity systems. In this case, the recognition ligands functionalizing the insulator layer are systematically labeled. This is illustrated especially in the case of DNA or RNA analysis, in which the complementary recognition ligands are labeled with biotin (cf. column 14, lines 49 to 63 of
Martin Jean-Rene
Souteyrand Eliane
Brusca John S.
Dennison, Schultz & Dougherty
Ecole Centrale de Lyon
Kim Young
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