Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-03
2002-08-27
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C204S412000, C205S775000, C427S126300, C427S126500
Reexamination Certificate
active
06440662
ABSTRACT:
The present invention relates to an improved sensor for electronically detecting a binding reaction between molecular structures or a pair of chemical substances, such as oligonucleotides, antigens, enzymes, peptides, antibodies, DNA and RNA fragments.
The present invention further provides a new production method for this improved sensor.
Techniques and sensors for detecting molecular structures and specific substances such as enzywes, peptides, oligonucleotides, antigens antibodies, DNA and RNA fragments in a solution sample are known in the art. In a specific class of sensors, use is made of the principle of measuring the impedance between two electrodes. The absence or presence of DNA-molecules or antibodies or antigens between the electrodes affects the permittivity and/or the conductivity between the electrodes. Various techniques were proposed to measure the presence and/or concentration of a given analyte in a sample solution by using a binding substance element having specific affinity for the analyte. Such specific binding reactions occur e.g. between enzymes and their substrates, antibodies and antigens, between DNA-DNA, between RNA-DNA, or other molecular structures.
Stoner et al in “Adsorption of blood proteins on metals using capacitance techniques”,
J. Phys. Chem
., 74, Mar. 5, 1970, describe a differential capacity measurement for evaluation of protein adsorption on metalic electrodes.
Arwin et al. in U.S. Pat. No. 4,072,576, use an adsorbed polypeptide substrate and establish a capacitive method for the measurement of enzymatic activity and the immunological interaction assay.
Giaever in U.S. Pat. No. 4,054,646, teaches an electrical method that measures the presence of antibodies in a solution, by coating a metallic substrate with an antigen. After the incubation of the electrodes with the sample solution, he measures capacitively the thickness of the molecular sheet, i.e. he distinguishes between mono- and bimolecular layer, by using a mercury drop as a second electrode.
Newman in Patent application W087/03095 discloses a capacitive sensor for chemical analysis and measurement. Said sensor can be used to detect a broad range of analytes including bacteria, viruses, antibodies, antigens, enzyme substrates and hormones. A thin insulating layer is coated on the surface of conductors and a substrate to form an open capacitor. A biospecific binding agent is immobilized on the surface of the insulating layer between the conductors. The dielectric constant of the biospecific binding agent is altered by binding of the analyte being detected with the biospecific binding agent. A similar sensing principle is disclosed in U.S. Pat. No. 5,114,674.
Battailard et al, 1988
, Anal.Chem
., 60, 2374-2379 and more recently, Klein et al, 1995
, Sensors and Actuators
, B 2627, pp. 474-476, show that a metal-semiconductor-insulator device can be used in a similar way as a MIS (metal-insulator-semiconductor) capacitor. The device is immersed in a solution together with a second, reference electrode. By measuring the ac capacity between the metallic layer of the device and the reference electrode, when dc biasing voltages are simultaneously applied, the fiat band voltage of the system is in fact measured in a similar way as in the case of a MIS capacitor. It was proven that the flat band voltage can be modulated by species adsorbed at the insolation-liquid interface. On this principle work the ISFET, ion-sensitive-field-effect-transistor and GENFET, gene-sensitive-field-effect-transistor.
U.S. Pat. No. 4,219,335, issued to Richard Ebersole discusses the use of immune reagents labeled with reactance tags. These tags can be detected ellectrically since they alter the dielectric, conductive or magnetic properties of the test surface.
Simlarly, EP 0 241 771, issued to S. J. Mroczkowski, teaches the detection of metal labeled antibodies by conductometric measurements. When antigens are immobilised inbetween two electrodes, the specific interaction with a metal-labeled antibody is measured by means of resistance decrease of the interelectrode medium.
M. Malmros in U.S. Pat. No. 4,334,880 describes conductivity variation of a semiconductive polymeric layer inbetween two planar electrodes. The said polymer incorporates, in a way or another, certain molecules able to recognize specific analytes. The recognition process induces conductivity changes of the polymeric layer.
Further variations on this central idea of impedimetric sensing appear in the art, EP 0 543 550, EP 0241 771, U.S. Pat. No. 4,453,126, GB 2,137,361, U.S. Pat. No. 3,999,122. Essentially in an impedimetric sensor, certain molecules are immobilised on the top, in between or both on the top and in between a pair of electrodes. Said molecules ‘recognise’ a specific analyte when exposed to a sample solution This recognition process eventually ends up, directly or indirectly, m conductivity and/or permitivity alteration of the space in the neighbourhood of the electrodes. Finally, by measuring the impedance between the two electrodes, a measure of the recognition process can be established.
The problem associated with the so called sensors as referred to above is that to have a good resolution, the immoblised layer should be perfectly homogenous and should not contain holes, which is hard to achieve.
With the advent of the microelectronic technology, there is a continuous effort to use it in order to develop micro-sensors. Sensors realised with microelectronic technology offer advantages such as low-cost production, increased reproducibility of the production process, uniformity, accurateness of detection, and flexibility in development. Such microelectronic sensors can comprise a multitude of individual test sites with reproducible, uniform electrical properties, whereby enhancing the detection sensitivity of the sensor. The test sites can be made with dimensions of the order of the dimensions of the molecules that have to be detected. The spatial limitations are the fabrication technology resolution and the sensitivity of the device which is dictated by the state of the art in instrumentation and the density of probes. A configuration can also be realised wherein the individual test sites can each yield a different type of signal according to the particular molecule which is to be detected in said test site.
Another important characteristic of the microelectronic technology is its planarity: the microelectrodes patterned this way are essentially flat elements. This feature is not a strong point in the impedimetric devices. In a planar impedimetric structure the electric field lines expand more above the device surface and out of the region of intrest in comparison to real 3-D structures. This is a major drawback especially when the region of interest is limited in space, i.e. it is an enzymatic or polymeric membrane or an adsorbed molecular layer at the surface of the structure. Any field line depassing this region of interest, introduces in the impedimetric response a shunting impedance which can be considered as noise for the measurement.
Still, depending on the electrodes geometry, i.e. dimensions and interspacing, a big majority of the total signal is enclosed in a certain region above the surface of the device as shown in FIG.
1
. From the same figure one can deduce that miniaturisation, i.e. L decrease, is crucial in obtaining impedimetric planar structures that probe the space in the very close neighbourhood of the device. An illustration of the dimension down scaling was given by DeSilva et al
DeSilva et al in 1995
, Biosensors & Bioelectronics
, 10, pp. 675-682 report a new biosensing structure that combines a covalent antibody immobilization technique with a simple impedance response method. The biosensor was fabricated by covalently binding anti-SEB antibodies onto an ultra-thin, island-like, electrically continuous, Pt film deposited onto a silicon chip. They register an impedance decrease when the specific interaction with SEB takes place.
However, the reproducibility is low due to the somewhat random behavior o
Baert Kris
Gerwen Peter Van
Rossau Rudi
Brusca John S.
Innogenetics N.V.
Knobbe Martens Olson & Bear LLP
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