| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 20, 2002 |
| PATENT TITLE |
Capacity affinity sensor |
| PATENT ABSTRACT | This invention describes a capacity affinity sensor based on self-assembled monolayers on an electrode with immobilized recognition elements available to analyte in the surrounding solution. Additional insulation is provided by auxiliary self-assembled molecules. The sensor has exceptional sensitivity and wide operating range due to these parts of the invention. It is versatile because different kinds of recognition elements can be immobilized directly on the surface of the measuring electrode. The electrode then becomes selective to those molecules in the solution, the analytes, that show affinity to the recognition element on the surface. Compared to capacitive sensors described before those described here shows at least a 1000-fold better sensitivity because of the properties of the layer binding the recognition element |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 15, 2000 |
| PATENT CT FILE DATE | September 2, 1998 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | March 25, 1999 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Taira et al., Electrode modification by long-chain, dialkyl disulfide reagent having terminal dinitrophenyl group and its application to impedimetric immunosensors. Anal. Sci., 9, 199-206, 1993.* Duan et al., Separation-free sandwich enzyme immunoaaaays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies. Anal. Chem., 66, 1369-1377, May 1994.* Livache et al., Preparation of a DNA matrix via an electrochemically directed copolmerization of pyrrole and oligonucleotides bearing a pyrrole group. Nucleic Acids Res., 22, 2915-2921, 1994.* Bryant et al., Surface raman scattering of self-assembled monolayers formed from 1-alkanethiols at Ag. J. Am. Chem. Soc., 113, 3629-3637, May 1991. |
| PATENT CLAIMS |
What is claimed is: 1. A capacity affinity sensor for determining the presence of and/or the quantity of a compound of interest in a liquid sample, comprising: a noble metal surface; and a first immobilized and electrically insulating layer having a first measurable capacitance, wherein the first immobilized and electrically insulating layer comprises a first self-assembled monolayer-forming compound having coupled thereto an affinity compound, and a second self-assembled monolayer-forming compound, the noble metal surface being at least 99% covered with the first immobilized and electrically insulating layer; wherein the affinity compound, upon contact with the liquid sample, is capable of specifically binding the compound of interest so as to form a second immobilized and electrically insulating layer having a second measurable capacitance. 2. The capacity affinity sensor of claim 1 wherein the noble metal is gold, silver, copper, platinum or palladium. 3. The capacity affinity sensor of claim 1 wherein the noble metal surface is provided by a piece of the noble metal. 4. The capacity affinity sensor of claim 3 wherein the piece of a noble metal is in the shape of a rod. 5. The capacity affinity sensor of claim 1 wherein the noble metal surface is provided as a layer of the noble metal coating a piece of an electrically insulating material. 6. The capacity affinity sensor of claim 5 wherein the electrically insulating material is glass or quartz. 7. The capacity affinity sensor of claim 1 wherein the first self-assembled monolayer-forming compound is D/L-thioctic acid, activated with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide. 8. The capacity affinity sensor of claim 1 wherein the second self-assembled monolayer-forming compound is a thiol comprising 3-25 carbon atoms in a straight saturated chain. 9. The capacity affinity sensor of claim 8 wherein the thiol is 1-dodecanethiol. 10. The capacity affinity sensor of claim 1 wherein the affinity compound is an antibody, a monoclonal antibody, an antibody fragment or a F(ab').sub.2 fragment. 11. The capacity affinity sensor of claim 1 wherein the affinity compound is a nucleic acid. 12. The capacity affinity sensor of claim 1 wherein the affinity compound is a single-stranded DNA compound. 13. The capacity affinity sensor of claim 1 wherein the sensor is adapted to determine the presence of human chorionic gonadotripin hormone (HCG), interleukin-2, human serum albumin, atrazine, or a DNA sequence. 14. A method for qualitatively or quantitatively determining the presence of a compound of interest in a liquid sample, comprising the steps of: (a) contacting the sensor of claim 1 with a reference liquid not containing the compound of interest and determining the capacitance of the sensor; (b) contacting the sensor with a sample suspected of containing the compound of interest so as to bind the compound of interest and determining the capacitance of the sensor having the compound of interest bound thereto; and (c) calculating the difference between the capacitance measured in step (a) and the capacitance measured in step (b) and optionally calculating the amount of the compound of interest by using prerecorded calibration data. 15. The method of claim 14 wherein the compound of interest is human chorionic gonadotropin hormone (HCG), interleukin-2, human serum albumin, atrazine, or a DNA sequence. 16. A capacity affinity sensor for determining the presence of and/or the quantity of a compound of interest in a liquid sample, comprising: a noble metal surface; and a first immobilized and electrically insulating layer having a first measurable capacitance, wherein the first immobilized and electrically insulating layer comprises a first self-assembled monolayer-forming compound and a second self-assembled monolayer-forming compound, the noble metal surface being at least 99% covered with the first immobilized and electrically insulating layer; wherein upon contact with the liquid sample, the first self-assembled monolayer-forming compound is capable of specifically binding the compound of interest so as to form a second immobilized and electrically insulating layer having a second measurable capacitance. 17. A method for qualitatively or quantitatively determining the presence of a compound of interest in a liquid sample, comprising the steps of: (a) contacting the sensor of claim 16 with a reference liquid not containing the compound of interest and determining the capacitance of the sensor; (b) contacting the sensor with a sample suspected of containing the compound of interest so as to bind the compound of interest and determining the capacitance of the sensor having the compound of interest bound thereto; and (c) calculating the difference between the capacitance measured in step (a) and the capacitance measured in step (b) and optionally calculating the amount of the compound of interest by using prerecorded calibration data. 18. The method according to claim 17 wherein the compound of interest is human chorionic gonadotropin hormone (HCG), interleukin-2, human serum albumin, atrazine, or a DNA sequence. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
Detecting interactions between molecules forms the basis of many analytical methods. The interaction can be detected and quantified through a number of schemes, e.g. precipitation, separation or through different marker molecules or reactions. Such an example is the development of immunoassays during the last three decades, which has revolutionized determination of drugs and hormones in clinical and pharmaceutical chemistry as well as contaminants in the environmental area. Almost all immunomethods require labels attached either to the antibody or the antigen. Another example is the binding between a DNA-probe and its complementary DNA-strand or DNA-fraction. A number of receptors or the complementary molecule can be studied using the same approach. There are a number of disadvantages associated with labels. It they are radioactive the work has to be carried out under strict safety regime and handling of waste is costly. The use of enzymes as labels requires an additional time-consuming incubation step. Common for all labels are that they require a synthetic coupling to either an antigen or an antibody or generally to the recognition element or the analyte. A big label may change the affinity between the molecules which is of particular concern when an assay is performed by. competition between an analyte from the sample and an added labeled molecule. Many affinity interactions cannot be studied because of this. Recognition of DNA-binding through the use of electrochemical intercalators shows low sensitivity. Many attempts have therefore been made to detect the binding itself by potentiometric [Taylor, R. F.; Marenchic, I. G.; Spencer, R. H. Anal. Chim. Acta 1991, 249, 67-70], piezoelectric [Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336], or optical measurements [Lof.ang.s, S. Pure Appl. Chem. 1995, 67, 829-834]. Attempts have previously been made to use capacitance measurements for detecting molecular interactions without the use of labels. A molecule with affinity for the analyte should be immobilized on a conducting electrode surface so that it can interact with the analyte in solution in such a way that the interaction causes a change in capacitance. This principle has been used in immunochemistry, by immobilization to oxide surfaces [Bataillard, P.; Gardies, F.; Jaffrezic-Renault, N.; Martelet, C.; Colin, B.; Mandrand, B. Anal. Chem. 1988, 60, 2374-2379] or for recognition of DNA-sequences [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.; Lawrence, M. Eurosensors X, The 10th European Conference on Solid-State Transducers, 1996, Leuven, Belgium]. Self-assembled monolayers of thiols, sulfides and disulfides on gold electrodes have been widely studied and long-chain alkanethiols are known to form insulating well-organized structures on gold substrates [Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc 1987, 109, 3559-3568]. The binding formed between the sulphur atom and gold is very strong and the formed self-assembled monolayers (SAM's) are stable in air, water and organic solvents at room temperature [Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335]. It has been suggested that microcontact printing [Mrksich, M.; Whitesides, G. M. Tibtech 1995, 13, 228-235] and photolithography [Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992, 114, 4432-4433] can be used to pattern surfaces with functionalized self-assembled monolayers for biosensor production with low cost for a diversity of applications, but until now it has not been possible to produce direct affinity sensors with high sensitivity. Terrettaz et al, Langmuir 1993, 9, 1361-1369, discloses a sensor, e.g. for assaying cholera toxin, where the ganglioside GM1 has been bound to a SAM layer. The detection limit for capacitance measurements using the sensor is somewhere within the range from 10.sup.-6 to 10.sup.-9 M. The article states that capacitance measurements are unsuitable for assaying cholera toxin because the capacitance changes were too small, and hence, the sensitivity is too low. Self-assembled monolayers of thiols on gold, with antigenic terminating groups have been reported before, but they had coverages of only 14, 19 or 31% for different electrodes [Taira, H.; Nakano, K.; Maeda, M.; Takagi, M. Anal. Sci. 1993, 9, 199-206]. The lowest measured value in the article was at an antibody concentration of 10 ng/ml, which can be compared to 1 pg/ml of antigen measured with our invention (See Example 1). The higher sensitivity obtained with our electrode can be explained by that the gold surface is first covered with a self-assembled monolayer of a thiol, sulphide or disulphide giving a high coverage of the surface, therafter the recognition element is immobilized on the surface and as the last step the surface is plugged with another thiol. The saturation seems to occur at similar concentrations in the two cases if the larger bulk of the antibody compared to the antigen is taken into account. This comparison thus supports the arguments given above that a dense layer is of great importance for a high sensitivity. DNA-probes have been immobilized e. g to SiO.sub.2 and a sensitivity of 10 ng/ml was obtained [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.; Lawrence, M. Eurosensors X, The 10th European Conference on Solid-State Transducers, 1996, Leuven, Belgium]. A peptide bound to an alkylthiol was also immobilized as a self-assembled layer on gold, but the antibody concentration was in this case in the mg/ml range making it a less succesful sensor [Rickert, J.; Wolfgang, G.; Beck, W.; Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768]. One of these previous approaches are illustrated in the patent EP 244326. The recognition element is bound to an insulating layer on top of a conducting substrate, the insulating layer typically being an oxide. The oxide layer has to be thick, typically 70 nm on silicon, in order to be stable and sufficiently insulating, resulting in a low sensitivity. It is difficult to obtain good surface coverage on oxides and the recognition elements are not well ordered. Rojas, M.; Koniger, R.; Stoddart, F.; Kaifer, A.; J. Am. Chem. Soc. 1995, 117, 336-343 discloses an assay method for determining ferrocene in a sample using cyclodextrin. All hydroxy groups of cyclodextin are substituted by thiol groups, and the modified cyclodextrins are chemically adsorbed to a gold surface. Empty spaces on the gold surface between the adsorbed modified cyclodextrin molecules are filled with adsorbed pentanethiols. The lowest ferrocene concentration determined is 5 .mu.M. There is always a need for improvements of analysis techniques. Especially when assaying biochemical compounds it is often necessary to be able to determine concentrations below 1 ng/ml. SUMMARY OF THE INVENTION It has now turned out that unexpectedly good capacity affinity sensors, suitable for determining the presence of a certain compound of interest by capacitance measurements using an electrode which can be produced by a method comprising the steps of: a) providing a piece, of a noble metal where said piece optionally can be a rod or, alternatively a piece of insulating material such as glass, silica or quartz, on which a noble metal is sputtred or printed; b) providing a first SAM-forming molecule comprising a coupling group and/or an affinity group specifically binding said compound of interest; c) contacting the piece in step a) with the first SAM-forming molecule in step b), thereby obtaining a self-assembling monolayer on said noble metal surface; d) in case the first SAM-forming molecule does not comprise an affinity group, contacting said self-assembling monolayer on said noble metal piece with an affinity molecule specifically binding said compound of interest, thereby coupling the affinity molecule to the self-assembling monolayer; and e) contacting the piece obtained in step c) or d) with a second SAM-forming molecule, thereby obtaining a noble metal surface that is at least 90%, preferably at least 97% covered with a self-assembling monolayer. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
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