Capacity affinity sensor

Details Capacity affinity sensor Capacity affinity sensor

2000-06-15

2002-08-20

Whisenant, Ethan C. (Department: 1655)

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

C435S283100, C435S285200, C435S287100, C530S350000, C536S023100

Reexamination Certificate

active

06436699

ABSTRACT:

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 [Löfås, 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
10
th 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
−6
to 10
−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
2
and a sensitivity of 10 ng/ml was obtained [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.; Lawrence,
M. Eurosensors X, The
10
th 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.; Königer, 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 &mgr;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 m

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