Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1999-10-13
2001-06-19
Prouty, Rebecca E. (Department: 1652)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C435S004000, C435S005000, C435S006120, C435S007200, C435S007300, C435S007710, C435S007720, C435S007800, C435S007900, C435S007910, C435S007930, C435S007600, C435S007400, C435S007920, C205S793500, C205S778000, C205S779000, C205S077000, C422S068100, C422S082010, C422S082020
Reexamination Certificate
active
06248229
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel methods and compositions for the detection of analytes based on changes in the nuclear reorganization energy, &lgr;, of electron transfer process.
BACKGROUND OF THE INVENTION
Electron transfer reactions are crucial steps in a variety of biological transformations ranging from photosynthesis to aerobic respiration. Studies of electron transfer reactions in both chemical and biological systems have led to the development of a large body of knowledge and a strong theoretical base, which describes the rate of electron transfer in terms of a definable set of parameters.
Electronic tunneling in proteins and other biological molecules occurs in reactions where the electronic interaction of the redox centers is relatively weak. Semiclassical theory reaction predicts that the reaction rate for electron transfer depends on the driving force (−&Dgr;G°), a nuclear reorganization parameter (&lgr;), and the electronic-coupling strength (H
AB
) between the reactants and products at the transition state, according to the following equation:
k
ET
=(4&pgr;
3
/h
2
&lgr;k
B
T)
½
(H
AB
)
2
exp[(−&Dgr;G°+&lgr;)
2
/&lgr;k
B
T]
The nuclear reorginzation energy, &lgr;, in the equation above is defined as the energy of the reactants at the equilibrium nuclear configuration of the products. There are two components of &lgr;; “outer sphere” effects (&lgr;
o
) and “inner sphere” effects (&lgr;
i
). For electron transfer reactions in polar solvents, the dominant contribution to &lgr; arises from the reorientation of solvent molecules in response to the change in charge distribution of the reactants. The second component of A comes from the changes in bond lengths and angles due to changes in the oxidation state of the donors and acceptors.
It is an object of the present invention to provide methods for the detection of target analytes exploiting changes in the solvent reorganization energy of electron transfer reactions.
SUMMARY OF THE INVENTION
In accordance with the above objects, the present invention provides methods of detecting a target analyte in a test sample. The method comprises binding an analyte to a redox active complex. The redox active complex comprises a solvent accessible transition metal complex having at least one coordination site occupied by a polar coordination group and a binding ligand which will bind the target analyte. The complex is bound to an electrode. Upon binding, a solvent inhibited transition metal complex is formed and electron transfer is detected between the solvent inhibited transition metal complex and the electrode. The methods also include applying at least a first input signal to the solvent inhibited transition metal complex.
In a further aspect, the invention provides methods of detecting a target analyte in a test sample comprising associating an analyte with a redox active complex. The redox active complex comprises a solvent inhibited transition metal complex, and a binding ligand which will bind the target analyte. Upon association, a solvent accessible transition metal complex is formed, which is then detected.
In an additional aspect, the invention provides methods of detecting a target analyte in a test sample comprising associating an analyte with a redox active complex. The complex comprises a solvent inhibited transition metal complex, a binding ligand which will bind the target analyte, and an analyte analog. The complex is bound to an electrode, and upon association, a solvent accessible transition metal complex is formed, which is then detected.
In a further aspect, the invention provides compositions comprising an electrode with a covalently attached redox active complex. The complex comprises a binding ligand and a solvent accessible redox active molecule, which has at least one, and preferably two or three coordination sites occupied by a polar coordination group, one or more of which may be a water molecule.
In a further aspect, the present invention provides an apparatus for the detection of target analytes in a test sample, comprising a test chamber comprising a first and a second measuring electrode. The first measuring electrode comprises a covalently attached redox active complex comprising a solvent accessible transition metal complex, preferably having at least three coordination sites occupied by a polar coordination group, and a binding ligand. The apparatus further comprises an AC/DC voltage source electrically connected to the test chamber, and an optional signal processor for detection.
DETAILED DESCRIPTION
The present invention provides methods and compositions for the detection of target analytes using changes in the solvent reorganization energy of transition metal complexes upon binding of the analytes, to facilitate electron transfer between the transition metal complex and an electrode. This invention is based on the fact that a change in the oxidation state of a redox active molecule such as a transition metal ion, i.e. upon the acceptance or donation of an electron, results in a change in the charge and size of the metal ion. This change in the charge and size requires that the surrounding solvent reorganize, to varying degrees, upon this change in the oxidation state.
For the purposes of this invention, the solvent reorganization energy will be treated as the dominating component of &lgr;. Thus, if the solvent reorganization energy is high, a change in the oxidation state will be impeded, even under otherwise favorable conditions.
In conventional methodologies using electron transfer, this solvent effect is minimized by using transition metal complexes that minimize solvent reorganization at the redox center, generally by using several large hydrophobic ligands which serve to exclude water. Thus, the ligand for the transition metal ions traditionally used are non-polar and are generally hydrophobic, frequently containing organic rings.
However, the present invention relies on the novel idea of exploiting this solvent reorganization energy to serve as the basis of an assay for target analytes. In the present invention, transition metal complexes that are solvent accessible, i.e. have at least one, and preferably more, small, polar ligands, and thus high solvent reorganization energies, are used. Thus, at initiation energies less than the solvent reorganization energy, no significant electron transfer occurs. However, upon binding of a generally large target analyte, the transition metal complexes becomes solvent inhibited, inaccessible to polar solvents generally through steric effects, which allows electron transfer at previously inoperative initiation energies.
Thus, the change in a transition metal complex from solvent accessible to solvent inhibited serves as a switch or trigger for electron transfer. This thus becomes the basis of an assay for an analyte. Closs and Miller have shown that there is a decrease in lambda in nonpolar solvents in their work on Donor(bridge)Acceptor electron transfer reactions in solution. (Closs and Miller, Science, 240, 440-447, (1988). This idea also finds conceptual basis in work done with metmyoglobin, which contains a coordinated water molecule in the hexacoordinate heme iron site and does not undergo self-exchange very rapidly (rate constant k
22
1M
−1
s
−1
). Upon chemical modification, the heme becomes pentacoordinate, removing the water, and the self-exchange rate constant increases significantly (rate constant k
22
1×10
4
M
−1
s
−1
); see Tsukahara, J. Am. Chem. Soc. 111:2040 (1989).
Without being bound by theory, there are two general mechanisms which may be exploited in the present invention. In a preferred embodiment, the binding of a target analyte to a binding ligand which is sterically close to a solvent accessible transition metal complex causes one or more of the small, polar ligands on the solvent accessible transition metal complex to be replaced by one or more coordination atoms supplied by the target analyte, causing a decrease in the solvent reor
Clinical Micro Sensors, Inc.
Flehr Hohbach Test Albritton & Herbert LLP
Monshipouri M.
Prouty Rebecca E.
Silva, Esq. Robin M.
LandOfFree
Detection of analytes using reorganization energy does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Detection of analytes using reorganization energy, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of analytes using reorganization energy will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2499682