Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1998-06-26
2004-01-27
Warden, T. Jill (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060
Reexamination Certificate
active
06682648
ABSTRACT:
FIELD OF INVENTION
The present invention relates to an electrochemical method and an associated microchip-based apparatus that can be used to afford voltammetric or amperiometric detection for monitoring immunochemical and/or molecular biology procedures.
BACKGROUND OF INVENTION
To assess the-utility of any chemical reaction, whether it be inorganic, organic or biochemical, the composition and relative quantities of reactants and products must be determined while the reaction is in progress or at its equilibrium endpoint. One specific means of affecting such monitoring utilizes biologic or non-biologic molecules capable of binding to either reactant or product molecules in a structure restricted manner. These analytic techniques are, in general, referred to as immunochemical, in reference to the selective recognition and binding capacity of immunoglobulins, even though substances other than antibodies may serve as recognition molecules. The terms receptor and ligand have been used to more generally describe this area of analytic art. Although diversely applied in basic organic and biochemistry, these techniques have seen their most prolific development in the field of clinical medicine, and relevant generalized principles for measuring the progress of reactions immunochemically, although applicable to many other scientific pursuits, can be illustrated using examples from this field. Here, a multitude of immunochemically formatted tests have been developed for measuring virtually any biologic molecule of clinical importance. Such analytic procedures represent the cornerstones for laboratory studies in toxicology, endocrinology, immunology, serology, microbiology, and enzymology, to name but a few.
The most frequently utilized methods at present, are the enzyme-linked immunosorbant assays (ELISAs). These procedures are applicable to a wide variety of fields such as biotechnology, environmental protection and public health. The performance of these conventional state of the art calorimetric methods of detection suffer from the infirmities of having requirements for optical clarity, photomultiplication, signal digitalization or analog quantitation and transmission, viscosity or background chromogenic neutrality.
In the immunochemistry field, for example, enzyme immunoassays (EIA) and, more particularly enzyme-linked immunosorbent assays (ELISA) are well known in the art and have become important and relatively cost-efficient tools of clinical laboratories for detecting traces of foreign substances, such as antigens or antibodies in body fluids and tissues. (See, e.g. Immunoassay, Diamandis, E. and Christopoulos, T. eds., (1996); Clausen, J., Immunochemical Techniques for the Identification and Estimation of Macromolecules (Laboratory Techniques in Biochemestry and Molecular Biology) Vol. 1 (1989); Tijssen, P., Practice and Theory of Enzyme Immunoassays (Laboratory Techniques in Biochemistry and Molecular Biology), (1985); Principles and Practice of Immunoassay, 2d ed., Price, C. and Newman, D., eds. (1997)—which are hereby incorporated by reference in their entirety.)
Such immunoassays, while generally reliable, depend on sophisticated and extremely expensive optical processes to report their results. Such optical processes are cumbersome because they are expensive, require a clean and unsoiled measurement chamber and their visually rendered signals prevent precise quantitation of results in a simple manner. State of the art optical systems have several drawbacks, in that they generally require optical clarity, photo multiplication, signal digitalization or analog quantitation and transmission, as well as compatible viscosity and/or a neutral optical background. Transparent support media, aqueous or otherwise, may become fouled or turbid and prevent or render difficult any accurate analyses utilizing optical reporters.
Attempts have been made to provide systems other than optical ones to detect antigens in body fluids. Duan, C. et al., “Separation-Free Sandwich Enzyme Immunoassays Using Microporous Gold Electrodes and Self-Assembled Monolayer/Immobilized Capture Antibodies,” Analytical Chemistry, 66/9:1369-77 (1994) discloses a separation-free system aimed at simplifying conventional immunoassay protocols utilizing a gold-plated microporous-membrane which serves as the solid phase for a noncompetitive sandwich-type immunoassay as well as a working electrode of an amperiometric detection system. A capture monoclonal antibody is covalently immobilized by a conventional chemical bonding agent at the gold plated side of the membrane. A model analyte protein as well as an alkaline phosphatase labeled antibody are incubated simultaneously with the immobilized capture antibody. Surface bound antibody is then separately detected from any excess conjugate in the sample by the introduction of an enzyme substrate, such as 4-aminophenol phosphate, from the backside of the membrane which is not gold-plated. The substrate seeps through the membrane and encounters the bound enzyme antibody conjugate at the gold-plated surface. Aminophenol is thus enzymatically generated and detected by oxidation at the gold electrode, the magnitude of the current being a measure of the concentration of analyte in the sample. However, the sensitivity of the system disclosed in Duan is very low, requiring a 20 nA signal compared to 0.1 nA in the present invention. This translates to a 50 times sensitivity advantage when considering actual protein detection limits. The system described by Duan was only capable of detecting protein (human chorionoic gonadotropin) down to a level of 500 ng/l, whereas the novel methodology herein described has shown a 10 ng/l protein detection limit.
In another experiment reported in Meyerhoff, M. et al., “Novel Nonseparation Sandwich-Type Electrochemical Enzyme Immunoassay System for Detecting Marker Proteins in Undiluted Blood,” Clinical Chemistry, 41/9:1378-1384 (1995), a similar microporous membrane was utilized in a non-separation sandwich-type electrochemical enzyme immunoassay system for detecting marker proteins in undiluted blood. However, this method is limited to prostate specific antigen (PSA) measurement in blood. The method described in this reference demonstrates no additional sensitivity when compared to the aforementioned article by Duan. Rather it simply describes the application of the technique to the measurement of an additional protein moiety (prostate specific antigen, PSA). Niwa, O. et al., “Small-Volume Voltammetric Detection of 4-Aminophenol with Interdigitated Array Electrodes and Its Application to Electrochemical Enzyme Immunoassay,” Analytical Chemistry, 65:1559-1563 (1993) have reported on the use of an interdigitated array (IDA) micro-electrode cell in small-volume voltammetric detection of 4-aminophenol. However, Niwa used only alkaline phosphatase and used a sensor with a relatively small sensing area measuring 2×2 mm and relatively large electrodes of width of 3 to 5 &mgr;m, spaced 2 or 5 &mgr;m from each other. Furthermore, their detection range was from 10 to 1,000 ng/ml for mouse IgG molecules above 1,000 nmol/l for p-aminophenol, which is about 100 times less sensitive than the present invention and does not make his technique viable for clinical applications with respect to disease-specific antibody detection and quantification.
H. T. Hang et al., in Anal. Him. Acta, 214:187-95 (1988) describes a system for the electrochemical detection of low molecular weight digoxin in the context of an immunoassay, but registers only currents generated by the oxidation of p-aminophenol.
Likewise in the molecular biology field, it is equally important to determine the composition and relative quantities of reactants and products while the reaction is in progress or at its equilibrium endpoint. One specific means of affecting such monitoring utilizes biologic or non-biologic labeling or reporter molecules capable of binding either reactant or product molecules in a structure-restricted manner. Many procedures commonly performed in the field of molecular
Hintsche Rainer
MacPhee Robert D.
Seitz Rene
Taylor Clive R.
Noguerda Alex
University of Southern California
Warden T. Jill
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