Devices and methods for detecting analytes using...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

Reexamination Certificate

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C422S051000, C422S051000, C422S051000, C422S067000, C422S068100, C204S193000, C204S280000, C204S400000, C204S403060, C204S412000, C435S004000, C435S007100, C435S007710, C435S007720, C435S007930, C435S007940, C435S287100, C435S970000, C435S975000, C436S501000, C436S519000, C436S518000, C436S524000, C436S538000

Reexamination Certificate

active

06670115

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to devices comprising electrosensors containing capture reagents, their preparation thereof, and their use for detecting, preferably, quantitative measurement, of analyte in a liquid sample. In particular, the invention relates to an enzyme electrosensor, e.g., electroimmunosensor, device for electrochemical detection and preferably, real-time measurement, which is suitable for use at point-of-care settings by unskilled personnel.
BACKGROUND OF THE INVENTION
There is an increasing public awareness of the need for diagnostics to determine levels of various components in human fluids, such as blood or serum. Of particular interest are tests designed for non-expert use that produce rapid and quantitative results.
Immunoassays have been widely used for the detection of antigens and antibodies. The most commonly used immunoassays are enzyme immunoassays (EIAs). The importance of EIAs, particularly in clinical analyses, medical diagnostics, pharmaceutical analyses, environmental control, food quality control, and bioprocess analyses, lies in their high sensitivity and specificity, which allow the detection of a wide spectrum of analytes in various sample matrices.
EIAs are commonly referred to as either heterogeneous (necessitating free antigen separation from those that have been bound to antibody) or homogeneous (requiring no separation or washing steps during the assay). Also, EIAs can be either competitive or non-competitive, depending on the availability of antibody binding sites. Conventional EIAs are convenient for analysis of great numbers of samples on a routine basis and are widely used in a broad spectrum of applications. However, these methods require multiple washing and incubation steps to implement, and can be utilized in high volume only by complex and expensive analytical equipment. The need for multiple washing and incubation steps has also limited the development of portable point-of-care analytical devices that can be used to perform assays in decentralized locations.
In recent years, efforts have been made to overcome the limitations of heterogeneous EIAs and to search for homogeneous, rapid, and separation-free immunoassays that can be readily conducted at the point of care. Fast and simple EIA tests capable of detecting a single analyte with a color change that can be visually interpreted have been developed. Based on the techniques of immobilizing antigen or antibody on a solid-phase support, assay formats such as dipsticks, test tubes, and wicking membrane test cartridges have been used to provide fast results for analytical conditions where a simple qualitative (yes
o) answer is clinically relevant. These membrane-based assays have gained increasing popularity in many areas of clinical chemistry. They not only form the basis of the majority of home use tests, but also are rapidly gaining use in the physician's office and hospital lab. These tests are widely accepted and increasingly used for detection of pregnancy, strep throat, and bacteria, as well as for prediction of ovulation. Examples of such assays are described in U.S. Pat. Nos. 5,622,871, 4,703,017, 5,468,647, 5,622,871, and 5,798,273. However, most of these rapid tests are incapable of performing sensitive and quantitative detection. As a result, medical diagnoses that require quantitative measurement of the target analyte remain within the domain of the complex immunoassay analyzers in the centralized laboratory.
A major trend in the development of rapid immunoassays is the move toward quantitative testing. The use of membrane-based immunoassays has been proposed for quantitative measurement of analytes. As a specific example, U.S. Pat. No. 5,753,517 describes a quantitative immuno-chromatographic assay utilizing antibody-coated particles, independent control particles, and capillary flow through a membrane. However, there are difficulties in developing such quantitative immunoassays based on membrane format for point-of-care diagnostic tests. Perhaps the most significant problems with the use of membrane-based immunoassays arise from requirements for the membrane that are contradictory. For example, immobilization of protein in the detection area requires that the membrane have a strong binding affinity for the protein, but transport of analyte and particles containing detection components demands that the membrane not bind to protein. Furthermore, factors commonly used for increasing the performance of the membrane assay are often mutually exclusive. For example, blocking reagents that reduce nonspecific interactions usually also reduce the amount of specific signal. In light of these competing requirements commonly seen in efforts to develop membrane-based immunoassays, it becomes clear that conventional membrane systems have limited advantages for use in quantitative immunoassays.
Accordingly, there is a need to develop improved assays, e.g., immunoassays, that can provide rapid, quantitative, and reliable results. The high sensitivity of electrochemical detection coupled with the inherent specificity of antibody-antigen reactions has resulted in a remarkable technique known as electrochemical immunoassay. The advantages of such assays include, among others, the ability to measure untreated samples in the presence of possible interfering substances, as well as the simplicity, and sensitivity associated with electrochemical detection.
Immunoassays employing amperometric electrochemical detection have been applied to the determination of analytes in fluid samples. An immunoassay device using amperometric detection to perform diagnostic tests for analytes in body fluids is described, as a specific example, in U.S. Pat. No. 5,830,680. The device includes an electrochemical detection system for a separation-free sandwich-type immunoassay, in which a protein analyte such as human chorionic gonadotropin (hCG) is sandwiched between a capture antibody immobilized on a microporous membrane gold electrode and an alkaline phosphatase-labeled antibody. Although such a device offers a separation-free feature, the time required for manipulating and incubating the sample limit the use of such assays for rapid diagnostic testing.
A method employing liposomes for signal production and electrochemical detection in immunoassays is described in U.S. Pat. No. 5,756,362. In these assays, liposomes that encapsulate an electroactive marker are conjugated with an analyte. A test device first allows incubation of a sample containing an analyte with a binding material specific for the analyte and the analyte-liposome conjugate. Following incubation, the mixture solution is allowed to traverse through an absorbent material strip to reach an electrochemical measurement portion where the liposome is lysed by a lysing reagent to release the electroactive marker. The amount of marker released is then detected electrochemically and correlated with the amount of analyte in the sample.
In the methods described in U.S. Pat. No. 5,391,272, bioactive components are coated onto colloidal gold and subsequently coated onto a sensor. Detection of analyte is achieved by measuring current generated by an electroactive species bound to the sensor as part of an analyte/enzyme catalytic response. Although the method is suitable for detecting several types of analytes (e.g. hormones or herbicides), it involves separation and incubation steps in order to achieve desirable sensitivity.
Other immunoassays using electrochemical detection have to rely on methods conventional in heterogeneous immunoassays, such as lengthy incubation time and multiple washing steps to separate free antigen and detection reagent from bound ones. Although several groups have reported methods for performing non-separation amperometric immunoassays, to date there have been no reports describing an amperometric immunoassay that is simple, rapid, and does not require a separation step.
Accordingly, there is still a need in the art for assay devices and methods that provide simple, quantitative and real time dia

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