There is an increasing need for rapid, reliable, inexpensive “in the field” methods for detecting and measuring pathogens and biomarkers in living organisms as well as pollutants and contaminants in the environment and in food sources. Immunoassays comprise one category of specific binding bioassays, which generally rely on the affinity of naturally occurring receptors or antibodies for specific compounds. The specific binding pairs employed in immunoassays are either an antigen or a hapten, and the antibody produced in immune response to the antigen or hapten. Another type of bioassay relies on hybridization binding reaction between complementary single strands of DNA or RNA.
Bioassays are of great importance because of their specificity toward analytes present in complex mixtures, and their high sensitivity. Most bioassays involve the use of a fluorescent, chemiluminescent, electrochemiluminescent, enzyme, electrochemical, or radioactive tag on an immunoreactive species which serves as an indicator that an immunospecific reaction has occurred.
Applying a different classification, immunoassays can be divided into two broad categories of non-amplified assays and amplified assays. Non-amplified assays most often involve the use of a tag, such as fluorescent tag, chemical species tag, electrochemical tag, radioactive label, or the like on an immunoreactive species which serves as an indicator that an immunospecific reaction has occurred. Only one tag per occurrence of immunospecific binding is being activated or released for subsequent detection by optical, chemical, or electrochemical means or by detection of radiation. One of the main disadvantages of the non-amplified assays is low sensitivity of assays.
Amplified assays involve amplification of each binding act between the analyte and the immunoreagent. For example, enzyme-linked immunosorbant assays (ELISAs) involve the use of an enzyme covalently coupled to an immunoreactive reagent to serve as an indicator that an immunospecific reaction has occurred. The enzyme is linked to a secondary reagent, which is added to the assay after the initial immunochemical interaction between the analyte and the immunospecific group, such as an antibody. The enzyme is capable of catalyzing a number of concurrencies of color-changing reaction, generating several hundred turnover events of such reaction in a reasonable period of time. The sensitivity of ELISA is due to the number of turnover events the enzyme is capable of during an incubation period with a substrate that is cleaved to a colored product. While ELISA can be extremely sensitive, it is frequently a very time-consuming assay which is difficult to use in the field.
Another type of an amplified assay is electrochemiluminescence (ECL) based assay, where an electrochemical tag is covalently coupled to an immunoreactive reagent and reacts electrochemically to emit light signal to serve as an indicator that an immunospecific reaction has occurred. Yet another type of an amplified assay is an assay based on method of immunoanalysis which combines immobilized immunochemistry with the technique of flow injection analysis, and employs microscopic spherical microcapsules or sacs, such as animal erythrocytes, polymer microcapsules, liposomes, or similar structures as carriers of detectable reagents. For example, liposomes, or lipid vesicles, can be modified on their surface with analytical reagents, and carry in their internal volume a large number of fluorescent or electroactive tags. After the immunospecific reaction has occurred, the liposomes are disrupted or lysed by the contact with the liposome lysing agent and release a large amount of tags per each binding act. The presence of tags is then detected by chemical, optical, or electrochemical means.
Liposomes have previously been reported as useful components for amplified immunoassays. For example, McConnell et al., U.S. Pat. No. 3,887,698, describe the use of liposomes containing stable free radicals in an electron paramagnetic resonance monitored immunoassay. Mandle et al., U.S. Pat. No. 4,372,745, describe the use of liposomes as fluorescer containing microcapsules, useful in an immunoassay. This assay requires the use of a detergent such as, Triton X-100 to break the liposomes and release the fluorescent compound. Liposomes have also been employed as a tags carrier in an immunoassay described by Ullman et al., U.S. Pat. No. 4,193,983. Tags used in this assay included fluorescers, enzymes and chemiluminescent compounds.
Cole, U.S. Pat. No. 4,342,826, describes an immunoassay method which utilizes antigen-marked, enzyme-encapsulated liposomes which are immunospecifically ruptured in the presence of the cognate antibody and an active complement. The assay utilizes the homogeneous phase reaction between the antibody and complement to release the enzyme tag. U.S. Pat. Nos. 6,248,596; 6,159,745; 6,086,748; 5,958,791; 5,789,154; 5,756,362; 5,753,519; and 5,389,523; by Durst and co-authors, further developed Liposome-enhanced amplified immunoassays and test devices for implementation of these assays. The technology described by Durst et al. has a limited multiplexing potential.
The disadvantages of both ECL and Liposome-based assays is the possibility to detect only one type of pathogen or environmental contaminant in each sample. The simultaneous detection of many pathogens or contaminants in a multiplexing assay format is difficult as only very limited number of uniquely detectable amplification tags compatible with the detection means are available. Typically only one tag is available, such as specific fluorescent, electrochemical, chemical, or radioactive tag.
At the same time, ELISA assays can detect multiple pathogens or environmental contaminants on a specially prepared multiplexing plates, but ELISA is a very time-consuming assay which is difficult to use in the field.
US Patent Application 20040009944 by Tam et al. describes methylated oligonucleotides made immunostimulatory in vivo, by encapsulation of the nucleic acid in a lipid particle. This technology applies to drug development.
US Patent applications 20040110220 and 20040072231 by Mirkin et al. describe using custom oligonucleotides for multiplexing assays employing gold nanoparticles with attached oligonucleotides, which are later released and detected. Conjugating oligonucleotides to particles is a complicated process. There are limitations in the amount and size of oligonucleotides which can be conjugated to gold particles.
US Patent application 20030013091 by Dimitrov describes capturing a target pathogen with a long piece of single strand complementary DNA which has many repeating short oligonucleotide sequences. After that Dimitrov proposes to add fluorescent labels attached to complementary short oligonucleotide sequences. The labels hybridize to the long piece of DNA and thus create amplification. This technology has limited amplification potential. Multiplexing is also complicated as discerning many fluorescent labels from each other is necessary.
In many applications, there is a need to perform multiplexing assays for many pathogens, biomarkers, and/or environmental contaminants simultaneously, due to time constraints and limited amount of analyte sample available. The optimal assay system should be fast, reliable, highly sensitive, and quantitative.