In the application of the protein microspheres of the present invention to the field of bioassays, the protein microspheres carry on the surface affinity molecules for specific recognition of and binding to target molecules in a sample. Bioassays such as enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), immuno agglutination assays or DNA, RNA or genomic assays are well known and play an important role in the detection of analytes in research, the human and veterinary diagnostic field, forensic diagnosis, environmental analysis, food analysis and biodefence screening for dangerous substances in the air or in water.
Bioassays are based on the interaction of at least one labelled biomolecule with an analyte (target) to be detected. The label is the means for “visualizing” the interaction. Different kinds of labels are known and give their names to the various techniques mentioned above: enzymes in ELISAs, radio isotopes in RIAs, fluorophores in FIAs, or specific labels for Western, Southern or Northern blots. Other label types include liposomes, latex particles in immuno agglutination assays as well as dyes, mediators, gold particles.
The most important requirements for bioassays are analytical specificity and analytical sensitivity. Analytical specificity is determined by the affinity molecules, or biorecognition molecules, for example in the case of matching of the binding site of an antibody to its antigen (analyte) or the hybridisation of two complementary nucleic acid strands. Analytical sensitivity of a bioassay is also influenced by the biorecognition molecules due to the affinity constant of its biointeraction with the target species. The label acts as a marker indicating that a reaction has taken place between the target and the affinity molecule and can be measured with different techniques:
(i) optically by the measurement of the absorption of a dye or the fluorescent light emitted by fluorophores, or the luminescent light emitted by luminescent or chemiluminescent compounds, or measurement of turbidity caused by the light scattering of agglutinated latex particles;
(ii) radioactively by the measurement of radio isotopes;
(iii) electrochemically by the measurement of mediators or electroactive substances; or
(iv) magnetically by the measurement of magnetic force.
(v) piezoelectrically by the measurement of changes in mass.
Radioimmunoassays, using radio isotopes as labels, are still regarded by many as the most sensitive method. This very powerful technique was introduced in 1959 by Yalow and Berson and represented a new era in analytical chemistry, diagnostics and medicine. Nevertheless, this technique has the disadvantage that the risk of harmful contamination of people and the environment cannot be eliminated altogether because of the radioactive isotopes used.
In the meantime, non-radioactive methods have been developed and improved with the aim of reaching comparable analytical sensitivity. The importance of optical methods based on fluorescence, luminescence and absorption spectroscopy has strengthened over time and is still growing.
ELISA technology uses enzymes as markers to amplify the signal. After the bioassay is performed, the biointeraction of the analyte and the probe is amplified by the production of a high number of dye molecules by one enzyme marker molecule. Enzymes such as like glucose oxidase (GOD, EC 1.1.3.4.), alkaline phosphatase (AP, EC 3.1.3.1) or peroxidase (POD, EC 1.11.1.7) may be used, with turnover numbers of 2000 substrate molecules per second (s−1), 5000 s−1 and 10000 s−1, respectively. Drawbacks of the ELISA technique are the high number of steps involved in the procedure and the length of time needed for substrate incubation.
Fluorescence methods have also been employed in bioassays for many years and continue to be of high interest. All fluorescence based techniques ensure a good sensitivity and a low detection limit of 10−4 to 10−18 M. Special techniques, e.g., “time resolved fluorescence”, chemi- and bioluminescence or techniques based on the energy transfer between a donor and an acceptor molecule can reach detection limits of 10−15 to 10−18 M.
The fluorescence-immunoassays known in the prior art use low molecular weight fluorescent labels with reactive functional linker groups (SOUTHWICK, P. L., et al., Cytometry, 11, pp. 418-430, 1990, MUJUMDAR, R. B., et al., Bioconjugate Chemistry, 4, pp. 105-111, 1993, MUJUMDAR, R. B., et al., Cytometry, 10, pp. 11-19, 1989), fluorescent and dye coloured particles (U.S. Pat. Nos. 4,837,168 and 6,013,531 and international patent application no. WO 95/08772) or fluorophore spiked dendrimers (DE 197 03 718).
It is also known to employ marker-loaded liposomes for signal amplification in immunoassays (U.S. Pat. Nos. 5,756,362, 4,874,710 and 4,703,017). In practice, the sensitivity of these methods is limited by the amount of marker substance which can be incorporated into the liposomes in solubilized form. A further drawback of using labelled liposomes is the limited stability of liposomes.
As mentioned above, it is important in bioassay development to achieve a very high analytical sensitivity, defined as the degree of signal response for a certain change in analyte concentration (slope of the calibration curve). In immunochemical determinations, whether sandwich or competitive assay types, the analytical sensitivity is dependent on the concentration range.
Another two factors affecting the analytical sensitivity are the quantity of sample necessary for the determination and the overall reaction time for the result. The higher the sample volume, and the longer the applied overall reaction time, the lower the concentration of analyte which can be detected and measured. However, in many practical situations, there is not a sufficient volume of sample available (e.g., in pharmaceutical research) or the component is distributed in a very large volume of sample (e.g., antibiotic residues in milk or a biodefensive substance in the air). Therefore, a key challenge is to detect and/or determine very small quantities of substances in the available small sample volumes, or to detect and/or determine a substance distributed at very low concentration in a large sample volume.
Consequently, the applied technology must have high analytical sensitivity, especially in the very low concentration ranges.
In order that the analytical sensitivities of the various known technologies can be compared objectively at very low concentration ranges, CLSI (Clinical and Laboratory Standards Institute and/or NCCLS in the US) has defined analytical sensitivity using 3 terms: Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ). The data for these parameters are estimated and used for comparisons between the various technologies.
To achieve high analytical sensitivities, different biolabel systems have been developed to effect signal amplification, such as enzymes biolabels, organic microcrystal biolabels and colloidal gold labels, etc.
In enzyme biolabel systems, enzyme molecules convert substrates into products with optical or electro-chemical properties. Due to a high turnover rate, such as with horseradish peroxidase, or due to a very large linear enzymatic reaction, such as with alkaline phosphatase, huge amounts of product (signal) can be generated to achieve amplification.
Another approach is the so-called “enzyme cycling” technique to amplify the detection signal which improves the assay sensitivity.
A new class of label utilizing solid particles of signal-generating substances has been disclosed in European published patent application no. EP 1309867. Billions of signal generating molecules present in each solid particle can be released instantly upon exposure to a releasing reagent to create a “Supernova Effect”.
The signal amplification principle of enzyme systems is based on the conversion of enzyme substrates to generate signals that are being released into the bulk phase. The signal amplification principle of solid particle systems is based on generation and release of a large number of signal molecules into the bulk phase of a reaction chamber. However, the release of the signal molecules to the bulk phase results in a partial dilution of the signal molecule concentration and affects the analytical sensitivity.
Also known is a signal amplified bioassay using colloidal gold labelling. Taton et al. (T. Andrew Taton, Chad A. Mirkin, Robert L. Letsinger, “Scanometric DNA Array Detection with Nanoparticle Probes”, Science, 289(8) 1757-1760, 2000) report a signal amplification method based on colloidal gold followed by silver enhancement, in which the colloidal gold promotes the reduction of silver(I) onto the gold particle surfaces, resulting in the accumulation of a large amount of silver metal onto the colloidal gold label. The silver enhancement approach can detect concentrations of oligonucleotides as low as 5 nanomoles per liter.
Another approach of using colloidal gold labels for amplified bioassays is based on bioaffinity-induced aggregation of colloidal gold, which results in a colour change from red to blue that can be observed with the naked eye. The bioaffinity-induced aggregation approach can detect oligonucleotides at concentration levels of 10 femtomoles per liter. The signal amplification principle of colloidal gold labelling is based on the accumulation or aggregation of signal molecules into a concentrated small volume. By comparing the signal amplification ability of the above mentioned label systems, the colloidal gold amplification system with signal molecule accumulation—fixed by another affinity molecule—at a focused area on a solid carrier (e.g., in lateral flow devices) can achieve high analytical sensitivity.