Sensitive Detection of Cardiac Biomarkers Using a Magnetic Microbead Immunoassay.

To achieve improved sensitivity in cardiac biomarker detection, a batch incubation magnetic microbead immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I. A sandwich immunoassay was performed in a simple micro-centrifuge tube allowing full dispersal of the solid capture surface during incubations. Following magnetic bead capture and wash steps, samples were analyzed in the presence of a manipulated magnetic field utilizing a modified microscope slide and fluorescent inverted microscope to collect video data files. Analysis of the video data allowed for the quantitation of myoglobin, heart-type fatty acid binding protein and cardiac troponin I to levels of 360 aM, 67 fM, and 42 fM, respectively. Compared to the previous detection limit of 50 pM for myoglobin, this offers a five-fold improvement in sensitivity. This improvement in sensitivity and incorporation of additional markers, along with the small sample volumes required, suggest the potential of this platform for incorporation as a detection method in a total sample analysis device enabling multiplexed detection for the analysis of clinical samples.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.

Emerging technologies for biomedical analysis.

Options for biomedical analysis continue to evolve from many fields of study, employing diverse detection and quantification methods. New technologies in this arena focus on improving the sensitivity of analysis and the speed of testing, as well as producing systems at low cost which can be used on site as a point-of-care device for telemedicine applications. In this article, the most important original experimental platforms as well as current commercial approaches to biomedical analysis are critically chosen and reviewed, covering January 2010 to January 2014. While literature is quite broad and numerous, there is clear emphasis on biological recognition and imaging for the most impactful works. The analytical approaches are discussed in terms of their utility in diagnostics and biomedical testing.

Recent developments in emerging microimmunoassays.

Creative and novel microimmunoassay approaches continue to proliferate across many platforms originating from several fields of study. These efforts are aimed at improving one or more metrics for clinical tests, including improved sensitivity, increased speed, reduced cost, smaller sample size, the ability to analyze multiple antigens in parallel and ease of use. Many approaches focus on the production of microarrays that accomplish standard assays in parallel, or mobile solid-support formats to overcome issues of high background noise and long incubation times. In this article, innovative developments beyond existing commercial tests in the microimmunoassay arena are reviewed, covering January 2008 to April 2012. These developing experimental platforms are discussed in terms of their ability to augment or replace current commercial approaches.