Exploiting fluorescence for multiplex immunoassays on protein microarrays

Abstract

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Lanthanide light for biology and medical diagnosis

“…hybridization of complementary nucleic acids can be detected with such microarrays. Nucleic acid and protein microarrays are becoming popular techniques in luminescent immunoassays for proteomics and diagnostics [76]. The full range of LLB-based analyses is of course available for these technologies and relevant protocols start to be proposed [68,77] both for well-type analysis featuring solid-state arrays [78] and for sophisticated microarrays [68,77].…”

Multiplexed microarrays based on optically encoded microbeads

“…This approach has attractive features, such as: straight forward labeling process, easy detection of large-scale samples, and compatibility with a variety of biological chemistries, localized signals, rapid signal acquisition and high sensitivity. In the following section we provide an overview of various encoding methods (Jun et al 2012; Resch-Genger et al 2008; Sukhanova andNabiev 2008; Herbáth et al 2014).…”

“…Moreover, high densities of labeling can cause fluorescence quenching. QDs are bright fluorophores which have narrow emission peaks and large Stokes shifts; these properties make them good candidates for labeling because of minimal overlap with barcodes, reduced spectral crosstalk, and possibility for label multiplexing (as well as barcode) (Leng et al 2015; Resch-Genger et al 2008; Herbáth et al 2014).…”

“…This result could be analyzed by microscope imaging or flow cytometry (Nie et al 2006; Lim et al 2011). Also there are some other label free techniques applicable in optical microbead systems such as SPR, carbon nanotubes (CNTs) and nanowires (Herbáth et al 2014).…”

Creating a high impact journal for research in fluorescence

“…We hope readers have found the journal to be a source of interest and inspiration and would welcome your feedback on topics you would like to see covered. Recent reviews have featured luminescent lanthanide reporters [1], protein microarray technology [2] and fluorescent probes and nanoparticles for intracellular sensing of pH values [3]. It is gratifying to know that the review articles we have published so far have been of interest, with review articles in the journal having already been downloaded over 9500 times.…”