A critical comparison of protein microarray fabrication technologies

Abstract

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

“…The pin is then moved to the appropriate location on the chip and then a light contact between the pins and the surface results in the deposition of typically nano liters of the protein liquid onto the chip surface. The morphology and size of the resulting spot depends on the drop volume, the concentration of the protein and other non-volatile compound, on the surface tension of the droplet, the drying conditions and the surface properties of the chip (Romanov et al, 2014). These printers are easy to use and very flexible concerning the properties of the print solution (different viscosities or the presence of charges).…”

“…These printers are easy to use and very flexible concerning the properties of the print solution (different viscosities or the presence of charges). However, they suffer from the following drawbacks (Romanov et al, 2014):…”

Integrating plasmonic diagnostics and microfluidics

“…Quite a few patterning methods have been developed and commonly used, such as microwriting, 14 microstamping, 15 and micromachining. 16 We describe here another strategy which utilizes addressable electrochemical control of the sensing elements to fabricate sensor arrays with a variety of supramolecular interfacial architecture.…”

Analysis of protein microarrays by FTIR imaging

“…Microarrays supplies useful tools for clinical studies (vaccine development, disease diagnosis, drug screening for example) as well as an important approach to give advancements in analytical studies [16].…”