Protein Microarray Immobilization via Epoxide Ring‐Opening by Thiol, Amine, and Azide

Seyed Mohammad Mahdi Dadfar,Michael Hirtz,Vanessa Trouillet,Sylwia Sekula‐Neuner

doi:10.1002/admi.202002117

Seyed Mohammad Mahdi Dadfar, Michael Hirtz + Show 2 more

Open Access

https://doi.org/10.1002/admi.202002117

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Abstract

AbstractImmobilization of functional molecules in ordered microarrays has vast importance for many applications in sensing and diagnostics. Here, the immobilization of small molecule compounds and protein in microchannel cantilever spotting (μCS) on epoxy‐terminated glass surfaces is studied via ring‐opening of epoxides by thiol, amine, and azide, with the purpose of creating microscale patterns for sensing applications. For this, glass surfaces carrying epoxy groups from silanization are functionalized with microarrays by μCS with different fluorescent and nonfluorescent dyes containing thiol, amine, or azide groups. Experiments with the fluorophores reveal that all routes result in successful immobilization in a short time. Furthermore, from these experiments, optimal reaction conditions including time, temperature, catalyst type, and catalyst amount are determined. The feasibility of these routes in sensing applications is examined by means of protein binding experiments. Comparing the fluorescence intensity values of different biotin carrying spot arrays immobilized on the epoxy‐terminated glass surfaces, after incubating the surfaces with fluorescent‐labeled streptavidin, reveal the highest surface density of immobilized biotin for the amine route in comparison to the thiol and azide routes. The obtained results from this work can inform the design and fabricating of protein biosensors as well as other biomedical and diagnostic applications.

Highlights

As a first check of successful functionalization prior to the further experiments, contact angle measurements were done on the hydroxyl- and epoxy-terminated glasses over the course of four weeks
To prevent ink spreading during spotting of microarrays, the water contact angle (WCA) should not be too low
We studied and compared other immobilizing routes including strain-promoted azide–alkyne cycloaddition (SPAAC), thiol–yne coupling (TYC), and thiol–ene Michael addition (TEMA) for the generation of covalently bound microarrays on functionalized glass surfaces, revealing a relative efficiency of reactions as: TEMA > TYC > SPAAC.[32,33]

Summary

Results and Discussion

As different fluorophores used for surface coupling have different functional groups, different emission spectra, and different intensities even for the same surface concentration of immobilized molecules, another strategy should be employed for the direct comparison of these reactions.[32,33] For this, via epoxide ring-opening routes, biotin-bearing compounds including biotin-thiol, biotin-amine, and biotin-azide were spotted on the epoxy-terminated glasses. We studied and compared other immobilizing routes including strain-promoted azide–alkyne cycloaddition (SPAAC), thiol–yne coupling (TYC), and thiol–ene Michael addition (TEMA) for the generation of covalently bound microarrays on functionalized glass surfaces, revealing a relative efficiency of reactions as: TEMA > TYC > SPAAC.[32,33] Comparing the obtained results in the current study with the TEMA route (yielding a fluorescence intensity of (3953 ± 210) a.u. for fluorescently labeled streptavidin)[33] indicates a more efficient biotin immobilization by the ring-opening of epoxy by thiol, amine, and azide

Conclusion

Experimental Section

Data Availability Statement