Producing Covalent Microarrays of Amine‐Conjugated DNA Probes on Various Functional Surfaces to Create Stable and Reliable Biosensors

Abstract Oligonucleotide‐based microarrays are ideal tools for genetic testing and diagnostics due to their stability, ease of synthesis, and high specificity for the target of interest. This study reports on the effectiveness of several coupling strategies to covalently attach single‐stranded nucleic acids to surfaces, focusing on the robustness of the attachment in various environmental conditions, such as pH and temperature. Various characteristics of DNA microarrays produced using amine‐conjugated DNA probes on five different functional surfaces, commonly used for immobilization of biomolecules, namely, epoxy, carboxyl, amine, aldehyde, and N ‐hydroxysuccinimide‐coated substrates, are investigated. Immobilization efficiency, changes in surface energy, as well as the stability of the conjugated DNA upon exposure to various environmental conditions are measured. Finally, in order to study the postimmobilization viability of the developed biosensors, microarrays of synthetic RNA cleaving probes (DNAzyme) are immobilized onto these surfaces and their functionality is evaluated through their ability to detect Escherichia coli at various temperatures. Results show that epoxy‐coated plastic surfaces are most ideal for the creation of DNA‐based biosensing chips. This study provides a guideline for producing oligonucleotide‐based microarrays on glass and plastic substrates and can be used for developing microarray‐based biosensors, suitable for long‐term storage.