Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection

Abstract

The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.

Holographic Recording of Unslanted Volume Transmission Gratings in Acrylamide/Propargyl Acrylate Hydrogel Layers: Towards Nucleic Acids Biosensing

“…Thin films of hydrogels composed of AA, MBA and PA were prepared by adapting the described protocol [ 12 , 23 ]. AA and MBA acted as the skeletal monomer and crosslinker, respectively.…”

“…PA is an additional monomer that provides the hydrogel with alkyne groups that are available to be functionalized with thiolated moieties by photo-click chemistry. We have previously optimized the relationship between monomers and crosslinkers (i.e., the crosslinking degree) needed to generate transparent and moldable hydrogels [ 23 ]. In addition, in the same study, we also fabricated hydrogels by photochemical and thermal activation.…”

“…Thermal activation (i.e., using KPS as activator) yielded hydrogels with more homogenous and porous networks. Accordingly, for this work, we have chosen the previously optimized (see materials and methods section) and characterized composition and activator [ 23 ]. Once synthesized, the morphology of the lyophilized (AM/PA) hydrogel was characterized by SEM microscopy ( Figure 1 a).…”

“…For that, starting from a VTG with a DE of 20% in water, they were conditioned in SSC1x, and their diffraction diminished to one half. Then, VTGs conditioned in SSC1x were incubated with 5 μM solution of an oligonucleotide probe bearing a thiol group in THF/Ac-TCEP 1:1, and irradiated for 30 min, according to the protocol described for biofunctionalization [ 23 ]. After washing with SSC1x for several hours, the diffraction efficiency decreased further by 40%.…”

“…This work focused on the optimization of the recording process of unslanted volume transmission gratings (VTGs) in Acrylamide/Propargyl Acrylate (AM/PA) hydrogel layers. The composition of the hydrogel had already been optimized in a previous work, in which hydrogels were surface micropatterned and utilized as biosensors [ 23 ]. By using this hydrogel composition, the bioreceptor elements (thiol-modified DNA probes) can be covalently immobilized via thiol-yne click reaction both before and after the fabrication of the volume grating.…”