Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

Abstract

The fabrication of 3D hydrogel microarrays for DNA analytics that allow simple visual signal readout for on-site applications is described. A convenient one-step polymerization of the hydrogel including in situ capture oligonucleotide immobilization is accomplished by using N,N'-dimethylacrylamide/polyethylene glycol (PEG1900 )-bisacrylamide monomers. The implementation of an acylphosphine-oxide photoinitiator even allows polymerization at daylight, whereas other approaches require exposure with light in the UV-range. This minimizes the risk of UV-caused DNA damages within the capture DNA-strand that could adversely affect the subsequent hybridization step. The porous network of these gel segments allows DNA as well as protein penetration. Thus, the successful in-gel DNA hybridization is monitored by the deposition of silver nanoparticles. These metal particles allow naked eye signal readout.

Fast‐Track, One‐Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification

“…Since the polymerized gels hinder diffusion and reactions inside the gel volume, a convenient one‐step procedure for polymerization, functionalization, and immobilization on the surface is highly desirable. The presented hydrogel preparation method fulfill these requisites, allowing for a gel‐functionalization with DNA capture probes in parallel with hydrogel array production in 20 min. Two sequences of the 16S rDNA region (Eco1, Eco4, Table ) were chosen for the E. coli specific detection.…”

“…This greatly reduces the swelling forces so that the link between gel and surface remain stable and the hydrogels stay immobilized. Additionally, the supplemented glycerol increases the porosity of the hydrogel network and ensures fast diffusion for the DNA macromolecules . Furthermore, no additional time‐ or cost‐intensive surface activation processes are needed to create robust immobilized hydrogels that withstand numerous washing steps.…”

“…In‐gel immobilization of these capture probes, that are complementary to sequence parts of PCR products (Eco1 complementary to 1.5 kb fragment, Eco4 complementary to 100 bp fragment) and a non‐complementary (NC) capture probe was realized. Acrydite‐modified capture probes enabled the formation of gels twice as fast as amine‐ and thiol‐modified probes . Here the comparably chemistry of polymerization and immobilization of acrylamide units favors the reaction.…”

“…This system refers as solid phase PCR and implies a reaction only on the surface of the hydrogels, since diffusion and functionality of the polymerase inside the gel is hindered. It was shown that only the polymer formed with the longer polyethyleneglycol (PEG) 1900BIS next to BIS allowed efficient enzyme diffusion and functionality inside the gel volume …”

“…It was shown that only the polymer formed with the longer polyethyleneglycol (PEG) 1900BIS next to BIS allowed effi cient enzyme diffusion and functionality inside the gel volume. [ 21 ] To the best of our knowledge, a DNA hybridization during PCR amplifi cation as a further time-saving mechanism has not been studied. Thus, a combination of fl uid phase PCR while in gel hybridization was proceeded in one tube.…”