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

Abstract

A timesaving and convenient method for bacterial detection based on one-step, one-tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one-pot synthesis, where N,N'-dimethylacrylamide/polyethyleneglycol(PEG1900 )-bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA-functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10(1) Escherichia coli cells.

Smart and Functionalized Development of Nucleic Acid‐Based Hydrogels: Assembly Strategies, Recent Advances, and Challenges

“…With the increasing demands regarding food safety evaluation and risk analysis, DNA hydrogels in various forms have been applied for the rapid detection of food contaminants, while performing different functions in biosensors. Beyer et al [194] coupled arrayed 3D hydrogel decorated chips with the PCR technique to amplify the target gene for bacterial detection. The hydrogel array, which consisting of 3D hydrogel droplets with high gel porosity and polymerized under visible light, provided a larger surface area than 2D surfaces for functionalization, further promoting the accessibility of gel-immobilized DNA molecules (Figure 7C).…”

“…The smart and functionalized assays conducted by biosensors containing NAHs have been expanded for different types of analytes in vitro or in vivo. In addition to well‐studied miRNA and some heavy metal ions, the hydrogel‐based biosensors have been designed specifically for protein, [ 75 ] bacteria, [ 194 ] and biotoxin [ 194 , 195 , 196 ] detection. In a study by Zhang et al., [ 75 ] Y‐shaped DNA and an aptamer linker were self‐assembled to fabricate a pure DNA hydrogel, entrapping AuNPs as indicator agents for the subsequent visual detection ( Figure 7 A ).…”

“…Beyer et al. [ 194 ] coupled arrayed 3D hydrogel decorated chips with the PCR technique to amplify the target gene for bacterial detection. The hydrogel array, which consisting of 3D hydrogel droplets with high gel porosity and polymerized under visible light, provided a larger surface area than 2D surfaces for functionalization, further promoting the accessibility of gel‐immobilized DNA molecules (Figure 7C ).…”

Construction of a CRISPR‐Biolayer Interferometry Platform for Real‐Time, Sensitive, and Specific DNA Detection

“…[1][2][3] These advances have led to the development of specific nucleic acid detection methods and molecular diagnostic assays. [4][5][6] Molecular diagnostics is a powerful tool applied in various fields, such as diseases, food safety, and environmental monitoring, [7][8][9][10] Some of these approaches play critical roles in the clinical setting by providing physicians with information to guide precise treatment, monitor patient's health status and improve patient outcome. [11][12][13] Nucleic acid detection methods, such as polymerase chain reaction (PCR), gene sequencing and in situ hybridisation, have been constructed and applied in molecular diagnostics to detect target sequences.…”

Autoinducer Sensing Microarrays by Reporter Bacteria Encapsulated in Hybrid Supramolecular‐Polysaccharide Hydrogels

“…The detection of bacteria and bacterial infections is an area of intensive research activities, not only in recent years. Among the various new formats for rapid, sensitive and selective bacteria detection, vesicle based approaches, autonomously reporting hydrogel films, as well as hydrogel dot microarrays have garnered particular attention. These formats mainly rely on the detection and reporting of secreted bacterial enzymes, DNA, and virulence factors .…”