Hydrogel‐based biosensors have drawn considerable attention due to their various advantages over conventional detection systems. Recent studies have shown that hydrogel biosensors can be excellent alternative systems to detect a wide range of biomolecules, including small biochemicals, pathogenic proteins, and disease specific genes. Due to the excellent physical properties of hydrogels such as the high water content and stimuli‐responsive behavior of cross‐linked network structures, this system can offer substantial improvement for the design of novel detection systems for various diagnostic applications. The other main advantage of hydrogels is the role of biomimetic three‐dimensional (3D) matrix immobilizing enzymes and aptamers within the detection systems, which enhances their stability. This provides ideal reaction conditions for enzymes and aptamers to interact with substrates within the aqueous environment of the hydrogel. In this review, we have highlighted various novel detection approaches utilizing the outstanding properties of the hydrogel. This review summarizes the recent progress of hydrogel‐based biosensors and discusses their future perspectives and clinical limitations to overcome.

Trail polymerization enables a significant enhancement of the DhITACT system. DhITACT-Trail (DNA hydrogel formation by isothermal amplification of complementary targets trail polymerization) offers a robust diagnosis of target RNA strands in pseudo-serum specimen. This system requires minimum liquid handling as compared to conventional analysis. In addition, a definitive diagnostic result can be achieved within 30 min by an optical detection.

DNA hydrogel formation by isothermal amplification of complementary targets in microfluidic channels (DhITACT) is a new platform for rapid and accurate detection of infectious pathogens. DNA hydrogel is formed in situ within microfluidic channels by the isothermal rolling circle amplification process upon the selective binding of target strands from the biological fluid. Once the volume of DNA hydrogel sufficiently enlarges, it can selectively block the matching channels with target pathogens.

Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

H. Lee and co‐workers demonstrate, on page 3513, the novel platform for rapid and accurate detection of infectious pathogens. “DNA hydrogel formation by isothermal amplification of complementary target in fluidic channels” (DhITACT) enables the naked‐eye detection of ebola and Bacillus Anthracis using a microfluidic array chip by selective blockage of the matching channels through in situ hydrogel formation when target pathogen strands are present.