An enzyme-amplified lateral flow strip biosensor for visual detection of microRNA-224

An enzyme-based dual-labeled nanoprobe is designed to fabricate a sensitive enzyme-amplified lateral flow biosensor for visual detection of mircoRNA-224 (miRNA-224). The recognition DNA probe (detection probe) and signal amplification enzyme (Horseradish peroxidase, HRP) are immobilized on gold nanoparticle (GNPs) surface, simultaneously. The capture DNA probes are immobilized on the test zone of the lateral flow biosensor. When miRNA-224 is present, the enzyme-based dual-labeled nanoprobes will be captured by forming the "sandwich structure" on the test zone of the lateral flow biosensor, enabling the visual detection for miRNA-224. Sensitivity is amplified by applying the 3,3,5,5-tetramethylbenzidine enzymatic substrate (TMB/H2O2 enzymatic substrate) onto the test zone. The enzymatic reactions between the HRP and the TMB/H2O2 enzymatic substrate will produce blue products, which deposit on the nanoprobe surface to enhance the visual effect and the corresponding response intensities of the test zone. This enzyme-amplified lateral flow biosensor shows a low limit of detection (LOD) (7.5 pM) toward miRNA-224 in the buffer solution, which is improved by 10-fold than that of the single-labeled lateral flow biosensor. This biosensor has been successfully used for the detection of the target miRNA-224 detection in A549 cell lysate.