Novel Signal Amplification Approach for HRP-based Colorimetric Genosensors Using DNA Binding Protein Tags

The need for sensitive detection of DNA is growing as more specific DNA sequences are being correlated to gene markers for disease diagnosis, food safety and other security related applications. Detection in hybridization-based assays is usually achieved with target-specific ssDNA probes conjugated directly to enzyme labels like HRP that provide signal amplification or with nanoparticles functionalized with DNA and multiple HRP molecules. In order to overcome some of the drawbacks presented by these approaches, we developed a unique DNA sensing platform based on an HRP–DNA binding protein tag conjugate and a hybrid ssDNA–dsDNA detection probe. Specifically, in this work we describe the preparation and characterization of an HRP conjugate with scCro DNA binding protein tag and its application for the detection of a model ssDNA target sequence. By using the HRP–scCro conjugate together with a hybrid detection probe containing three scCro‐specific dsDNA binding sites, we demonstrate an improvement by over 3-fold in both sensitivity and limit of detection of high-risk human papillomavirus (HPV16), compared to the standard ssDNA–HRP conjugate. These results show that the HRP–DNA binding protein tag conjugate can be used as an alternative and universal tool for signal enhancement in enzyme-linked assays suitable for integration in point-of-care systems.