Despite significant advances in nanopore nucleic acids sequencing and sensing, proteins detection remains challenging due to the complexity of inherent protein molecular properties (i.e., net charges, polarity, molecular conformation & dimension) and sophisticated environmental parameters (i.e., biofluids), resulting in unsatisfied electrical signal resolution for proteins detection such as poor accessibility, selectivity and sensitivity. The selection of an appropriate electroanalytical approach is strongly desired which should be capable of offering easily detectable and readable signals regarding proteins particularly depending on the practical application.

Recently, researchers in Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences (CIGIT, CAS) proposed an advanced method for efficient nanopore electrical detection of antigen proteins. In the study, a molecular sandwich-based DNAzyme catalytic reaction cooperated nanopore detecting approach was designed. Especially, this approach is given the easy use of Mg2+ catalyzed DNAzyme (10-23) toward nucleic acids digestion for efficient antigen protein examination. Its applicability within the proposed strategy operates by initial formation of a molecular sandwich containing capture antibody-antigen-detection antibody for efficiently entrapment of target proteins (herein taking HIV p24 antigen for example) and immobilized on magnetic beads surface. After that, the DNAzyme was linked to the detection antibody via biotin−streptavidin interaction. In the presence of Mg2+, DNAzyme catalytic reaction was triggered to digest nucleic acids substrates and release unique cleavage fragments as reporters capable of transducing easier detectable nucleic acids as substitute of complicated and difficulty-yielded protein signals, in a nanopore. Notably, experimental validation confirms the detecting stability and sensitivity for target antigen referenced with other antigen proteins, meanwhile demonstrates the detection efficacy in human serum environment at very low concentration (LoD ~ 1.24 pM). This DNAzyme cooperated nanopore electroanalytical approach denotes an advancement in protein examination, may benefit in vitro test of proteinic biomarkers for disease diagnosis and prognosis assessment.  

This work is invited and published in Faraday Discussions (DOI https://doi.org/10.1039/D4FD00146J ) by Committee Chair Prof. Dr. Yi-tao Long for the forthcoming discussion event “New horizons in nanoelectrochemistry Faraday Discussion”, whereas the attention will focus on nanopore electroanalytical chemistry to understand dynamic single-molecule reactions and sensitive detection of nucleic acid polymers, proteins, viruses, and so on. Lebing Wang (Graduate student of Chongqing University of Posts and Telecommunications) is the first-author, Prof. Dr. Youwen Zhang（Rutgers University, USA）and Prof. Dr. Liang Wang (CIGIT, CAS) are corresponding authors. This work was supported by the National Key Research and Development Program of China, Chongqing Talents-Exceptional Young Talents Project, The Youth Innovation Promotion Association of Chinese Academy of Sciences & Chongqing Zhongke Dexin Biotechnology Co. Ltd.

Figure 1. Molecular sandwich-based DNAzyme catalytic reaction enables efficient nanopore electrical detection for antigen proteins.