Surface blocking is a well-known process for reducing unwanted nonspecific adsorption in sensor fabrication, especially important in the emerging field where DNA/RNA applied. Bovine serum albumin (BSA) is one of the most popular blocking agents with an isoelectric point at pH 4.6. Although it is widely recognized that the adsorption of a blocking agent is strongly affected by its net charge and the maximum adsorption is often observed under its isoelectric form, BSA has long been perfunctorily used for blocking merely in neutral solution, showing poor blocking performances in the optical-fiber evanescent wave (OFEW) based sensing toward DNA target. To meet this challenge, we first put forward the view that isoelectric BSA (iep-BSA) has the best blocking performance and use an OFEW sensor platform to demonstrate this concept. An optical-fiber was covalently modified with amino-DNA, and further coupled with the optical system to detect fluorophore labeled complementary DNA within the evanescent field. A dramatic improvement in the reusability of this DNA modified sensing surface was achieved with 120 stable detection cycles, which ensured accurate quantitative bioassay. As expected, the iep-BSA blocked OFEW system showed enhanced sensing performance toward target DNA with a detection limit of 125 pM. To the best of our knowledge, this is the highest number of regeneration cycles ever reported for a DNA immobilized optical-fiber surface. This study can also serve as a good reference and provide important implications for developing similar DNA-directed surface biosensors.
Split aptamers (SPAs) are a pair of oligonucleotide fragments generated by cleaving a long parent aptamer. SPAs have many compelling advantages over the parent aptamer such as sandwich target binding, optimized concise structure, and low cost. However, only a limited number of SPAs have been developed so far, because the traditional theory restricts the splitting to the functionally dispensable site that many parent aptamers do not possesses. In this work, we challenge the traditional mechanism and hypothesize that SPAs can also be generated by splitting the parent aptamer at the functionally essential site while still preserving the biorecognition capability. To prove our hypothesis, we discoveried three SPAs with Broken initial small-molecule binding Pockets ( BP SPAs) and validate their binding capability both in wet lab and in silico. An allosteric binding mechanism of BP SPAs, in which a new binding pocket is formed upon the target binding, is revealed by all-atom microsecond-scale molecular dynamics simulations. Our findings will greatly promote discovery of new SPAs and their applications in molecular sensing in many fields.
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