| With the continuous growth of the population,the demand for clean water is increasing.However,the current water pollution is serious,especially the existence of pathogenic bacteria in water sources,which leads to the occurrence of many diseases.Therefore,it is very important to develop a real-time,rapid and highly sensitive method for detecting pathogenic bacteria and their biomarkers.Among the current detection methods,biosensor has been widely studied because of its advantages of convenience,rapidity and low cost.Nanomaterials that release cargo molecules such as fluorophore,dye and protein upon exposure to different types of stimuli have broad application prospect in the field of biosensors.Strategies that speed up the on-command release of proteins(e.g.,enzymes)from stimuli-responsive materials are intrinsically necessary for biosensing applications,especially point-of-care testing,as they will achieve fast readouts with catalytic signal-amplification.However,current materials that release enzymes are challenging to work with because they usually exhibit response times on the order of hours up to days.Functional DNA superstructures has high biocompatibility,high loading capacity and editability,which is expected to realize the rapid release of protein.Herein,in this paper,a fast-responding gating system using protein-encapsulating functional DNA superstructures(denoted as protein@3D DNA)was constructed,the mechanism of loading and releasing protein was analyzed,and the rapid detection of toxin B(Clostridium difficile marker)was realized by combining with paper based microfluidic chip.The details are as follows:(1)A protein-encapsulating functional DNA superstructures was synthesized.Using a circular DNA as a template,DNA long strands were synthesized by rolling circle amplification(RCA)under the induction of?29 DNA polymerase,which combined with magnesium pyrophosphate precipitates generated in the reaction,and finally generated3D DNA.Proteins were directly embedded into 3D DNA during the one-pot rolling circle amplification process.SEM images and TEM images indicated the formation of internal porous structures of HRP@3D DNA.CFM images showed that monodisperse particles were homogenously luminescent following excitation.The high loading capacity of 3D DNA on proteins was also verified,the maximum loading capacity was determined to be(4.3±0.3)×10~4F-HRP molecules per particle.(2)The properties of protein@3D DNA loading and releasing protein were investigated.First,it was verified that protein loading and release are closely related to the size of the protein and its interaction with 3D DNA.Secondly,the release kinetics of the gated system was investigated,the specific DNA-DNA interactions provide a means of triggering release.Then,the aptamer was combined into 3D DNA,and it was proved that the aptamer-target complex(such as small molecule ATP and protein platelet-derived growth factor)could also achieve protein release,thus demonstrating the universality of this system.(3)A simple paper device by employing protein@3D DNA was constructed for colorimetric detection of toxin B.By simple steps,toxin B can be detected quickly and specifically.A small amount of buffer containing toxin B(15μL)was added into the reaction zone,the highly sensitive quantitative detection of toxin B can be realized through the color signal in the detection zone.The detection sensitivity of toxin B was up to 0.1 n M within 16 min.This paper elaborates the whole process from the design of stimuli-responsive materials,preparation of materials,feasibility test of releasing protein and mechanism analysis.To a certain extent,the relationship between protein release and target,3D DNA and protein is clarified,which provides more new ideas for the research and application of biosensors. |
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