Controllable Molecular Transport In Complex Biochemical System And Its Application In Biosensing

The controllable transport of low-molecular-weight compounds,proteins and nucleic acids among the functional sites is critically important to the complex biological network systems.With the development of modern biological technology and the improvement of chemical synthesis,artificial biochemical systems based on biomolecules such as proteins and nucleic acids have been widely applied in biosensing,drug therapy and bioenergy fields.Therefore,the precisely controllable mass transport is of great significance to the construction of complex biochemical systems,biomimetic devices and even artificial living systems.A variety of biological functional units are located at different functional sites of complex biochemical systems in the form of homogeneous or heterogeneous spatial distribution,and the molecular transport among the functional sites results in the cooperative and efficient functioning of the biochemical system.The development of nanotechnology and micromanufacturing technology has realized effective mass transport in artificial biochemical systems with precise design of biorecognition system,elegant nanoreactor and microfluidic devices.However,these techniques are limited by complicated fabrication processes and insufficient fabrication accuracy,and are mainly applied in simple biochemical processes,rather than complex biochemical systems.Therefore,developing precise and intelligent mass transport system for multiple molecules among multiple sites in complicated biochemical systems has become one of the key issues in this field.In this thesis,firstly,we analyzed the basic process of mass transport in biochemical reaction systems.Secondly,we combined DNA nanotechnology and three-dimensional microfluidic chip to design biochemical reaction system with multiple biomolecules precisely assembled.Finally,the intelligent control of mass transfer in complex biochemical systems is realized,and multiple smart and sensitive biosensors are also developed.The main results are as follows:(1)Based on the interface regulation of DNA probes to biomolecules' assembly,we realized the ideal assembly of multiples enzymes on gold electrode surface with fine interenzymes distance,making our strategy far better than traditional methods.Based on that,we optimized the transport efficiency of the intermediate product in the cascade reactions.The improved intermediate product transport efficiency brought high cascade reaction efficiency,which is about 10 to 100 folds higher than that on unregulated interface.The highly efficient enzyme cascade system was further applied for the ultrasensitive detection of sarcosine,a marker of prostate cancer,which is expected to achieve accurate diagnosis of prostate cancer.(2)We constructed a gravity guided molecular threading system on a three-dimensional origami paper chip for molecular transporting in biomolecules heterogeneous distributed biochemical system.Based on this,we have realized highly directed and ordered molecular transport between multiple sites.The molecular threading system could break through the transport distance limitation(~centimeters)on traditional paper chips and increased the efficiency of long-distance transport efficiency from 0 to 25%.Applying the molecular threading system on biomolecules transport in complex cascade reaction system,the total reaction efficiency could be enhanced about 1000 folds on framework nucleic acids assembled interface.Finally,we developed a single-step electrochemical DNA sensor for quantitative point of care analysis with 1 picomolar sensitivity in 60 minutes.(3)In order to realize the intelligent control of mass transport in biochemical system,we designed a kind of stimulus responsive DNA hydrogel by hybridization chain reaction.Based on the natural size selectivity and phase separation characteristics of hydrogels,we achieved substrate size selective catalysis by cascaded enzymes encapsulated in the hydrogel.By integrating the ATP responsive units into the DNA hydrogel,hydrogel structure changing in response to ATP was further used in the intelligent control of substrate transport and enzyme catalytic rate.Finally,this ATP-responsive DNA hydrogel was used to specifically capture and separate circulating tumor cells.Even 10 tumor cells could be detected in whole blood by our method.Moreover,in response to ATP,the released circulating tumor cells maintained integrated molecular information and the ability of proliferation and growth.The controllable capture and release of living tumor cells is expected to be applied for individualized treatment and drug screening of tumors.