Creation Of Smart Microfluidic Chips Based On Molecular Switches And Their Applications In Bioanalysis

Lots of work has been focused on controlling the microcosmic world with macrocosmic methods,and subsequently to control the macrocosmic change of the substance property by changing their micro-structures.Among these work,it's becoming more and more attractive to control the properties reversibly through manipulating the structure,compose,conformation and oxidation state and so on.The reversible change is similar to "on/off" switching.With this kind of operation,we can control the properties of substance,to realize intelligent manipulation and reduplicate usage.Therefore,the research of substance and systems with "on/off" property is bewitching.In order to construct these kinds of systems,it's needed to construct special microcosmic structures on the atom or molecule scale.To use atoms and molecules as basic unit to design and assembly with a "bottom-up" style,we can get devices with special "on/off" property,which are called "molecular switches"."Molecular switches" are chemical systems with organized structures and "on/off" function.They have two stable states,between which they can switch under the external stimulus.The external stimulus could be photo,electrical potential, temperature,chemical energies(pH,specific molecules or ions).When some of these conditions change,the electrons would be rearranged,leading to the change of molecular shape,formation of deformation of chemical bond.And accordingly,these changes will result in the change of wettability,color,solubility,etc.,which could be useful for information convection.Based on the interaction of micro-electronics and bioengineering,it becomes possible to construct molecular devices with molecular switches.Smart surface is a kind of well organized,compositive,planar molecular switches.It's a surface with controlled "on/off" ability.Usually,smart surfaces are fabricated from polymer,self-assemble monolayer(SAM),special metallic oxide nano materials.The environmental conditions could inspire the property change of smart surface include temperature,light,electric field,pH,solvents and so on.The molecules composing the surface can behave conformational,configurational switching upon the stimuli,and therefore induce reversible changes in wettability, optics,electricity,etc.Microfluidic devices are the core parts of micro total analysis system(μ-TAS) and effective means for bioseparation,bioassay,environmental inspection,medical diagnosis.Because it's easy to manipulate it,and it's sensitive,fast and low reagent consumption,it's convenient to realize automatization and on-line measurements with the microfluidic devices.Microfluidic chips are well researched and widely used microfluidic devices.Many kinds of microfluidic chips have been designed and fabricated according to different applications,including sensors,reaction containers and so on.This thesis is about to fabricate two kinds of smart surface based on supermolecules,inclusive complexes,self-assembly technique,and temperature-sensitive polymers.Furthermore,expand these surfaces to protein program adsorption,separation and reproducing of antibody.Thirdly,to make smart microfluidic chips with these surfaces.One of the smart surfaces is electricity-sensitive smart surface.It's a low-density SAM.This layer is a potential controlled hydrophilic/hydrophobic switch.Its wettability could change under the applied electric potential.Subsequently,it can reversibly adsorb and release avidin or streptavidin protein under the potential control.The other is a temperature-sensitive surface,with thermosensitive polymer PNIPAAm,we got bioconjugate between PNIPAAm and anti-BSA antibody,and fabricated a temperature-sensitive and reproducible smart surface.This approach realizes the regeneration of the immune surface,so it can be used again and again.Meanwhile,it supplies a new research platform for protein-protein interaction.Furthermore,we introduce these smart surfaces into microfluidic chips to prepare smart chips with "on/off" property.And also these chips were successfully applied in controlled protein adsorption/release and separation.This kind of microfluidic chip is cheap,low energy and reagent consumption,easily measured.So it's a successful example of fabricating smart microfluidic devices with SAM.This paper contains 6 chapters which are outlined below:In Chapter 1,the research of molecular switches,smart surface and microfluidic chips are reviewed.Molecule self-assembly and polymer are the main two ways to get molecular switches.SAM is a stable,well organized monolayer from the spontaneous interaction between the molecules and the substrate.It's easy to introduce kinds of functional groups to the surface with this method,which supplies a good foundation for smart surface with different functions.Thermosensitive polymers can behave property change upon temperature change.PNIPAAm is a typical and widely studied thermosensitive polymer.They are widely used in drug release,immune assay and cell culture.In Chapter 2,fabricating potential-sensitive smart surface based on self-assembly technique,and its application in controlled protein adsorption are investigated. Cyclodextrin was used to control the density of the monolayer to get low-density 16-mercaptohexadecanoic acid monolayer(MHA LD-SAM).Referring to high-density MHA SAM,the surface coverage for LD-SAM fromα-,β-,γ-cyclodextrin shows an obvious decrease from 100%to 61.2%,45.3%and 29.2%, respectively.By applying different potentials to this surface,the MHA molecule could behave conformational transformation and accordingly the wettability of the surface could change.The achievement of the smart surface(MHA LD-SAM) was proved with MALDI-TOF-MS,quartz crystal microbalance(QCM),NMR,cyclic voltammetry(CV) and impedance(IMP).Contact angle measurement was used to prove the wettability change of the surface.The prepared smart surface was also used for controlled protein adsorption,which was characterized with QCM and fluorescence measurements.In Chapter 3,the electric potential-sensitive surface was implemented into microfluidic chip to develpe two kinds of smart chips,which were applied in controlled protein adsorption.With the cheap material,PMMA,we made chips using hot-imprinting method.The prepared microfluidic chips were with a channel in the centre,and on the channel surface a layer of gold was modified,on which the followed fictionalizations were implemented.We fabricated two chips,the one is COOH-chip with terminal carboxyl groups on the channel surface,and the other is NH₂-chip with terminal amino groups on the channel surface.They can adsorb positive and negative potentials under potential control,respectively.Chapter 4 is about the application of the smart microfluidic chips in protein adsorption/release and separation.Fluorescence,laser induced fluorescence(LIF) and confocal fluorescence microscopy(CFFM) are used to demonstrate the separation. For COOH-chip,it can capture more than 80%avidin from the Avidin/Streptavidin mixture upon external potential;as for NH₂-chip,it can also separate more 80% Streptavidin from the Avidin/Streptavidin mixture.And COOH-chip was used to separate low concentrate Avidin from the Avidin/Streptavidin mixture with a molar ratio of 1:1000 of Avidin:Streptavidin.The results were satisfying.So this electric potential-sensitive smart microfluidic chips is a possible alternative method for concentrating low-abundance proteins,which is very important for proteomics research.Chapter 5 describes establishing the PNIPAAm-antibody surface that could supply the most effective dissociation of the antigen and regeneration of antibody into reuse of the immunosensors.As a model antibody-antigen system,bovine serum albumin(BSA) and the corresponding antibody anti-BSA were chosen.A reversible PNIPAAm-antibody conjugate surface was established by triggered control of external temperature.This took advantage of the thermally tuable conformation changes for the PNIPAAm-conjugated antibody surface,and could be used for switchable antigen association and dissociation.The temperature controlling strategy could realized the regeneration of the immunosensor on which immobilized anti-BSA antibodies retain the activity and specificity necessary to carry out more than 30 reproducible assays for BSA.The dissociation reaches 89%,which can compare with the general recovery methods.The controlled binding and unbinding were monitored by QCM,CFFM,native electrophoresis,laser induced fluorescence,and electrochemical impedance.Chapter 6 is the summary of this thesis and the future prospects for related research.This thesis reports the investigation about several smart surface and devices based on molecular switches.With self-assembly technique,thermosensitive polymer, and different environmental stimulus,hydrophilic/hydrophobic surface switches, temperature-responsive immune surface were fabricated successfully,as well as microfluidic chips from these smart surfaces.And they were successfully used in controlled protein separation and reversible immune recognition.