The Construction And Mechanism Investigation Of Functional Recognition Systems Based On Biomolecules/Nanomaterials

Molecular recognition is one of main themes in the development ofsupramolecular chemistry, which is defined as the process that a subject moleculeselectively combined with a object one to have some new special function. Thecombining manners are weak interactions containing those non-convalent bonds, suchas hydrogen bonds, metal coordination, π-π stacking interactions, Van der Waals forceand hydrophobic interactions et. al. The specific recognition process will result inphysical properties changes as optical or electrical properties, or chemical propertiesof the system. Accompanied with these structural or behavioral variations, therelating storage, transfer and expression of chemical informations will occur. Basedon the system changes induced by molecular recognition process we can furtherdesign and construct selective chemo/biosensors, which could be applied to recognize,separate and detect ions, small molecules and/or macro biomolecules in theenvironment and/or biosystems.Due to the unique physical and chemical properties, nanomaterials likenanoparticles which can be used as scaffold and/or response units, have a greatpotential to be applied to the molecular recognition and detection. The physiologicalfunction in living body relies on not only the biochemical reaction, but also the highspecific and affinity recognition and the related combination between biomolecules.Because of the excellent recognition mode and properties showing in living body, thebiomolecules provide us a good goal to learn, imitate and utilize. By combining thenanoparticles and biomolecules, we have designed and constructed three functionalsystems, and investigated the process and mechanism of relating molecularrecognition. Three parts are involved in this thesis: (1). A new nanosensor for chromium(III) based on the functional goldnanoparticles has been constructed. Firstly, we synthesized the sodium citrateprotected gold nanoparticles, which was then modified by a ofter used colorimetricagent for the detection of thiols in biological samples,5,5′-dithiobis (2-nitrobenzoicacid)(DTNBA). DTNBA contains a disulfide bond, which could be easily brokenand formed Au-S bond with gold nanoparticles after mixing it with gold nanoparticles.In the near neutral aqueous solutions, the functional gold nanoparticles(TNBA-AuNPs) could selectively recognize Cr3+over other16metal ions. TheUV-vis extinction spectrum of TNBA-AuNPs changes were induced by Cr3+: theintensity of extinction peak at524nm decreased, while that at650nm increased withthe addition of Cr3+. TNBA-AuNPs also displayed higher sensitive response to Cr3+with a detection limit of93.6ppb. Further research on the involved mechanismshowed that the changes of UV-vis extinction spectrum was acribed to the Cr3+-induced aggregation of TNBA-AuNPs, which consequently changed the surfaceplasmon resonance of TNBA-AuNPs. The responses of TNBA-AuNPs to Cr3+comemainly from the carboxyl group in DTNBA as the shield of it could not induce anyaggregation of TNBA-AuNPs after Cr3+incorporation; in addition, the nitryl group inDTNBA was important to the selectivity of TNBA-AuNPs to Cr3+, as in the absenceof it several metal ions could induced aggregation of TNBA-AuNPs at lowconcentration level. In addition, we also studied the effect of TNBA surface densityon the gold nanoparticle. The results showed that onec the surface density of TNBAon AuNP was higher than2000, there was no notable differences for the response ofTNBA-AuNPs to Cr3+.(2). Based on the high affinity and specific interaction between melittin andphosphocholine, we constructed a f rster resonance energy transfer (FRET) system,where the CdSe/ZnS quantum dot was used as donor and a fluorescent molecule (Cy3) was used as acceptor. Further investigations were extended to its application on theprotein-membrane interaction and the detection of trypsin in solutions. Such aprotocol based on the high specific and affinity interaction between melittin andnatural/artificial membrane supplied a way to construct more functional systems likethis. In this part, firstly, we encapsulated the CdSe/ZnS QDs using phosphocholinebased on the hydrophobic interaction to transfer them to water phase and formed thephosphocholine encapsulated QDs. To achieve it, two different routes were designedto construct the FRET system:(a) Melittin was added and combined to thephosphocholine encapsulated QDs firstly, with the insertion of the N-terminal regionof melittin into the membrane and the exposion of its C-terminal region outside thelipid bilayer; then the addition of Cy3succinimidyl ester into the system and thefollowing reaction with amino group would form a FRET functional system (b) Thephosphocholine encapsulated QDs was mixed with the Cy3-prelabeled melittin, andthen the FRET functional system would form. Further, we investigated theapplication of such FRET functional system as:(a) Due to FRET efficiency dependingon the distance between the donor and acceptor, we calculated the distance betweenQDs and Cy3through FRET efficiency. The method constructing FRET functionalsystem based on the interaction between melittin and phosphocholine could be used tocalculate the distance from membrane-protein to membrane and study their interactionby changing the encapsulation layer or the membrane proteins.(b) The FRETfunctional system was also used to detect the activity of trypsin based on the specifichydrolysis of trypsin on melittin, and a detection limit of0.2μM was achieved.(3). By labeling a fluorescent molecule, Cy3, on C-phycocyanin (C-PC), we haveconstructed a FRET system (Cy3/C-PC), and also investigated the effect ofhydrostatic pressure on the fluorescence and FRET behaviors of it. Undercompression, the Cy3fluorescence in Cy3/C-PC increased firstly and then decreased, while the C-PC fluorescence decreased gradually and disappeared finally. Throughcomparing such fluorescence changes with pressure-induced emission changes onsolo Cy3, C-PC and Cy3/BSA, we proposed that there were three possible issuesinduced by pressure at least could affect the fluorescence of Cy3/C-PC system:(a)Responses of solo Cy3and C-PC itself to pressure. Under compression, theviscosity of the water was increased, which increased the barrier of bond rotation inthe excited Cy3and reduced the efficiency of trans→cis isomerization from the firstexcited state, and finally the Cy3fluorescence was increased. On the other hand, thehigh pressure broke the conformation of C-PC, which induced the decrease offluorescence on C-PC.(b) The dye-protein interaction. In elevating the pressure,the dye-protein interaction was increased, depreciating the increasing rate of Cy3.And at higher pressure, the dye-protein interaction was enhanced by thepressure-induced conformational changes, therefore, Cy3fluorescence started todecrease.(c) FRET efficiency between Cy3and C-PC. Under compression, theFRET efficiency was enhanced, which was illustrated by the lower increasing rate anda more notable decrease of Cy3in Cy3/C-PC system in compare to that of Cy3/BSA.Meanwhile, the decreasing rate of C-PC in Cy3/C-PC system was lower than that ofsolo C-PC. Further, through the pressure-induced changes of UV-vis absorptionspectra on C-PC, we calculated the overlap integral between the Cy3emission andC-PC absorption and found that the distance from Cy3to C-PC chromophore wasshortened, which induced the increase of FRET efficiency finally. C-PC is one ofimportant components in light-harvesting complex in cyanobacteria and eukaryoticred algae, which construct phycobilisomes with phycoerythrin and allophycocyaninthrough weak interactions between them. This work is a preparatory work of usingFRET effect to investigate the protein-protein recognition and interactions, which ishelpful to introduce nanomaterials and construct functional recognition system. In conclusion, we have successfully constructed three different functionalsystems based on biomolecules/nanomaterials, which have been applied to either ionsrecognition, detection of enzyme activity or protein-proteion interactions. And wehave also performed substantially studying of the mechanism in the process ofconstruction and functional application.