Investigation Of Protein-and DNA-based Interactions Using Surface Plasmon Resonance And Related Technologies

Surface plasmon resonance(SPR)is a powerful optical technique for measuring the thickness and structure of ultrathin adsorbate layers and for evaluating affinity of biomolecules at metal film/solution interface. SPR interrogates biomolecular interactions without the necessity of labeling the analytes.Moreover,the analysis is sensitive and can be conducted in real time.However,there are certain inherent limitations in conventional SPR analysis.On one hand,the analysis becomes very difficult when small molecules are assayed or weak affinity reactions are investigated.On the other hand,at issues are whether the preimmobilized biomolecules onto the sensor surface can retain their native structures and functionality O ur work aims at addressing these two questions.To improve the sensitivity of SPR dectection,three approaches have been implemented.Firstly,much attention was paid to the sensitivity of SPR instrument itself.A highly sensitive SPR spectrometer equipped with a bicell detector was constructed and combined with a microbore flow injection device.As a result,the angle resolution was as small as 10^-5degree.Such a resolution is lower than that available on most commercial SPR instrument.Secondly,optimal immobilization conditions of biomolecules were investigated.For example, 11-mercaptoundecanoic acid(MUA)instead of carboxylmethylated dextran was used to immobilize metallothionein,and the approch afforded a much low concentration detection level for metal ions.The detection level for Cd²⁺(0.1μM or 15 ppb)compares well with other well-established sensitive analytical techniques.The reason is due to compactness of the preformed MUA layer and our highly sensitive SPR. Thirdly,an enzyme-catalyzed precipitation reaction is used to amplify the SPR signal.In the presence of H₂O₂,the horseradish peroxidase enzyme catalyzes oxidation of 4-chloro-1-naphthol(CN)to form a precipitate on the sensor surface.The precipitated film significantly lowers the detection level,allowing detection of oligodeoxynucleotide(ODN)target concentration as low as 10 fM.To verify whether the native structure and function of biomolecules may alter after their immobilization on SPR chip surface,a solution-based,viz,affinity capillary electrophoresis(ACE),is used to confirm the binding constant K_b obtained by SPR.The K_b value between ferulic acid(FA)and bovine serum albumin(BSA)determined from the SPR measurements((5.1±0.6)×10⁴ M^-1)is in excellent agreement with the value obtained by the ACE mobility ratio assay((5.6±0.4)×10⁴ M^-1). The consistency of measurements between ACE and SPR,a technique addressing interfacial processes,confirms that immobilization of the protein onto surfaces does not alter its interaction with other molecules.Finally,three different immobilization methods are carried out to investigate the binding between antimicrobial agent(triclosan)and enoyl-acyl carrier protein(ACP)reductase(FabI).The similar K_b values indicate that all of the immobilization methods do not alter the interaction of this protein with a potential antimicrobial molecule(triclosan).CE is utilized for the qualitative and quantitative analysis of the protein/ligand complex recovered after the SPR analysis.Our method provides a new route for screening potential antimicrobial agents.