Application And Investigation Of New Analytical Properties Of R-phycoerythrin

R-phycoerythrin?R-PE?is a natural,non-toxic,stable and water-soluble light-harvesting pigment protein extracted from algae.R-PE has a conservative and relatively stable structure,bright photoluminescence,good biocompatibility,and high pH stability,and excellent optical properties such as high absorption coefficient,high fluorescence quantum yield and large Stokes shift.These excellent properties make R-PE a promising model protein and fluorescent marker in biological analysis and biomedicine.R-PE has an inherent affinity towards deoxyribonucleic acid?DNA?.It can bind to DNA through non-covalent electrostatic interaction,hydrogen bond,and hydrophobic interaction.Therefore,exploring the non-covalent interaction between R-PE and DNA will help to expand the application of R-PE as a DNA sensing and analysis tool.However,as far as we know,the non-covalent interaction of R-PE-DNA has rarely been reported.In addition,there are abundant amino acid residues on the surface of R-PE.Previous studies have suggested that o-phthalaldehyde?OPA?,the main component of medical disinfectants,can react with amino groups under certain conditions,and the fluorescence intensity of the derivatives can be used to monitor the concentration of OPA.Therefore,we exploited the excellent optical properties of R-PE and rich amino acid residues on the surface of R-PE to develop a novel ratio fluorescent probe for OPA detection.On the basis of the excellently physical and chemical properties and unique optical properties of R-phycoerythrin,two fluorescent probes based on R-PE were constructed to detect DNA and OPA,respectively.The details are as follows:1.In this section,based on the non-covalent interaction between R-phycoerythrin?R-PE?and DNA mediated by Poly dimethyl diallyl ammonium chloride?PDADMAC?,a simple and rapid fluorescent biosensor for DNA detection was developed.In the presence of PDADMAC,R-PE can effectively adsorb BHQ2-labeled single-stranded DNA?BHQ2-ssDNA?to form a stable R-PE/ssDNA complex.The fluorescence of R-PE can be significantly quenched by fluorescence resonance energy transfer?FRET?between R-PE and BHQ2.After the emergence of DNA target,BHQ2-ssDNA was released from the surface of R-PE due to the formation of rigid double strands,and the fluorescence of R-PE was also restored.The linear detection range of the fluorescent biosensor is 0.5¹00nM,the detection limit is 0.17 nM,the sensitivity is high,and the single base recognition has good selectivity,which can be used for the detection of actual samples.The sensor is natural,non-toxic,biocompatible and stable,and does not need additional cleaning steps and long incubation time in the measurement process,so it is suitable for rapid analysis.This method provides a new perspective for the design of DNA analysis methods and a promising platform for nucleic acid detection and molecular sensing.2.In this section,based on the derivatization of R-phycoerythrin with o-phthalaldehyde?OPA?,a novel fluorescence ratio probe for the detection of OPA was constructed.After unfolding by SDS,R-phycoerythrin exposes all primary amino groups,which reacts with OPA in an alkaline environment in the presence of?-mercapto ethanol??ME?.In this sensing system,with the increase of OPA concentration,the fluorescence signal of R-phycoerythrin weakens gradually,whereas the blue fluorescence signal is gradually enhanced.A strong-weak fluorescence change significantly enhanced the sensitivity of the fluorescence probe.This probe can selectively detect OPA within the linear range of 5¹00?g·mL^-1 with the detection limit of 1.7?g·mL^-1.Furthermore,the color change of solution,originating from the reaction between R-PE and OPA,provides a simple and fast method to detect OPA by the naked eye,which extended the application of the probe in the detection of OPA.