Construction Of Surface Plasmon Resonance Imaging And Its Study On The Screening Of Nucleic Acid Aptamers

Surface plasmon resonance imaging(SPRi)sensing analysis is one of the commonly used methods of life analysis chemistry.It has real-time,label-free,high-throughput,high-sensitivity detection characteristics.The dynamic information acquisition about molecular interaction shows a huge advantage.The screening process of nucleic acid aptamers was a cumbersome,time-consuming and complex molecular group recognition process.It is one of the research frontiers in recent years by applying real-time and label-free technology to nucleic acid aptamer screening technology.The application of SPRi to nucleic acid aptamer screening is important for its rapid discovery of new aptamer molecules.In this work,the surface plasmon resonance imaging instrument was constructed,and the sensitivity of the instrument detection signal and biomolecule detection ability were studied.Some analytical methods based on SPRi screening for nucleic acid aptamers have been developed,enabling rapid discovery of new nucleic acid aptamer molecules.The main research results are as follows:1.Construction and performance characterization of intensity modulated surface plasmon resonance imagingIn this chapter,we built an intensity modulated surface plasmon resonance imaging based on Krestchmann.By selecting a wide-spectrum wavelength low-temperature drift LED as the incident light source,and using a collimating lens,a narrow-band filter,a polarizing plate high refractive index prism,a low-magnification optical lens,and a high-resolution cooling CCD camera as components.The auxiliary assembly of precise positioning of these optical devices was realized by using three-dimensional printing technology,thereby completing the construction of a surface plasma imaging instrument.Then use different refractive index solutions to test the sensitivity and signal resolution of the instrument.The sensitivity of the instrument reaches 3.126 × 104 A.U./RIU,and the signal resolution can reach 2.259 ×10'7 RIU.The spatial resolution of the instrument is characterized by a TEM copper mesh with different mesh numbers,and the instrument is capable of achieving a spatial resolution of approximately 15 ?m.Finally,the instrument detects the interaction between biomolecules(mouse IgG/goat anti-mouse IgG)and achieves a detection sensitivity of 4 nM.The SPRi has high-throughput and high-sensitivity detection capability in the field of bioassay.2.Silver Decahedral Nanoparticles Empowered SPR Imaging-SELEX for High Throughput Screening of Aptamers with Real-Time AssessmentWe constructed a real-time screening method for nucleic acid aptamers of lactoferrin based on surface plasmon resonance imaging(SPRi)signal amplification by decahedral silver nanoparticle(Ag10-NPs).Eight parallel channel microfluidic chips were combined with SPRi sensor chip,the target protein(lactoferrin protein(Lac))was immobilized in the positive channel,and the mixed control proteins(?-lactalbumin(?),beta-lactoglobulin(?),casein(casein)and bovine serum albumin(BSA))were immobilized in the negative channel.Ag10-NPs was conjugated with an ssDNA library(lib)(Ag10-NPs-lib)that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence.Introduction of the Ag10-NPs-lib to the SPRi flow channels drastically increased the sensitivity of SPRi signal for real-time monitoring of SELEX.The work allows rapid screening of potential targets,and yields nine aptamers with high affinity(nanomolar range)for Lac after only six-rounds of selection.The aptamer Lac 13-26 was then further tested by SPRi,and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac.The novel SPRi-SELEX method demonstrated here showed many advantages of real-time evaluation,high throughput,and high efficiency.3.Elimination terminal fixed region screening and high-throughput kinetic determination of aptamer for lipocalin-1 by surface plasmon resonance imagingA highly efficient method for eliminating terminal fixed region interference of aptamer with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles probe(Ag10-A10-RP(15))capture and block the terminal fixed region candidates.Proteins microarray chip was developed by immobilization of target protein(Lipocalin-1(LCN-1))and control proteins(Human serum albumin(HSA),Bovine serum albumin(BSA)and Holo-transferrin)on the biochip surface.The nucleic acid pool was first incubated with target and then captured by hybridization with Ag10-A10-RP(15).The work allows rapid screening of aptamer elimination fixed-region interference,and the kinetic constants of candidate sequences between LCN-1 can be quickly determined using SPRi technology.Eventually,ten aptamers with high affinity and specific for LCN-1 after only fifth-round of selection was acquired.4.Screening of lactoferrin double aptamers by surface plasmon resonance imaging combined with microarrayNucleic acid aptamer probes for different immune sites in lactoferrin molecules were screened based on SPRi combined with decahedral nanosilver signal amplification strategy.Based on the principle of biomolecular recognition,a method for label-free,high-throughput screening of double immune sites has been developed.Anti-lactoferrin nucleic acid aptamers were immobilized on the surface of the SPRi sensor chip.The lactoferrin was introduced and captured by the aptamer on the surface of chip,and then the second nucleic acid strand was introduced.The binding signal of SPRi was observed to determine the recognition event of the second immune site.The second SPRi signal was generated only when the site of lactoferrin interaction with the second nucleic acid molecule was different from the first immune site.Based on this principle,we finally screened a pair of lactoferrin nucleic acid aptamer pairs(Lac-8 and Lac-25)and developed a lactoferrin fluorescence detection method using the selected pair of nucleic acid aptamers.