Study On The Construction Of Deoxyribozyme Functionalized Au NP@Pt Probe And The High-fidelity Detection Of L-histidine

L-histidine is a semi-essential amino acid,which is of vital importance for infants’ growth and development.However,infants need to uptake of L-histidine from the outside because the amount of L-histidine synthesized in babies’ body can not meet the needs of infants’ growth and metabolism.For non-breastfed babies,infant formula powder with enough L-histidine is important for their growth.In order to ensure the food quality and safety,it is of great significance to develop an efficient and accurate detection method to measure the content of Lhistidine in milk powder.The concentional methods for L-histidine detection involve largescale instruments,high detecting cost,and skilled professionals,which are not suitable for daily detection.Deoxyribozyme(DNAzyme)is a class of single-stranded DNA molecule with high specificity for substrate recognition and high catalytic capacity.They are often combined with nanomaterials to construct nanobiosensors,and have shown great potential in the field of food quality and safety testing.However,the stability of nanobiosensor structure has always been a limiting factor in terms of its application in complex systems.In the previous research of our group,we found that Au NP@Pt nanoparticles have similar optical properties and large specific surface area to Au NP,and the Pt-S bond is resistant to thiol interference,providing an ideal material for the construction of highly stable nucleic acid-functionalized nanoprobes.Based on this,this thesis proposes to construct DNAzyme-functionalized Au NP@Pt nanoprobes in order to substantially improve their stability in complex biothiol-rich systems.On this basis,the rapid and high-fidelity detection for L-histidine in milk powder was carried out using the DNAzymefunctionalized Au NP@Pt nanoprobe developed above.The specific work is as follows:1.Construction and performance investigation of DNAzyme-functionalized Au NP@Pt probesBecause the Pt-S bond is resistant to thiol interference,Au NP@Pt nanoparticles were prepared in this work,and the DNAzyme showing cross-linking with-SH and fluorophore comodified by Pt-S bond was used to construct DNAzyme-functionalized Au NP@Pt probe.Fluorescence spectroscopy experiments show that Au NP@Pt nanoparticles have similar optical properties to Au NP,which can significantly quench the fluorescence signal of Cy3.Compared with free DNAzyme,the substrate catalytic activity of DNAzyme-functionalized Au NP@Pt probe was not affected.The DNAzyme-functionalized Au NP@Pt nanoprobes showed good stability after treatment with whey protein,the main component of sulfhydryl-rich formula milk powder,and the fluorescence only increased by 2 times within 12 h.In contrast,the DNAzyme-functionalized Au NP probe based on the Au-S effect produced the same fluorescence signal recovery in only 1 h.This study provides ideas for the development of highly stable DNAzyme probes.2.DNAzyme-functionalized Au NP@Pt probe for high-fidelity detection of L-histidineDeveloping a simple,rapid and accurate L-histidine detecting method for quality assessment of infant formula is of great significance for ensuring food quality and safety.This chapter uses the DNAzyme-functionalized Au NP@Pt probe constructed in the previous chapter to carry out high-fidelity detection of L-histidine.When there is no L-histidine in the detection system,the fluorescence is quenched due to the close proximity of the fluorophore on the DNAzyme and the Au NP@Pt nanoparticles.When L-histidine is detected present in the system,L-histidine can induce the catalytic domain of DNAzyme to fold to form a specific secondary structure,and then catalyze the cleavage of the RNA site in the substrate sequence,generating and releasing short chains containing fluorescent groups.The fluorescence recovered because the fluorophore group was far away from Au NP@Pt nanoparticles.The experimental results showed that the linear range of the method for the detection of L-histidine was 0-100 μM,and the detection limit was 3.87 μM.In addition,the probe has good selectivity and can significantly distinguish common amino acids from L-histidine.The detection results of spiked infant formula showed that the recovery rates were 97.87%-103.58%,indicating that the detection accuracy of this method was high.In this work,a simple and high-fidelity Lhistidine analysis method was developed,which provided an important means for on-site instant assessment of food quality such as milk powder.