Study On Quantitative Method Of NGAL Protein Based On Surface-Enhanced Raman Spectroscopy

Neutrophil gelatinase-associated lipocalin(NGAL)is a secretory glycoprotein that can stably exist in fluid samples such as serum,plasma,and urine.The NGAL level in bodily fluids is correlated with acute kidney injury,acute infections,tumors and other diseases.Currently,NGAL is widely used as an early diagnostic biomarker for acute kidney injury.The development of a sensitive,specific,wide dynamic range,reproducible,reliable,simple,high-throughput and automated biomarker detection method is crucial for laboratory research and clinical application.Western-blotting(WB),radioimmunoassay(RIA),enzyme-linked immunosorbent assay(ELISA),chemiluminescence immunoassay(CLIA),and particle-enhanced turbidimetric immunoassay(PETIA),as well as corresponding instruments,have been used for NGAL quantification,greatly promoting the basic research and clinical application of NGAL.Due to the wide range of concentration variation of NGAL in body fluids(ranging from low ng/m L to tens of mg/m L),expanding the linear detection range of NGAL quantification methods has been one of the focuses of this field.Moreover,the detection time of NGAL as a biomarker for acute kidney injury has an important impact on its clinical application value.Surface-enhanced Raman spectroscopy(SERS)has the advantages of rapid,high sensitivity,small sample volume,and non-destructive detection,and its application in the detection of biomolecules has received increasing attention.Based on the demand for NGAL quantification and the preliminary research foundation of the research team,this paper attempts to establish a SERS-based NGAL protein quantification method.The main research content and results are as follows:(1)Establish a solid-phase quantification method for NGAL protein based on SERS.Firstly,NGAL antibody and silicon wafer were covalently coupled by sequentially modifying the silicon wafer with hydroxylation,alkylation and aldehyde treatment to produce the NGAL detection chip.At the same time,4-nitrothiophenol(4-NTB)was used as a Raman reporter molecule,Ag@Au core-shell nanoparticles were used as Raman enhancement substrates and NGAL aptamers were used as specific recognition molecules to prepare the SERS detection probe Ag-NTB@Au-apt for NGAL.Then,a SERS solid-phase quantification method for NGAL was established by the preliminary optimization of the detection system.The methodological study results showed that the linear detection range is 0.1～1000 ng/m L(the lowest detection limit is0.054 ng/m L),the recovery rate is 86.84% and the intra-batch and inter-batch differences are 19.34% and 33.06% respectively.This indicates that the method has a wide linear detection range and high detection sensitivity.On the other hand,this method has disadvantages such as long detection time(over 2 hours)and large intrabatch and inter-batch variability.Although this method has significant advantages such as a wide detection range and high sensitivity,it still needs to be further optimized to shorten the detection time and improve the reproducibility.(2)Development of a liquid-phase quantitative method for NGAL protein based on SERS.Firstly,NGAL aptamer-modified gold nanoparticles(Au NPs)and silver nanoparticles(Ag NPs)were prepared.NGAL protein competitively binds to the aptamer on Au NPs with Raman reporter molecule Malachite Green(MG).Au NPs and Ag NPs serve as SERS substrates for MG and the Raman intensity of MG was used as the parameter to quantify NGAL.Then,a SERS-based liquid-phase quantitative method for NGAL was established through preliminary optimization of sample incubation time,MG concentration,centrifugation conditions,nanoparticle closure and other conditions.The methodological study results showed that the linear detection range is 0.15～5pg/m L with a detection limit of 0.133 pg/m L,the recovery rate is 87.513%,the detection time is about 1.5 hours and the intra-batch and inter-batch differences are12.10% and 31.03% respectively.These data indicate that this method can significantly improve detection sensitivity and shorten detection time,but its detection linear range is narrow and there is a large inter-batch difference.Therefore,there is still much room for improvement in this method.In summary,this paper has established two surface-enhanced Raman spectroscopy(SERS)-based NGAL protein-specific quantitative methods.The establishment and improvement of these methods will enrich the NGAL quantification methods,contributing to NGAL laboratory research and clinical applications.In addition,this paper’s research provides experimental reference and theoretical basis for the application of SERS technology in biomarker detection.