Determination Of Glycosylation And Phosphorylation Levels Of Neurofilament Light Chain Protein In Serum

Neurodegenerative diseases are a major group of diseases that threaten human health.Synaptic disruption and neuroinflammation are the main pathological features of neurodegenerative diseases.Neurofilament light chain proteins（NFL）play an important role in the neuroskeleton,synaptic structure and function.The short head structural domain of NFL contains shared sites of glycosylation and phosphorylation and plays an important role in the function and structure of neurofilaments,which are potential markers of neurodegenerative diseases.Based on this,the main research aim of this thesis is to develop electrochemical immunosensors for the simultaneous detection of total NFL and its glycosylation and phosphorylation levels.This makes possible the accurate diagnosis of relevant neurodegenerative diseases.The design of the amplification strategy is the biggest highlight in our sensing design ideas.We combine two amplification pathways,solution and interface,with two mutually independent sources of signal amplification.This ensures the simultaneous and accurate detection of different functional regions in the same protein molecule.Specific studies include:（1）An electrochemical immunosensor was constructed for the detection of NFL content and its glycosylation level.The sensor uses mesoporous silica nano（MSN）composites(Ab₂@MSN/Cu²⁺)and horseradish peroxidase-labelled wheat germ agglutinin（WGA-HRP）as immunoprobes for the simultaneous detection of total NFL（t NFL）and glycosylated NFL（O-NFL）levels.In this method,copper ion amplification is the surface amplification pathway,while the signal for oxygen reduction originates from the solution.The sensor showed good sensitivity and selectivity for both t NFL and O-NFL with linear ranges of 1-25 pg·m L^-1and 0.25-25 pg·m L^-1,respectively,and detection limits of 0.13 pg·m L^-1 and0.11 pg·m L^-1,respectively.Spiked recoveries of patient serum samples at ten-fold dilution were in the range of 96-105%,indicating that the sensor still has good precision and accuracy in real-world sample detection.The t NFL and O-NFL levels were measured in patients with brain injury（including patients with cerebral thrombosis（CT）,Alzheimer’s disease（AD）and Parkinson’s disease（PD））and in the healthy population.The results showed that all brain injured patients had different t NFL levels from the healthy population,while the difference between t NFL levels in patients with CT and PD was not significant.At the same time,O-NFL levels were significantly different in the serum of the healthy population and the three types of brain injury patients.The results suggest that O-NFL levels are a more effective marker of neurodegenerative disease than t NFL.（2）Phosphorylation and glycosylation are two major pathways of post-translational modification of proteins that directly affect their physiological functions.Here,we constructed an electrochemical immunosensor for the detection of NFL content and its glycosylation and phosphorylation levels.It is important to note that only one of the two post-translational modifications may be present for the same protein due to competing sites for glycosylation and phosphorylation of NFL.Based on this,the sensor uses horseradish peroxidase-labelled NFL antibody（Ab₂-HRP）,MSN composite(WGA@MSN/Cu²⁺)and titanium phosphate composite(Zn²⁺@Ti P)as probes for the determination of t NFL,O-NFL and phosphorylated NFL（P-NFL）levels.In this method,Cu and Zn ion amplification is the surface amplification pathway,while the signal for oxygen reduction originates from solution.The sensor has good sensitivity and selectivity for t NFL,O-NFL and P-NFL determination with a linear range of 1-30 pg·m L^-1 and detection limits of 0.18 pg·m L^-1,0.21 pg·m L^-1and 0.19 pg·m L^-1 respectively.The recoveries obtained with this sensor ranged from 95%to 104%in tenfold dilutions of patient serum samples.The sensor was used to measure t NFL,O-NFL and P-NFL levels in patients with CT,AD and PD patients versus healthy donors.The results showed that the decrease in O-NFL levels in patients with neurodegenerative disease was accompanied by a significant increase in phosphorylation levels compared to healthy individuals.However,phosphorylation levels in patients with CT were similar to those in healthy subjects.Clearly,combining phosphorylation levels and glycosylation levels of the NFL can further improve the accuracy of the diagnosis of neurodegenerative diseases.