Study On Piezoelectric Sensor Analysis For The Detection Of Phosphoproteins

Protein plays a vital role in the life events.It is the executor of physiological functions and a direct manifestation of life phenomena.Many mechanisms that study physiology or pathology are also based on protein levels.Reversible phosphorylation of proteins is an important post-translational modification in the human body,and the occurrence of many diseases is closely related to abnormal phosphorylation.Identification of phosphorylated proteins is an important part of proteomics research,where mass spectrometry is an important tool for the analysis of phosphorylation sites.However,due to the diversity of phosphorylated proteins,the short time of natural phosphorylation sites,and relatively low abundance,it is difficult to detect phosphorylation sites.Phosphorylated proteins need to be enriched before mass spectrometry analysis to remove interference from non-phosphoproteins to increase the relative content of phosphoproteins for subsequent mass spectrometric analysis.However,these methods still have some shortcomings,including time-consuming,complicated sample pretreatment and expensive instruments.Therefore,it is essential to establish a sensitive,accurate and rapid method for trace analysis of phosphorylated proteins.In this work,a simple,efficient and sensitive quartz crystal microbalance sensor was developed for trace determination of phosphorylated proteins.The quartz crystal microbalance sensor was constructed by aminated nano-titanium dioxide membrane on the gold plate surface modified with thiol undecanoic acid,realizing selective and quantitative determination for phosphorylated proteins.Thiol undecanoic acid acted as carrier for immobilizing aminated nano-titanium dioxide and it was immobilized on the surface of a quartz crystal gold plate with a gold-sulfur bond reaction.Aminated nano-titanium dioxide had a significant impact in adsorpting phosphorylated proteins specifically thanks to its large surface area and thus the sensitivity of the sensor can be improved.And the amino group-containing titanium oxide and the carboxyl group-containing decyl undecanoic acid can be assembled together by an amide reaction making the piezoelectric frequency response signal more stable.In the experiment,aminated nano-titanium dioxide was characterized by atomic force microscopy(AFM)and prepared electrodes were characterized by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS).Meanwhile,multiple factors that may affect the performance of the sensor were studied and optimized.Under the optimal experimental conditions,the piezoelectric frequency response signal was proportional to the concentration of phosphorylated proteins in the range of 0.001 mg·mL-1 to 1.0 mg·mL-1 with a low detection limit of 5.31 ng·mL-1(S/N=3).The constructed sensor exhibited good sensitivity,selectivity and favorable stability and was used to detect phosphorylated proteins in real samples.Traditional MALDI-TOF/MS method was employed to validate the accuracy of the sensor for detection results.And the results showed that there was no significant difference between the results of quartz crystal microbalance sensor and MALDI-TOF/MS method,indicating enormous potential of the constructed sensor in the future of food safety detection.Therefore,this constructed sensor can be used in phosphorylated proteins detection.