Application Of Multifunctional Peptides In SPD-L1 Sensitive Detection

Cancer is one of the major causes of death.Soluble programmed death-ligand 1(sPD-L1)has become an important biomarker for early screening of cancer and prediction of immunotherapy.It can be divided into free PD-L1 and PD-L1 positive exosomes(PD-L1~+exosomes)two categories.Therefore,monitoring of two sPD-L1in serum,respectively,can provide data support for personalized medical treatment of patients.However,the concentration of sPD-L1 in clinical samples is too low to achieve sensitive detection.Surface plasmon resonance(SPR)sensor and electrochemical sensor own the merits of easy operation,small sample and low cost,which have been used for the detection of biomarkers.In recent years,designable multifunctional peptides can freely combine the functions of targeting,antifouling,anchoring and self-assembly to improve the capture efficiency.Based on this,two kinds of multifunctional peptides were designed to construct SPR sensor and electrochemical sensor for sensitive capture of sPD-L1,and had been successfully applied to the detection of sPD-L1 in clinical samples.The main contents of this research are as follows:1.Multifunctional peptides based on intracellular domain-specific for sensitive detection of sPD-L1The free PD-L1(sPD-L1)in serum retains the complete structure of PD-L1 and can combine with PD-L1 inhibitors to reduce the therapeutic effect of immunotherapy.Therefore,quantitative detection of serum sPD-L1 is very important for predicting the curative effect and monitoring the disease progress.All the reported sPD-L1 probes are extracellular domain binding molecules with overlapping binding sites,which are not conducive to efficient capture of sPD-L1.Based on this,a multifunctional peptide SIBP composed of specific recognition sequence and anchoring sequence was designed and applied to the SPR detection of sPD-L1 in serum.The recognition sequence of SIBP can specifically target and capture the intracellular region of sPD-L1.Moreover,the combination of SIBP and Anti-PD-L1 avoids the overlap of binding sites,which eliminate the interference of steric hindrance and improve the capture efficiency of SPR sensor.The anchoring sequence of SIBP can form a dense antifouling monolayer on the surface of gold nanoparticles(Au NPs),which can effectively prevent the non-specific adsorption,and ensure the selectivity of the sensor.The results showed that the sensor had a good linear relationship in the range from 10ng/m L to 2000 ng/m L,while the detection limit was 1.749 ng/m L,and cancer patients and healthy donors in clinical samples had been successfully distinguished.Therefore,the SPR sensor designed in this experiment is expected to be used in the clinical detection of sPD-L1.2.Tree-like antifouling multifunctional peptides based on multivalent interactions for sensitive detection of PD-L1~+exosomesPD-L1 positive exosome(PD-L1~+exosomes)is a kind of sPD-L1 and has become an important biomarker for the prediction of immunotherapy.However,the low concentration of PD-L1~+exosomes in serum greatly limited the detection.Moreover,the complex components in serum samples can easily cause non-specific response of the sensor,which leads to false positive results,and reduces the sensitivity of detection.Inspired by this,a tree-like multifunctional antifouling peptide TMAP composed of anchoring sequence as"root",antifouling sequence as"branches"and recognition sequence as"leaves"was designed for the electrochemical detection of PD-L1~+exosomes.The anchoring sequence ensures that TMAP can form a dense monolayer on the interface,which thickens the hydration layer of the antifouling sequence and further enhances the antifouling performance of the sensor.In addition,the recognition sequence of TMAP forms multivalent interaction with PD-L1~+exosomes through different“branches”,which improves the capture efficiency of PD-L1~+exosomes.TMAP and silver nanoclusters as dual signal amplification can significantly enhance the sensor’s sensitivity and selectivity.The sensor showed a good linear relationship in the spiked samples,and can successfully distinguish healthy donors from cancer patients in clinical samples.It is proved that this electrochemical sensor based on TMAP can realize the sensitive detection of PD-L1~+exosomes,which provides an idea for the application of multifunctional peptides in the field of biosensing.