Microfluidic Phage Display For Peptide Screening

Molecular recognition is the molecular basis of physiological and pathological activities.Phage display is one of the powerful tools for studying molecular recognition,which plays an important role in basic research,targeted therapy and delivery,molecular diagnosis and in vivo/in vitro imaging.Microfluidics have been widely used in biology,chemistry,medicine and their intersecting fields by virtue of its small reaction system,high transfer efficiency,precise fluid manipulation,easy integration and automation.There have been many innovative strategies based on microfluidics for pahge display innovation.However,the current peptide screening by phage display still faces the problems of time-consuming and labor-intensive process,low success rate,and low efficiency.Based on the above problems,this thesis started with traditional phage display methods,and proposed a variety of microfluidic platforms to develop more rigorous,automated,and more efficient methods for ligand screening by phage display.The main parts of this thesis are as follows:1.Screening for specific peptide against Glypican-1 by phage displayGlypican-1(GPC1)is an extracellular vesicle biomarker for pancreatic carcinoma,whose expression is related to the size and growth of tumor and survival rate.The screening of GPC1 specific peptide is of great significance for the early diagnosis,mechanism understanding and functional study of pancreatic carcinoma.In this chapter,A specific GPC1 peptide WJX6 was identified with high affinity and specificity through three rounds of screening,which has realized peptide-based specific recognition of tumor cells.The highly specific GPC1 peptide will contribute to the development of new diagnosis methods for pancreatic carcinoma and molecular studies of disease development.2.Stringent peptide ligand screening based on microfluidic frequency-increasing collision by phage displayBased on the issues of multiple rounds and low success rate in the current biopanning process,from the perspective of increasing the screening stringency,this chapter used deterministic lateral displacement(DLD)microfluidic system to obtain more enriched library through fewer rounds based on frequency-increasing collisions.DLD is used to enhance the screening stringency of the washing step in the traditional screening process,and the rest of the process is consistent with the traditional screening.It has been verified that DLD has exhibited about 10 times higher efficiency for nonspecific adsorption removal,compared with the maxium removal of traditional method,thus reaching higher enrichment in every round.In addition,compared with the third round library of the traditional method,the DLD method required only two rounds to obtain a more convergent secondary library with higher affinity.Finally,a high-affinity EphA2 specific peptide GHH was obtained,with a dissociation constant of 388 nM,has exhibited excellent cell selectivity and cell permeability as a promising molecular tool for drug delivery.In summary,DLD method has reduced the number of screening rounds,improved the success rate of new ligand screening,and also increased the possibility of obtaining high-quality ligands.3.Automated peptide ligand screening platform based on digital microfluidicsFrom the perspective of automation,this chapter proposes an automated biopanning platform(Auto-Panning)based on digital microfluidic technology,dispensed with labor-intensive process of peptide screening.By integrating magnetic separation module and temperature control module on the digital microfluidic platform,multiple rounds of ’counter selection-positive selection-washing-elution-amplification’were automatedly processed.Benifited from the continuous liquid handling,three rounds of biopanning can be completed within 16 hours,avoiding the weekly manual operation in the traditional approach.Through Auto-Panning,a bioactive EphA2 peptide ligand HPF was screened,which successfully proved the feasibility and potential of the platform in the field of ligand screening.In addition,Auto-Panning reduced the reagent consumption to only 3%of the traditional method.The liquid control based on software is more flexible and user-friendly than the complicated pump/valve control in traditional micro fluidics.As a highly automated platform,AutoPanning will further promote the development of molecular screening and accelerate the discovery of new ligands.4.High-throughput monoclonal screening platform of phage displayScreening is not the end of the ligand discovery,after which a large number of monoclonal characterization is still needed to hit the high-affinity sequences.The laborintensive but low-throughput characterization process further limits the new ligand discovery.From the perspective of cascading screening and characterization,this chapter used droplet microfluidics to construct a monoclonal screening platform,i.e.double monoclonal Display(dm-Display).The processes of monoclones separation,amplification,target recognition,and affinity characterization have been integrated into one droplet,so that high-affinity clones can be directly identified and separated from a highly diverse phage display library,avoiding cumbersome enrichment and affinity identification steps in traditional biopanning.In this chapter,we have screened the specific peptide ligand against transferrin receptor(TfR,CD71)and Glypican-1(GPC1)within only 24 hours.Based on its monoclonal manipulation strategy,it can further bring impetus to other display platforms for ligand screening to discover new drugs,vaccines,or therapeutic reagents.