The Development Of Biosensors Based On Drug Screening For Anti Helicobacter Pylori Infection

Helicobacter pylori(H.pylori)is a pathogenic bacterium that colonizes the gastric mucosa and the surface of gastric epithelial cells of more than half of the world’s population.Its persistent infection is considered to be the main non-genetic factor leading to various gastric lesions including gastric cancer.With the increase of drug resistance worldwide,the cure rate of traditional antibiotic therapy continues to decline,and it is urgent to develop against H.pylori infection drugs based on new targets.However,the limitations of traditional screening technology delayed the discovery process of new effective drugs.As an interdisciplinary technology of biology,chemistry,electricity and other disciplines,biosensor has the advantages of fast,accurate,high sensitivity and low detection limit.It has shown its application potential in the field of drug research and development.In this paper,the key bioactive molecules in the survival,colonization,and pathogenicity stages of H.pylori were heterologously expressed,respectively.Then three novel biosensors were designed and constructed using these biomolecules as recognition elements.Finally,the application potential of the developed biosensor for screening new drugs against H.pylori infection was evaluated.The type and intensity of action of the tested compounds were analyzed based on enzymatic reaction kinetics and enzyme inhibition theory.In addition,the interaction mechanism between the candidate compounds and their corresponding biological targets were further explored at the amino acid level through molecular docking.The main research results are as follows:(1)Urease is the key molecule for H.pylori to resist gastric acid environment.Based on the heterologous expression of H.pylori urease b subunit(HPUb),the HPUb/Platinum nanoparticles(Pt)/Nanoporous gold(NPG)/Glassy carbon electrode(GCE)biosensor was constructed by layer-by-layer assembly.The two-step catalytic reaction based on the specific hydrolysis of urea by urease and the efficient electrocatalytic oxidation of NH3 by Pt nanoparticles has realized the detection of urea and the rapid screening of urease inhibitors.The results showed that the inhibition constants of the tested compounds on HPUb were Ki,AHA=5.0397mmol/L,Ki,hydroxyurea=6.2012mmol/L,Ki,mcthylurea=0.3038mmol/L,Ki,acetamide=0.3866mmol/L,Ki,formamide=2.4747mmol/L,respectively.This indicated that the inhibitory intensity order of these inhibitors was:methylurea>acetamide>formamide>AHA>hydroxyurea.In addition,the interaction analysis at the amino acid level through molecular docking simulation revealed the important role of the carbonyl and primary amine groups of the inhibitor molecular structure and the GLY279,ASP362,and ALA365 in the HPUb active pocket in the process of urease inhibition.Combining the inhibition constant of urease inhibitors with the molecular binding mechanism of the urease inhibitor and HPUb,it can be inferred that the non-polar long-chain modification of one of the primary amino groups based on the molecular structure of urea is a development direction of high-efficiency urease inhibitors.(2)AlpB outer membrane protein,a important component in H.pylori biofilm formation,was selected as a biological recognition element to screen anti-biofilm drugs.A novel AlpB/colloidal gold(CG)/NPG/Nafion-reduced graphene oxide(rGO)/GCE biosensor was constructed based on the heterologous expression of AlpB.A detectable electrical signal is generated by affecting the adhesion of AlpB outer membrane proteins.The prepared AlpB-based biosensor not only successfully identified six anti-biofilm drugs:S-(Carboxymethyl)-L-cysteine(SCC),allicin,curcumin,erythromycin,rifampicin,and N-Acetyl-L-Cysteine(NAC),but also evaluated the sensitivity and action intensity of different anti-biofilm drugs binding to AlpB by interaction kinetics analysis.The sensitivity order of AlpB to the six anti-biofilm drugs was:allicin>erythromycin>SCC>curcumin>rifampicin>NAC and the action intensity of the six anti-biofilm drugs on AlpB was:rifampicin>NAC>allicin>erythromycin>SCC>curcumin.Among them,allicin had the most significant AlpB sensitivity,and rifampicin showed the greatest intensity of action.In addition,the molecular docking results showed that the six anti-biofilm drugs might exert their anti-biofilm effects by spontaneously binding to the conserved region of AlpB protein.(3)The ability of local peptidoglycan layer cleavage of the lytic transglycosylases Cag4 is the basis for maintaining the carcinogenic ability of H.pylori based on the secretion of Cytotoxin-associated gene A(CagA).The Cag4/NPG/Nafion-rGO/NPG/GCE biosensor based on signal conversion strategy of enzyme-inorganic co-catalysis was constructed using the heterologously expressed H.pylori 26695 Cag4 as the biological recognition element for screening Cag4 allosteric regulators.In the detection process,Cag4 first cleaves the β-1,4 glycosidic bond between N-acetyl-β-D-glucosamine(NAG)and N-acetyl-β-D-muramic acid(NAM)of peptidoglycan,the released NAG is then electro catalytic oxidized by NPG to generate the response current.The results showed that chitosan or carboxymethyl chitosan was a mixed Cag4 inhibitor combining non-competition with uncompetition.The inhibition constants were Ki’Chitosan=0.88909 mg/mL and Ki’Carboxymethyl chitosan=1.13480 mg/mL,respectively.Surprisingly,D-(+)-cellobiose showed the activation effect of Cag4 on cell wall lysis by decreasing the Ka value by 29.7%and increasing the Vmax value by 71.3%.In addition,molecular docking revealed the importance of the polarity of the C2 substituent group with glucose as the main structure in the Cag4 allosteric regulator.In this paper,three different biosensors were designed and constructed to achieve the screening of new drugs against H.pylori infection that target the key biological active molecules in the survival,colonization,and pathogenesis stages of H.pylori respectively.Among them,methyluria,allicin,and chitosan have the value of further research.The relationship between the structure and function of competitive urease inhibitors,anti-biofilm drugs,and Cag4 allosteric modulators was preliminarily clarified by combining the results of biosensor screening and molecular docking simulation analysis.This study provides a rapid and efficient platform for screening potential drugs against H.pylori infection,and lays a theoretical foundation and new ideas for the research and development of new drugs targeting these three biological targets.