Studies On Paper-Based Analysis Of Pseudomonas Aeruginosa Using Bacteriophage Tail Fiber Protein As Recognition Element

Rapid and sensitive detection of pathogenic bacteria is an effective way for diagnosing infectious diseases and guiding antibiotic use.Due to the abuse of antibiotics,resistant bacteria become a severe threat to global public health.Conventional bacterial detection methods require long-time analysis and high-cost manipulation,which fail to offer timely diagnosis information in clinic.Therefore,it is essential to develop effective,sensitive and affordable bacteria detection methods.Bacteriophages are the most abundant and ubiquitous virus on the earth,which can attach onto the surface of host bacteria and infect them.Bacteriophage tail fiber protein(TFP)is a functional protein which plays a major role in recognizing host bacteria.In our works,the gene sequence of TFP from a Pseudomonas aeruginosa(P.aeruginosa)bacteriophage Pa P1 was obtained and then ligated with the gene of small ubiquitin-like modifier(SUMO)tag to express SUMO tagfused TFP(STFP).The fusion protein shows significantly improved aqueous solubility in comparison with the original recombinant TFP,thus can be an ideal recognition element.Based on the recognition capacity of STFP toward P.aeruginosa,novel strategies for P.aeruginosa detection and antibiotic susceptibility test were established.The works are described as following:(1)Expression,purification and biological properties of STFPBacteriophage TFP is a functional protein which shows significant selectivity for anchoring bacterial cells.In this study,we obtained the gene fragment of TFP from bacteriophage Pa P1.In order to improve the water solubility of TFP,SUMO tag was fused to the N-terminal of TFP,then the obtained gene fragment was inserted between Nde I and Hind III of p ET30 a.The recombinant plasmid p ET30a-STFP was transformed into Escherichia coli expression system for expression and purification of STFP with Ni-NTA affinity column.The molecular weight of the obtained STFP was determined to be around 80 KDa,which is identical to the theoretically estimated value.The biological experimental results demonstrate that the fused SUMO tag has no interference on the binding capacity of STFP.Meanwhile,STFP has nonlytic activity and shows broadspectrum recognition ability for P.aeruginosa when compared with bacteriophage Pa P1.(2)Development of lateral flow assay(LFA)strip with STFP for rapid detection of P.aeruginosaFirstly,the nitrocellulose(NC)membrane sprayed with TFP and STFP was stained with Ponceau S for visual observation of protein distribution.It showed that STFP can be uniformly distributed onto NC membrane to form a homogeneous test line while TFP cannot.Therefore,STFP with good distribution property was sprayed on NC membrane as capture molecule.Au Co nanoparticles with chemiluminescence(CL)enhancement property were synthesized by one-step reduction method and labeled with aptamer as signal tracer.Thus,P.aeruginosa can be semi quantified with accumulated color band on test line and quantified with CL signal catalyzed by Au Co NPs.The concentration range for quantification was 3.3 × 10~2 CFU/m L to 3.3 × 10~7 CFU/m L,and the detection limit was 48 CFU/m L.The developed method showed good selectivity for P.aeruginosa and the recoveries were between 81% and 112% in cerebrospinal fluid,physiological salt solution,drinking water.We cultured P.aeruginosa in pear juice and then assayed with LFA method and traditional plate counting method in parallel,the relative errors of two methods were-12.5%-3.8%.The whole process for detection of P.aeruginosa can be completed within 20 min,showing attractive merit of rapidness for point-of-care testing.(3)Detection and antimicrobial susceptibility test of P.aeruginosa using STFP modified paper-based analytical deviceIn this study,a paper-based analytical device(PAD)with microzone array was fabricated on a filter paper using a handheld and lightweight stamp.PAD was modified with chitosan,and immobilized with streptavidin by chitosan-glutaraldehyde crosslinking reaction.STFP was reacted with NHS-biotin to obtain biotinylated STFP,which can be immobilized in PAD by biotin-streptavidin affinity system.For sensor construction,STFP was used as capture molecule,fluorescein isothiocyanate labeled antimicrobial peptide was used as signal probe.In the presence of P.aeruginosa,a sandwich complex was formed on the PAD,and P.aeruginosa can be quantified by the fluorescence signal.The results showed that the detection range of target bacteria was 1.0 × 10~3 CFU/m L to 1.0 × 10~7 CFU/m L,and the detection limit was 137 CFU/m L.The method showed good specificity and can be applied for the detection of P.aeruginosa in serum,urine and milk.The antimicrobial susceptibility test results of P.aeruginosa for imipenem,meropenem,cefepime,amikacin and gentamicin were obtained by PAD,which were consistent with the traditional broth dilution method.The established PAD are inexpensive,sensitive and accessible,showing great application potential for pathogens detection,especially in resource-limiting areas and countries.