A New Biosensor Based On The Recognition Of Phages And The Signal Amplification Of Organic-inorganic Hybrid Nanoflowers For Discriminating And Quantitating Live E.coli Bacteria In Urine

ObjectiveRapid and accurate detection of live Escherichia coli?E.coli?in urine is indispensable for the diagnosis of urinary tract infection in clinical practice.In the present study,a new electrochemical biosensor was developed to quantify live E.coli in urine accurately and rapidly,by uniting the specific recognition of T4 phages and the signal amplification of organic-inorganic hybrid nanoflowers?GOx&HRP-Cu₃?PO₄?₂?and the capture probe antimicrobial peptide?AMP?magainin I.The capability of this constructed biosensor was evaluated by detecting live E.coli.Methods1.Preparation of organic-inorganic hybrid nanoflowers:CuSO₄·5H₂O,HRP and GOx were added into PBS simultaneously.After incubation at room temperature for 12 h,the nanoflowers precipitates were collected.Then the chemical structure and property of hybrid nanoflowers was characterized.2.Fabrication the compound of nanoflowers&AuNPs&Thi&phage:The T4 phage and thionine?Thi?were connected to hybrid nanoflowers through Au-NH bond,using the AuNPs as a bridge.Then the compound was collected and its status was characterized.3.Fabrication of the electrochemical biosensor:AMP magainin I was fixated on the Au electrode surface by Au-S bond firstly.When E.coli appeared,the compound of nanoflowers&Au NPs&Thi&phage can be combined to electrode surface.Then the electrochemical biosensor can be started to quantitate E.coli.4.Optimizing the experimental conditions:In order to reach optimal analytical performance,several important parameters were optimized,including the concentration of AMP magainin I,the immobilisation time of AMP magainin I on Au electrode,the pH of working solution and the incubation time between nanoflower compound and E.coli.5.Evaluation analytical property of the proposed electrochemical biosensor:Under the optimal experimental conditions,the proposed biosensor was used to quantitate different concentrations of E.coli.And some independent experiments were performed to assess its repeatability,specificity,linearity range,limit of detection and stability.6.The recovery experiment of urine specimen:Different concentrations of E.coli?1.5×10³ CFU/mL,1.5×10⁵ CFU/mL,1.5×10⁷CFU/mL?were prepared with healthy urine samples.These urine samples were quantified by the proposed electrochemical biosensor and traditional method?culturing and aerobic plate counts?.Results1.The character results of hybrid nanoflower on morphology and spectroscopy:?1?The high-resolution SEM image demonstrated that hierarchical peony-like flower morphology with good monodispersity,which was assembled from numerous nanoplates;?2?The chemical components of HRP and GOx were found by FT-IR in hybrid nanoflowers;?3?XRD verified that the hybrid nanoflowers were well crystallized and its crystal structure was similar to Cu₃?PO₄?₂.?4?The XPS demonstrated that P,Cu,C and O were the main components of the hybrid nanoflowers.2.The character results of nanoflowers&AuNPs&Thi&phage by electron microscope:?1?The unconnected nanoflowers were smooth and had uncontaminated petal-like structures.?2?After reacting with AuNPs,several granular substances were observed by high-resolution TEM image,which were considered to be AuNPs.?3?After reacting with phages,the uneven surface was observed on the petal-like structure of nanoflowers by SEM.3.The results of optimizing experimental:?1?The optimal concentration of AMP magainin I was 10?g/mL at the range of 5-15?g/mL;?2?The optimal immobilisation time of AMP magainin I on the Au electrode was 1 h at the range of 0.5-12.0 h;?3?The optimal pH of working solution was 5.0 at the range of pH 3.0-8.2;?4?The optimal incubation time between nanoflower compound and E.coli was 20 min at the range of 5-25min.4.The results of property evaluation:?1?Repeatability:The different concentrations of E.coli?1.5×10² CFU/mL,1.5×10⁵ CFU/mL,1.5×10⁸CFU/mL?were measured repeatedly with the proposed electrochemical biosensor,and the RSD values were 3.7%,3.3%and 2.8%,respectively?n=10?;?2?Specificity:The obvious current response was observed only with the existence of live E.coli.Current responses of these interfering bacteria and dead E.coli were almost negligible;?3?Linearity range and limit of detection:The biosensor had good linearity between the current signal intensity and the logarithm concentrations of E.coli from 15-1.5×10⁸CFU/mL.The linear regression equation was I??A?=1.06logc+1.10?R²=0.99?with a very low detection limit of 1 CFU/mL.?4?Stability:After the biosensor was stored 5 d,10 d and 15 d at 4?,compared with its initial response,the current response was 97.4%–97.6%,94.1%–94.5%and91.5%–92.0%,respectively?n=3?.5.The results of recovery experiment:The recovery rate of the presented biosensor was identical to that of plate count results.These showed that the accuracy of the proposed biosensor was acceptable.ConclusionA new electrochemical biosensor was proposed successfully based on the recognition of phages and the signal amplification of organic-inorganic hybrid nanoflowers.The proposed electrochemical biosensor had good repeatability,specificity and stability.Without any intricate pre-treatments,the electrochemical biosensor can be used to detect live E.coli with a very low detection limit and wide detection range.Additionally,the electrochemical biosensor can quantify live E.coli within only 140 min.The recovery of the presented biosensor was identical to that of plate count results.These showed that the accuracy of the proposed biosensor was acceptable.Therefore,it was possible for the proposed biosensor to quantify live E.coli in practical application.This electrochemical biosensor showed great potential application in diagnosing and monitoring UTI in clinical settings.