Fast Detection Of E.coli By A Novel Fluorescent Sensor Based On A Fret System Between UCNPs And GO@Fe₃O₄ In Urine Specimens

ObjectiveBacterial urinary tract infection is a common infectious disease,80%of which are caused by Escherichia coli.Traditional detection methods are time-consuming,which is not conducive to select the valid antimicrobial agents in clinical practice.Therefore,it is urgent to establish a new method to provide the reliable laboratory evidence for the rapid and sensitive diagnosis of urinary tract infection.This study intends to use the principle of FRET between UCNPs and GO@Fe₃O₄nanocomposites to construct a new fluorescent biosensor for detection of Escherichia coli rapidly and sensitively in urine specimen.Furthermore,the analytical performance of this biosensor was evaluated on the basis of optimizing the key parameters of the newly-built sensor.Methods1.Synthesis of UCNP nanoparticles:First,the OA-capped UCNPs were synthesized by a hydrothermal method under an argon atmosphere;then,polyacrylic acid was added and react with OA-capped UCNPs overnight to synthesize water-soluble UCNPs.Its morphology was characterized by transmission electron microscope,and its chemical composition was characterized by energy spectrometer.2.Synthesis of UCNP nanoprobes:UCNPs-PAA and E.coli aptamer were mixed in HEPES solution and incubated at 37°C for 240 min.And then they were washed twice with deionized water by centrifugation（10625×g）.Subsequently,mPEG-NH₂was added to the UCNPs-Apt solution and reacted at 37°C for 120 min.After washing twice with deionized water,the mPEG-UCNP-Apt nanoprobes were dispersed in the HEPES buffer and stored at 4°C for future usage.Zeta potential was used to characterize the synthesis process of mPEG-UCNP-Apt nanoprobes.3.Construction of the biosensor:GO@Fe₃O₄could bind to the single-stranded DNA throughπ-πbonds while the UCNPs-Apt and GO@Fe₃O₄were incubated at 37°C,therefore,the fluorescence of UCNPs was quenched.When Escherichia coli existed,the affinity between Escherichia coli and aptamer was greater than that of GO@Fe₃O₄and single-stranded nucleic acid,it could pull UCNPs-Apt away from the surface of GO@Fe₃O_4,consequently,the fluorescence of UCNPs was recovered,which achieved the quantitative detection of Escherichia coli.4.Optimize experimental conditions:the key parameters were optimized to improve the detection performance of the biosensor,it includes the concentration ratio of UCNPs-Apt and GO@Fe₃O₄,the incubation time of UCNPs-Apt and GO@Fe₃O₄.5.Evaluation of the analytical performance of the biosensor:the repeatability,specificity,linearity range,and the limit of detection of the sensor were evaluated through measuring the Escherichia coli solution with different concentrations.6.Recovery test:Escherichia coli were added to urine specimens with different concentrations,which were detected by the constructed sensor.Finally,the recovery rate was calculated.7.Clinical sample detection:20 urine samples were detected by the constructed sensors.And the consistency with the results of bacterial culture was compared.Results1.Characterization of UCNPs:Transmission electron microscopy revealed that UCNPs were dispersed in aqueous solution with an average size of about 200 nm in uniformed shape and size;energy spectrometer analysis showed that the main elements of UCNPs included Y,Yb,F,Na,Tm.2.Characterization of the UCNPs nanoprobes:Fluorescence spectrophotometer results showed that the fluorescence intensity of UCNPs nanoprobes was not decreased significantly before and after aptamer modification;After mPEG-NH₂coated,the fluorescence intensity was only reduced slightly,it showed the good stability of UCNP fluorescence.Zeta potential results showed that when aptamers were modified on UCNPs,the surface charge of UCNPs decreased;and after coated with mPEG-NH₂to reduce non-specific adsorption,the surface charge of UCNPs increased correspondingly due to the presence of mPEG-NH₂.These results indicated that UCNPs nanoprobes were successfully synthesized.3.Optimization of experimental conditions:the optimal conditions for the key parameters were:the final concentration of GO@Fe₃O₄is 0.60mg/mL,and the final incubation time of GO@Fe₃O₄and UCNPs was 30min.4.Analytical performance of the biosensor:the relative standard deviations were 0.10%,1.27%,4.43%,7.90%,7.27%（n=3,the concentration was 1.0×10³CFU/mL,respectively,1.0×10⁴CFU/mL,1.0×10⁵CFU/mL,1.0×10⁶CFU/mL,1.0×10⁷CFU/mL）.This method had good specificity,it response to Escherichia coli only.And the linear range was 1.0×10³CFU/mL-1.0×10⁷CFU/mL;the limit of detection was 467 CFU/mL.5.Recovery test:the urine samples were spiked with Escherichia coli.And the final concentrations were 1.0×10³CFU/mL and 1.0×10⁵CFU/mL,respectively.The recovery was 101.70%and 95.60%,respectively.6.Clinical sample detection:20 urine samples were detected by the constructed sensors.The coincidence rate was 95%,it indicates that this method has clinical application value.ConclusionIn this study,a novel biosensor based on UCNPs and GO@Fe₃O₄ nanocomposite was successfully constructed.The sensor has a wide linearity range,high sensitivity,good specificity,and can be used to detect Escherichia coli in urine samples.This method can complete the detection of Escherichia coli within 30 minutes,which provide laboratory evidence for the diagnosis of urinary tract infections.