Establishment And Evaluation Of Visual Detection Methods Based On Nanocomposites For Salmonella Typhimurium

Salmonella typhimurium（S.typhimurium）is one of the most common foodborne pathogens.S.typhimurium can cause foodborne diseases through contaminated food,with symptoms of acute gastroenteritis such as diarrhea and abdominal pain and can be severe and life-threatening in infants and the elderly with weakened immune systems.Therefore,timely detection can effectively prevent and control foodborne disease outbreaks.Traditional detection methods have some shortcomings.For example,plate culture method is time-consuming and labor-intensive,polymerase chain reaction（PCR）has complex nucleic acid extraction process,enzyme-linked immunosorbent assay（ELISA）is expensive and low sensitive,which greatly limit their application in field detection or remote areas.It is important to develop simple,sensitive,portable,and accurate methods for detecting S.typhimurium.Noble metal nanocomposites have been a hotspot for biosensor development on account of their unique properties and multifunctional properties.In this study,four rapid,sensitive,and accurate visual detection strategies for S.typhimurium were constructed by utilizing the optical properties or catalytic functions of nanocomposites.The optical signals were acquired and semi-quantitatively analyzed by smartphone.This study provided theoretical basis and technical support for the detection of foodborne pathogens,which is the focus of food safety.Part I Establishment and evaluation of the colorimetric detection method based on Fe₃O₄@Ag nanoprobes functionalized with aptamersObjective:To establish simple and rapid paper chip-based colorimetric methods for detecting S.typhimurium,aptamer functionalized Fe₃O₄@Ag nanoprobes（apt-Fe₃O₄@Ag NPs）were prepared to capture S.typhimurium and etched to produce Ag⁺ions to inhibit urease activity.A simple and rapid colorimetric analysis method was established with urea/phenol red test paper.Methods:1.Fe₃O₄ nanoparticles were synthesized by hydrothermal method.Fe₃O₄@Ag nanoparticles were synthesized by the solution-phase reduction method.The thiol modified S.typhimurium aptamers were conjugated to the prepared apt-Fe₃O₄@Ag NPs.The above nanomaterials were characterized by Transmission Electron Microscope（TEM）,X-Ray Diffraction（XRD）,Ultraviolet and visible spectrum（UV-vis spectrum）,Fourier transform infrared spectroscopy（FTIR）,and Vibrating sample magnetometer（VSM）.2.Neutral cotton filter paper was used as test paper material.The test paper was prepared by modifying filter paper with urea and phenol red.3.Computer software Image J was used to analyze the test paper image and calculate the gray value.The concentration of H₂O₂,the etching time and the reaction time of urease and Ag⁺ions in the detection system were optimized.4.To evaluate this detection method,the limit of detection（LOD）,detection range,and specificity were studied.5.To evaluate the storage lifetimes,the nanoprobes and test paper with different storage time were used to detect S.typhimurium by this method.6.To evaluate the testing effect of the spiked sample,the standard recovery experiment of milk samples was studied.Results:1.The TEM,XRD,UV-vis spectrum,FTIR and VSM characterization results showed that apt-Fe₃O₄@Ag NPs were synthesized successfully and could specifically capture S.typhimurium.2.The best experimental conditions as following:the concentration of H₂O₂ was100 m M,etching time was 10 min,and the reaction time of urease and Ag⁺ions was10 min.3.The detection time of the established visual sensor for S.typhimurium was about1 h,the LOD was 48 CFU/m L,the linear equation was y=-7.249x+151.500,R²=0.985（x is the log scale of the concentration of S.typhimurium,and y is the gray value of the central effective region of the test paper）.The detection range was 1×10²～1×10⁶CFU/m L,and this method has good specificity.4.The stability results of the nanoprobes showed that apt-Fe₃O₄@Ag NPs and the test paper had good detection effect and good stability in several months.5.The recoveries of the detection method in milk spiked samples were 92.48%～95.99%,the relative standard deviations（RSDs）were lower than 10%,suggesting that the established detection method had good accuracy.Conclusions:1.A simple,rapid,and accurate colorimetric detection method based on apt-Fe₃O₄@Ag NPs combined with urea/phenol red test paper was established.2.The apt-Fe₃O₄@Ag NPs can capture S.typhimurium,and the Ag⁺ions generated by the etching of the outer silver shell can be used as urease inhibitors.The recognition and reaction bifunction of the nanoprobes was used to simplify the detection procedure.The problem of complex detection operation and unstable results caused by the use of two or more nanomaterials in the traditional“sandwich”strategy was solved.3.The decomposition of urea by urease and the color reaction of phenol red indicator can be both completed on urea/phenol red test paper,further simplifying the operation process.In addition,the cost of urea/phenol red dipstick is low.4.This strategy had high sensitivity,good specificity,simple operation and could be used for the detection of milk samples.The nanoprobe and test paper had good storage stability.5.The test results could be obtained and analyzed by smartphones and transformed and analyzed by Image J software,independent of large instruments and professionals.Meanwhile,The paper used for qualitative and semi-quantitative analysis has good portability and is suitable for field detection in the field of public health.Part II Establishment and evaluation of the colorimetric detection method based on CTAB‐induced supramolecular assembly ofβ-CD-AuNPsObjective:To establish fast and stable colorimetric detection method for S.typhimurium,β‐cyclodextrin‐coated gold nanoparticles（β-CD-AuNPs）supramolecular self-assembly aggregation triggered by exadecyl trimethyl ammonium bromide（CTAB）was developed to produce the colorimetric signals.Methods:1.β-CD-AuNPs were synthesized by one-step method and characterized by TEM,UV-vis and FTIR.The ability to resist ions interference was tested using 1 M Na Cl and compared with bare AuNPs.2.The hydration particle size of single-stranded DNA-CTAB（ss DNA-CTAB）complex was determined using DLS.UV-vis spectroscopy was used to investigate the self-assembled aggregation ofβ-CD-AuNPs induced by CTAB and to analyze the feasibility of the scheme.3.The concentration of CTAB and aptamers,incubation time for aptamers reacted with CTAB,and coloration time were optimized.4.To evaluate this detection strategy,the LOD,detection range,and specificity were studied.5.To evaluate the storage lifetime ofβ-CD-AuNPs,theβ-CD-AuNPs with different storage time were used to detect S.typhimurium by this method.6.To evaluate the testing effect of the spiked sample,the standard recovery experiment of milk samples was studied.Results:1.The results of TEM,UV-vis and FTIR characterization ofβ-CD-AuNPs indicated thatβ-CD were modified on the AuNPs successfully.2.DLS demonstrated the binding of ss DNA and CTAB.When the concentration of CTAB was 200 m M,the hydration particle size of ss DNA-CTAB complex was about150 nm.Then,the self-assembled aggregation ofβ-CD-AuNPs induced by CTAB was based on host-guest chemistry and was not affected by Na Cl.3.The best experimental conditions as following:the concentration of aptamers was 30μM,the concentration of CTAB was 200 m M,the incubation time was 30 min,and the coloration time was 10 min.4.The detection time of the established method for S.typhimurium was about 90min,the linear equation was y=0.206x+0.248,R²=0.970(x is the the log scale of the concentration of S.typhimurium,and y is A_650nm/A_520nm value of the solution),and the LOD was 13 CFU/m L.The linear range was 1×10²～1×10⁷ CFU/m L.In addition,the method had good specificity.5.The stability test results ofβ-CD-AuNPs showed thatβ-CD-AuNPs had good detection effect and good stability in several months.6.The recoveries of milk samples were 93.55%～111.32%,and the RSDs were lower than 10%.The method had good accuracy.Conclusions:1.A rapid,accurate and stable colorimetric method for the detection of S.typhimurium based on the CTAB-induced supramolecular self-assembly ofβ-CD-AuNPs was established.2.The CTAB inducedβ-CD-AuNPs self-assembled aggregation was verified to be dependent on the host-guest chemical interaction between CTAB andβ-CD.Different from the traditional aggregation of AuNPs,the self-assembly aggregation process was more stable.The problem of instability of AuNPs aggregation-based colorimetric analysis was solved.3.It was verified that CTAB has dual functions in this strategy:（1）binding with aptamer to transform the concentration of target bacteria,（2）inducingβ-CD-AuNPs self-assembled aggregation to output coloration detection signal.4.This colorimetric sensor had good stability,high sensitivity,simple operation,and could detect milk samples.Part III Establishment and evaluation of the colorimetric assay method based on COF-AuNPs functionalized with aptamersObjective:A simple,rapid,accurate and stable colorimetric assay method for S.typhimurium was established utilizing intrinsic peroxidase-like activity of AuNPs embedded spherical covalent organic framework functionalized with aptamers（apt-COF-AuNPs）.Methods:1.COF with spherical structure was prepared as the skeleton of AuNPs,COF-AuNPs were synthesized and modified with aptamers to obtain apt-COF-AuNPs.The synthesized materials were characterized by TEM,XRD,Energy-dispersive X-ray spectroscopy（EDS）,FTIR and Zeta potential.2.The peroxide-like activity of apt-COF-AuNPs was evaluated by enzymatic reaction kinetic assays with the peroxidase substrate（TMB and H₂O₂）,and the kinetic parameters were determined by UV-vis and calculated according to the Michaelis-Menten equation.The catalytic activity at different temperature,p H and storage time were measured to evaluate the stability of apt-COF-AuNPs.3.The incubation time of apt-COF-AuNPs and S.typhimurium and the catalytic reaction time of TMB-H₂O₂ system catalyzed by apt-COF-AuNPs were optimized.4.To evaluate this detection strategy,the LOD,detection range,and specificity were studied.5.To evaluate the practicability of this method,milk samples were used for the test of actual samples.6.The novel pre-concept smartphone application program（app）was used to obtain images,to calculate the Blue channel values of the samples,and to classify the danger level according to the Blue values.Results:1.The nanomaterials were characterized by TEM,XRD,EDS,FTIR and Zeta potential.The results showed that the materials were successfully prepared and apt-COF-AuNPs could specifically capture S.typhimurium.2.The Michaelis constant and maximum reaction rate were calculated and compared by enzymatic reaction kinetics assay.The results indicated that both apt-COF-AuNPs and COF-AuNPs had high affinity for the substrate,and the affinity decreased significantly after the capture of S.typhimurium.In addition,COF-AuNPs had better temperature,p H and storage stability than bare AuNPs.3.The experimental conditions were selected as follows:the incubation time of apt-COF-AuNPs and S.typhimurium was 30 min,and the shielding effect was the best.The reaction time of apt-COF-AuNPs catalyzed TMB-H₂O₂ system was 5 min,and the color development effect was the best.4.The detection time of the established visual sensor for S.typhimurium was about45 min,the LOD was 7 CFU/m L,and the linear equation was y=-0.083x+0.876,R²=0.981(x is the the log scale of the concentration of S.typhimurium,and y is A_650nmvalue of the solution).The linear range was 10～1×10⁷ CFU/m L.In addition,the method had good specificity.5.Compared with the gold standard plate counting method,the recovery of milk samples was 109.37%and the RSD was 7.23%.The method had good accuracy and practicability.6.According to the developed smartphone app,the images were obtained,and the danger level of samples was analyzed,which showed good accuracy with the results of UV-vis spectrometer.Conclusions:1.Apt-COF-AuNPs probes with high peroxidase-like activity was synthesized,showing excellent stability and strong anti-interference ability.As the framework of AuNPs,COF can not only adsorb the surrounding substrates to obtain better catalytic effect,but also inhibit the aggregation of AuNPs and maintain the stability of the nanozyme activity.2.Based on the peroxidase-like activity of apt-COF-AuNPs,a fast,stable,and sensitive colorimetric detection method was established for S.typhimurium combined with smartphone app.3.Apt-COF-AuNPs have dual functions of recognition and catalysis.They not only can specifically capture S.typhimurium,but also can be used to catalyze the TMB-H₂O₂ system to generate colorimetric signals.4.The test results could be directly distinguished by the naked eye,which was suitable for field detection.Combined with the developed app,the danger level can be interpreted to obtain semi-quantitative analysis results.Part IV Establishment and evaluation on the fluorescence detection method based on AuNPs-MCOF functionalized with aptamersObjective:A simple,rapid,sensitive,and stable fluorescence strategy for the detection of S.typhimurium was established using highly emissive AuNPs-doped magnetic COF functionalized with aptamers（apt-MCOF-AuNPs）.Methods:1.The core-shell structured MCOF nanoparticles were synthesized by using Fe₃O₄as magnetic core and 1,3,5-tris（4-aminophenyl）benzene and 2,5-dimethoxyterephthalaldehyde as COF ligands.MCOF-AuNPs were obtained by in-situ reduction method.Finally,MCOF-AuNPs were modified by aptamer to obtain apt-MCOF-AuNPs.The synthesized materials were characterized by TEM,EDS,FTIR,Zeta potential,VSM and fluorescence spectra.The fluorescence stability of MCOF-AuNPs was evaluated,including the effects of ionic strength,p H and storage time on the fluorescence emission intensity.2.By measuring fluorescence intensity,the fluorescence quenching effect of different metal ions on MCOF-AuNPs was compared.The fluorescence lifetime and X-ray photoelectron spectroscopy（XPS）were used to study the mechanism of MCOF-AuNPs fluorescence quenching by Fe³⁺ions.3.The concentration of apt-MCOF-AuNPs,the incubation time of apt-MCOF-AuNPs and S.typhimurium,the concentration of Fe³⁺ions,and the reaction time of apt-MCOF-AuNPs quenched by Fe³⁺ions were optimized.4.To evaluate this detection strategy,the LOD,detection range,precision and specificity were studied.5.The storage stability and recyclability of apt-MCOF-AuNPs were evaluated by fluorescence measuring.6.Test chicken,egg,and milk samples were used to evaluate the practicability of this method for the test of actual samples.7.The novel smartphone app was used to obtain images,to calculate the Hue values of the samples,and to classify the danger level according to the Hue values.Results:1.The nanocomposites were characterized by TEM,EDS,FTIR,Zeta potential,VSM and fluorescence spectra.The results showed that apt-MCOF-AuNPs could capture S.typhimurium specifically.The fluorescence stability results showed that MCOF-AuNPs had high anti-interference ability,and the ionic strength（0.25～1.25 M Na Cl）,p H（2～8）and storage time（1～5 weeks）had little influence on the fluorescence intensity.2.By comparing the fluorescence quenching effect of different metal ions on MCOF-AuNPs,the results showed that Fe³⁺ions can specifically quench the fluorescence of MCOF-AuNPs.After added Fe³⁺ions,the fluorescence lifetime of MCOF-AuNPs decreased,so it was a dynamic quenching process.The XPS results suggested that there is an interaction between amino groups and Fe³⁺ions in MCOF-AuNPs.The above results indicated that the quenching mechanism was photoinduced electron transfer（PET）.3.The best conditions were as follows:the concentration of apt-MCOF-AuNPs was 1 mg/m L,the incubation time of apt-MCOF-AuNPs and S.typhimurium was 30min,the concentration of Fe³⁺ions was 1 M,and the reaction time of apt-MCOF-AuNPs quenched by Fe³⁺ions was 3 min.4.The detection time of the established visual sensor for S.typhimurium was about33 min,the LOD was 4 CFU/m L,and the linear equation was y=0.386x+0.688,R²= 0.973（x is the the log scale of the concentration of S.typhimurium,and y is and the F/F₀value of the solution）.The linear range is 10～1×10⁷ CFU/m L.In addition,the method has good specificity.Meanwhile,intra-day and intra-day precision results showed that the RSDs was less than 6%.The method had good precision and accuracy.5.The results of probe stability and cycling experiments showed that apt-MCOF-AuNPs had good detection effect and good stability in several weeks.Apt-MCOF-AuNPs had good reuse performance and can be recycled for 3 times,which effectively reduced the detection cost.6.Compared with the gold standard plate counting method,the recoveries were99.08%～102.79%,and the RSDs were all less than 3%.The method was accurate and practical.7.According to the developed smartphone app,the images were obtained,and the danger level of samples were analyzed.The results were in good accurancy with those of the fluorescence spectrometer.Conclusions:1.The apt-MCOF-AuNPs nanoprobes with high and stable fluorescence emission were synthesized,which could recognize S.typhimurium.The mechanism of high fluorescence emission of MCOF-AuNPs is that Fe₃O₄ as the magnetic core reduced the aggregation induced quenching caused byπ-πstacking of COFs,and the local field enhancement effect and non-radiative plasmonic coupling effect of AuNPs improve the radiative transition efficiency of ground and excited state molecules.2.Based on the fluorescence characteristics of apt-MCOF-AuNPs,a fast,accurate,economical,and sensitive fluorescence detection method was established for S.typhimurium.The detection method had high sensitivity and good accuracy,so it has great potential to be developed for laboratory detection.3.The stability of this nanoprobe was good,it could be recycled.The detection process was simple and does not rely on large instruments and professionals,and it was suitable for on-site qualitative and quantitative analysis.Combined with the developed app,the danger level can be interpreted to obtain semi-quantitative analysis results.