Research On Detection Methods Of Food Safety Hazard Factors Based On Aptamer Recognition And Chemiluminescent Functionalized Nanomaterials

Chemiluminescenct?CL?functionalized nanoparticles can combine the unique optical properties,magnetic properties,excellent catalytic activity of nanoparticles with CL properties of the CL reagents.The combination of nanoparticles and CL regents can not only add CL property to nanoparticles but also enhance the sensitity of CL system.It is of great significance in the fields of biology,medicine and food safety detection.As a new type of molecular recognition element,aptamer possesses characteristics of good specificity,wide range of targets,and easy to be chemically modified and has been widely used in food safety detection.Antibiotic residues and contamination of food borne pathogens are important hazard factors in food safety,so is of great importance for monitoring the contamination level of antibiotic residues and food borne pathogens.In this thesis,the work focused on the application of novel CL functionalized nanoparticels and aptamer in food safety detection.By combining magnetic separation technique and rolling circle amplification?RCA?technique,some rapid,sensitive and reliable aptasensors were established for the detection of antibiotic residues and food borne pathogens.This thesis provides new methods for enriching and improving food safety technology.Firstly,N-?aminobutyl?-N-?ethylisoluminol??ABEI?functionalized flowerlike gold nanoparticles?AuNFs??ABEI-AuNFs?with good dispersity and excellent CL property were synthesized by reducing HAuCl4 with ABEI and chitosan.P-iodophenol?PIP?was added to form ABEI-H2O2-PIP steady-state chemiluminescence?CL?system.Compared with traditional luminol based detection methods with a flash type CL,steady-state CL system can significantly extend the detection time to 20 min.Also the steady-state CL system lays the foundation for the subsequent CL based aptasensor.Secondly,the application of ABEI-AuNFs together with Fe₃O₄ magnetic nanoparticles?MNPs?and aptamer in the detection of chloramphenicol?CAP?was studied.Aptamer was conjugated with Fe₃O₄ MNPs to form capture probes and thiolated hybridized complementary strand was modified on the surface of ABEI-AuNFs to form signal probes.CAP and the complementary sequence of the aptamer can competitively combine with the aptamer thus forms a competitive format.Fe₃O₄ MNPs and signal probes complexes can be obtained by magnetic separation and the CL signals can be determined after adding H2O2 and PIP.Based on ABEI-AuNFs labeling,aptamer recognition and magnetic separation,a novel and sensitive analytical method has been successfully applied for the detection of f CAP in the range of 0.01-0.20 ng/m L and the detection limit was 0.01 ng/mL.Also the recoveries of CAP were between 94.8 and 103.1% indicating that the proposed method was successfully applied to measure CAP in milk samples.Thirdly,on the basis of the successful realization of single component detection with ABEI-AuNFs as labels,we made a preliminary exploration on the application of ABEI-AuNFs in multiplex detection.The microtiter plates were used as solid carrier and three antibiotics were used as analytical models,the Oxytetracycline?OTC?,tetracycline?TET?and kanamycin?Kana?aptamer were fixed on the microtiter plates by avidin.And thiolated hybridized complementary strand?cDNA?was modified on the surface of ABEI-Au NFs to form signal probes.Based on the competitive format,OTC,TET and Kana can be simultaneous detected.The ranges are 0.05 to 5 ng/m L,0.05 to 5 ng/mL,and 0.005 to 0.5 ng/mL,and the detection limits are 0.02,0.02 and 0.002 ng/m L,respectively.Also the recoveries of OTC,TET and Kana were between 96.04 and 102.66% indicating that the proposed method was successfully applied to measure OTC,TET and Kana in milk samples.Fourthly,the sensitization of the ABEI-AuNFs based CL system was studied.On the one hand,the CL intensity of ABEI-AuNFs was enhanced,on the other hand,rolling circle amplification technique was introduced.Fe₃O₄ MNPs are functionalized with aptamer to form capture probes.Meanwhile,thiolated cDNA was modified on the surface of ABEI-AuNFs to form signal probes.And the RCA product can specifically bind the target by specific DNA sequence design.In the presence of Salmonella typhimurium?S.typhimurium?that can be first captured by the capture probes then combine with RCA products to form a sandwich complex.Co²⁺/ABEI-AuNFs-cDNA signal probes were then assembled on the RCA products to produce and enhance CL signals.The “capture probe-target-RCA product?signal probes?” complexes can be obtained by magnetic separation and the CL signals can be determined after adding H2O2 and PIP.The correlation between the CL signal and the logarithmic concentration of S.typhimurium was found to be linear within the range of 32 cfu/mL to 3.2×106 cfu/m L.The limits of detection of the developed method were found to be 10 cfu/mL for S.typhimurium.The method was also used to detect S.typhimurium in real pork samples.Fifthly,on the basis of sensitization of the ABEI-AuNFs CL system,the application of ABEI-AuNFs in chemiluminescence resonance energy transfer?CRET?system was studied and homogeneous CL detection system was established.In this design,thiolated cDNA modified ABEI-AuNFs were designed as donor and WS2 nanosheet was designed as acceptor.Combining RCA technique,the CRET system was constructed according to the difference binding ability of WS2 nanosheet between single strand DNA and double strand DNA.Staphylococcus aureus?S.aureus?was used as target bacteria.The correlation between the CL signal and the logarithmic concentration of S.aureus was found to be linear within the range of 50 cfu/m L to 1.5×105 cfu/m L.The limits of detection of the developed method were found to be 15 cfu/m L for S.aureus.The method was also used to detect S.aureus in real pork samples.In summary,the well prepared ABEI-AuNFs with excellent CL property and biological modification can be used as an alternative CL nanolabels in the detection of food hazard factor including antibiotic residues and food borne pathogens.By combination of aptamer recognition,RCA technique,the novel magnetic separation and microtiter plates was well as homogeneous CRET based CL detection systems were estabilised,which has the advantages of high specificity,high sensitivity and good reliability.This thesis extends the application range of CL detection,and provides technical support for food safety.