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摘要： 冷冻是消费者用于延长肉类储藏期的一种常用方法，也是研究人员常用于肉类品质评估和加工的前道工序.目前尚无完善记录表明，冷冻对烹饪后的禽胸肉质的感官品质会产生影响.本研究的目的是比较新鲜与冷冻-解冻鸡胸肉的感官质量剖面属性.鸡胸肉块在宰后24 h内从胴体上分割，分别以新鲜状态直接烹饪或置于-20℃冷冻.冷冻样品采用三种不同解冻方式：冷冻后直接取出烹饪解冻（0 h）；在20℃水中预解冻2 h后烹饪（2 h）；或在4℃预解冻24 h后烹饪（24 h）.对照组则为新鲜鸡胸肉直接烹饪.肉块加热到78℃时用于评价感官质量属性.该实验由经过训练的描述型品评员采用0～15强度标度法进行评价.结果显示：不同的处理方式并没有引起任何风味特征和5种质构特征（内聚力、硬度、多汁、肉块大小、肉块湿度）上的差异（P>0.05）.然而，不同处理方式引起了团块内聚性、分解率和咀嚼性上的显著差异（P<0.05）：冻后直接烹饪（0 h）和解冻24 h烹饪（24 h）的鸡肉块的团块内聚性评价要明显高于预解冻2 h的样品.冻后直接烹饪样品（0 h）的分解率和咀嚼性显著高于新鲜对照组和预解冻2 h的样品.本研究结果表明，冷冻-解冻对鸡胸肉的感官风味品质无影响，但可能改变烹饪后鸡胸肉的质构特征.因此，冷冻-解冻对肉类质构的影响取决于烹饪前的解冻方法.
关键词： 鸡胸肉； 感官； 风味； 质地； 冷冻-解冻
Abstract： A novel immunoassay system based on magnetic fluorescent probes was established to detect 2.4 dichlorophenoxyacetic acid （2，4 D） residue in liquid system in food and agricultural products.The composites of anti 2，4 D antibody bound to Fe3O4@SiO2 NH2 was employed as the solid phase as well as magnetic probe.The composites composed of 2，4 D OVA labeled with CdTe@SiO2 NH2 as the fluorescent probe was used to produce fluorescent signal.2，4 D and its fluorescent probe competed binding the antibody on the surface of the magnetic probe.The optimization of 2，4 D OVA dosage，coupling PH and reaction time in preparing the fluorescent probe were investigated.It showed that in the synthesis of fluorescent probe 8.2 was the optimal pH，70 min was the optimal coupling time，500 μL amount of 2，4 D OVA.The standard curve was obtained with the concentration of 2，4 D and the maximum fluorescence intensity.The detection limit of the assay was gotten and it was 3.55×10-8.One reaction step and one washing step were needed.The assay significantly shortened the testing time and amplified the detection signal compared with classic ELISA.
Key words： immunoassay； fluorescence probe； magnetic probe； 2，4 D； quantum dots
CLC number： TS 207Document code： AArticle ID： 1000 5137（2014）06 0566 07
1Introduction
2，4 Dichlorophenoxyacetic acid is one of the most widely used herbicides and plant growth regulator in agriculture，forestry and right of way applications.2，4 D has been used and monitored for several decades.It has been reported it concerned with the occurrence of cancer in humans[1]，endocrine disrupting activities[2]，acute congestion and degenerative changes in central nerve system[3].Presently，an increasing number of countries strictly limited the residues of 2，4 D in grain，fruit，and vegetables.Thus，it is essential to develop a more efficient method for detection of 2，4 D. Several methods have been described for the determination of 2，4 D，such as Thin layer chromatography，chromatography （GC），high performance liquid chromatography （HPLC），gas chromatography mass spectrometry （GC/MS） and liquid chromatography mass spectrometry （HPLC/MS）[4-7].However，they are always complicated operation，time consuming，even require costly and bulky instrumentation.Immunoassay is a common useful assay for biochemical analysis.The strong，specific binding of an antibody to its antigen has been widely exploited in biochemical studies，sensor design，clinical diagnostics，environmental monitoring，and food safety.
Semiconductor quantum dots （QDs） exhibit a lot of unique optical properties compared with organic fluorescent dyes such as sharp emission band with broad excitation，size controlled tunable maximum wavelength of emission，higher fluorescence intensity，and strong resistance to photo bleaching，which can be exploited in biological imaging and bio conjugation[8-12].Magnetic nanoparticles （NPs） show some special magnetic properties including superparamagnetism，lower Curie temperature，and higher susceptibility，which have been widely used in magnetic storage media，catalysis，magnetic separation，and biomedicine.Composite nanoparticles of Fe3O4@SiO2 and CdTe@SiO2 can enhance the biocompatibility and hydrophilicity of single nanoparticles，thus they can be used as powerful tools for biochemical analysis and sensitive detection in environmental safety.
In this research，a new immunoassay system based on magnetic fluorescent probes dedicated to 2，4 D rapid determination was systemically investigated.The antibody，labeled with Fe3O4@SiO2 NH2 which serves as solid phase，is employed as magnetic probe.The 2，4 D OVA，bond with CdTe@SiO2 NH2 is used as fluorescent probe.2，4 D standard solution and fluorescent probe competed for the same antibody active sites.Furthermore，the optimization of dosage of 2，4 D OVA，coupling PH and reaction time in preparation of the fluorescent probe were also studied.
2Materials and methods
2.1Reagents and instruments
Ethanol，2，4 D，1 （3 Dimethylaminopropyl） 3 ethylcarbodiimide hydrochloride （EDC·HCl，98.5%），N Hydroxy succinimide（NHS，98%），3 aminopropyltriethoxysilane （APTS）（98%），ammonium hydroxide，Tris （hydroxymethyl） aminomethane （Tris），HCl，（3 （2 aminoethylaminol） propylmethyldmethoxysilane） （AEAPS），tetraethyl orthosilicate （TEOS），Bovine serum albumin （BSA） and other reagents were of analytical grade and purchased from Aladdin.Red CdTe QDs （Em=634 nm），anti 2，4 D polyclonal antibody （The antibody titer by ELISA method was 1∶12 000，IC50 was 50 ng/mL ）and complete antigen （2，4 D OVA） were produced in our laboratory.Double distilled water was used through the process.Fluorescence spectrophotometer was from Varian company （America），3 18k high speed refrigerated centrifuge was from Sigma （America），KQ 250 ultrasonic cleaner was from Kunshan Ultrasonic Instruments Co.，Ltd （China），RH KT/C Magnetic stirrers was from IKA （Germany），DHP 9082 cultural incubator with constant temperature was from Shanghai Yiheng Scientific instruments Ltd （China）. 2.2Preparation of 2，4 D magnetic probe
2.2.1Synthesis of Fe3O4@SiO2 NH2 composite particles
Fe3O4@Organic Layer complex microspheres were firstly synthesized by thermal decomposition method.Single magnetic microspheres coated with 30 nm of silica shell could be obtained with the reverse micro emulsion system.Then the Fe3O4@SiO2 magnetic composites were modified with AEAPS and Fe3O4@SiO2 NH2 composite particles were prepared[17].
2.2.2Immobilization of anti 2，4 D antibody on Fe3O4@SiO2 NH2 composite particles
3.0 mg of Fe3O4@SiO2 NH2 composite particles was adequately dissolved in 3.0 mL of phosphate buffered saline （PBS pH=8.2，0.2 mol/L），then the supernatant was discarded by means of magnetic separation.The pH value of the solution was adjusted twice by Tris HCl buffer solution （pH=8.2，50 mmol/L）.Subsequently，200 μL of anti 2，4 D antibody was added into the mixture and incubated at 37℃ for 30 min.After separating magnetically and washing with PBS for two times，excess functional groups on Fe3O4@SiO2 NH2 composite was blocked by buffer （PBS of 3% BSA） and then stored at 4℃ overnight.The product was magnetically separated from free antibody and washed with PBST （PBS of 0.05% Tween 20） for three times.Finally the sample was re suspended boric acid buffer solution （pH=8.2，0.2 mol/L） and stored at 4℃ before used （Scheme 1b）.
2.3Preparation of 2，4 D fluorescent probe
2.3.1Synthesis of CdTe@SiO2 NH2 composite particles
The red CdTe nanocrystals （Em=634 nm） prepared under 105℃ was used to synthesis CdTe@SiO2 NH2 composite particles.12 mL of CdTe QDs was added in 72 ml of ethanol and 15.2 mL of double distilled water.After dispersing adequately，the mixtures were added by 8 mL of ammonium hydroxide and stirred for 10 min.Subsequently，1 mL of TEOS was added in and stirred for 30 min.Finally，40 μL of APTS was introduced under vigorous stirring for 4 h.The resulting product was centrifuged at 12 000 r·min-1 for 3 min and washed with double distilled water and ethanol for three times，respectively.The sample was redissolved in double distilled water as a stock solution[16].
2.3.2Conjugation of 2，4 D OVA to CdTe@SiO2 NH2
1.0 mL of CdTe@SiO2 NH2 solution was added into 10.0 mL of boric acid buffer （0.2 mol/L） with dispersing adequately.4 mg of EDC·HCl and 3 mg of NHS were dissolved in the mixtures and incubated at 37℃ for 10 min.Then 2，4 D OVA was added into the mixtures and stirred for about 1 h.After centrifuging at 5 000 r·min-1 for 3 min，the precipitation （CdTe@SiO2 NH2 2，4 D OVA） was dispersed in boric acid buffer （Scheme 1a）. 2.4Optimization of reaction parameters in preparation of fluorescent probe
Several parameters were investigated systematically in order to synthesis the fluorescent probe with optimal conditions，including the dosage of 2，4 D OVA，PH value of boric acid buffer and coupling time.
2.5Establishment of the 2，4 D immunoassay system based on magnetic fluorescent probes
300 μL of fluorescent probe was added into 2，4 D standard solution of different concentrations and dispersed adequately.Subsequently，1.0 mL of magnetic probe was added into the mixture and incubated at 37℃ for 40 min.2，4 D standard solution and fluorescent probes competed binding the antibody on the surface of the magnetic probes.After magnetic separation，the magnetic part was washed three times with boric acid buffer solution to remove the residual fluorescent probe.Finally，the product was re suspended in Boric acid buffer solution （0.2 mol/L，pH 8.20） and the fluorescent signal was monitored by the fluorescence photometer.Establishment of the immunoassay system was presented in Scheme 1c.
3Results and Discussion
3.1Optimization of the dosage of 2，4 D OVA in preparation of fluorescent probe
As described in Section 2.3.2，2，4 D OVA at different dosage （100，300，500，700 μL） was conjugated with CdTe@SiO2 NH2 composite particles.Fluorescent signal was monitored by the fluorescence photometer to determine the maximum dosage of combination.
Figure 1 illustrated the fluorescence spectra of the fluorescent probes in different dosage of 2，4 D OVA.As the dosage of 2，4 D OVA increasing，the fluorescence intensity of the fluorescent probe gradually enhanced and subsequently，remaind stable when the dosage of 2，4 OVA arrived at 500 μL.Hence，500 μL amount of 2，4 D OVA was employed in the synthesis of fluorescent probe.
3.2Optimization of PH value in preparation of fluorescent probe
3.3Optimization of coupling time in preparation of fluorescent probe
Effect of coupling time on the fluorescent intensity of fluorescent probe was examined and optimized.30，40，50，60，70，80 min were applied in the conjugation from 2，4 D OVA to CdTe@SiO2 NH2 （Figure 3）.The fluorescent intensity of fluorescent probe reached a maximum when reaction time arrived at 70 min.So that 70 min was the optimal coupling time for preparation of 2，4 D fluorescent probe.
Figure 3Effect of different coupling time on fluorescent intensity of fluorescent probe
3.4Characterization of the immunoassay based on magnetic fluorescent probes The composites by the anti 2，4 D antibody labeled with Fe3O4@SiO2 NH2 served as solid phase，was employed as magnetic probe.The 2，4 D OVA，composite bonded with CdTe@SiO2 NH2 was employed as fluorescent probe.2，4 D standard solution and fluorescent probe competed binding the antibody on the surface of the magnetic probe.The complex of 2，4 D magnetic probe and magnetic fluorescent probe were formed，respectively.Only the complex of magnetic fluorescent probe could produce a fluorescent signal which could indicate the presence of 2，4 D.If there was no 2，4 D in the detection system，the complex of magnetic fluorescent probe was formed and in this case，the fluorescent intensity reaches a maximum （Figure 4A）.Otherwise，2，4 D would competes with the fluorescent probe，which would weaken the fluorescent signal.In addition，the higher concentration of 2，4 D，the weaker intensity of fluorescent signal would be （Figure 3B F）.
For the immunoassay based on magnetic fluorescent probes，the standard curve was obtained with the concentration of 2，4 D as the abscissa and the maximum fluorescence intensity as the ordinate （Figure 5）.The regression equation was y=-0.570 3x+182.371 4 （R2=0.996 59），which indicated that the fluorescent intensity was inversely proportional to the concentration of 2，4 D standard solution.However，deviation of the result would occur when the fluorescent signal approaches the maximum or the minimum.Therefore，the effective value should range from 10%～90% of the maximum.The detection limits （LOD） of 2，4 D in this assay was 35.5×10-9 based on the curve.
Only about 1.0 h was needed to finish the procedure of the immunoassay based on magnetic fluorescent probes with one reaction step and one washing step.However，to fulfill the procedure of the traditional ELISA，four reaction steps and there washing steps are needed and moreover more than 2.0 h were needed.Comparing with the flat solid phase，the easy separated and re dispersed nature allowed for a ″in solution″ reaction，which also significantly shorten the reaction time and amplify the detection signal.Meanwhile，the LOD of 2，4 D ELISA kit is 5×10-8 and that of 2，4 D colloidal gold strip is 2.1×10-5 in the market.It indicated the assay was also feasible.
4Conclusion
In conclusion，this work established a novel immunoassay system for rapid detection of 2，4 D residues in liquid system.The composite of Fe3O4@SiO2 NH2，labeled 2，4 D antibody served as a solid phase carrier instead of ordinary coated microplate.The composite of CdTe@SiO2 NH2，bonded with 2，4 D OVA was used to produce fluorescence signal as the fluorescence probe.It showed that 8.2 was the optimal pH，70 min was the optimal coupling time，500 μL amount of 2，4 D OVA in the synthesis of fluorescent probe.The standard curve was obtained with the concentration of 2，4 D and the maximum fluorescence intensity.The regression equation was gotten：y=-0.570 3x+182.3714 （R2=0.996 59）.The detection limit of the assay was 3.55×10-8.For the assay，one reaction step and one washing step were needed.The procedure could be finished within 1.0 h without complicated preparing procedures by competing reaction，and the detection limit is 3.55×10-8.Moreover，the optimized parameters in preparation of fluorescent probe had been obtained.The results demonstrates that this immunoassay based on magnetic fluorescent probes was a timesaving and feasible assay and would be a reliable tool for rapid detection of 2，4 D residues. References：
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（责任编辑：顾浩然，冯珍珍）
关键词： 鸡胸肉； 感官； 风味； 质地； 冷冻-解冻
Abstract： A novel immunoassay system based on magnetic fluorescent probes was established to detect 2.4 dichlorophenoxyacetic acid （2，4 D） residue in liquid system in food and agricultural products.The composites of anti 2，4 D antibody bound to Fe3O4@SiO2 NH2 was employed as the solid phase as well as magnetic probe.The composites composed of 2，4 D OVA labeled with CdTe@SiO2 NH2 as the fluorescent probe was used to produce fluorescent signal.2，4 D and its fluorescent probe competed binding the antibody on the surface of the magnetic probe.The optimization of 2，4 D OVA dosage，coupling PH and reaction time in preparing the fluorescent probe were investigated.It showed that in the synthesis of fluorescent probe 8.2 was the optimal pH，70 min was the optimal coupling time，500 μL amount of 2，4 D OVA.The standard curve was obtained with the concentration of 2，4 D and the maximum fluorescence intensity.The detection limit of the assay was gotten and it was 3.55×10-8.One reaction step and one washing step were needed.The assay significantly shortened the testing time and amplified the detection signal compared with classic ELISA.
Key words： immunoassay； fluorescence probe； magnetic probe； 2，4 D； quantum dots
CLC number： TS 207Document code： AArticle ID： 1000 5137（2014）06 0566 07
1Introduction
2，4 Dichlorophenoxyacetic acid is one of the most widely used herbicides and plant growth regulator in agriculture，forestry and right of way applications.2，4 D has been used and monitored for several decades.It has been reported it concerned with the occurrence of cancer in humans[1]，endocrine disrupting activities[2]，acute congestion and degenerative changes in central nerve system[3].Presently，an increasing number of countries strictly limited the residues of 2，4 D in grain，fruit，and vegetables.Thus，it is essential to develop a more efficient method for detection of 2，4 D. Several methods have been described for the determination of 2，4 D，such as Thin layer chromatography，chromatography （GC），high performance liquid chromatography （HPLC），gas chromatography mass spectrometry （GC/MS） and liquid chromatography mass spectrometry （HPLC/MS）[4-7].However，they are always complicated operation，time consuming，even require costly and bulky instrumentation.Immunoassay is a common useful assay for biochemical analysis.The strong，specific binding of an antibody to its antigen has been widely exploited in biochemical studies，sensor design，clinical diagnostics，environmental monitoring，and food safety.
Semiconductor quantum dots （QDs） exhibit a lot of unique optical properties compared with organic fluorescent dyes such as sharp emission band with broad excitation，size controlled tunable maximum wavelength of emission，higher fluorescence intensity，and strong resistance to photo bleaching，which can be exploited in biological imaging and bio conjugation[8-12].Magnetic nanoparticles （NPs） show some special magnetic properties including superparamagnetism，lower Curie temperature，and higher susceptibility，which have been widely used in magnetic storage media，catalysis，magnetic separation，and biomedicine.Composite nanoparticles of Fe3O4@SiO2 and CdTe@SiO2 can enhance the biocompatibility and hydrophilicity of single nanoparticles，thus they can be used as powerful tools for biochemical analysis and sensitive detection in environmental safety.
In this research，a new immunoassay system based on magnetic fluorescent probes dedicated to 2，4 D rapid determination was systemically investigated.The antibody，labeled with Fe3O4@SiO2 NH2 which serves as solid phase，is employed as magnetic probe.The 2，4 D OVA，bond with CdTe@SiO2 NH2 is used as fluorescent probe.2，4 D standard solution and fluorescent probe competed for the same antibody active sites.Furthermore，the optimization of dosage of 2，4 D OVA，coupling PH and reaction time in preparation of the fluorescent probe were also studied.
2Materials and methods
2.1Reagents and instruments
Ethanol，2，4 D，1 （3 Dimethylaminopropyl） 3 ethylcarbodiimide hydrochloride （EDC·HCl，98.5%），N Hydroxy succinimide（NHS，98%），3 aminopropyltriethoxysilane （APTS）（98%），ammonium hydroxide，Tris （hydroxymethyl） aminomethane （Tris），HCl，（3 （2 aminoethylaminol） propylmethyldmethoxysilane） （AEAPS），tetraethyl orthosilicate （TEOS），Bovine serum albumin （BSA） and other reagents were of analytical grade and purchased from Aladdin.Red CdTe QDs （Em=634 nm），anti 2，4 D polyclonal antibody （The antibody titer by ELISA method was 1∶12 000，IC50 was 50 ng/mL ）and complete antigen （2，4 D OVA） were produced in our laboratory.Double distilled water was used through the process.Fluorescence spectrophotometer was from Varian company （America），3 18k high speed refrigerated centrifuge was from Sigma （America），KQ 250 ultrasonic cleaner was from Kunshan Ultrasonic Instruments Co.，Ltd （China），RH KT/C Magnetic stirrers was from IKA （Germany），DHP 9082 cultural incubator with constant temperature was from Shanghai Yiheng Scientific instruments Ltd （China）. 2.2Preparation of 2，4 D magnetic probe
2.2.1Synthesis of Fe3O4@SiO2 NH2 composite particles
Fe3O4@Organic Layer complex microspheres were firstly synthesized by thermal decomposition method.Single magnetic microspheres coated with 30 nm of silica shell could be obtained with the reverse micro emulsion system.Then the Fe3O4@SiO2 magnetic composites were modified with AEAPS and Fe3O4@SiO2 NH2 composite particles were prepared[17].
2.2.2Immobilization of anti 2，4 D antibody on Fe3O4@SiO2 NH2 composite particles
3.0 mg of Fe3O4@SiO2 NH2 composite particles was adequately dissolved in 3.0 mL of phosphate buffered saline （PBS pH=8.2，0.2 mol/L），then the supernatant was discarded by means of magnetic separation.The pH value of the solution was adjusted twice by Tris HCl buffer solution （pH=8.2，50 mmol/L）.Subsequently，200 μL of anti 2，4 D antibody was added into the mixture and incubated at 37℃ for 30 min.After separating magnetically and washing with PBS for two times，excess functional groups on Fe3O4@SiO2 NH2 composite was blocked by buffer （PBS of 3% BSA） and then stored at 4℃ overnight.The product was magnetically separated from free antibody and washed with PBST （PBS of 0.05% Tween 20） for three times.Finally the sample was re suspended boric acid buffer solution （pH=8.2，0.2 mol/L） and stored at 4℃ before used （Scheme 1b）.
2.3Preparation of 2，4 D fluorescent probe
2.3.1Synthesis of CdTe@SiO2 NH2 composite particles
The red CdTe nanocrystals （Em=634 nm） prepared under 105℃ was used to synthesis CdTe@SiO2 NH2 composite particles.12 mL of CdTe QDs was added in 72 ml of ethanol and 15.2 mL of double distilled water.After dispersing adequately，the mixtures were added by 8 mL of ammonium hydroxide and stirred for 10 min.Subsequently，1 mL of TEOS was added in and stirred for 30 min.Finally，40 μL of APTS was introduced under vigorous stirring for 4 h.The resulting product was centrifuged at 12 000 r·min-1 for 3 min and washed with double distilled water and ethanol for three times，respectively.The sample was redissolved in double distilled water as a stock solution[16].
2.3.2Conjugation of 2，4 D OVA to CdTe@SiO2 NH2
1.0 mL of CdTe@SiO2 NH2 solution was added into 10.0 mL of boric acid buffer （0.2 mol/L） with dispersing adequately.4 mg of EDC·HCl and 3 mg of NHS were dissolved in the mixtures and incubated at 37℃ for 10 min.Then 2，4 D OVA was added into the mixtures and stirred for about 1 h.After centrifuging at 5 000 r·min-1 for 3 min，the precipitation （CdTe@SiO2 NH2 2，4 D OVA） was dispersed in boric acid buffer （Scheme 1a）. 2.4Optimization of reaction parameters in preparation of fluorescent probe
Several parameters were investigated systematically in order to synthesis the fluorescent probe with optimal conditions，including the dosage of 2，4 D OVA，PH value of boric acid buffer and coupling time.
2.5Establishment of the 2，4 D immunoassay system based on magnetic fluorescent probes
300 μL of fluorescent probe was added into 2，4 D standard solution of different concentrations and dispersed adequately.Subsequently，1.0 mL of magnetic probe was added into the mixture and incubated at 37℃ for 40 min.2，4 D standard solution and fluorescent probes competed binding the antibody on the surface of the magnetic probes.After magnetic separation，the magnetic part was washed three times with boric acid buffer solution to remove the residual fluorescent probe.Finally，the product was re suspended in Boric acid buffer solution （0.2 mol/L，pH 8.20） and the fluorescent signal was monitored by the fluorescence photometer.Establishment of the immunoassay system was presented in Scheme 1c.
3Results and Discussion
3.1Optimization of the dosage of 2，4 D OVA in preparation of fluorescent probe
As described in Section 2.3.2，2，4 D OVA at different dosage （100，300，500，700 μL） was conjugated with CdTe@SiO2 NH2 composite particles.Fluorescent signal was monitored by the fluorescence photometer to determine the maximum dosage of combination.
Figure 1 illustrated the fluorescence spectra of the fluorescent probes in different dosage of 2，4 D OVA.As the dosage of 2，4 D OVA increasing，the fluorescence intensity of the fluorescent probe gradually enhanced and subsequently，remaind stable when the dosage of 2，4 OVA arrived at 500 μL.Hence，500 μL amount of 2，4 D OVA was employed in the synthesis of fluorescent probe.
3.2Optimization of PH value in preparation of fluorescent probe
3.3Optimization of coupling time in preparation of fluorescent probe
Effect of coupling time on the fluorescent intensity of fluorescent probe was examined and optimized.30，40，50，60，70，80 min were applied in the conjugation from 2，4 D OVA to CdTe@SiO2 NH2 （Figure 3）.The fluorescent intensity of fluorescent probe reached a maximum when reaction time arrived at 70 min.So that 70 min was the optimal coupling time for preparation of 2，4 D fluorescent probe.
Figure 3Effect of different coupling time on fluorescent intensity of fluorescent probe
3.4Characterization of the immunoassay based on magnetic fluorescent probes The composites by the anti 2，4 D antibody labeled with Fe3O4@SiO2 NH2 served as solid phase，was employed as magnetic probe.The 2，4 D OVA，composite bonded with CdTe@SiO2 NH2 was employed as fluorescent probe.2，4 D standard solution and fluorescent probe competed binding the antibody on the surface of the magnetic probe.The complex of 2，4 D magnetic probe and magnetic fluorescent probe were formed，respectively.Only the complex of magnetic fluorescent probe could produce a fluorescent signal which could indicate the presence of 2，4 D.If there was no 2，4 D in the detection system，the complex of magnetic fluorescent probe was formed and in this case，the fluorescent intensity reaches a maximum （Figure 4A）.Otherwise，2，4 D would competes with the fluorescent probe，which would weaken the fluorescent signal.In addition，the higher concentration of 2，4 D，the weaker intensity of fluorescent signal would be （Figure 3B F）.
For the immunoassay based on magnetic fluorescent probes，the standard curve was obtained with the concentration of 2，4 D as the abscissa and the maximum fluorescence intensity as the ordinate （Figure 5）.The regression equation was y=-0.570 3x+182.371 4 （R2=0.996 59），which indicated that the fluorescent intensity was inversely proportional to the concentration of 2，4 D standard solution.However，deviation of the result would occur when the fluorescent signal approaches the maximum or the minimum.Therefore，the effective value should range from 10%～90% of the maximum.The detection limits （LOD） of 2，4 D in this assay was 35.5×10-9 based on the curve.
Only about 1.0 h was needed to finish the procedure of the immunoassay based on magnetic fluorescent probes with one reaction step and one washing step.However，to fulfill the procedure of the traditional ELISA，four reaction steps and there washing steps are needed and moreover more than 2.0 h were needed.Comparing with the flat solid phase，the easy separated and re dispersed nature allowed for a ″in solution″ reaction，which also significantly shorten the reaction time and amplify the detection signal.Meanwhile，the LOD of 2，4 D ELISA kit is 5×10-8 and that of 2，4 D colloidal gold strip is 2.1×10-5 in the market.It indicated the assay was also feasible.
4Conclusion
In conclusion，this work established a novel immunoassay system for rapid detection of 2，4 D residues in liquid system.The composite of Fe3O4@SiO2 NH2，labeled 2，4 D antibody served as a solid phase carrier instead of ordinary coated microplate.The composite of CdTe@SiO2 NH2，bonded with 2，4 D OVA was used to produce fluorescence signal as the fluorescence probe.It showed that 8.2 was the optimal pH，70 min was the optimal coupling time，500 μL amount of 2，4 D OVA in the synthesis of fluorescent probe.The standard curve was obtained with the concentration of 2，4 D and the maximum fluorescence intensity.The regression equation was gotten：y=-0.570 3x+182.3714 （R2=0.996 59）.The detection limit of the assay was 3.55×10-8.For the assay，one reaction step and one washing step were needed.The procedure could be finished within 1.0 h without complicated preparing procedures by competing reaction，and the detection limit is 3.55×10-8.Moreover，the optimized parameters in preparation of fluorescent probe had been obtained.The results demonstrates that this immunoassay based on magnetic fluorescent probes was a timesaving and feasible assay and would be a reliable tool for rapid detection of 2，4 D residues. References：
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