Study On The New Technology Of Detecting Magnetic Microspheres Based On Amino Functionalized Microspheres

Magnetic nanoparticle is a new type of functional material. It has many advantages（magnetic response, efficient, rapid separation, etc.） and has been widely used in the extraction of nucleic acids, immunoassay, cell separation, chiral separation enrichment areas in recent years. Magnetic nanoparticle with different inorganic or organic functional groups has special properties, which can meet the needs of different applications.Based on the previous wok of our laboratory, amino functionalized magnetic nanoparticles（MNP-NH₂） with a core of Fe3O4 was prepared.Based on MNP-NH₂ and silver absorption of efficiently, we constructed a new detection technique of homocysteine（Homocysteine, HCY）.Based on the adsorption of MNP-NH₂ and Pb²⁺, We constructed a new purification method for Pb²⁺ in water samples.The research is not only a further application scope of magnetic nanoparticles,but also a useful exploration of atomic absorption spectrophotometer（atomic absorption spectrophotometer, AAS） in the non-metallic elements detection.The main research includes:1. The Pb²⁺adsorption and purification technology based on MNP-NH₂ was established.The experiment was carried out to optimize the binding condition of MNP-NH₂ and Pb²⁺.The adsorption mechanism was preliminarily studied by the adsorption isotherm. The pH, adsorption time, the dosage of MNP-NH₂, adsorption capacity of MNP-NH₂, adsorption isotherm was studied. The concentration of elution agent,elution time, and the use times of MNP-NH₂ were studied by MNP-NH₂ regeneration experiment. The interference ions in water samples were studied. The established method had been used on different water samples.Results show:（1）Adsorption experiment: In the range of pH3.0⁵.0, the adsorption efficiency of MNP-NH₂ on Pb²⁺ was ranged from 59.1% to 69.8%. when pH < 3.0or pH> 5.0 the adsorption rate was low and pH 5.0 was selected at next experiment; Pb²⁺and MNP-NH₂ can be combined in a relatively short time and reached equilibrium and 10 min was chosed as the adsorption time; 10 mg MNP-NH₂ can be absorbed101.4 ng Pb²⁺; The adsorption rate of Pb²⁺ increased with the dosage of MNP-NH₂ as it is less than 20.0mg. when the dosage of MNP-NH₂ is greater than 20.0mg the adsorption rate did not changed with increasing of MNP-NH₂; According to theadsorption isotherm fitting, the adsorbed Pb²⁺is to a flat state arrangement and almost no competitive solvent.（2）MNP-NH₂ Regeneration Experiment: selection of volume fraction of 5% HNO3 as eluent; Elution time had no effect on desorption rate, in order to reduce the time, 5min was choosed as elution time; After using 10 times for adsorption of Pb²⁺, adsorption efficiency beginning to fall. The MNP-NH₂ under the optimum experimental conditions can be reused 10 times.（3） Interference experiment:Studied the effects of K +, Na +, Cl-, NO3-, SO42-, Cr3 +, Zn2 +, Ca2 +, Mg2 +, Al3+, Cu2+,Mn2+, Fe3+, Ni2+ on adsorption. The results show that these ions exist has no effect on the adsorption;（4） Sample determination: Under the optimized experimental conditions, the purify efficiency of Pb²⁺ in different water samples was studied by the experimental results. The effect of the adsorption times on the adsorption was studied,selected the tap water, water from Haihe, plant sewage water treatment as water samples. the adsorption efficiency was 60%,80% and 94% when adsorption times ranged from one to three.The MNP-NH₂ has a good effect on the adsorption and purification of Pb²⁺ in water.2. Study on a new detection method of HCY based on competition of MNP-NH₂ by AAS.MNP-NH₂ under certain conditions has good adsorption with Ag+, and the –SH of HCY also has very strong binding capacity with Ag+.The research use the characteristic that the binding ability of Ag+ and MNP-NH₂ is weaker than that of Ag+and-SH. At first, amino functionalized magnetite nanoparticles compound silver composite（MNP-NH₂- Ag） was prepared.Then, HCY was add, Ag+ was released into solution due to-SH compete binding. After magnetic separation, the concentration of HCY was determined by determination of Ag+ with AAS. Various experimental conditions was optimized, including the method to determine Ag+ by AAS, the preparation of MNP-NH₂-Ag（Ag+ concentration, ammonia dosage, the pH of lotion,reaction buffer pH, the time silver mixed liquid reacted with MNP-NH₂-Ag）, the combine of MNP-NH₂-Ag with HCY（the pH of lotion, MNP-NH₂-Ag and HCY reacted time）, the interference test and so on were optimized.Results show:（1） The method to determine silver has been improved based on drinking water national standard（GB5750-2006）. The determination conditions:the wavelength 338.3nm, drying temperature of 150 ℃ for 10 s, ashing temperature of500℃ for 23 s, atomization temperature of 1800℃ for 3s, sample volume in 10μL,AAS determination of silver with good linearity in the range of 0.0¹00.0 μg/L;（2）The use level of ammonia in the 2.0 to 2.5mL response signal tends to be stable.2.5mL was chosen as the most suitable use level of ammonia to prepare [Ag（NH3）2] +;When the pH of MNP-NH₂-Ag lotion is 12.0, the response signal reach the largest.when above 12.0, the response signal then begin to decrease. pH12.0 was select as the best pH of MNP-NH₂-Ag lotion; In the range of pH6.5⁹.5, no change in response signal, the pH value had no influence on the experiment.Because the low pH will affect the existing state of HCY/cysteine（CYS）, pH9.0 was selected as the most suitable value; When the range of silver concentration changed from 5mg/L to7.5mg/L the signal reaches the maximum and stable.5mg/L silver standard solution was choosed to prepare MNP-NH₂-Ag; The time of silver mixed with Na2HPO4 had no affect to experiment; The time of silver mixed liquid reacted with MNP-NH₂-Ag also had no affcet and 10 min was chosed as the appropriate time.（3） Experiments showed that the-SH of CYS in the reaction system also had response at the same time.We optimized the responnse between HCY、CYS and MNP-NH₂-Ag. The pH of the reaction system had a impact on the response on determination of HCY/CYS. At pH11.0, CYS and HCY had the same response signal with MNP-NH₂-Ag. The phosphate buffer of pH11 was selected as the MNP-NH₂-Ag lotion; the reaction time of MNP-NH₂-Ag with HCY did not affect the response signal and 30 min was chosen as the best reaction time.3.A method for quantitative determination of HCY in blood samples by AAS was established.The method was applied to determine HCY in real samples based on part one.The pretreatment conditions of serum sample were optimized. With the vitamin B6,serine conditions, cystathionine-beta-synthetase（ CBS） can make HCY inversion into a free thiol L-cystathionine and CYS does not occur conversion characteristic, so as to determine the HCY contentration. The detection of HCY in blood samples was established by two step method.Various factors influence the determination such as the effect of sodium borohydride,high molecular weight protein removal methods were optimized.Experiments on the interference test, the sample determination working curves, LOD,the linear range of the experiments, recovery rate, and comparison method were study.Results show:（1）The two sulfur bond of HCY and CYS complex can be opened by adding sodium borate; The interfere of sodium borate can be avoid by adding acid to reaction solution; 3KD filter and high-speed centrifugation methods can remove the effects of high molecular proteins. However,centrifugal processing with good repeatability and can also make mixed liquor separation becomes clear, so we chose the 10000 rpm high-speed centrifugal as the method of removal high molecular proteins.（2）Interference experiment show that there is no response when the other amino acids and MNP-NH₂-Ag react. HCY response signal was significantly higher than those of other amino acids and can exclude the interference of other kinds of amino acid. Its application in the detection of HCY in blood samples, the detection limit is 3.4μmol/L. the detection scope is 0.0-500.0μmol/L. recovery rate is between97%-120%; Research for determination of HCY concentration and enzyme immunoassay for detection of serum HCY compared differences in less than 7%, the method has good accuracy and precision.