纳米催化共振散射光谱分析

共振散射光谱技术是利用荧光分光光度计的同步扫描技术建立起来的一项光谱分析新技术,具有简便、快速、灵敏度高等优点,在蛋白质、核酸、无机离子等痕量分析中得到应用.结合本课题组近年来的研究工作,本文综述了共振散射光谱技术在免疫纳米催化、核酸适配体纳米催化分析中的应用.     

非标记纳米银探针催化共振散射光谱检测痕量ATP

在pH 7.8 Tris-HCl缓冲液中, 三磷酸腺苷(adenosine triphosphate, ATP)的核酸适体(Apt 1)与其互补链(Apt 2)结合生成双链DNA (double-strand DNA, dsDNA). 此dsDNA不能稳定纳米银(AgNP), NaCl可致AgNP聚集, 在500 nm波长处产生一个较强的共振散射峰. 加入ATP后, ATP与dsDNA中的Apt 1结合形成较稳定的发夹结构结合物并释放出可稳定AgNP的Apt 2. 随着ATP浓度(16.5～1650 nmol/L)增加, 生成的Apt 2增加, 被Apt 2稳定的AgNP即AgNP-Apt 2结合物增加, 聚集的AgNP减少, 500 nm处的共振散射值线性减小. 该适配体反应中的AgNP-Apt 2对葡萄糖-铜(II)微粒反应具有较强的催化作用, 其产物氧化亚铜微粒在610 nm处有一较强共振散射峰. 随着ATP浓度增大, 反应液中AgNP-Apt 2增多, 催化作用增强, 610 nm处的共振散射峰增强. ATP浓度在4.95～165 nmol/L范围内与共振散射增大值ΔI_{610 nm}呈线性关系, 检出限为1.8 nmol/L ATP. 据此建立了灵敏度高、选择性好、简便快速检测ATP的共振散射光谱新方法.     

Resonance scattering spectral detection of ultratrace IgG using immunonanogold-HAuCl_4-NH_2OH catalytic reaction

Nanogold particles of 10 nm were used to label goat anti-human IgG (GIgG) to obtain nanogold-labeled GIgG (AuGIgG). In a citrate-HCl buffer solution of pH 2.27,AuGIgG showed a strong catalytic effect on the reaction between HAuCl4 and NH2OH to form big gold particles that exhibited a resonance scatter-ing (RS) peak at 796 nm. Under the chosen conditions,AuGIgG combined with IgG to form immuno-complex AuGIgG-IgG that can be removed by centrifuging at 16000 r/min. AuGIgG in the centrifuging solution also showed catalytic effect on the reaction. On those grounds,an immunonanogold catalytic RS assay for IgG was designed. With addition of IgG,the amount of AuGIgG in the centrifuging solution decreased; the RS intensity at 796 nm (I796 nm) decreased linearly. The decreased intensity ΔI796 nm was linear with respect to the IgG concentration in the range of 0.08-16.0 ng·mL-1 with a detection limit of 0.02 ng·mL-1. This assay was applied to analysis of IgG in sera with satisfactory sensitivity,selectivity and rapidity.     

Resonance scattering spectral detection of ultratrace IgG using immunonanogold-HAuCl<Subscript>4</Subscript>-NH<Subscript>2</Subscript>OH catalytic reaction

Nanogold particles of 10 nm were used to label goat anti-human IgG (GIgG) to obtain nanogold-labeled GIgG (AuGIgG). In a citrate-HCI buffer solution of pH 2.27, AuGIgG showed a strong catalytic effect on the reaction between HAuCl₄ and NH₂OH to form big gold particles that exhibited a resonance scattering (RS) peak at 796 nm. Under the chosen conditions, AuGIgG combined with IgG to form immunocomplex AuGIgG-IgG that can be removed by centrifuging at 16000 r/min. AuGIgG in the centrifuging solution also showed catalytic effect on the reaction. On those grounds, an immunonanogold catalytic RS assay for IgG was designed. With addition of IgG, the amount of AuGIgG in the centrifuging solution decreased; the RS intensity at 796 nm (I _{796 nm}) decreased linearly. The decreased intensity ΔI _{796 nm} was linear with respect to the IgG concentration in the range of 0.08–16.0 ng · mL⁻¹ with a detection limit of 0.02 ng · mL⁻¹. This assay was applied to analysis of IgG in sera with satisfactory sensitivity, selectivity and rapidity. Supported by the National Natural Science Foundation of China (Grant No. 20667001), Natural Science Foundation of Guangxi Province (Grant No. 0728213), and the Foundation of New Century Ten-Hundred-Thousand Talents of Guangxi Province     

A rapid and sensitive immunonanogold resonance scattering spectral probe for complement 3

Gold nanoparticles in size of about 10 nm was used to label goat anti-human complement 3 (anti-C3) to obtain a sensitive and selective immunoresonance scattering spectral probe for C3. It was based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na₂HPO₄–citric acid buffer solutions and in presence of polyethylene glycol (PEG). The resonance scattering (RS) intensity at 560 nm enhanced greatly with C3. Well linear relationships between the enhanced RS intensity (I_RS) and the C3 concentration in the range of 8.33–200 ng ml⁻¹ were obtained, with a detection limit of 2.8 ng ml⁻¹ and the limit of quantification 8.51 ng ml⁻¹ for C3. The convenient and selective and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.     

A highly sensitive and selective immunonanogold resonance scattering spectral assay for sudan I

A sensitive and selective resonance scattering spectral (RSS) assay was proposed for the determination of sudan I (SDI), using 10 nm nanogold to label the antibody against sudan I (anti-SDI Ab) to obtain a RSS probe for SDI. The immunonanogold reaction between nanogold-labelled anti-SDI Ab and SDI took place in pH 4.92 KH₂PO₄–Na₂HPO₄ buffer solution and in the presence of polyethylene glycol (PEG)-6000, and the intensity of resonance scattering peak at 580 nm decreased greatly. The decreased intensity ΔI_{580 nm} was proportional to the concentration of SDI in the range of 0.23–45.0 ng mL^?1. The linear regression equation was calculated as ΔI_580nm = 1.20c + 2.01 (R = 0.9975, n = 6), with a detection limit (3σ) of 0.13 ng mL^?1. The SDI in egg samples was assayed, with satisfactory results.     

Silver nanoparticle labeled immunoresonance scattering spectral assay for trace fibrinogen

Silver nanoparticles in size of 8.0 nm was prepared by the trisodium citrate and used to label goat anti-human fibrinogen. In the pH 5.8 Na2HPO4-NaH2O4 buffer solution (PBS) and in the presence of polyethylene glycol (PEG) and KCl, the immune reaction between silver-labeled goat anti-human fibrinogen and fibrinogen took place and led the resonance scattering intensity at 465 nm (I465) to decreasing. The I465 decreased intensity was linear to the fibrinogen concentration in the range from 0.067 to 1.67 μg/mL, with a detection limit of 0.024 μg/mL. This method was applied to determination of fibrinogen in human plasma, with satisfactory results.     

Immunonanogold-catalytic resonance scattering spectral assay of trace human chorionic gonadotrophin

Gold nanoparticles in diameter of 10 nm were used to label rabbit anti-human chorionic gonadotrophin (RhCG) antiserum to obtain a resonance scattering spectral probe (AuRhCG) for human chorionic gonadotrophin (hCG). The immunoreaction between AuRhCG and hCG take place to form hCG–AuRhCG immunocomplex in pH 5.0 citric acid–Na₂HPO₄ buffer solution. The immunocomplex solutions were centrifuged to obtain the supernatant solution. The AuRhCG in the supernatant solution exhibited strong catalytic effect on the particle reaction between Ag⁺ and hydroquinone to produce gold–silver composite particles in pH 3.4 citric acid–trisodium citrate buffer solution. There is a stronger resonance scattering (RS) peak at 423 nm for the particles. With the addition of hCG, the AuRhCG in the supernatant solution decreased, and the RS intensity at 423 nm decreased. The decreased RS intensity ΔI_423 nm was proportional to the concentration of hCG in the range of 2.5–208.3 mIU/mL with a detection limit of 0.83 mIU/mL. This method has been applied to the determination of hCG in urine samples, with satisfactory results.     

纳米金共振散射光谱法测定痕量乐果

在H2SO4介质中,有机磷乐果和钼酸钠、酒石酸锑钾反应,生成淡黄色的有机磷锑钼三元杂多酸,纳米金在726 nm处产生一个较强的共振散射峰。抗坏血酸可将该有机磷锑钼杂多酸还原为有机磷锑钼杂多蓝,导致726 nm处共振散射峰的强度降低。乐果质量浓度在0.014～1.66μg/mL范围内与共振散射光强度降低值ΔI呈良好的线性关系,其回归方程为ΔI=155.4ρ+1.4,相关系数为0.9970,方法的检出限为3.9 ng/mL,该法可用于废水中乐果的测定。     

高灵敏选择性检测凝血酶的适体纳米银共振散射光谱探针

采用四氢硼钠制备了较稳定的纳米银,并用凝血酶(TB)适体修饰纳米银制各了识别凝血酶的适体纳米银探针.在pH 7.0的Tris-HCl缓冲溶液及KCl存在下,适体纳米银探针与凝血酶特异结合生成G-四分体和纳米银聚集体,导致体系在480 nm处的共振散射峰增强.随着凝血酶浓度的增大,生成的纳米银聚集体越多,共振散射强度线性...     

适体修饰纳米金催化-共振散射光谱测定凝血酶

用核酸适体修饰纳米金制备了识别凝血酶(TB)的适体修饰纳米金(AptAu)共振散射光谱探针. 在pH 7.40 的Na₂HPO₄-NaH₂PO₄缓冲溶液中及NaCl, KCl存在下, AptAu探针中的适配体特异识别凝血酶, 生成稳定的G-四分体和大粒径的纳米金聚集体. 经微孔滤膜过滤后, 纳米金聚集体被分离, 以滤液中未反应的AptAu作催化晶种, 在20.0 μg/mL HAuCl₄-5.01 mmol/L HCl-1.83 mg/mL CTMAB-50.1 μg/mL VC条件下, 催化维生素C (VC)还原HAuCl₄生成较大粒径的金颗粒, 体系在600 nm处有一共振散射峰. 随着凝血酶浓度的增大, 滤液中AptAu浓度降低, 催化作用减弱, 600 nm处的共振散射峰降低, 其降低值ΔI_{600 nm}与凝血酶浓度在6.40×10^－3～0.150 U/mL范围内存在良好线性关系, 回归方程为ΔI＝1.26×10³C＋1.50, 相关系数为0.999, 检出限为1.30×10^－3 U/mL. 该法用于定量分析人血浆中凝血酶, 结果满意.     

A new and selective and sensitive nanogold-labeled immunoresoance scattering spectral assay for trace prealbumin

A gold-labeled immunoresonance scattering spectral probe for trace prealbumin (PA) was prepared by using gold nanoparticles in size of 9.0 nm to label goat anti-human prealbumin polyclonal antibody. The immune reaction between the gold-labeled antibody and prealbumin took place in pH 7.6 Na₂HPO₄-NaH₂PO₄ buffer solution. In the presence of polyethylene glycol PEG-10000, the labeled gold nanoparticles were released and aggregated which brought the resonance scattering intensity (I_RS) at 580 nm to enhance greatly. The ΔI_RS is proportional to the prealbumin concentration in the range from 16.67 to 666.67 ng/mL, with a detection limit of 4.1 ng/mL. This simple, sensitive and selective assay was applied to determination of prealbumin in human plasma, with satisfactory results.     

Resonance Scattering Detection of Trace Hg2+ Using Aptamer‐modified AuPd Nanoalloy Probe as Catalyst

The 5 nm AuPd nanoalloy in mole ratio of Au:Pd=32:1 was prepared, using sodium citrate as the stabilizing agent and NaBH₄ as the reductant. The AuPd nanoalloy was modified by the aptamer to prepare an aptamer‐ AuPd (AptAuPd) probe for resonance scattering (RS) detection of 5.0–1250 nmol/L Hg²⁺. The AptAuPd‐Hg²⁺ aptamer reaction solution was filtrated by membrane, and the AptAuPd in the filtrate exhibited strong catalytic effect on the slow NiP particle reaction between NiCl₂ and NaH₂PO₂, and the NiP particles showed a RS peak at 508 nm. The RS intensity decreased when Hg²⁺ concentration increased. The decreased RS intensity was linear to Hg²⁺ concentration in the range of 0.5–1250 nmol/L. The RS assays were used to determine Hg²⁺ in real samples, with good results.     

双链DNA裂解-纳米金共振散射光谱探针检测痕量UO2^2＋

在pH 5.5的2-（N-吗啉）-乙磺酸缓冲液中,底物DNA和酶DNA杂交形成双链DNA（dsDNA）.当加入UO 2＋后,dsDNA中的底物DNA链被裂解,释放的裂解链DNA吸附在金纳米粒子（GN）表面,未吸附裂解链DNA的GN在NaCl存在下发生聚集,在610 nm处有一最强的共振散射峰.随着UO 2＋的浓度增大,...     

Nanosilver resonance scattering spectral method for determination of hydroxyl radical and its application

Silver nanoparticles were prepared under a microwave high-pressure condition using citric acid sodium as a reducer while the excess citrate was removed under high speed centrifugation. There is a resonance scattering (RS) peak at 470 nm for silver nanoparticles. In a pH 4.0 HAc-NaAc buffer solution, hydroxyl radicals from the Fenton reaction can oxidize silver nanoparticles to Ag , resulting in the RS intensity decreasing. The decreased RS intensity at 470 nm (△I 470 nm) is linear with respect to the concentration of H2O2 (C) in the range of 0.27-7.56 μmol/L with a detection limit of 0.23 μmol/L. Its regression equation is △I 470 nm = 24.3 C 13.8 with a correlation coefficient of 0.9959. This method was applied to screening the antioxidants with satisfactory results.     

青霉素类药物的共振光散射光谱分析

本文研究了青霉素钠-碱性品红缔合物的共振散射光谱,建立了pH =7.5的C-L缓冲溶液中,共振光散射法测定青霉素钠的新方法.线性回归方程△IRu=1.74+5.17C,相关系数R=0.9981,检测限3.16×10-7mol/L.该方法简单快速,灵敏度高,对实际样品进行了测定,得到了令人满意的结果.     

Aptamer-modified AuRe Nanoalloy Probe for Trace Hg2+ Using Resonance Scattering as Detection Technique

The AuRe nanoalloy particles in molar ratio of 9:1 were prepared by sodium borohydride procedure, and modified by single strand DNA (ssDNA) to prepare an aptamer AuRe nanoprobe (AuRessDNA) for Hg²⁺. In the pH 7.0 Na₂HPO₄‐NaH₂PO₄ buffer solution and in the presence of NaCl, Hg²⁺ interacted with AuRessDNA to form double‐stranded T‐Hg²⁺‐T mismatched and release AuRe nanoparticles that aggregate to large AuRe nanoparticles clusters causing the resonance scattering (RS) peak red shifting and the RS intensity enhanced linearly. On those grounds, 0.067–33.3 nmol·L^?1 Hg²⁺ can be detected rapidly by the aptamer‐modified AuRe nanoparticles RS assay, with a detection limit of 0.04 nmol·L^?1 Hg²⁺. If the aggregated AuRe particles were removed by membrane filtration, the excess AuRessDNA in the filtration solution exhibits catalytic effect on the new Te particle reaction between Na₂TeO₄ and SnCl₂. As the concentration of Hg²⁺ increased, the AuRessDNA nanoparticles in the filtrate solution decreased, the RS intensity at 734 nm decreased linearly. The Hg²⁺ concentration (c) in the range of 0.00133–0.267 nmol·L^?1 was linear to the decreased RS intensity (ΔI_734nm), with a regression equation of ΔI= ?786.4c?4.4, a correlation coefficient of 0.9975, and a detection limit of 0.9 pmol·L^?1 Hg²⁺. This method was applied to the detection of Hg²⁺ in water samples, with satisfactory results.     

Study on the resonance Rayleigh scattering spectra and resonance non-linear spectra of congo red-amikacin system and its analytical application

The interaction between congo red (CR) and amikacin (AMK) was studied by resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS) and second-order scattering (SOS) combining with absorption spectrum. In a weak acidic medium, CR combined with AMK to form an ion association complex with the composition ratio of 1∶1 by electrostatic interaction, hydrophobicity and charge transferring effect. As a result, the new spectra of RRS, FDS, and SOS appeared and their intensities were enhanced greatly. The maximum wavelengths of RRS, FDS and SOS were located at 563 nm, 475 nm and 940 nm, and the scattering intensities were proportional to the concentration of AMK. These three methods have very high sensitivities, and the detection limits were 4.0 ng·mL?1 for RRS, 3.6 ng·mL?1 for FDS and 1.9 ng·mL?1 for SOS, respectively. At the same time, the methods have better selectivity. A new method for the determination of trace amounts of AMK with congo red by resonance scattering technique has been developed. The recovery for the determination of AMK in blood serum and urine sample was between 95.5% and 105.5%. In this study, the properties, such as enthalpy of formation, charge distribution and mean polarizability, were calculated by AM1 quantum chemistry method. In addition, the reaction mechanism and the reasons for the enhancement of scattering spectra were discussed.     

检测痕量纳米金的纳米催化光度法

在EDTA—NaOH介质中,金纳米微粒对盐酸联氨还原硫酸铜生成铜微粒这一慢反应具有较强的催化作用．铜微粒在750nm处产生一个吸收峰．随着纳米金浓度的增大,750nm处的吸光强度线性增大．对于粒径为10、30、50nm的纳米金,其线性范围、回归方程、检出限分别为0．12～1．68、0．36～2．80、1．00～5．00nmol／L,△A750nm=0．3205CAu＋0．0076、△A750nm=0．2201CAu＋0．0056、△A750nm=0．1150CAu＋0．0066,0．05、0．20、0．50nmol／L Au．分别对0．50、1．00nmol／L纳米金（d=10nm）平行测定10次,求得其相对标准偏差分别为4．2％、3．5％．     

核酸适体修饰纳米金共振散射光谱探针快速检测痕量Pb~(2+)

以柠檬酸三钠做稳定剂,用硼氢化钠还原氯金酸制备了粒径为5nm的纳米金.用铅离子核酸适体aptamer保护纳米金获得了检测铅离子的适体纳米金(aptamer-NG)共振散射光谱探针.在pH7.0的Na2HPO4-NaH2PO4缓冲溶液中及30mmol·L-1NaCl存在下,aptamer-NG稳定而不聚集.Pb2+可与该探针中的aptamer形成非常稳定的G-四分体结构,并释放出纳米金.在NaCl作用下纳米金聚集形成较大的微粒,导致552nm处共振散射峰强度增大.Pb2+浓度在0.07～42nmol·L-1范围内与552nm处共振散射强度增大值ΔI成线性关系,其回归方程为ΔI=12.0c+9.2,线性相关系数为0.9965,方法检出限为0.03nmol·L-1Pb2+.该方法用于水样中铅离子检测,结果与石墨炉原子吸收光谱法结果一致.