WO2020098182A1 - 基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法 - Google Patents

基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法 Download PDF

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WO2020098182A1
WO2020098182A1 PCT/CN2019/077071 CN2019077071W WO2020098182A1 WO 2020098182 A1 WO2020098182 A1 WO 2020098182A1 CN 2019077071 W CN2019077071 W CN 2019077071W WO 2020098182 A1 WO2020098182 A1 WO 2020098182A1
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cds
paracetamol
cuncs
arginine
fluorescence
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金辉
桂日军
卜祥宁
付永鑫
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青岛大学
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6489—Photoluminescence of semiconductors

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  • the invention belongs to the technical field of preparation of nanocomposite materials and fluorescent sensors, and in particular relates to a preparation method of a paracetamol ratio fluorescent sensor based on a copper nanocluster / carbon dot / arginine composite.
  • the prepared sensor can be used for high sensitivity of paracetamol And highly selective quantitative detection.
  • Paracetamol also known as acetaminophen, is a commonly used phenolic analgesic drug with anti-inflammatory and antipyretic effects. Paracetamol is also a popular over-the-counter analgesic and antipyretic. Small doses of paracetamol are beneficial to the human body, but overdose can cause health problems, such as kidney failure and liver necrosis. Excessive consumption of paracetamol is one of the main causes of liver failure. The development of simple, accurate and quantitative detection methods for paracetamol in human serum is of great significance for human health monitoring.
  • Li Junhua et al. Built a paracetamol electrochemical sensor based on nanocomposite modified electrodes (Li Junhua, Liu Mengqin, Zhang Fuxing, Xu Zhifeng, Deng Peihong, Tang Siping, Liu Xing. A CoFe 2 O 4 NWs / RGO nanocomposite and its prepared paracetamol electrochemical Sensor.
  • Tan Xuecai et al. Reported molecularly imprinted electrochemical analysis methods for the determination of paracetamol (Tan Xuecai, Sun Yuexin, Yu Huicheng, Huang Zaiyin, Liu Shaogang, Lei Fuhou.
  • the analysis and detection methods for paracetamol mainly include electrochemical analysis and fluorescence analysis, but these methods only rely on a single signal output, and the single signal intensity is easily interfered by factors such as background, reagents, system and environmental conditions, causing fluctuations in the measurement results .
  • dual-signal ratio processing is used to obtain the signal intensity ratio, which has a self-calibration function, which can effectively eliminate the influence of self and background signals, and improve the accuracy and reliability of the detection results.
  • the present invention reports a paracetamol ratio fluorescent sensor based on copper nanoclusters / carbon dots / arginine complex.
  • Red fluorescent copper nanoclusters were prepared using DNA as a template, which combined with blue fluorescent carbon dots CDs through electrostatic adsorption and hydrogen bonding. After adding arginine, CuNCs / CDs / arginine complexes were formed. Adding arginine significantly reduced the blue fluorescence of CDs, and then added paracetamol. Arginine and CDs were separated due to the specific binding of arginine and paracetamol. The blue fluorescence of CDs quenched gradually recovered, and the addition of arginine and paracetamol will not Significantly caused the fluorescence change of CuNCs.
  • the purpose of the present invention is to overcome the above-mentioned defects of the prior art, design a simple method, low cost, high sensitivity and good selectivity based on a copper nanoclusters / carbon dots / arginine complex paracetamol ratio fluorescent sensor ⁇ ⁇ ⁇ Preparation method.
  • the present invention relates to a preparation process of a paracetamol ratio fluorescence sensor based on copper nanoclusters / carbon dots / arginine complex including the following steps:
  • a method for preparing a paracetamol ratio fluorescent sensor based on a copper nano-cluster / carbon dot / arginine complex characterized in that the method specifically includes the following steps:
  • the product solution was dialyzed using a dialysis bag with a molecular weight cut-off of 1000 Da to remove unreacted experimental materials.
  • the solution in the dialysis bag was removed by rotary evaporation to remove 90% of the liquid.
  • the remaining liquid was vacuum dried to obtain CuNCs, which were stored or dispersed in the solution at 4 ° C in the dark Prepare CuNCs dispersion for future use;
  • the red fluorescent CuNCs described in step (1) have an average size of 1 to 5 nm;
  • the blue fluorescent CDs described in step (2) have an average size of 1 to 5 nm;
  • the mass concentration of CuNCs is 1-10 mg mL -1
  • the mass concentration of CDs is 1-10 mg mL -1
  • the molar concentration of arginine is 1-100 ⁇ M
  • the linear detection range of the molar concentration of paracetamol described in step (4) is 0.01 to 500 ⁇ M, and the detection limit is 10 to 50 nM.
  • the effect of the present invention is to report a paracetamol ratio fluorescent sensor based on copper nanoclusters / carbon dots / arginine complex.
  • Red fluorescent copper nanoclusters CuNCs were prepared using DNA as a template, which combined with blue fluorescent carbon dots CDs through electrostatic adsorption and hydrogen bonding. After adding arginine, CuNCs / CDs / arginine complexes were formed. Adding arginine significantly reduced the blue fluorescence of CDs, and then added paracetamol. Arginine and CDs were separated due to the specific binding of arginine and paracetamol.
  • the fluorescence signal of CuNCs as the reference signal and the fluorescence signal of CDs as the response signal the linear relationship between the CDS and CuNCs fluorescence emission peak intensity ratio I CDs / I CuNCs and the concentration of paracetamol was fitted to construct a paracetamol ratio fluorescence sensor.
  • the method of the invention has simple operation, low cost, easily available raw materials, strong anti-interference ability of the ratio signal, good accuracy, high sensitivity and selectivity, and can be developed into a novel ratio fluorescence sensor for paracetamol Efficient detection.
  • FIG. 1 is a schematic diagram of the preparation of the paracetamol ratio fluorescent sensor based on the copper nanoclusters / carbon dots / arginine complex and the principle of paracetamol detection according to the present invention
  • FIG. 2 (a) is a ratio fluorescence sensor of the present invention for measuring the fluorescence emission spectrum of the corresponding sensor system at different paracetamol concentrations;
  • Figure 2 (b) shows the ratio of CDs / I CuNCs fluorescence intensity of CDs and CuNCs corresponding to different paracetamol concentrations. The linear relationship between different ratios and the concentration of paracetamol is fitted.
  • the solution in the dialysis bag was removed by rotary evaporation to remove 90% of the liquid.
  • the remaining liquid was dried in vacuum to obtain CuNCs, which were stored or dispersed in the solution at 4 ° C in the dark to prepare CuNCs dispersion. Standby, where the average size of CuNCs is 2nm.
  • CDs Preparation of CDs: Weigh 1.5g of chitosan, add 1.5mL of acetic acid dropwise to fully mix and dissolve, then add 3mL of polyvinylamide, and after stirring for 10min, add 25.5mL of double distilled water to dilute to obtain a homogeneous dispersion, which is dispersed
  • the solution was transferred to a micro-pressure autoclave with a polytetrafluoroethylene liner, and the reaction was stirred at 200 ° C for 5 hours.
  • the product solution was dialyzed using a dialysis bag with a molecular weight cut-off of 1000 Da to remove unreacted experimental materials, and the solution in the dialysis bag was removed by rotary evaporation.
  • CDs which are stored or dispersed in a solution at 4 ° C in the dark, to prepare a dispersion of CDs for use.
  • the average size of CDs is 3 nm.
  • Preparation of CuNCs / CDs / arginine complex Under magnetic stirring, add the aqueous dispersion of CDs dropwise to the aqueous dispersion of CuNCs to form a homogeneous mixture of CuNCs / CDs, then add the refined drops dropwise to this mixed solution
  • the aqueous solution of lysine forms a homogeneous mixture of CuNCs / CDs / arginine complexes, in which the concentrations of CuNCs, CDs and arginine are 5 mg mL -1 , 2 mgmL -1 and 50 ⁇ M, respectively.
  • Example 2 The preparation process of the paracetamol ratio fluorescent sensor and the principle diagram of the ratio fluorescence detection of paracetamol in this example.
  • the process steps for preparing CuNCs and CDs are the same as those in Example 1.
  • the average size of CuNCs is 3 nm and the average size of CDs is 4 nm.
  • Other specific process steps are as follows:
  • Preparation of CuNCs / CDs / arginine complex Under magnetic stirring, add the aqueous dispersion of CDs dropwise to the aqueous dispersion of CuNCs to form a homogeneous mixture of CuNCs / CDs, then add the refined drops dropwise to this mixed solution Aqueous lysine solution forms a homogeneous mixture of CuNCs / CDs / arginine complexes, in which the concentrations of CuNCs, CDs and arginine are 5 mg mL -1 , 5 mg mL -1 and 20 ⁇ M, respectively.
  • Example 3 The preparation process of the paracetamol ratio fluorescent sensor and the principle diagram of the ratio fluorescence detection of paracetamol in this example.
  • the process steps for preparing CuNCs and CDs are the same as those in Example 1, wherein the average size of CuNCs is 3 nm and the average size of CDs is 5 nm.
  • Other specific process steps are as follows:
  • Preparation of CuNCs / CDs / arginine complex Under magnetic stirring, add the aqueous dispersion of CDs dropwise to the aqueous dispersion of CuNCs to form a homogeneous mixture of CuNCs / CDs, then add the refined drops dropwise to this mixed solution Aqueous lysine solution forms a homogeneous mixture of CuNCs / CDs / arginine complexes, in which the concentrations of CuNCs, CDs and arginine are 2 mg mL -1 , 5 mg mL -1 and 10 ⁇ M, respectively.

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Abstract

基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法。红荧光铜纳米簇CuNCs通过静电吸附和氢键作用与蓝荧光碳点CDs结合，加入精氨酸后形成CuNCs/CDs/精氨酸复合物。加入精氨酸导致CDs蓝荧光减弱，再加入扑热息痛，因其与精氨酸特异性结合导致精氨酸与CDs分离，CDs蓝荧光恢复，精氨酸和扑热息痛不会引起CuNCs荧光显著改变。以CuNCs荧光为参比，CDs荧光为响应，拟合CDs/CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建扑热息痛比率荧光传感器。与现有技术相比，方法操作简单，比率信号抗干扰能力强，准确性好，灵敏度和选择性高，可作为一种新颖的比率荧光传感器用于扑热息痛的高效检测。

Description

基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法 技术领域：
本发明属于纳米复合材料与荧光传感器的制备技术领域，具体涉及一种基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其制备的传感器可用于扑热息痛的高灵敏度和高选择性定量检测。
背景技术：
扑热息痛又名对乙酰氨基酚，是一种常用的酚类止痛药物，具有抗炎解热作用。扑热息痛也是一种流行的非处方镇痛药和解热药，小剂量扑热息痛对人体是有益的，但过量服用会引起健康问题，比如肾衰竭、肝坏死等。扑热息痛的过量服用是导致肝功能衰竭的主要原因之一，开发对人体血清中扑热息痛的简单、精准和定量检测方法，对于人体健康的监测具有重要意义。
当前，针对扑热息痛检测的分析技术已经被陆续开发和研究了，包括电化学法、比色法、化学发光法、荧光法等。文献检索发现，Song等构建了一种氧化石墨烯改性玻碳电极用于扑热息痛电化学行为测定(Jinchun Song,Ji Yang,Junfen Zeng,Juan Tan,Li Zhang.Graphite oxide film-modified electrode as an electrochemical sensor for acetaminophen.Sensors and Actuators B-Chemical,2011,155,220–225)；Alam等采用主体-客体相互作用的β-环糊精和氧化还原性质的多壁碳纳米管实现了痕量对乙酰氨基酚的灵敏检测(Arif Ul Alam,Yiheng Qin,Massimo Catalano, Luhua Wang,Moon J.Kim,Matiar M.R.Howlader,Nan-Xing Hu,M.Jamal Deen.Tailoring MWCNTs and β-Cyclodextrin for Sensitive Detection of Acetaminophen and Estrogen.ACS Applied Materials&Interfaces,2018,10,21411-21427)。
李俊华等基于纳米复合物修饰电极构建扑热息痛电化学传感器(李俊华,刘梦琴,张复兴,许志锋,邓培红,唐斯萍,刘兴.一种CoFe 2O 4NWs/RGO纳米复合材料及其制备得到的扑热息痛电化学传感器.中国发明专利.公开号CN106248766A)；谭学才等报道了测定扑热息痛的分子印迹电化学分析方法(谭学才,孙月新,余会成,黄在银,刘绍刚,雷福厚.扑热息痛分子印迹电化学传感器及其制备方法.中国发明专利.公开号CN102735729A)；丁亚平等基于氨基酸修饰CdTe纳米粒子构建荧光探针用于扑热息痛定量检测(丁亚平,陆雅翔,李丽,罗立强,程瑜.利用CdTe纳米荧光探针测定扑热息痛的方法.中国发明专利.公开号CN102519921A)。
有关扑热息痛的分析检测方法主要包括电化学分析法和荧光分析法，但这些方法仅仅依赖单一信号输出，而单一信号强度易受背景、试剂、系统和环境条件等因素的干扰，引起测定结果的波动。相比而言，采用双信号比值处理来获得信号强度比率，具备了自校准功能，可有效消除自体和背景信号的影响，提高检测结果的准确性和可靠性。基于此，本发明报道了一种基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器。以DNA为模板制备红荧光铜纳米簇CuNCs，通过静电吸附和氢键作用与蓝荧光碳点CDs结合，加入精氨酸后形成CuNCs/CDs/精氨酸复合物。加入精氨酸导致CDs蓝荧光显著减弱，再加入扑热息痛，因精氨酸与扑热息痛特异性结合导致精氨酸与CDs分离，CDs淬灭的蓝 荧光逐渐恢复，精氨酸和扑热息痛的加入不会显著引起CuNCs荧光改变。以CuNCs荧光为参比信号，CDs荧光为响应信号，拟合CDs和CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建扑热息痛比率荧光传感器。截止目前，尚未有采用CuNCs/CDs/精氨酸复合物来构建比率荧光传感器，以及采用比率荧光方法来检测扑热息痛的国内外文献和专利的报道。
发明内容：
本发明的目的在于克服上述现有技术存在的缺陷，设计一种方法简单、成本低廉、灵敏度高、选择性好的一种基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法。
为了实现上述目的，本发明涉及的一种基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备工艺包括以下步骤：
1.基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，该方法具体包括以下步骤：
(1)CuNCs的制备：用超纯水溶解DNA干燥粉末配制DNA溶液，用含3-吗啉丙烷磺酸与NaCl的缓冲液稀释至pH 7.5，3-吗啉丙烷磺酸与NaCl浓度分别为10mM和15mM，向上述DNA混合溶液中加入抗坏血酸，抗坏血酸最终浓度调节至2mM，在室温和磁力搅拌下，逐滴加入CuCl 2，CuCl 2最终浓度调节至1mM，混合溶液在避光处反应20min，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CuNCs，在4℃避光条件下储存或分散在溶液中制备CuNCs分散液备用；
(2)CDs的制备：称取1.5g脱乙酰壳聚糖，滴加1.5mL乙酸充分混合溶解，再加入3mL聚乙烯酰胺，充分搅拌10min后加入25.5mL二次蒸馏水稀释得到均质分散液，将此分散液转入聚四氟乙烯内胆的微型高压反应釜中，在200℃下搅拌反应5h，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CDs，在4℃避光条件下储存或分散在溶液中制备CDs分散液备用；
(3)CuNCs/CDs/精氨酸复合物的制备：在磁力搅拌下，向CuNCs水分散液中逐滴加入CDs水分散液，形成CuNCs/CDs均质混合液，然后向此混合液中逐滴加入精氨酸水溶液，形成均质CuNCs/CDs/精氨酸复合物混合液；
(4)在室温和磁力搅拌下，向CuNCs/CDs/精氨酸复合物混合液中加入一定量的扑热息痛形成均质混合液，在避光处孵化5min后测定不同扑热息痛浓度下均质混合液的荧光发射光谱，拟合CDs和CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建扑热息痛比率荧光传感器。
步骤(1)中所述的红荧光CuNCs其平均尺寸为1～5nm；
步骤(2)中所述的蓝荧光CDs其平均尺寸为1～5nm；
步骤(3)中所述的均质混合液中CuNCs质量浓度为1～10mg mL -1，CDs质量浓度为1～10mg mL -1，精氨酸摩尔浓度为1～100μM；
步骤(4)中所述的扑热息痛摩尔浓度的线性检测范围为0.01～500μM，检测极限为10～50nM。
本发明的效果是：报道了一种基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器。以DNA为模板制备红荧光铜纳米簇CuNCs， 通过静电吸附和氢键作用与蓝荧光碳点CDs结合，加入精氨酸后形成CuNCs/CDs/精氨酸复合物。加入精氨酸导致CDs蓝荧光显著减弱，再加入扑热息痛，因精氨酸与扑热息痛特异性结合导致精氨酸与CDs分离，CDs淬灭的蓝荧光逐渐恢复，精氨酸和扑热息痛的加入不会显著引起CuNCs荧光改变。以CuNCs荧光为参比信号，CDs荧光为响应信号，拟合CDs和CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建扑热息痛比率荧光传感器。与现有技术相比，本发明方法操作简单，成本低廉，原料易得，比率信号抗干扰能力强，准确性好，灵敏度和选择性高，可发展成为一种新颖的比率荧光传感器用于扑热息痛的高效检测。
附图说明：
图1为本发明涉及的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备与扑热息痛检测的原理示意图；
图2(a)为使用本发明的比率荧光传感器测定不同扑热息痛浓度下对应的传感器体系的荧光发射光谱；
图2(b)为不同扑热息痛浓度对应CDs和CuNCs荧光发射峰强度比率I CDs/I CuNCs,拟合不同比率值与扑热息痛浓度之间的线性关系。
具体实施方式：
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1：
本实施例涉及的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其制备工艺和比率荧光检测扑热息痛的原理示 意图如图1所示，具体工艺步骤如下：
CuNCs的制备：用超纯水溶解DNA干燥粉末配制DNA溶液，用含3-吗啉丙烷磺酸与NaCl的缓冲液稀释至pH 7.5，3-吗啉丙烷磺酸与NaCl浓度分别为10mM和15mM，向上述DNA混合溶液中加入抗坏血酸，抗坏血酸最终浓度调节至2mM，在室温和磁力搅拌下，逐滴加入CuCl 2，CuCl 2最终浓度调节至1mM，混合溶液在避光处反应20min，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CuNCs，在4℃避光条件储存或分散在溶液中制备CuNCs分散液备用，其中CuNCs平均尺寸为2nm。
CDs的制备：称取1.5g脱乙酰壳聚糖，滴加1.5mL乙酸充分混合溶解，再加入3mL聚乙烯酰胺，充分搅拌10min后加入25.5mL二次蒸馏水稀释得到均质分散液，将此分散液转入聚四氟乙烯内胆的微型高压反应釜中，在200℃下搅拌反应5h，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CDs，在4℃避光条件储存或分散在溶液中制备CDs分散液备用，其中CDs平均尺寸为3nm。
CuNCs/CDs/精氨酸复合物的制备：在磁力搅拌下，向CuNCs水分散液中逐滴加入CDs水分散液，形成CuNCs/CDs均质混合液，然后向此混合液中逐滴加入精氨酸水溶液，形成均质CuNCs/CDs/精氨酸复合物混合液，其中CuNCs，CDs和精氨酸的浓度分别为5mg mL -1，2mgmL -1和50μM。
在室温和磁力搅拌下，向CuNCs/CDs/精氨酸复合物混合液中加入一定量的扑热息痛形成均质混合液，在避光处孵化5min后测定不同扑 热息痛浓度下均质混合液荧光发射光谱(如图2(a)所示)，拟合CDs/CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系(如图2(b)所示)，构建出扑热息痛比率荧光传感器，其中扑热息痛摩尔浓度的线性检测范围为0.02～400μM，检测极限为20nM。
实施例2：本实施例涉及的扑热息痛比率荧光传感器的制备工艺和比率荧光检测扑热息痛的原理示意图，CuNCs和CDs制备的工艺步骤同实施例1，其中CuNCs平均尺寸为3nm，CDs平均尺寸为4nm。其它具体工艺步骤如下：
CuNCs/CDs/精氨酸复合物的制备：在磁力搅拌下，向CuNCs水分散液中逐滴加入CDs水分散液，形成CuNCs/CDs均质混合液，然后向此混合液中逐滴加入精氨酸水溶液，形成均质CuNCs/CDs/精氨酸复合物混合液，其中CuNCs，CDs和精氨酸的浓度分别为5mg mL -1，5mg mL -1和20μM。
在室温和磁力搅拌下，向CuNCs/CDs/精氨酸复合物混合液中加入一定量的扑热息痛形成均质混合液，在避光处孵化5min后测定不同扑热息痛浓度下均质混合液荧光发射光谱，拟合CDs/CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建出扑热息痛比率荧光传感器，其中扑热息痛摩尔浓度的线性检测范围为0.01～100μM，检测极限为10nM。
实施例3：本实施例涉及的扑热息痛比率荧光传感器的制备工艺和比率荧光检测扑热息痛的原理示意图，CuNCs和CDs制备的工艺步骤同实施例1，其中CuNCs平均尺寸为3nm，CDs平均尺寸为5nm。其它具体工艺步骤如下：
CuNCs/CDs/精氨酸复合物的制备：在磁力搅拌下，向CuNCs水分 散液中逐滴加入CDs水分散液，形成CuNCs/CDs均质混合液，然后向此混合液中逐滴加入精氨酸水溶液，形成均质CuNCs/CDs/精氨酸复合物混合液，其中CuNCs，CDs和精氨酸的浓度分别为2mg mL -1，5mg mL -1和10μM。
在室温和磁力搅拌下，向CuNCs/CDs/精氨酸复合物混合液中加入一定量的扑热息痛形成均质混合液，在避光处孵化5min后测定不同扑热息痛浓度下均质混合液荧光发射光谱，拟合CDs/CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建出扑热息痛比率荧光传感器，其中扑热息痛摩尔浓度的线性检测范围为0.02～500μM，检测极限为15nM。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (5)

  1. 基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，该方法具体包括以下步骤：
    (1)CuNCs的制备：用超纯水溶解DNA干燥粉末配制DNA溶液，用含3-吗啉丙烷磺酸与NaCl的缓冲液稀释至pH 7.5，3-吗啉丙烷磺酸与NaCl浓度分别为10mM和15mM，向上述DNA混合溶液中加入抗坏血酸，抗坏血酸最终浓度调节至2mM，在室温和磁力搅拌下，逐滴加入CuCl 2，CuCl 2最终浓度调节至1mM，混合溶液在避光处反应20min，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CuNCs，在4℃避光条件下储存或分散在溶液中制备CuNCs分散液备用；
    (2)CDs的制备：称取1.5g脱乙酰壳聚糖，滴加1.5mL乙酸充分混合溶解，再加入3mL聚乙烯酰胺，充分搅拌10min后加入25.5mL二次蒸馏水稀释得到均质分散液，将此分散液转入聚四氟乙烯内胆的微型高压反应釜中，在200℃下搅拌反应5h，产物溶液采用截留分子量1000Da透析袋透析，除去未反应实验原料，将透析袋中溶液采用旋转蒸发除去90％液体，剩余液体用真空干燥得到CDs，在4℃避光条件下储存或分散在溶液中制备CDs分散液备用；
    (3)CuNCs/CDs/精氨酸复合物的制备：在磁力搅拌下，向CuNCs水分散液中逐滴加入CDs水分散液，形成CuNCs/CDs均质混合液，然后向此混合液中逐滴加入精氨酸水溶液，形成均质CuNCs/CDs/精氨酸复合物混合液；
    (4)在室温和磁力搅拌下，向CuNCs/CDs/精氨酸复合物混合液中加入一定量的扑热息痛形成均质混合液，在避光处孵化5min后测定不同扑热息痛浓度下均质混合液的荧光发射光谱，拟合CDs和 CuNCs荧光发射峰强度比率I CDs/I CuNCs与扑热息痛浓度间的线性关系，构建扑热息痛比率荧光传感器。
  2. 根据权利要求1所述的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，步骤(1)中所述的红荧光CuNCs其平均尺寸为1～5nm。
  3. 根据权利要求1所述的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，步骤(2)中所述的蓝荧光CDs其平均尺寸为1～5nm。
  4. 根据权利要求1所述的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，步骤(3)中所述的均质混合液中CuNCs质量浓度为1～10mg mL -1，CDs质量浓度为1～10mg mL -1，精氨酸摩尔浓度为1～100μM。
  5. 根据权利要求1所述的基于铜纳米簇/碳点/精氨酸复合物的扑热息痛比率荧光传感器的制备方法，其特征在于，步骤(4)中所述的扑热息痛摩尔浓度的线性检测范围为0.01～500μM，检测极限为10～50nM。
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CN115121803B (zh) * 2021-03-11 2024-04-30 上海交通大学医学院附属仁济医院 一种基于dna框架结构的多聚纳米团簇的合成方法
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