WO2021223317A1 - 基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法 - Google Patents

基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法 Download PDF

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WO2021223317A1
WO2021223317A1 PCT/CN2020/100532 CN2020100532W WO2021223317A1 WO 2021223317 A1 WO2021223317 A1 WO 2021223317A1 CN 2020100532 W CN2020100532 W CN 2020100532W WO 2021223317 A1 WO2021223317 A1 WO 2021223317A1
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sqds
lactic acid
auncs
aqueous solution
bsa
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金辉
姜晓文
桂日军
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青岛大学
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N2021/6432—Quenching

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  • the invention belongs to the technical field of preparation of novel fluorescent nanocomposites and ratio fluorescent nano probes, and specifically relates to a method for preparing ratio fluorescent lactic acid probes based on a novel nanocomposite system and an enzyme-catalyzed reaction.
  • the prepared probes can be used in biology Highly sensitive and selective quantitative detection of lactic acid in fluid samples.
  • Lactic acid is one of the final products of glycolysis in the human body. When oxygen is insufficient, reduced coenzyme I will accumulate, which in turn activates the glycolysis pathway, leading to the production of lactic acid.
  • Human lactic acidosis is one of the most common types of metabolic acidosis. The increase in blood lactic acid concentration is closely related to respiratory diseases and diabetic ketonemia. In addition, bacterial meningitis, brain trauma, etc. can also cause an increase in the concentration of lactic acid in the cerebrospinal fluid. Based on this, the implementation of quantitative detection of lactic acid concentration in human biological fluid samples is of great significance for early screening, accurate diagnosis and clinical treatment of lactic acid-related diseases.
  • the previous literature For the detection of lactic acid with traditional instrumental analysis techniques, the previous literature generally has problems such as harsh conditions, complicated procedures, time-consuming operations, high costs, and lack of specificity.
  • the previous literature For the detection of lactic acid by biochemical probe and sensor technology, the previous literature has adopted a single-signal output mode, which relies on the accurate measurement of the intensity of a single response signal.
  • the measurement of response signal intensity will be interfered by internal and external factors, such as changes in the surrounding environment, instrument efficiency, system measurement errors, probe and sensor material usage, etc., which may cause inconsistencies in the measurement results. Stability and random fluctuations weaken the feasibility and practicability of the detection technology.
  • the dual signal peak intensity ratio method is used to detect lactic acid, which not only has the advantages of biochemical probe and sensor technology, but also overcomes the problems of unstable and random measurement results.
  • the ratio method takes the ratio of the peak intensity of the double signal to the response signal output, and has a built-in calibration function, which effectively avoids the interference of the background signal and improves the accuracy of the detection result.
  • the invention discloses a method for preparing a ratio fluorescent nano probe which is novel in design, simple and efficient, and can be used for quantitative detection of lactic acid.
  • the purpose of the present invention is to overcome the above-mentioned problems in the prior art, and develop a novel and simple and efficient method for preparing a fluorescent lactic acid probe based on a novel nanocomposite system and an enzyme-catalyzed reaction ratio.
  • the prepared probe can be used for Highly sensitive and selective quantitative detection of lactic acid in biological fluid samples.
  • the present invention relates to a method for preparing a fluorescent lactic acid probe based on the ratio of a novel nanocomposite system and an enzyme-catalyzed reaction.
  • the preparation method includes the following steps:
  • SQDs Preparation of sulfur quantum dots SQDs: Weigh 1g sublimed sulfur, 2mL polyethylene glycol PEG400 and 3g sodium hydroxide, respectively add 50mL double distilled water under magnetic stirring to form a homogeneous mixed solution; transfer to 100mL In a polytetrafluoroethylene-lined mini-high pressure reactor, the reaction was stirred at 70°C for 6 hours; after the reaction, 2 mL of the product solution was taken, and 2 mL of hydrogen peroxide aqueous solution with a mass concentration of 7 wt% was added to it, and stirred well to mix evenly. Prepare SQDs water dispersion for use;
  • BSA/AuNCs Preparation of bovine serum albumin-stabilized gold nanoclusters BSA/AuNCs: prepare 40mg/mL BSA aqueous solution, 5mmol/L chloroauric acid aqueous solution, 0.5mol/L sodium hydroxide aqueous solution and 2mol/L mercaptopropionic acid aqueous solution; Under magnetic stirring, add 5mL chloroauric acid aqueous solution and 0.5mL sodium hydroxide aqueous solution to 5mL BSA aqueous solution in sequence to form a homogeneous mixture; then add 0.5mL mercaptopropionic acid aqueous solution, and incubate the mixture at 4°C for 1h; After the end, the product solution is transferred to a dialysis bag with a molecular weight cutoff of 5000kDa, and the dialysis process is performed for 24 hours to remove unreacted raw materials and small molecular impurities; the dialyzed solution is rotary
  • the effect of the present invention is to disclose a method for preparing a fluorescent lactic acid probe based on the ratio of a novel nano composite system and an enzyme-catalyzed reaction.
  • the outer electronic structure of Fe 3+ is 3d 5 4s 0 , and the 5 d orbitals are half-filled, resulting in higher
  • Figure 1 The preparation method of fluorescent lactic acid probe based on the ratio of novel nanocomposite system and enzyme-catalyzed reaction and the principle schematic diagram of quantitative detection of lactic acid;
  • Fig. 1 The method for preparing a fluorescent lactic acid probe based on the ratio of a novel nanocomposite system and an enzyme-catalyzed reaction and the principle schematic diagram of quantitative detection of lactic acid related to this embodiment are shown in Fig. 1.
  • the specific preparation steps are as follows:
  • SQDs Preparation of sulfur quantum dots SQDs: Weigh 1g sublimed sulfur, 2mL polyethylene glycol PEG400 and 3g sodium hydroxide, respectively add 50mL double distilled water under magnetic stirring to form a homogeneous mixed solution; transfer to 100mL PTFE In an ethylene-lined mini-high pressure reactor, the reaction was stirred at 70°C for 6 hours; after the reaction, 2 mL of the product solution was taken, and 2 mL of hydrogen peroxide aqueous solution with a mass concentration of 7 wt% was added to it. Stir fully to mix evenly to obtain SQDs Water dispersion for use;
  • BSA/AuNCs Preparation of bovine serum albumin-stabilized gold nanoclusters BSA/AuNCs: prepare 40mg/mL BSA aqueous solution, 5mmol/L chloroauric acid aqueous solution, 0.5mol/L sodium hydroxide aqueous solution and 2mol/L mercaptopropionic acid aqueous solution; stir under magnetic force Next, add 5mL chloroauric acid aqueous solution and 0.5mL sodium hydroxide aqueous solution to 5mL BSA aqueous solution in turn to form a homogeneous mixture; then add 0.5mL mercaptopropionic acid aqueous solution, and incubate the mixture for 1h at 4°C; after the reaction, the product The solution was transferred to a dialysis bag with a molecular weight cutoff of 5000kDa and treated for 24 hours to remove unreacted raw materials and small molecular impurities; the dialyzed solution was rotary evaporated to remove
  • This embodiment relates to a method for preparing a fluorescent lactic acid probe based on the ratio of a novel nanocomposite system and an enzyme-catalyzed reaction and a schematic diagram of the principle of quantitative detection of lactic acid as shown in Fig. 1.
  • SQDs, SQDs/MOF and BSA/AuNCs The preparation steps are the same as in Example 1, and other specific preparation steps are as follows:
  • This embodiment relates to a method for preparing a fluorescent lactic acid probe based on the ratio of a novel nanocomposite system and an enzyme-catalyzed reaction and a schematic diagram of the principle of quantitative detection of lactic acid as shown in Fig. 1.
  • SQDs, SQDs/MOF and BSA/AuNCs The preparation steps are the same as in Example 1, and other specific preparation steps are as follows:

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Abstract

本发明公开了基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法，制备硫量子点内包覆的金属有机骨架复合物SQDs/MOF，牛血清白蛋白稳定的金纳米簇BSA/AuNCs，配制SQDs/MOF、BSA/AuNCs、乳酸氧化酶LOD和Fe2+的混合溶液体系，加入乳酸LA后，LOD催化LA生成H2O2，H2O2催化Fe2+成Fe3+，Fe3+引发的电子转移导致AuNCs荧光减弱，而包覆在MOF结构中的SQDs荧光变化甚微，拟合AuNCs与SQDs荧光发射峰强度比率IAuNCs/ISQDs与LA浓度对数之间的线性关系，构建新型比率荧光纳米探针，用于乳酸的高灵敏和选择性定量检测。

Description

基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法 技术领域
本发明属于新型荧光纳米复合物和比率荧光纳米探针的制备技术领域，具体涉及一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法，该制备的探针可用于生物流体样品中乳酸的高灵敏和选择性定量检测。
背景技术
乳酸(Lactic acid，LA)是人体内糖酵解的最终产物之一，当氧气不足时，会出现还原型辅酶I积累，进而启动了糖酵解途径，导致乳酸的产生。人体乳酸性酸中毒是一种最常见的代谢性酸中毒类型，血液中乳酸浓度的升高与呼吸系统疾病、糖尿病酮血症等密切相关。此外，细菌性脑膜炎、脑外伤等也可以引起脑脊液中乳酸浓度的升高。基于此，执行人体生物流体样品中乳酸浓度的定量检测，对于乳酸相关疾病的早期筛查、精准诊断和临床治疗都具有重要意义。
在先前的文献资料中，已经开发了不同的分析技术用于乳酸的检测，如比色法、分光光度法、高效液相色谱法、气相色谱法、酶法、电化学法等。例如，韦真博等制备了聚金属酞菁-碳纳米管复合材料修饰电极，用于电催化乳酸和乳酸检测(韦真博；杨亚男；康志伟；王俊；程邵明，一种检测乳酸浓度的复合材料修饰电极的制备方法及应用，中国发明专利公开号CN110186966A)；Sidra Amin等制备氧化镍纳米颗粒修饰的玻碳电极，用于构建非酶电化学传感器检测乳酸(Sidra Amin,Aneela Tahira,Amber Solangi,Raffaello Mazzaro,Zafar Hussain Ibupoto,Alberto Vomiero,A sensitive enzyme-free lactic acid sensor based on NiO nanoparticles forpractical applications,AnalyticalMethods,2019,11,3578–3583)。
对于传统仪器分析技术检测乳酸，先前文献普遍存在条件苛刻，程序复杂，操作费时，成本较高，缺乏特异性等问题。对于生化探针和传感器技术检测乳酸，先前文献均采用了单信号输出模式，依赖于单一响应信号强度的精确测量。在实际样品中检测乳酸的过程中，响应信号强度的测量会受到内外和外因素的干扰，如周围环境变化，仪器工作效率，系统测量误差，探针和传感器材料用量等，引起测量结果的不稳定和随机波动，进而削弱了检测技术的可行性和实用性。
相比先前报导的乳酸检测方法，采用双信号峰值强度比率法检测乳酸，既具备了生化探针和传感器技术的优势，又能克服测量结果不稳定和随机波动的问题。比率法以双信号峰值强度的比值为响应信号输出，具备内置校准功能，有效避免了背景信号的干扰，提升了检测结果的准确度。截止目前，尚未有采 用双信号峰值强度比率法来定量检测乳酸的国内外文献和专利报道。本发明公开了一种设计新型，简单高效，可用于乳酸定量检测的比率荧光纳米探针的制备方法。制备硫量子点SQDs内包覆的金属有机骨架MOF复合物，即SQDs/MOF；制备牛血清白蛋白BSA稳定的金纳米簇BSA/AuNCs；将SQDs/MOF的水分散液与BSA/AuNCs的水分散液混合，再依次加入乳酸氧化酶LOD和亚铁离子Fe 2+，最终形成均质混合液。向混合液中加入乳酸LA，引起AuNCs荧光减弱而SQDs荧光变化甚微，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数之间的线性关系，可构建用于乳酸定量检测的新型比率荧光纳米探针。
发明内容
本发明的目的在于克服上述现有技术存在的问题，发展了一种设计新颖和简单高效的基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法，该制备的探针可用于生物流体样品中乳酸的高灵敏和选择性定量检测。
为实现上述目的，本发明涉及的一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法，其制备方法包括以下步骤：
(1)硫量子点SQDs的制备：称取1g升华硫，2mL聚乙二醇PEG400和3g氢氧化钠，在磁力搅拌下分别加入50mL二次蒸馏水中，形成均质混合溶液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应6h；反应结束后取2mL产物溶液，向其中加入2mL质量浓度为7wt％的过氧化氢水溶液，充分搅拌以混合均匀，制得SQDs水分散液备用；
(2)硫量子点内包覆的金属有机骨架复合物SQDs/MOF的制备：将4mL SQDs水分散加入20mL的2-甲基咪唑的乙醇溶液中，磁力搅拌10min以形成均质混合液；加入15mL硝酸锌水溶液，磁力搅拌30min，制得含有沉淀物的混合液；在3000rpm转速下离心20min，沉淀物用乙醇和二次蒸馏水交替洗涤两次，经冷冻干燥得到干燥产物SQDs/MOF，在室温避光处保存备用；
(3)牛血清白蛋白稳定的金纳米簇BSA/AuNCs的制备：配制40mg/mL BSA水溶液，5mmol/L氯金酸水溶液，0.5mol/L氢氧化钠水溶液和2mol/L巯基丙酸水溶液；在磁力搅拌下，向5mL BSA水溶液中依次加入5mL氯金酸水溶液和0.5mL氢氧化钠水溶液，形成均质混合液；再加入0.5mL巯基丙酸水溶液，混合液在4℃下孵育1h；反应结束后产物溶液转入5000kDa截留分子量的透析袋中，透析处理24h，除去未反应的原料和小分子杂质；透析后的溶液经旋转蒸发除去大部分溶剂，然后冷冻干燥处理，得到干燥产物BSA/AuNCs；
(4)分别配制浓度为5～10mg/mL的SQDs/MOF水分散液，浓度为5～10mg/mL的BSA/AuNCs水分散液；在磁力搅拌下，将5～10mL的SQDs/MOF水分散液与5～10mL的BSA/AuNCs水分散液进行充分混合，形成均质混合液； 向混合液中加入浓度为10mmol/L硝酸盐铁水溶液1～5mL，然后加入1～2mL浓度为5～10U/mL乳酸氧化酶LOD溶液，充分搅拌混合均匀，形成混合溶液体系；向该混合溶液体系中加入乳酸LA，其中LA浓度调整为0.1μmol/L至20mmol/L，测量“混合溶液体系+乳酸”的荧光发射光谱，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数LgC LA之间的线性关系，构建用于乳酸定量检测的新型比率荧光纳米探针；该探针对LA浓度的线性检测范围为0.1μmol/L至20mmol/L，检测限为0.05～0.5μmol/L。
本发明的效果是公开了一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法。水热制备硫量子点SQDs；SQDs与2-甲基咪唑及硝酸锌共混，一锅合成硫量子点内包覆金属有机骨架复合物SQDs/MOF；水相制备牛血清白蛋白稳定金纳米簇BSA/AuNCs；配制SQDs/MOF、BSA/AuNCs、LOD和Fe 2+混合溶液体系；加入乳酸LA，乳酸氧化酶LOD催化氧化LA生成丙酮酸和过氧化氢H 2O 2，H 2O 2催化氧化Fe 2+成Fe 3+；Fe 3+外层电子结构为3d 54s 0，5个d轨道是半填充的，导致较高电荷密度和较强吸电子能力；当Fe 3+与BSA/AuNCs共存时，Fe 3+快速吸附在BSA/AuNCs表面，发生非辐射电子转移，电子从BSA/AuNCs激发态到Fe 3+出现的d轨道，故Fe 3+引发的电子转移引起了BSA/AuNCs荧光猝灭(响应信号)；AuNCs表面巯基丙酸的功能基团与Fe 3+结合，形成有效的结合位点，而Fe 3+与BSA之间仅仅存在较弱的配位结合作用；SQDs包覆在MOF结构中，其荧光几乎不受周围物质的影响(参比信号)；拟合AuNCs与SQDs荧光发射峰强度比率I AuNCs/I SQDs与LA浓度对数之间的线性关系，可构建新型比率荧光纳米探针，用于生物流体样品中乳酸的高灵敏和选择性定量检测。
附图说明
图1.基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法及其定量检测乳酸的原理示意图；
图2.在乳酸LA不同存在浓度下，测量“混合溶液体系+乳酸”荧光发射光谱，拟合AuNCs与SQDs荧光发射峰强度比率I AuNCs/I SQDs与LA浓度对数LgC LA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
本实施例涉及的一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法及其定量检测乳酸的原理示意图如图1所示，具体制备步骤如下：
硫量子点SQDs的制备：称取1g升华硫，2mL聚乙二醇PEG400和3g氢氧化钠，在磁力搅拌下分别加入50mL二次蒸馏水中，形成均质混合溶液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应6h；反应结束后取2mL产物溶液，向其中加入2mL质量浓度为7wt％的过氧化氢水溶液，充分搅拌以混合均匀，制得SQDs水分散液备用；
硫量子点内包覆的金属有机骨架复合物SQDs/MOF的制备：将4mL SQDs水分散加入20mL的2-甲基咪唑的乙醇溶液中，磁力搅拌10min以形成均质混合液；加入15mL硝酸锌水溶液，磁力搅拌30min，制得含有沉淀物的混合液；在3000rpm转速下离心20min，沉淀物用乙醇和二次蒸馏水交替洗涤两次，经冷冻干燥得到干燥产物SQDs/MOF，在室温避光处保存备用；
牛血清白蛋白稳定的金纳米簇BSA/AuNCs的制备：配制40mg/mL BSA水溶液，5mmol/L氯金酸水溶液，0.5mol/L氢氧化钠水溶液和2mol/L巯基丙酸水溶液；在磁力搅拌下，向5mL BSA水溶液中依次加入5mL氯金酸水溶液和0.5mL氢氧化钠水溶液，形成均质混合液；再加入0.5mL巯基丙酸水溶液，混合液在4℃下孵育1h；反应结束后产物溶液转入5000kDa截留分子量的透析袋中，透析处理24h，除去未反应的原料和小分子杂质；透析后的溶液经旋转蒸发除去大部分溶剂，然后冷冻干燥处理，得到干燥产物BSA/AuNCs；
分别配制浓度为5mg/mL SQDs/MOF水分散液，浓度为5mg/mL BSA/AuNCs水分散液；在磁力搅拌下，将5mL SQDs/MOF水分散液与5mL BSA/AuNCs水分散液进行充分混合，形成均质混合液；向混合液中加入浓度为10mmol/L硝酸盐铁水溶液1mL，然后加入1mL浓度为5U/mL乳酸氧化酶LOD溶液，充分搅拌混合均匀，形成混合溶液体系；向该混合溶液体系中加入乳酸LA，其中LA浓度调整为1μmol/L至10mmol/L，测量“混合溶液体系+乳酸”荧光发射光谱，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数LgC LA之间的线性关系(如图2所示)，构建用于乳酸定量检测的新型比率荧光纳米探针；该探针对LA浓度的线性检测范围为1μmol/L至10mmol/L，检测限为0.4μmol/L。
实施例2
本实施例涉及的一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法及于定量检测乳酸的原理示意图如图1所示，其中的SQDs、SQDs/MOF和BSA/AuNCs的制备步骤同实施例1，其它具体制备步骤如下：
分别配制浓度为6mg/mL SQDs/MOF水分散液，浓度为6mg/mL BSA/AuNCs水分散液；在磁力搅拌下，将6mL SQDs/MOF水分散液与6mL BSA/AuNCs水分散液进行充分混合，形成均质混合液；向混合液中加入浓度为10mmol/L硝酸盐铁水溶液2mL，然后加入1.2mL浓度为6U/mL乳酸氧化酶 LOD溶液，充分搅拌混合均匀，形成混合溶液体系；向该混合溶液体系中加入乳酸LA，其中LA浓度调整为0.1μmol/L至1mmol/L，测量“混合溶液体系+乳酸”荧光发射光谱，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数LgC LA之间的线性关系，构建用于乳酸定量检测的新型比率荧光纳米探针；该探针对LA浓度的线性检测范围为0.1μmol/L至1mmol/L，检测限为0.08μmol/L。
实施例3
本实施例涉及的一种基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法及于定量检测乳酸的原理示意图如图1所示，其中的SQDs、SQDs/MOF和BSA/AuNCs的制备步骤同实施例1，其它具体制备步骤如下：
分别配制浓度为8mg/mL SQDs/MOF水分散液，浓度为8mg/mL BSA/AuNCs水分散液；在磁力搅拌下，将8mL SQDs/MOF水分散液与8mL BSA/AuNCs水分散液进行充分混合，形成均质混合液；向混合液中加入浓度为10mmol/L硝酸盐铁水溶液4mL，然后加入1.5mL浓度为8U/mL乳酸氧化酶LOD溶液，充分搅拌混合均匀，形成混合溶液体系；向该混合溶液体系中加入乳酸LA，其中LA浓度调整为1μmol/L至15mmol/L，测量“混合溶液体系+乳酸”荧光发射光谱，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数LgC LA之间的线性关系，构建用于乳酸定量检测的新型比率荧光纳米探针；该探针对LA浓度的线性检测范围为1μmol/L至15mmol/L，检测限为0.5μmol/L。
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 (1)

  1. 基于新型纳米复合体系与酶催化反应的比率荧光乳酸探针的制备方法，其制备方法包括以下步骤：
    (1)硫量子点SQDs的制备：称取1g升华硫，2mL聚乙二醇PEG400和3g氢氧化钠，在磁力搅拌下分别加入50mL二次蒸馏水中，形成均质混合溶液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应6h；反应结束后取2mL产物溶液，向其中加入2mL质量浓度为7wt％的过氧化氢水溶液，充分搅拌以混合均匀，制得SQDs水分散液备用；
    (2)硫量子点内包覆的金属有机骨架复合物SQDs/MOF的制备：将4mL SQDs水分散加入20mL的2-甲基咪唑的乙醇溶液中，磁力搅拌10min以形成均质混合液；加入15mL硝酸锌水溶液，磁力搅拌30min，制得含有沉淀物的混合液；在3000rpm转速下离心20min，沉淀物用乙醇和二次蒸馏水交替洗涤两次，经冷冻干燥得到干燥产物SQDs/MOF，在室温避光处保存备用；
    (3)牛血清白蛋白稳定的金纳米簇BSA/AuNCs的制备：配制40mg/mL BSA水溶液，5mmol/L氯金酸水溶液，0.5mol/L氢氧化钠水溶液和2mol/L巯基丙酸水溶液；在磁力搅拌下，向5mL BSA水溶液中依次加入5mL氯金酸水溶液和0.5mL氢氧化钠水溶液，形成均质混合液；再加入0.5mL巯基丙酸水溶液，混合液在4℃下孵育1h；反应结束后产物溶液转入5000kDa截留分子量的透析袋中，透析处理24h，除去未反应的原料和小分子杂质；透析后的溶液经旋转蒸发除去大部分溶剂，然后冷冻干燥处理，得到干燥产物BSA/AuNCs；
    (4)分别配制浓度为5～10mg/mL的SQDs/MOF水分散液，浓度为5～10mg/mL的BSA/AuNCs水分散液；在磁力搅拌下，将5～10mL的SQDs/MOF水分散液与5～10mL的BSA/AuNCs水分散液进行充分混合，形成均质混合液；向混合液中加入浓度为10mmol/L硝酸盐铁水溶液1～5mL，然后加入1～2mL浓度为5～10U/mL乳酸氧化酶LOD溶液，充分搅拌混合均匀，形成混合溶液体系；向该混合溶液体系中加入乳酸LA，其中LA浓度调整为0.1μmol/L至20mmol/L，测量“混合溶液体系+乳酸”的荧光发射光谱，通过拟合AuNCs与SQDs的荧光发射峰强度比率即I AuNCs/I SQDs与LA浓度对数LgC LA之间的线性关系，构建用于乳酸定量检测的新型比率荧光纳米探针；该探针对LA浓度的线性检测范围为0.1μmol/L至20mmol/L，检测限为0.05～0.5μmol/L。
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