Preparation of Graphene Oxide Nanosheets and Their Portable Colorimetric Determination of Biothiols
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摘要:
目的 开发一种能够实时、快速、可视化检测生物硫醇（MEs）的方法，为疾病的早期诊断和评估提供有力手段。 方法 通过改良的Hummers方法制备具有优异的类过氧化物酶活性（POD-like）的片状氧化石墨烯（2D GO），并将比色法与智能手机集成构建快速可视化检测MEs的传感策略。 结果 2D GO能够催化过氧化氢（H2O2）分解产生具有强氧化性的羟基自由基（·OH），进而将无色3，3'，5，5'-四甲基联苯胺（3，3'，5，5'-tetramethylbenzidine，TMB）氧化为蓝色的ox-TMB，结合MEs的强还原作用对“2D GO+TMB+H2O2”比色传感体系显色效果的抑制作用实现了对MEs的快速可视化检测。 结论 基于2D GO所构建的比色平台具有较宽的线性检测范围（10～ 1000 μmol/L）以及良好的检测限（LOD < 7 μM），并成功用于测定胎牛血清样品中的MEs，回收率表现良好。Abstract:Objective To develop a method capable of real-time, rapid, and visual detection of MEs , providing a powerful tool for early diagnosis and assessment of diseases. Methods In this study, flake graphene oxide （2D GO） with excellent peroxidase-like activity （POD-like） was prepared by a modified Hummers' method, and a sensing strategy for the rapid visual detection of MEs was constructed by integrating colorimetry with a smartphone. Results 2D GO can catalyse the decomposition of hydrogen peroxide （H2O2） to produce highly oxidative hydroxyl radicals （·OH）, which oxidize the colourless 3, 3', 5, 5'-tetramethylbenzidine （TMB） into blue ox-TMB. The strong reducing effect of MEs on the "2D GO + TMB + H2O2" colorimetric sensing system enables rapid visual detection of MEs. Conclusion The colorimetric platform constructed based on 2D GO has a wide linear detection range （10- 1000 μmol/L） and a good detection limit （LOD < 7 μM）, and was successfully used for the determination of MEs in foetal bovine serum samples, with satisfactory recovery rates.-
Key words:
- Graphene oxide /
- Biothiols /
- Colorimetric sensing /
- Peroxide mimics enzymes
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图 2 材料的表征及类酶活性验证
A：2D GO的TEM图像；B：不同溶液体系的紫外-可见吸收光谱图，从上到下依次为a. TMB+H2O2+2D GO；b. TMB+H2O2；c. TMB；d. TMB+GO。
Figure 2. Characterisation of the materials and validation of enzyme-like activity
图 3 实验条件的优化
A：pH值；B：温度；C：H2O2浓度；D：2D GO浓度。
Figure 3. Optimisation of experimental conditions
图 4 米氏动力学研究
A和C：反应速度随H2O2或TMB浓度的变化曲线；B和D：H2O2或TMB相应的Lineweaver-Burk双倒数曲线。
Figure 4. Michaelis kinetics studies
图 5 检测性能的评估
A～C：TMB+H2O2+2D GO体系中加入不同浓度MEs的紫外-可见吸收光谱图（插图为不同浓度MEs与ΔA的线性关系图）；D～F：不同浓度MEs与通过智能手机辅助计算欧氏距离EDs的相关系数图。
Figure 5. Evaluation of detection performance
表 1 米氏常数（Km）和最大反应速率（Vmax）的比较
Table 1. Comparison of Michaelis constants （ Km ） and maximum reaction rates （ Vmax ）
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表 2 不同方法检测MEs的比较
Table 2. Comparison of different methods for detecting MEs
催化剂 方法 物质 线性范围 检出限 参考文献 mBrB 毛细管电泳 GSH 7.5～100 µmol/L 1.41 μmol/L [24] TCNQ 和 GO协同 电化学分析法 GSH 0.25～124.3 μmol/L，
124.3 μmol/L～1.67 mmol/L0.15 μmol/L [25] MNPG 荧光法 GSH 0.2～20 μmol/L 0.05 μmol/L [26] 7 氟苯并-2-氧杂 -1，
3-二唑-4-磺酸铵高效液相色谱 Cys、Hcy、
CysGly、GSH0.5 ～15 μmol/L 0.10 μmol/L [27] BrDMC 质谱法 GSH 1.0～100.0 μmol/L 0.4 μmol/L [28] 2D GO 比色法 Hcy 10～ 1000 μmol/L0.66 μmol/L 本工作
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表 3 胎牛血清加标实验中MEs检测结果
Table 3. Detection results of MEs in fetal bovine serum spiking assays
样品 加标量 （μmol/L） 紫外-可见光谱法 手机可视化比色法 检测（μmol/L） 回收率（%） RSD（n=3，%） 检测（μmol/L） 回收率（%） RSD（n=3，%） GSH 0 55.20 − 4.3 7.80 − 4.9 50 101.90 96.90 7.0 60.99 105.5 2.6 400 458.74 100.8 3.3 411.82 101.0 0.5 800 851.42 99.6 6.5 871.99 107.9 1.0 Cys 0 13.55 − 4.3 11.15 − 4.9 50 60.20 94.20 7.9 65.90 107.8 4.3 400 411.47 99.50 7.7 403.83 98.20 1.4 800 817.99 100.5 6.3 716.78 88.40 0.5 Hcy 0 10.40 − 7.2 11.50 − 6.2 50 61.22 99.50 2 62.27 100.9 5.6 400 408.90 99.50 1.7 411 99.90 2.1 800 813.00 100.3 0.4 811.4 100.1 1.1
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