CN105466876B - 一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒 - Google Patents
一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于钯纳米颗粒可视化检测6‑巯基嘌呤的试剂盒,该试剂盒包括:具有过氧化物酶活性的钯纳米颗粒液体;3,3’,5,5’‑四甲基联苯胺‑乙醇溶液;过氧化氢水溶液;pH为4.0磷酸二氢钠‑磷酸氢二钠缓冲液。利用本发明的试剂盒能方便地用于人血清和人尿中6‑巯基嘌呤含量的测定,其具有灵敏度高、选择性好、检测限低。
Description
技术领域
本发明属于纳米技术和仿生技术领域,具体的是涉及一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒。
背景技术
6-巯基嘌呤,又称巯嘌呤,为嘌呤类抗代谢型抗癌药物,在体内通过阻断次黄嘌呤转变为腺嘌呤核苷酸及鸟嘌呤核苷酸而抑制核酸的合成,临床上主要用于治疗急性白血病、绒毛膜上皮癌和恶性***等[Cancer,1999,86(6),1080-1086]。服用6-巯基嘌呤会产生肝损伤和骨髓抑制等副作用。6-巯基嘌呤的活性因个体差异和血浆浓度而异[Biosens.Bioelectron.2013,41,844–847],对用药病人进行药物浓度监测可以指导临床合理用药且对于减少药物不良反应具有重要意义。因此,为了人类健康,建立一种廉价,高敏感性,高选择性的6-巯基嘌呤检测方法至关重要。到目前为止,6-巯基嘌呤的检测方法有电化学分析法、高效液相色谱法、质谱分析法、毛细管电泳法、拉曼光谱法、荧光光谱法等[Biosens.Bioelectron.2013,41,844–847;J.Electroanal.Chem.2012,665,63–69]。在这些方法中,电化学分析法被广泛地报道且许多修饰电极用于6-巯基嘌呤的检测,但是它们较差的重复性和复杂的电极修饰过程限制了他们在实际样品中的应用;虽然高效液相色谱法、质谱分析法、毛细管电泳法和拉曼光谱法检测6-巯基嘌呤时具有选择性且灵敏,但是它们需要昂贵的设备和有毒的溶剂且往往涉及复杂的样品预处理;于对荧光检测法来说,由于没有分离功能导致选择性较低,所以它的应用有限[Biosens.Bioelectron.2013,41,844–847]。
发明内容
本发明的目的是克服现有技术的不足,提供一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒。
本发明的技术方案概述如下:
一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒,包括:
1)具有过氧化物酶活性的钯纳米颗粒液体;
2)3,3’,5,5’-四甲基联苯胺-乙醇溶液;
3)过氧化氢水溶液;
4)pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液。
3,3’,5,5’-四甲基联苯胺-乙醇溶液的浓度优选为1-50mM。
过氧化氢水溶液的浓度优选为1-10M。
pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液的浓度优选为20-80mM。
其中,具有过氧化物酶活性的钯纳米颗粒是用下述方法制成:
1)脱氧核苷酸的退火:
将2.5-250μL 2-200μM的脱氧核苷酸水溶液、50-200μL 5-20mM pH为3.0-9.0的磷酸盐缓冲溶液加入1.5mL的离心管中并加入三蒸水使总体积为400μL,混匀;升温至90-100℃,维持5-10min,降至室温;
脱氧核苷酸的序列为
Gb,其中b=10-200;
Cc,其中c=10-200;
(GC)d,其中d=5-100;
(G2C)e,其中e=4-70;
(G3C)f,其中f=3-60;
(G4C)g,其中g=2-50;
(GC2)h,其中h=4-70;,
(GC3)i,其中i=3-60;
(GC4)j,其中j=2-50;
2)钯纳米颗粒的制备:
将8-16μL 200-400μM氯钯酸钠水溶液加入到步骤1)获得的退火的脱氧核苷酸水溶液中,在20-30℃下混匀,静置0.5-4h,加入6-24μL 0.6-2.4mM还原剂水溶液,混匀,在20-30℃下静置10-15h,得到具有过氧化物酶活性的钯纳米颗粒液体。
还原剂优选为硼氢化钠或二甲胺硼烷。
具有过氧化物酶活性的钯纳米颗粒的制备中所使用的磷酸盐缓冲溶液优选为磷酸二氢钠-磷酸氢二钠缓冲溶液、磷酸二氢钾-磷酸氢二钾缓冲溶液或磷酸二氢钠-磷酸缓冲溶液。
本发明的优点:
利用本发明的试剂盒能方便地用于人血清和人尿中6-巯基嘌呤含量的测定,其具有灵敏度高、选择性好、检测限低。
附图说明
图1是实施例1中合成的具有过氧化物酶活性的钯纳米颗粒的透射电镜图;
图2是实施例1中过氧化氢水溶液浓度为125mM,3,3’,5,5’-四甲基联苯胺乙醇溶液浓度分别为0.015mM、0.06mM、0.08mM、0.125mM、0.165mM、0.25mM的动力学曲线图;
图3为图2的双倒数曲线图。
图4是实施例1中3,3’,5,5’-四甲基联苯胺乙醇溶液浓度为0.125mM,过氧化氢水溶液浓度分别为15mM、30mM、50mM、75mM、100mM、125mM、150mM的动力学曲线图;
图5为图4的双倒数曲线图。
图6为试验例1的标准工作曲线图;
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
下面的实施例是为了使本领域的技术人员能够更好地理解本发明,但不对本发明作任何限制。
实施例1
一种具有过氧化物酶活性的钯纳米颗粒的制备方法,包括如下步骤:
(1)脱氧核苷酸C10的退火:
将25μL 20μM的脱氧核苷酸C10水溶液、100μL 10mM pH为5.0的磷酸二氢钠-磷酸氢二钠缓冲溶液加入1.5mL的离心管中并加入三蒸水使总体积为400μL,混匀;升温至95℃,维持5min,降至室温;
(2)钯纳米颗粒的制备:
将12μL 300μM氯钯酸钠水溶液加入到步骤(1)获得的退火的脱氧核苷酸水溶液中,在25℃下混匀,静置2h,加入15μL 1.5mM二甲胺硼烷水溶液,混匀,在25℃下静置12h,得到具有过氧化物酶活性的钯纳米颗粒液体。用透射电镜扫描获得平均粒径为4.8nm。(见图1)
检测:
测定钯纳米颗粒的过氧化物酶活性:
在6个4mL比色皿中,均加入9μL实施例1获得的具有过氧化物酶活性的钯纳米颗粒液体、750μL 10mM pH为4.0的磷酸二氢钠-磷酸氢二钠缓冲液、38.2μL 0.125M过氧化氢水溶液后,再向1-6个比色皿中依次加入15μL的0.015mM、0.06mM、0.08mM、0.125mM、0.165mM、0.25mM 3,3’,5,5’-四甲基联苯胺乙醇溶液,加入三蒸水使终体积为3000μL,混合反应10min,采用紫外分光光度计扫描652nm处的吸收光谱,得到吸光度,通过Lineweaver–Burk方程计算出其米氏常数。(见图2和图3)
具有过氧化物酶活性的钯纳米颗粒表现出高的酶活性,对3,3’,5,5’-四甲基联苯胺的Km值为0.051mM。
在7个4mL比色皿中,均加入9μL实施例1获得的具有过氧化物酶活性的钯纳米颗粒液体、750μL 10mM pH为4.0的磷酸二氢钠-磷酸氢二钠缓冲液、15μL 0.125mM 3,3’,5,5’-四甲基联苯胺乙醇溶液后,再向1-7个比色皿中依次加入38.2μL的15mM、30mM、50mM、75mM、100mM、125mM、150mM过氧化氢水溶液,加入三蒸水使终体积为3000μL,混合反应10min,采用紫外分光光度计扫描652nm处的吸收光谱,得到吸光度,通过Lineweaver–Burk方程计算出其米氏常数。(见图4和图5)
具有过氧化物酶活性的钯纳米颗粒表现出高的酶活性,对过氧化氢的Km值为29.5mM。
实施例2
一种具有过氧化物酶活性的钯纳米颗粒的制备方法,包括如下步骤:
(1)脱氧核苷酸G200的退火:
将2.5μL 2μM的脱氧核苷酸G200水溶液、50μL 5mM pH为3.0的磷酸二氢钾-磷酸氢二钾缓冲溶液加入1.5mL的离心管中并加入三蒸水使总体积为400μL,混匀;升温至90℃,维持10min,降至室温;
(2)钯纳米颗粒的制备:
将8μL 200μM氯钯酸钠水溶液加入到步骤(1)获得的退火的脱氧核苷酸水溶液中,在20℃下混匀,静置4h,加入6μL 0.6mM硼氢化钠水溶液,混匀,在20℃下静置15h,得到具有过氧化物酶活性的钯纳米颗粒液体。
用透射电镜扫描获得平均粒径为1.0nm。测定方法同实施例1,对3,3’,5,5’-四甲基联苯胺的Km值为0.003mM,对过氧化氢的Km值为481.4mM。
实施例3
一种具有过氧化物酶活性的钯纳米颗粒的制备方法,包括如下步骤:
(1)脱氧核苷酸C200的退火:
将250μL 200μM的脱氧核苷酸C200水溶液、200μL 20mM pH为9.0的磷酸二氢钠-磷酸溶液加入1.5mL的离心管中并加入三蒸水使总体积为400μL,混匀;升温至100℃,维持5min,降至室温;
(2)钯纳米颗粒的制备:
将16μL400μM氯钯酸钠水溶液加入到步骤(1)获得的退火的脱氧核苷酸水溶液中,在30℃下混匀,静置0.5h,加入24μL2.4mM二甲胺硼烷水溶液,混匀,在30℃下静置10h,得到具有过氧化物酶活性的钯纳米颗粒液体。
用透射电镜扫描获得平均粒径为1.9nm。测定方法同实施例1,对3,3’,5,5’-四甲基联苯胺的Km值为0.014mM,对过氧化氢的Km值为140mM。
用G10、(GC)5、(GC)100、(G2C)4、(G2C)70、(G3C)3、(G3C)60、(G4C)2、(G4C)50、(GC2)4、(GC2)70、(GC3)3、(GC3)60、(GC4)2、(GC4)50;分别替换实施例1中的C10,其它同实施例1,所获得的具有过氧化物酶活性的钯纳米颗粒的平均粒径见表1。并用同实施例1相同的方法测定对3,3’,5,5’-四甲基联苯胺的Km值,对过氧化氢的Km值,见表1。
表1
结论:本发明的方法制备的具有过氧化物酶活性的钯纳米颗粒对过氧化氢的Km值小,说明具有过氧化物酶活性的钯纳米颗粒与过氧化氢的亲和力高,反应速率快,催化活性高。
实施例4
一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒,包括:
1)实施例1制备的具有过氧化物酶活性的钯纳米颗粒液体;
2)25mM 3,3’,5,5’-四甲基联苯胺-乙醇溶液;
3)9.8M过氧化氢水溶液;
4)40mM pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液;
试验例1
利用实施例4的试剂盒检测6-巯基嘌呤:
在11个4mL比色皿中,均加入9μL实施例1制备的具有过氧化物酶活性的钯纳米颗粒液体、750μL 10mM pH为4.0的磷酸二氢钠-磷酸氢二钠缓冲液、38.2μL 0.125M过氧化氢水溶液、15μL 0.125mM 3,3’,5,5’-四甲基联苯胺-乙醇溶液后,再向1-11个比色皿中依次加入24μL浓度分别为:0nM、2nM、100nM、150nM、200nM、250nM、300nM、350nM、500nM、600nM、800nM 6-巯基嘌呤水溶液,加入三蒸水使终体积为3000μL,混合反应10min;(各试剂的浓度用试剂盒的试剂配制而成);
采用紫外分光光度计扫描652nm处的吸收光谱,并通过数码相机拍照记录不同浓度6-巯基嘌呤存在下反应液的颜色,获得颜色比较图像;计算不同浓度的6-巯基嘌呤在652nm处的吸光度获得其校准曲线A652=1.1316–0.0025[6-MP]。(见图6)A652吸光度数值与6-MP浓度在0~350nM具有很好的线性关系,对于6-巯基嘌呤检测限为3nM。
试验例2
利用实施例4的试剂盒检测人尿样品和人血清样品中的6-巯基嘌呤的含量:
在8个4mL比色皿中,均加入9μL具有过氧化物酶活性的钯纳米颗粒液体、750μL10mM pH为4.0的磷酸二氢钠-磷酸氢二钠缓冲液、15μL 0.125mM 3,3’,5,5’-四甲基联苯胺-乙醇溶液后,再向第1-第4个比色皿中分别加入:
6μL稀释10倍的人血清、
6μL稀释10倍的人血清、6μL 20nM6-巯基嘌呤、
6μL稀释10倍的人血清、6μL 40nM6-巯基嘌呤、
6μL稀释10倍的人血清、6μL 80nM6-巯基嘌呤;
在第5-第8个比色皿中分别加入:
6μL稀释10倍的人尿液、
6μL稀释10倍的人尿液、6μL 20nM6-巯基嘌呤、
6μL稀释10倍的人尿液、6μL 40nM6-巯基嘌呤、
6μL稀释10倍的人尿液、6μL 80nM6-巯基嘌呤;
再向每个管中加入:
加入38.2μL 125mM过氧化氢水溶液,加入三蒸水使终体积为3000μL,混合反应10min,采用紫外分光光度计扫描652nm处的吸收光谱,根据线性方程测定样品溶液,从而得出样品溶液中6-巯基嘌呤的含量。(见表2)
表2
实施例5
一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒,包括:
1)实施例2制备的具有过氧化物酶活性的钯纳米颗粒液体;
2)1mM 3,3’,5,5’-四甲基联苯胺-乙醇溶液;
3)1M过氧化氢水溶液;
4)20mM pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液。
测定方法同实施例4
实验证明,本实施例的试剂盒能够用于人血清和人尿中6-巯基嘌呤含量的测定。
实施例6
一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒,包括:
1)实施例2制备的具有过氧化物酶活性的钯纳米颗粒液体;
2)50mM 3,3’,5,5’-四甲基联苯胺-乙醇溶液;
3)10M过氧化氢水溶液;
4)80mM pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液。
测定方法同实施例4
实验证明,本实施例的试剂盒能够用于人血清和人尿中6-巯基嘌呤含量的测定。
Claims (4)
1.一种基于钯纳米颗粒可视化检测6-巯基嘌呤的试剂盒,其特征包括:
1)具有过氧化物酶活性的钯纳米颗粒液体;
2)3,3’,5,5’-四甲基联苯胺-乙醇溶液;
3)过氧化氢水溶液;
4)pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液。
2.根据权利要求1所述的试剂盒,其特征是所述3,3’,5,5’-四甲基联苯胺-乙醇溶液的浓度为1-50mM。
3.根据权利要求1所述的试剂盒,其特征是所述过氧化氢水溶液的浓度为1-10M。
4.根据权利要求1所述的试剂盒,其特征是所述pH为4.0磷酸二氢钠-磷酸氢二钠缓冲液的浓度为20-80mM。
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