CN108760700A - 一种荧光金纳米簇的制备及用于四环素及铜荧光探针 - Google Patents
一种荧光金纳米簇的制备及用于四环素及铜荧光探针 Download PDFInfo
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- CN108760700A CN108760700A CN201810531924.2A CN201810531924A CN108760700A CN 108760700 A CN108760700 A CN 108760700A CN 201810531924 A CN201810531924 A CN 201810531924A CN 108760700 A CN108760700 A CN 108760700A
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/07—Metallic powder characterised by particles having a nanoscale microstructure
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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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Abstract
本发明公开了一种荧光金纳米簇的制备及用于四环素及铜荧光探针。本发明以牛奶中分离得到的酪蛋白及碳量子点为合成金纳米簇的稳定剂及还原剂,通过一步法合成荧光金纳米簇(Au NCs)。Au NCs在350nm激发波长下,于433nm、702nm和1052nm处产生三发射,四环素在702nm处具有线性猝灭效果,向被猝灭之后的Au NCs体系中加入Cu2+,荧光恢复,且整个过程几乎不影响1052nm处的荧光强度;将四环素加入到Au NCs中,在365nm紫外灯下观察,Au NCs的红色荧光变为黄色荧光,再加入Cu2+后,黄色荧光逐渐消失,红色荧光逐渐恢复。由此建立了四环素和Cu2+的近红外比率荧光开关和紫外下可视化双信号检测。该方法荧光光谱在近红区,干扰小、特异性强、检测灵敏度高、操作方便,可同时进行四环素和Cu2+的检测。
Description
技术领域
本发明涉及化学分析检测技术领域,具体为一种荧光金纳米簇的制备及用于四环素及铜荧光探针。
背景技术
贵金属荧光纳米簇是一种新型荧光纳米材料,由几个到几百个原子组成,粒径约为 0.2~3.0 nm,接近电子的费米波长,呈现独特的光学、电学、化学等特性,在生物分析成像、环境监测等领域有广阔的应用前景。荧光金纳米簇( AuNCs) 具有较强的光致发光、良好的光稳定性、大的斯托克斯位移和高的荧光量子产率,是目前研究最广泛的贵金属荧光纳米簇,已被用于过氧化氢、金属离子、茶多酚等的检测。以牛血清白蛋白( BSA)为配体,在温和的条件下制备出具有红色荧光特性的蛋白质杂化荧光金纳米簇( AuNCs@BSA)。目前,已有多种有机物用于AuNCs的合成,如树枝状分子或高聚物( 聚酰胺、聚乙稀亚胺等) 、氨基酸( 脯氨酸、酪氨酸、半胱氨酸等) 、转铁蛋白、酶( 辣根过氧化物酶、胰蛋白酶、溶菌酶、胃蛋白酶等) 及一些小分子物质( 硫辛酸、二硫苏糖醇)等。因为BSA原料易得,且以BSA为配体合成AuNCs的过程简单、条件温和,所以AuNCs@ BSA在很多领域应用广泛。酪蛋白是牛奶中的主要蛋白质,含量约为2.6 g/100 mL,占牛奶中蛋白质总量的 80%。酪蛋白含有人体必需的 8种氨基酸,是一种含磷钙的结合蛋白。
碳量子点,如同石墨烯量子点及碳纳米点,是一类碳的新型碳材料。它具有优异的光学性能,可调的激发和发射行为,较高的荧光稳定性,较低的毒性和良好的生物相容性,在越来越多的领域中得到了广泛的应用。碳量子点表面丰富的羟基,使其成为金纳米簇合成中表面修饰很好的还原剂及稳定剂。目前,用酪蛋白及碳量子点作为还原剂及稳定剂合成金纳米簇的方法,未见报道,更未见合成材料用于两种荧光比率探针的报道。
本发明以富含多糖的石斛为碳量子原料,通过一步热分解法合成荧光碳量子点,采用碳量子点及酪蛋白作为合成荧光金纳米簇的还原剂和保护剂,合成荧光金纳米簇。在350nm的激发波长下,金纳米簇同时在蓝光区(443nm)和近红光区(714nm、1052nm)产生发射光谱,以荧光金纳米簇作为四环素及铜的荧光探针,构建了近红光区比率荧光传感器。方法新颖,特异性强,灵敏度高。
发明内容
本发明的目的在于提供一种以碳量子点及酪蛋白作为还原剂和保护剂合成荧光金纳米簇方法,并用于高灵敏、高选择性的四环素及铜的荧光探针。
本发明的目的是通过以下技术方案实现:
荧光金纳米簇合成包括以下步骤:将生鲜乳中分离得到的酪蛋白溶液及碳量子点和氯金酸溶液在37℃等体积混合,磁力搅拌反应1-2 min 后,逐滴加入 1 mol /L NaOH 溶液调节pH值至12-13,在37℃ 条件下搅拌反应12 h,溶液颜色由亮黄色变为深棕色。
所述的生鲜乳中分离得到的酪蛋白方法包括:取 50mL生鲜牛乳, 在恒温水浴中加热至40℃, 边搅拌边慢慢加入10%醋酸溶液,使牛奶pH =4.6-4.8, 放置冷却、澄清后,200目的尼龙布的布氏漏斗抽滤,依次用乙醇、乙醇和***的等体积混合液各30-50ml洗涤两次、30-50mL***分两次洗涤,最后真空过滤,自然干燥得白色的酪蛋白。
所述的碳量子点的合成方法包括:称取2-5g干燥的铁皮石斛粉末,分散于90-150mL 60-70℃的热水中并不断搅拌,待搅拌均匀呈糊状后再加入10-20mL无水乙醇,1-3mL乙二胺,搅拌均匀后,转移至聚四氟乙烯内衬反应釜于180℃加热9-12h,自然冷却后,离心,除去大颗粒杂质,过0.22μm滤膜后得到氮掺杂的碳量子点。将所得量子点置于60℃真空干燥箱中干燥24h,得碳量子点。
所述的离心是在转速8000-10000rpm下进行15-20min。
所述的酪蛋白、碳量子点、氯金酸的重量比例为15-20:5-10:1。
合成的荧光金纳米簇用于四环素及铜的荧光探针,具体方法包括:
(1)荧光金纳米簇的激发波长及发射波长确定:将合成的荧光金纳米簇溶解于水溶液中,在300到800nm进行扫描,荧光金纳米簇在350nm激发波长下,于433nm、702nm 和1052nm处产生三发射;
(2)近红外比率荧光开关分别检测四环素和Cu2+:在含有浓度范围0-20 μM的四环素溶液中,四环素对荧光金纳米簇荧光强度在702nm 处具有线性猝灭作用,Cu2+对猝灭之后的荧光金纳米簇体系有荧光恢复作用,且荧光增敏在0-30 μM范围成线性关系,整个过程几乎不影响1052nm 处的荧光强度,由此建立近红外比率荧光探针进行四环素和Cu2+的检测;
(3)Cu2+的可视检测:将四环素加入到荧光金纳米簇中,在365nm 紫外灯下观察,荧光金纳米簇的红色荧光变为黄色荧光,再加入Cu2+后,黄色荧光逐渐消失,红色荧光逐渐恢复,根据荧光红色的深浅进行Cu2+含量测定;
步骤(2)所述荧光金纳米簇用量为100-300μL;
步骤(3)所述荧光金纳米簇用量100-200 μL。
本发明的优点在于:
1.本发明采用酪蛋白及以富含多糖的石斛合成的碳量子为合成荧光金纳米簇的还原剂和保护剂,合成的荧光金纳米簇具有蓝光区(443nm)和近红光区(702nm、1052nm)的三个发射波长;
2、四环素对合成的荧光金纳米簇荧光强度在702nm 处具有线性猝灭效果,Cu2+对猝灭之后的荧光金纳米簇体系有荧光恢复作用,且荧光增敏成线性关系,整个过程几乎不影响1052nm 处的荧光强度,由此建立近红外比率荧光探针进行四环素和Cu2+的检测;
3、利用四环素对到荧光金纳米簇的猝灭作用,在365nm紫外光下,荧光金纳米簇的红色荧光变为黄色荧光,再由于Cu2+对荧光的恢复作用,红色荧光逐渐恢复,根据荧光红色的深浅进行Cu2+的可视测定;
4、近红外比率荧光探针进行四环素和Cu2+的检测发生在近红外区,干扰小、特异性强、灵敏度高,方法独特。
附图说明
图1为实施例1中合成荧光金纳米簇的扫描电镜图。
图2为实施例1中合成荧光金纳米簇的激发发射光谱示意图谱。
图3为四环素的比率荧光探针以相对荧光强度比值I702/I1052对四环素示意图谱。
图4为Cu2+的比率荧光探针以相对荧光强度比值I702/I1052对Cu2+浓度示意图谱。
具体实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明,但本发明的保护范围并不仅限于此。
实施例1:鸡蛋中四环素及铜的含量测定操作步骤如下:
(1)生鲜乳中酪蛋白的分离方法:取 50mL生鲜牛乳, 在恒温水浴中加热至40℃, 边搅拌边慢慢加入10%醋酸溶液,使牛奶pH =4.6-4.8, 放置冷却、澄清后,200目的尼龙布的布氏漏斗抽滤,依次用乙醇、乙醇和***的等体积混合液各30-50ml洗涤两次、30-50毫升***分两次洗涤,最后真空过滤,自然干燥得白色的酪蛋白;
(2)碳量子点(CDs)的合成:称取2-5g干燥的铁皮石斛粉末,分散于90-150mL 60-70℃的热水中并不断搅拌,待搅拌均匀呈糊状后再加入10-20mL无水乙醇,1-3mL乙二胺,搅拌均匀后,转移至聚四氟乙烯内衬反应釜于180℃加热9-12h,自然冷却后,10000rpm离心15min,除去大颗粒杂质,过0.22μm滤膜后得到氮掺杂的碳量子点,以硫酸奎宁为标准物,其荧光量子产率25%。将所得量子点置于60℃真空干燥箱中干燥24h,备用;
(3)荧光金纳米簇(AuNCs)合成:将步骤(1)得到的20mg /mL酪蛋白溶液3mL及步骤(2)得到的3mg/mL碳量子点10mL和3.4 mg /mL氯金酸溶液1 mL在37℃等体积混合,磁力搅拌反应1-2 min 后,逐滴加入 1 mol /L NaOH 溶液调节pH值至12-13,在37℃ 条件下搅拌反应12 h,溶液颜色由亮黄色变为深棕色;
(4)荧光金纳米簇的激发波长及发射波长确定:将合成的荧光金纳米簇溶解于水溶液中,在300到1200nm进行扫描,荧光金纳米簇的最大激发波长在350nm,最大发射波长分别在433nm、702nm及1052nm处;
(5)荧光金纳米簇对四环素的荧光响应:在含有浓度范围0-20 μM的四环素(TeT)溶液中,加入合成的荧光金纳米簇100μL,荧光光谱在350nm波长的光照激发下,探针以相对荧光强度比值I702/I1052对四环素浓度进行作图,得到线性关系I702/I1052= 0.03210+0.0191C(μM)(r= 0.9968),检出限为 0.015μM ( S /N = 3),用于定量检测四环素的浓度;
(6)荧光金纳米簇对Cu2+的荧光响应:在含有浓度15 μM的与100μL AuNCs的荧光猝灭TeT- AuNCs体系中,加入Cu2+,荧光光谱在350nm波长的光照激发下,荧光增敏在0-30 μM范围,探针以相对荧光强度比值I702/I1052对Cu2+浓度作图,得到线性关系I702/I1052= 0.2142+0.0067C(μM) (r= 0.9970),检出限为 0.05μM ( S /N = 3),用于定量检测Cu2+;
(7)Cu2+的可视检测:在含有浓度15 μM的与100μL AuNCs的荧光猝灭TeT- AuNCs体系中,加入10-100μM Cu2+,荧光光谱在365nm波长的光照激发下,黄色荧光逐渐消失,红色荧光逐渐恢复,根据红色荧光恢复的强弱进行Cu2+的可视检测;
(8)鸡蛋样品四环素测定:取蛋清50mL加入100mL甲醇-丙酮(1:1,V/V)提取试剂,匀质后于冰箱中静置3h,过滤除去沉淀后,在通风橱中减压蒸发浓缩至5mL,按步骤(5)进行四环素的比率荧光测定,代入线性关系,得0.33μM;
(9)鸡蛋样品铜测定:称取蛋清0. 4g于聚四氟乙烯消化罐中,分别加人浓HNO3 2.0mL和H2O2 1.0mL,放入微波炉内,置于5档 ( 100 %功率,650 W ) ,消化15min, 将提取样品消化液于50mL容量瓶中, 稀释至刻度。取5mL按步骤(6)进行Cu2+的比率荧光测定,代入线性关系,得3.75μM;同时按步骤(7)进行Cu2+的可视检测,结果一致。
实施例2:环境水样中四环素及铜的含量测定步骤为:
(1)生鲜乳中酪蛋白的分离方法::同实施例1步骤(1);
(2)碳量子点(CDs)的合成:同实施例1步骤(2);
(3)荧光金纳米簇(AuNCs)合成:同实施例1步骤(3);
(4)荧光金纳米簇的激发波长及发射波长确定:同实施例1步骤(4);
(5)荧光金纳米簇对四环素的荧光响应:同实施例1步骤(5);
(6)荧光金纳米簇对Cu2+的荧光响应:同实施例1步骤(6);
(7)Cu2+的可视检测:同实施例1步骤(7);
(8)环境水样中四环素测定:取环境水样5mL,按步骤(5)进行四环素的比率荧光测定,代入线性关系,未检出;
(9)环境水样中Cu2+测定:取环境水样5mL,按步骤(6)进行Cu2+的比率荧光测定,代入线性关系,得9.01μM;同时按步骤(7)进行Cu2+的可视检测,结果一致。
实施例3:牛奶样品中四环素及铜含量测定步骤为:
(1)生鲜乳中酪蛋白的分离方法::同实施例1步骤(1);
(2)碳量子点(CDs)的合成:同实施例1步骤(2);
(3)荧光金纳米簇(AuNCs)合成:同实施例1步骤(3);
(4)荧光金纳米簇的激发波长及发射波长确定:同实施例1步骤(4);
(5)荧光金纳米簇对四环素的荧光响应:同实施例1步骤(5);
(6)荧光金纳米簇对Cu2+的荧光响应:同实施例1步骤(6);
(7)Cu2+的可视检测:同实施例1步骤(7);
(8)牛奶样品四环素测定:准确称取脱脂奶0.1g于有刻度10 mL具塞试管中,加入4.00mL木瓜蛋白酶溶液,振荡,置65 ℃恒温箱中酶解过夜,次日取出酶解液冷却,以3000r/min离心 15 min,吸取上清液1 mL,按步骤(5)进行四环素的比率荧光测定,代入线性关系,得0.12μM;
(9)牛奶样品Cu2+测定:准确移取5.00 mL牛奶样品于高温消化管内,加入5.00 mL HNO3和2.00 mL HClO4,再加入一粒玻璃珠,管口放歪嘴漏斗,静置24 h,置电炉上加热至红色气体散尽,取下冷却,然后继续加热至冒浓白烟,冷却至室温,加入少量重蒸馏水,消化至样品变为澄清液体后取出冷却,加10%的HNO3溶液定容至50 mL。取5mL按步骤(6)进行Cu2+的比率荧光测定,代入线性关系,得1.05μM;同时按步骤(7)进行Cu2+的可视检测,结果一致。
由图1可知:合成荧光金纳米簇的扫描电镜图,其纳米粒径约为7nm。
由图2可知:本发明制备的荧光金纳米簇,在激发波长为433nm时,在702nm 和1052nm处有最大发射波长。
图3为四环素的比率荧光探针以相对荧光强度比值I702/I1052对四环素示意图谱,说明有好的线性关系;
图4为Cu2+的比率荧光探针以相对荧光强度比值I702/I1052对Cu2+浓度示意图谱,说明有好的线性关系。
Claims (6)
1.一种荧光金纳米簇的制备及用于四环素及铜荧光探针,其特征在于,荧光金纳米簇合成包括以下步骤:将生鲜乳中分离得到的酪蛋白溶液及碳量子点和氯金酸溶液在37℃等体积混合,磁力搅拌反应1-2 min 后,逐滴加入 1 mol /L NaOH 溶液调节pH值至12-13,在37℃ 条件下搅拌反应12 h,溶液颜色由亮黄色变为深棕色。
2.根据权利要求书1所述的生鲜乳中分离得到的酪蛋白方法包括:取 50mL生鲜牛乳,在恒温水浴中加热至40℃, 边搅拌边慢慢加入10%醋酸溶液,使牛奶pH =4.6-4.8, 放置冷却、澄清后,200目的尼龙布的布氏漏斗抽滤,依次用乙醇、乙醇和***的等体积混合液各30-50mL洗涤两次、30-50mL***分两次洗涤,最后真空过滤,自然干燥得白色的酪蛋白。
3.根据权利要求书1所述的碳量子点的合成方法包括:称取2-5g干燥的铁皮石斛粉末,分散于90-150mL 60-70℃的热水中并不断搅拌,待搅拌均匀呈糊状后再加入10-20mL无水乙醇,1-3mL乙二胺,搅拌均匀后,转移至聚四氟乙烯内衬反应釜于180℃加热9-12h,自然冷却后,离心,除去大颗粒杂质,过0.22μm滤膜后得到氮掺杂的碳量子点,将所得量子点置于60℃真空干燥箱中干燥24h,得碳量子点。
4.根据权利要求书1所述的酪蛋白、碳量子点、氯金酸的重量比例为15-20:5-10:1。
5.根据权利要求书3所述的碳量子点的合成方法,其特征在于:离心是在转速8000-10000rpm下进行15-20min。
6.一种荧光金纳米簇用于四环素及铜荧光探针,具体方法包括:
(1)荧光金纳米簇的激发波长及发射波长确定:将合成的荧光金纳米簇溶解于水溶液中,在300到800nm进行扫描,荧光金纳米簇在350nm激发波长下,于433nm、702nm 和1052nm处产生三发射;
(2)近红外比率荧光开关分别检测四环素和Cu2+:在含有浓度范围0-20 μM的四环素溶液中,四环素对荧光金纳米簇荧光强度在702nm 处具有线性猝灭作用,Cu2+对猝灭之后的荧光金纳米簇体系有荧光恢复作用,且荧光增敏在0-30 μM范围成线性关系,整个过程几乎不影响1052nm 处的荧光强度,由此建立近红外比率荧光探针进行四环素和Cu2+的检测;
(3)Cu2+的可视检测:将四环素加入到荧光金纳米簇中,在365nm 紫外灯下观察,荧光金纳米簇的红色荧光变为黄色荧光,再加入Cu2+后,黄色荧光逐渐消失,红色荧光逐渐恢复,根据荧光红色的深浅进行Cu2+含量测定。
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