CN114965392A - 一种基于NGQDs-MoS2荧光共振能量转移结合适配体检测GP73的方法 - Google Patents
一种基于NGQDs-MoS2荧光共振能量转移结合适配体检测GP73的方法 Download PDFInfo
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Abstract
一种基于NGQDs‑MoS2荧光共振能量转移结合适配体检测GP73的方法，以GP73适配体为识别探针，GP73适配体能够特异性识别和结合GP73蛋白，基于氮掺杂石墨烯量子点(NGQDs)‑GP73适配体和二硫化钼（MoS2）间的荧光共振能量转移原理，建立一种检测GP73的荧光适配体生物传感器，用以检测血清中GP73的含量。该方法操作流程简单方便、花费低，检测限低。
Description
技术领域
本发明属于光学传感技术领域，具体涉及一种基于荧光共振能量转移的GP73检测方法。
背景技术
高尔基体П型跨膜糖蛋白73（GP73）常用的检测方法是凝集素亲和质谱分析方法和酶联免疫吸附测定(enzyme linked immunosorbent assay，ELISA)等，存在价格高、适用环境差、基层单位不易开展的问题。公开号为CN107271674A的发明专利，公开了一种酶联免疫试剂盒，该方法存在成本较高，耗时较长，操作复杂等问题。荧光共振能量转移（FRET）是距离十分相近的两个荧光分子之间所产生的一种能量迁移现象，供体的荧光强度比它单独存在时要低的多（荧光猝灭），而受体发射的荧光却大大增强（敏化荧光）。开发新型低成本的FRET传感体系用于GP73肿瘤标记物的检测是一种新的发展方向。公开号为CN106226522A的发明专利，公开了一种基于磁微粒结合荧光标记GP73抗体的荧光共振能量转移检测GP73的方法，该方法存在抗体与GP73结合能力较弱，操作复杂等问题。因此，开发新型低成本的FRET传感体系用于GP73肿瘤标记物的检测是一种新的发展方向。
发明内容
本发明所要解决的技术问题是提供一种基于氮掺杂石墨烯量子点（NGQDs）和二硫化钼（MoS2）的荧光共振能量转移的GP73检测方法，以提高GP73的检测效率，提高灵敏度，该方法能达到1.29 ng/mL的检测限。
为了解决该技术问题，采用具有高荧光量子产率、荧光性能稳定的NGQDs作为荧光物质，使用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）将NGQDs表面的羧基活化，将氨基化GP73适配体（GP73Apt）与NGQDs通过氨基与羧基键进行脱水缩合结合，形成荧光标记的NGQDs-GP73Apt复合物。在NGQDs- GP73Apt溶液中加入MoS2溶液，NGQDs-GP73Apt复合物与MoS2通过范德华力结合在一起，发生荧光共振能量转移（FRET），使NGQDs-GP73Apt的荧光能量转移到了MoS2上，整个系统的荧光强度就会变低；加入GP73蛋白后，由于GP73Apt的特异性，GP73会优先与NGQDs-GP73Apt结合，适配体结构改变，从MoS2底面分离，抑制了荧光共振能量转移，从而使NGQDs-GP73Apt的荧光得到恢复。根据反应体系中荧光强度恢复程度的变化，建立GP73浓度和NGQDs-GP73Apt的荧光强度变化的线性关系，实现GP73灵敏度高、选择性好的定量检测。
本发明按照以下步骤进行：
步骤1：荧光共振供体NGQDs-GP73Apt的制备
(1)氮掺杂石墨烯量子点(NGQDs)的制备：将柠檬酸白色颗粒用氨水溶解，加热，反应完成后冷却，超纯水溶解，然后用NaOH调节pH值至7.0，离心，取上清液，用超纯水稀释得到特定浓度NGQDs溶液；
(2)荧光共振供体(NGQDs-GP73Apt)的制备：在NGQD溶液中加入EDC，将NGQDs表面的羧基活化，量取GP73Apt溶液，与NGQDs溶液等比例混合均匀，在孵育箱中孵育一定时间，得到NGQDs-GP73Apt溶液。
步骤2：荧光共振能量转移的反应体系的构建
(1)称量MoS2粉末，加入超纯水定容，放入超声波细胞破碎机中破碎，至MoS2粉末完全分散在超纯水中，即得MoS2分散液；
(2)将MoS2溶液和NGQDs-GP73Apt溶液混合，混匀后孵育，使NGQDs的荧光淬灭，形成NGQDs-GP73Apt-MoS2 FRET荧光体系。用荧光分光光度计进行测量，测定其荧光强度，记作F0。
步骤3：GP73工作曲线的绘制
(1)将不同浓度的标准GP73蛋白溶液加入步骤2中的NGQDs-GP73Apt-MoS2 FRET荧光体系，在一定温度下孵育反应一定时间，用荧光分光光度计进行检测，测定其荧光强度，记作F1；
(2)用(F1-F0)/F0作为纵坐标，GP73浓度作为横坐标，绘制工作曲线，计算出该方法的最低检测限。
步骤4：实际样品中GP73的检测
(1)将待测样品加入到步骤2中的NGQDs-GP73Apt-MoS2 FRET荧光体系，在一定温度下孵育反应一定时间，用荧光分光光度计进行检测，测定其荧光强度；
(2)根据步骤3所得到的GP73的工作曲线，计算待测样品中GP73的浓度。
进一步优选：
所述步骤1中GP73-Apt的DNA序列为5′-TGG CCT GCA TCA TCG TCT TG-NH2-3′；
所述步骤1中加热条件为210 ℃，6 h，在该条件下NGQD荧光量子产率为23%；
所述步骤1中NaOH溶液的浓度为1 mol/L；
所述步骤1中的EDC浓度为1.0 mg/mL；
所述步骤1中GP73Apt为1.5 μM/μL；
所述步骤1中活化时间为40 min，孵育时间为1 h；
所述步骤2中MoS2溶液浓度为1.0 mg/mL；
所述步骤2中MoS2溶液和NGQDs-GP73Apt溶液的体积比为2:1；
所述步骤2中孵育温度为25 °C，孵育时间为20 min；
所述步骤2、步骤3和步骤4中荧光分光光度计的激发波长为310 nm，发射波长400nm，在该条件下荧光强度较高；
所述步骤3和步骤4中孵育温度为25 °C，孵育时间为80 min。
其中，步骤1得到了一种发出蓝色荧光的NGQDs的纳米荧光材料和NGQDs-GP73Apt探针，为步骤2提供荧光共振能量转移反应体系的荧光能量供体。步骤2提供了MoS2，是荧光能量转移受体；利用NGQDs-GP73Apt中的核酸适配体碱基与MoS2通过范德华力和π-π共轭作用，使得NGQDs-GP73Apt和MoS2紧密接近，发生FRET现象，呈现出NGQDs-GP73Apt的荧光猝灭。步骤3 NGQDs-GP73Apt-MoS2 FRET荧光体系中存在GP73，由于GP73Apt优先结合GP73，由此成功削弱了NGQDs-GP73Apt和MoS2之间的相互作用力。因此，NGQDs-GP73Apt与MoS2分离，FRET过程被抑制，从而使NGQDs-GP73Apt的荧光恢复。步骤3的GP73的工作曲线为步骤4的实际样本中GP73浓度的测定提供计算依据。步骤1-4的实验结果证明了能够利用NGQDs-GP73Apt和MoS2间的荧光共振能量转移原理建立一种新的GP73的检测方法。
本发明与现有技术相比具有如下优点：
1、MoS2具有优良的荧光猝灭性质，NGQDs的荧光强度强且稳定，两者建立的传感器稳定性良好；MoS2对GP73适配体链的吸附能力强，对NGQDs的猝灭效果显著。
2、适配体和目标物之间的亲和力常常比抗原和抗体之间的亲和力强，这会提高检测灵敏度及检测范围。此外，适配体比抗体更易被化学方法标记和修饰，易以荧光共振能量转移原理构建荧光适配体传感器，检测过程操作简单，可以实现一步式的反应，检测时间更短且成本较低。
3、该体系采用以GP73适配体为识别探针检测GP73的方法，这种生物探测方法具有背景干扰小的特点，能够有效提高检测的精确度。该方法的最低检测限为1.29 ng/mL。
附图说明
图1 基于NGQDs-MoS2荧光共振能量转移结合适配体检测GP73原理图；
图2 A是NGQDs的透射电镜图；B是NGQDs-GP73Apt的透射电镜图；
图3 NGQDs和NGQDs-GP73Apt的荧光光谱图；
图4 不同GP73浓度下NGQDs-GP73Apt-MoS2 FERT系统的荧光恢复强度图及标准曲线。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
一种基于NGQDs-MoS2的荧光共振能量转移结合适配体检测GP73的方法，检测原理见图1。首先制备NGQDs，并用EDC将NGQDs表面的羧基活化40 min后，加入与活化后的NGQDs等比例的氨基修饰的GP73适配体振荡混合均匀后孵育；再加入MoS2构成荧光共振GP73适配体传感器，此时可以观察到荧光猝灭的现象，随后测量结合后的NGQDs-GP73Apt-MoS2的荧光强度F0并做记录；当用荧光共振GP73适配体传感器检测GP73蛋白时，GP73蛋白与GP73适配体特异性结合，使得MoS2游离出来，阻断了荧光供体NGQDs和荧光受体MoS2间的FRET体系，故而NGQDs的荧光得到恢复，随后测量结合后的NGQDs-GP73Apt的荧光强度F1并做记录，并通过计算得出GP73浓度，由此建立了一种GP73蛋白的检测方法。通过荧光分光光度计测量荧光强度的变化，可以有效的对GP73蛋白实现高灵敏定量分析。
实施步骤如下：
1、 NGQDs-GP73Apt的制备
（1）称取2.0 g柠檬酸白色颗粒（CA），溶解于0.3 mL氨水中，在干燥箱中以210 ℃的温度加热6小时。
（2）待冷却至室温，用1 mol/L的NaOH水溶液调节NGQDs悬浮液的pH值至7.0。
（3）以1200 rpm离心十分钟，保留上清液，然后将制备的NGQDs原液用超纯水稀释至200 mL，此时的NGQDs溶液为明黄色，且浓度为10.0 mg/mL。在4 ℃条件下进行保存。
图2是NGQDs和NGQDs-GP73Apt的透射电镜图，所制备的NGQDs的大小均匀，分散性良好，粒径在10 nm 左右。
（4）用pH=7.0的PBS缓冲液稀释NGQDs浓度为8.0 μg/mL，取100 μL 8.0 μg/mL的NGQDs溶液加入30 μL浓度为1.0 mg/mL的EDC溶液振荡混合均匀10 min，然后于25 ℃的超声振荡活化40分钟。取100 μL活化后的NGQDs溶液与100 μL 1 μM的GP73适配体振荡混合均匀，然后将混合液至于震荡培养箱中以25 ℃的温度孵育1个小时，得到NGQDs-GP73Apt信号探针。
图3是NGQDs和NGQDs-GP73Apt的荧光光谱图，二者的荧光光谱基本一致，NGQDs-GP73Apt的荧光强度较NGQDs的荧光强度高，说明NGQDs与GPC3Apt已成功连接。
、 荧光共振能量转移的反应体系的构建
（1）称量20mg的MoS2粉末，加入超纯水定容于20 mL，然后放入超声波细胞破碎机中破碎1h，等到MoS2粉末完全分散在超纯水中，即得到1.0 mg/mL的MoS2分散液。
（2）取200μL 1.0 mg/mL MoS2溶液和400μL NGQDs-GP73Apt混合，震荡混和均匀后在25℃下孵育20分钟，得到NGQDs-GP73Apt-MoS2 FERT系统。用荧光分光光度计进行扫描，固定激发波长为310 nm，测量其在400 nm处的荧光强度，记作F0。
、 GP73工作曲线的绘制
将步骤2中测定了荧光强度后的NGQDs-GP73Apt-MoS2 FERT系统均匀分成10组，然后按浓度梯度加入20 μL GP73蛋白溶液（0 ng/mL，2.5 ng/mL，5 ng/mL，10 ng/mL，15 ng/mL，20 ng/mL，40 ng/mL，60 ng/mL，80 ng/mL，100 ng/mL），震荡混和均匀后在25℃下反应80分钟，用荧光分光光度计进行扫描，固定激发波长为310 nm，测定其在400 nm处的荧光强度，记作F1。
不同GPC3浓度下NGQDs-GP73Apt-MoS2 FERT系统的荧光光谱图见图4，可看出NGQDs-GP73Apt-MoS2荧光适配体传感器的荧光恢复强度((F1-F0)/F0)与GP73浓度的呈正相关。当GP73蛋白浓度范围为2.5 ng/mL~100 ng/mL时，NGQDs-GP73Apt-MoS2荧光适配体传感器的荧光恢复值与GP73浓度之间的关系呈线性，工作曲线为Y=0.00146X+2.67119E-4，(其中Y代表荧光强度、X代表GP73蛋白的浓度)，相关系数为R2=0.99654，NGQDs-GP73Apt-MoS2荧光共振GP73适配体传感器的最低检测限度为1.29 ng/mL。
、 实际血清样本中GP73的检测
将浓度为10 ng/mL、20 ng/mL、60 ng/mL的GP73标准溶液与正常人血清样本以1:1的比例充分混合，制成混合液。然后用制备好的NGQDs-GP73Apt-MoS2荧光适配体传感器检测混合液的荧光恢复强度值。根据步骤3所得到的工作曲线Y=0.00146X+2.67119E-4，计算可得到对应的实际血清样本中GP73的浓度，检测结果见表1，回收率为98.87%-100.55%，而相对标准偏差在0.03%-1.65%之间，在肝癌标志物GP73的检测领域有潜在应用价值。
表1 实际血清中GP73检测结果
Claims (5)
1.一种非诊断目的基于NGQDs-MoS2荧光共振能量转移结合适配体检测GP73的方法，按以下步骤进行：
步骤1：荧光共振供体NGQDs-GP73Apt的制备
NGQDs的制备：将柠檬酸用氨水溶解，加热，反应完成后冷却，超纯水溶解，然后用NaOH调节pH值至7.0，离心，取上清液，用超纯水稀释得到特点定度NGQDs溶液；
NGQDs-GP73Apt的制备：将NGQDs表面的羧基活化，量取GP73Apt，与NGQDs混合均匀，在孵育箱中孵育一定时间，得到NGQDs-GP73Apt溶液；
步骤2：荧光共振能量转移的反应体系的构建
称量MoS2粉末，加入超纯水定容，放入超声波细胞破碎机中破碎，至MoS2粉末完全分散在超纯水中，即得MoS2分散液；
将MoS2溶液和NGQDs-GP73Apt溶液进行混合，孵育，使NGQDs的荧光淬灭，形成NGQDs-GP73Apt-MoS2 FRET荧光体系；用荧光分光光度计进行测量，固定激发波长为310 nm，测量其400 nm处荧光强度，记作F0；
步骤3：GP73工作曲线的绘制
将不同浓度的GP73蛋白加入NGQDs-GP73Apt-MoS2 FRET荧光体系，孵育，用荧光分光光度计进行检测，固定激发波长为310nm，测量其400 nm处荧光强度，记作F1；
用(F1-F0)/F0作为纵坐标，GP73浓度作为横坐标，绘制工作曲线，计算出该方法的最低检测限；
步骤4：实际样品中GP73的检测
将待测样品加入到步骤2中的NGQDs-GP73Apt-MoS2 FRET荧光体系，孵育，用荧光分光光度计进行检测，固定激发波长为310 nm，记录400 nm处的荧光强度；
根据步骤3所得到的GP73的工作曲线，计算待测样品中GP73的浓度。
2. 按照权利要求1所述的GP73检测方法，其特征在于：步骤1中所述破碎时间为1h；MoS2分散液浓度为1.0 mg/mL。
3.按照权利要求1所述的GP73检测方法，其特征在于：步骤2中所述孵育温度为25°C，孵育时间为20分钟。
4. 按照权利要求1所述的GP73检测方法，其特征在于：步骤2中所述GP73Apt为100μL 1μM。
5.权利要求1所述的GP73检测方法，其特征在于：步骤3和步骤4中所述孵育温度为25°C，孵育时间为80分钟。
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