CN116656781B - 一种用于检测反义寡核苷酸类药物的荧光探针及检测方法 - Google Patents
一种用于检测反义寡核苷酸类药物的荧光探针及检测方法 Download PDFInfo
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- CN116656781B CN116656781B CN202310830021.5A CN202310830021A CN116656781B CN 116656781 B CN116656781 B CN 116656781B CN 202310830021 A CN202310830021 A CN 202310830021A CN 116656781 B CN116656781 B CN 116656781B
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Abstract
本发明公开了一种用于检测反义寡核苷酸类药物的荧光探针及检测方法,所述荧光探针包括MNPs‑SSB分散液和RQDs‑CP分散液,所述MNPs‑SSB由磁球和修饰在磁球上的单链DNA结合蛋白构成;所述RQDs‑CP由红色荧光量子点和修饰在红色荧光量子点上的单链DNA构成;所述单链DNA的序列与待测反义寡核苷酸类药物的序列互补,所述单链DNA结合蛋白可特异性结合所述单链DNA。本发明的荧光探针可用于复杂生物样品中诺西那生钠的快速、灵敏、高通量的检测;本方法无需洗涤、离心等复杂繁琐的前处理,且不需要额外添加酶或底物等酶促反应,分析周期短,能够实现高灵敏性、高特异性、快速简便的高通量分析检测。为诺西那生钠及类似反义寡核苷酸药物的药代动力学研究及临床用药监测提供了技术支持。
Description
技术领域
本发明涉及一种荧光探针及检测方法,尤其涉及一种用于检测反义寡核苷酸类药物的荧光探针及检测方法。
背景技术
脊髓性肌萎缩(Spinal muscular atrophy,SMA)是一种罕见的常染色体隐性遗传神经肌肉疾病,由于运动神经元存活基因1(Survival motor neuron gene 1,SMN1)致病性变异(纯合缺失或突变),修饰基因SMN2产生的全长功能性SMN蛋白不足,最终导致脊髓前角a-运动神经元变性而致病。家族中有SMA患者的人群儿童、成人均可患病,发病率为万分之一,携带率为1/50~1/40。依照国际分型标准,按照发病年龄和可获得最大运动里程碑,将SMA从重到轻分为5个类型(0~4),其中0型SMA为出生前或出生时,无运动历程碑,仅存活数月。1型与2型SMA通常导致患儿的寿命严重缩短甚至死亡,3型与4型SMA虽然不致命,但会严重影响患者的运动功能,导致生活质量的急剧下降。诺西那生钠(Nusinersen Sodium,商品名Spinraza)是全球首个SMA精准靶向治疗药物,2016年由FDA批准上市。诺西那生钠是一种改性的反义寡核苷酸(ASO)药物,由18个核糖核苷酸残基组成的一段单链RNA(5’-UCA CUUUCA UAA UGC UGG-3’),其中核呋喃糖基环的2'-羟基被2'-O-2-甲氧基乙基取代,磷酸键被硫代磷酸酯键取代,以降低诺西那生钠对体外环境与体内广泛存在的核酸酶的降解能力。研究显示,诺西那生钠可修饰SMN2基因剪接,促进全长功能性SMN蛋白产生,显著改善SMA患者的生存以及运动、呼吸等重要功能,改变SMA疾病进程,且安全性和依从性良好。临床上采用鞘内注射的方式进行多剂量给药,由于寡合苷酸药物的半衰期较短且极易在肝肾中蓄积,血浆与脑积液中的诺西那生钠含量非常低,给诺西那生钠的药代动力学研究以及临床治疗药物的监测带来了巨大的挑战。
目前,已被报道的诺西那生钠检测方法主要有酶联免疫吸附法(Enzyme linkedimmunosorbent assay,ELISA)与电化学发光法(Electro ChemiluminescenceImmunoassay,ECL)。其中,用于诺西那生钠检测的ELISA技术是根据2002年Rosie等人报道的一种非竞争性杂交-连接酶联免疫吸附试验技术加以改进开发而来。该方法首先使用固相载体上的模板链捕获诺西那生钠,利用T4连接酶催化诺西那生钠-模板杂交双链的粘末端与地高辛修饰的连接探针连接,最后以碱性磷酸酶标记的地高辛抗体进行ELISA分析。该方法检测限约为1.5ng/m,涉及多重酶促反应,原理复杂,成本高昂,需要反复洗涤、孵育,重现性差,且实验周期长。诺西那生钠临床试验数据显示,诺西那生钠进入体内后绝大部分富集于肝、肾、脾等器官,血浆以及脑脊液中的浓度极低,且生物样品中的各种酶活物质及内源性物质对检测均有干扰。此外,与microRNA以及其他的反义寡核苷酸药物相比,诺西那生钠的寡核苷酸链长度较短,仅含有18个核苷酸残基,这使得杂交酶联免疫吸附试验(hELISA)以及一些信号放大策略难以应用于诺西那生钠的生物分析检测。电化学发光法是目前用于诺西那生钠检测的首选方法,灵敏度高(检测限低至0.015ng/mL),但是检测条件苛刻,仪器昂贵,且需要对电极进行复杂的改性,这严重限制了ECL法的应用。LC-MS/MS技术已经被广泛用于体内药物分析,寡核苷酸药物因极性较强且具有大量负电荷,通常需要在液相分离系统中加入离子对试剂以增强保留并改善色谱行为,然而离子对试剂的加入会导致显著的离子抑制效应,导致检测灵敏度较低,因此经典的LC-MS/MS技术并不适合于寡核苷酸药物的高灵敏检测。
发明内容
发明目的:本发明的目的是提供一种快速、灵敏、高通量的用于检测反义寡核苷酸类药物的荧光探针;本发明的另一目的是提供一种及检测反义寡核苷酸类药物的方法。
技术方案:本发明的一种用于检测反义寡核苷酸类药物的荧光探针,所述荧光探针包括MNPs-SSB分散液和RQDs-CP分散液,所述MNPs-SSB由磁球和修饰在磁球上的单链DNA结合蛋白构成;所述RQDs-CP由红色荧光量子点和修饰在红色荧光量子点上的单链DNA构成;所述单链DNA的序列与待测反义寡核苷酸类药物的序列互补,所述单链DNA结合蛋白可特异性结合所述单链DNA。
优选的,所述红色荧光量子点为CdTe@ZnS荧光量子点。
作为上述方案的进一步改进,所述CdTe@ZnS荧光量子点由以下步骤制备所得:
(1)镉源和还原型谷胱甘肽加溶剂溶解,获得镉前体溶液;
(2)锌源和还原型谷胱甘肽加溶剂溶解,获得锌前体溶液;
(3)NaHTe溶液注入镉前体溶液中,回流一段时间,在反应沸腾状态下,注入锌前体溶液,继续回流一段时间,反应结束,得到CdTe@ZnS荧光量子点。
作为上述方案的更进一步改进,所述RQDs-CP由以下步骤制备所得:
(1)CdTe@ZnS荧光量子点、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和单链DNA结合蛋白在缓冲溶液中反应一段时间;
(2)反应结束后,收集红色沉淀,即为RQDs-CP。
可选地,所述反义寡核苷酸类药物为诺西那生钠及其类似物;
优选的,所述反义寡核苷酸类药物为诺西那生钠,所述单链DNA序列为
5’-H2N-(CH2)6-CCA GCA TTA TGA AAG TGA-3’
所述单链DNA结合蛋白为T4 Gene 32Protein。
作为上述方案的更进一步改进,所述MNPs-SSB由以下步骤制备所得:
(1)羧基化磁性纳米颗粒洗涤后,分散于偶联缓冲液中,得到羧基化磁性纳米颗粒分散液;
(2)所述羧基化磁性纳米颗粒分散液与1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和单链结合蛋白混合,室温反应一段时间,反应结束后,进行磁分离,所得产物即为MNPs-SSB。
另一方面,本发明提供一种利用上述的荧光探针检测反义寡核苷酸类药物的方法,所述方法包括以下步骤:
(1)若干份RQDs-CP分散液中分别加入梯度浓度的反义寡核苷酸类药物,孵育一段时间后,加入MNPs-SSB分散液,继续孵育一段时间;孵育结束后进行磁分离,得到若干份上清溶液;
(2)分别测定若干份上清溶液的荧光强度,得到反义寡核苷酸类药物浓度与对应荧光强度的标准曲线;
(3)RQDs-CP分散液中加入待测样品,孵育一段时间后,加入MNPs-SSB分散液,继续孵育一段时间;孵育结束后进行磁分离,得到上清溶液;
其中,步骤(3)中的RQDs-CP分散液浓度、MNPs-SSB分散液浓度、孵育条件与步骤(1)保持一致;
(4)测定步骤(3)中上清溶液的荧光强度,并与步骤(2)中得到的标准曲线比对,得到待测样品中反义寡核苷酸类药物的浓度。
优选的,步骤(1)和步骤(3)中,RQDs-CP分散液中RQDs-CP的浓度为0.1~0.8mg/mL。
优选的,步骤(1)和步骤(3)中,RQDs-CP分散液与反义寡核苷酸类药物或待测样品的孵育温度为25~37℃。
优选的,步骤(1)和步骤(3)中,步骤(2)和步骤(4)中使用酶标仪测定荧光强度,测定条件为:在610nm处测定荧光强度,荧光光谱扫描范围500~700nm,激发波长为365nm。
优选的,所述方法对反义寡核苷酸类药物的线性检测范围为0.1~200nM。
针对诺西那生钠药物在复杂生物基质中检测分析中的难点,本发明提出了一种基于单链DNA结合蛋白特异性捕获寡核苷酸功能化荧光探针的分析策略,用于复杂生物样品中诺西那生钠的快速、灵敏、高通量的检测。首先,将单链DNA结合蛋白(Single-strandedbinding protein,SSB)通过酰胺键修饰在磁球(Magnetic nanoparticles,MNPs)表面,得到MNPs-SSB。与诺西那生钠互补的单链DNA(ssDNA)通过酰胺键修饰在红色荧光量子点(RQDs)表面,得到RQDs-CP的捕获探针(Capture probe,CP)。SSB能够特异性结合单链DNA(ssDNA),几乎不与双链DNA(dsDNA)、双链RNA(dsRNA)以及DNA-RNA杂交双链结合,即使与单链RNA结合,但是亲和力较低(约为单链DNA的1/10)。当样品中不存在诺西那生钠时,RQDs-CP的捕获探针能够与MNPs-SSB高效结合,经磁分离后上清液中的RQDs-CP较少,上清液的红色荧光较弱。当样品中存在诺西那生钠时,RQDs-CP的捕获探针与诺西那生钠形成DNA-RNA杂交双链,难以被MNPs-SSB捕获,此时上清液中的RQDs-CP含量增加,红色荧光增强。因此,上清液的红色荧光强度可反映样品中的诺西那生钠的浓度,通过建立荧光检测信号(F/F0)与诺西那生钠浓度的线性关系实现定量分析。
有益效果:与现有技术相比,本发明具有如下显著优点:本发明的荧光探针可用于复杂生物样品中诺西那生钠的快速、灵敏、高通量的检测;本发明在生物样品中的诺西那生钠在0.1~200nM的范围内,检测信号F/F0与诺西那生钠浓度之间具有良好的线性关系(R2=0.9152),常见小分子化合物以及诺西那生钠单碱基、二碱基、三碱基突变的相似序列的检测信号均明显低于诺西那生钠,表明此方法具有良好的特异性。此外,本方法无需洗涤、离心等复杂繁琐的前处理,且不需要额外添加酶或底物等酶促反应,分析周期短(仅为30min),能够实现高灵敏性、高特异性、快速简便的高通量分析检测。为诺西那生钠及类似反义寡核苷酸药物的药代动力学研究及临床用药监测提供了技术支持。
附图说明
图1为诺西那生钠检测原理图,其中(A)为RQDs-CP与诺西那生钠的杂交过程;(B)为不存在诺西那生钠时MNPs-SSB对RQDs-CP的捕获过程;(C)为诺西那生钠存在时MNPs-SSB对RQDs-CP与RQDs-CP-NS特异性捕获过程。
图2为实施例1-3中的产物的表征图,其中(A)为RQDs的荧光图谱与紫外图谱题(插图为RQDs分散液在可见光与365nm紫外灯下的照片);(B)为RQDs与RQDs-CP的紫外吸收光谱;(C)为RQDs与RQDs-CP的荧光谱图;(D)为RQDs(左)与RQDs-CP(右)分散液在365nm紫外灯下的照片;(E)为RQDs的高分辨透射电子显微镜(HRTEM)图像;(F)为RQDs的晶格条纹;(G)为RQDs的粒径分布图;
图3为实施例5中不同浓度MNPs-SSB对RQDs-CP的捕获效果图,其中(A)为RQDs-CP与不同浓度MNPs-SSB孵育后上清液在610nm处的荧光强度;内插图是RQDs-CP与不同浓度MNPs-SSB(mg/ml)孵育后上清液在365nm紫外灯下的照片;(B)为不同浓度诺西那生钠溶液(0~1000nM)经过孵育后上清液的荧光光谱;(C)为RQDs-CP浓度对荧光信号的影响;(D)为RQDs-CP与诺西那生钠孵育温度对荧光信号的影响;F与F0分别为20nM与0nM诺西那生钠样品经过孵育后上清液在610nm处的荧光强度。
图4为实施例6中生物样本分析的结果图,其中(A)为生物样本中的诺西那生钠的线性回归曲线;(B)为检测方法对小分子物质的选择性,其中NaCl、KCl、Na2SO4、NaH2PO4、Glucose浓度均为10mM;CaCl2、MgCl2浓度为1mM;还原型谷胱甘肽(GSH)、L-赖氨酸(L-Lys)、L-组氨酸(L-His)浓度均为0.1mM;诺西那生钠浓度为100Nm;(C)为检测方法对诺西那生钠相似序列的选择性其中选择性探针以及诺西那生钠浓度均为50nM。其中F与F0分别为干扰样品与阴性对照样品(超纯水)上清液在610nm处的荧光强度。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
本发明实施例提供一种用于检测反义寡核苷酸类药物的荧光探针,所述荧光探针包括MNPs-SSB分散液和RQDs-CP分散液,所述MNPs-SSB由磁球和修饰在磁球上的单链DNA结合蛋白构成;所述RQDs-CP由红色荧光量子点和修饰在红色荧光量子点上的单链DNA构成;所述单链DNA的序列与待测反义寡核苷酸类药物的序列互补,所述单链DNA结合蛋白可特异性结合所述单链DNA。
所述反义寡核苷酸类药物为诺西那生钠或与诺西那生钠类似寡核苷酸药物。可选地,所述反义寡核苷酸类药物为诺西那生钠。
本发明实施例以述反义寡核苷酸类药物为诺西那生钠进行具体举例说明。若反义寡核苷酸类药物为其他与诺西那生钠类似寡核苷酸药物,本领域技术人员可根据待测药物,设计相应的与待测药物序列互补的单链DNA,以及与单链DNA特异性结合的单链DNA结合蛋白。其中,所选的单链DNA结合蛋白与单链RNA的亲和力低于待测药物。
如图1所示,诺西那生钠检测原理如下:
将单链DNA结合蛋白通过酰胺键修饰在磁球表面,得到MNPs-SSB。与诺西那生钠互补的单链DNA(ssDNA)通过酰胺键修饰在红色荧光量子点(RQDs)表面,得到RQDs-CP的捕获探针。
如图1(A)所示,若待测样品中存在诺西那生钠,RQDs-CP与待测样品中的诺西那生钠杂交;如图1(B)所示,加入MNPs-SSB后,当样品中不存在诺西那生钠时,RQDs-CP的捕获探针能够与MNPs-SSB高效结合,经磁分离后上清液中的RQDs-CP较少,上清液的红色荧光较弱。如图1(C)所示,加入MNPs-SSB后,当样品中存在诺西那生钠时,RQDs-CP的捕获探针与诺西那生钠形成DNA-RNA杂交双链,难以被MNPs-SSB捕获,此时上清液中的RQDs-CP含量增加,红色荧光增强。因此,上清液的红色荧光强度可反映样品中的诺西那生钠的浓度,通过建立荧光检测信号(F/F0)与诺西那生钠浓度的线性关系实现定量分析。
本发明实施例涉及的生物材料如下:
单链DNA序列为5’-H2N-(CH2)6-CCA GCA TTA TGA AAG TGA-3’;所述单链DNA结合蛋白为T4 Gene 32Protein。
实施例1CdTe@ZnS荧光量子点(RQDs)的制备
本发明实施例提供一种CdTe@ZnS荧光量子点的制备方法,所述方法包括以下步骤:
(1)加入CdCl2·2.5H2O(0.8mmol)还原型谷胱甘肽(GSH,1.6mmol)以及100mL超纯水,用1M NaOH溶液调节至pH10.0,得到镉前体溶液。
(2)向三颈烧瓶中加入ZnSO4·7H2O(1.26mmol),GSH(1.26mmol)以及100mL超纯水,用1M NaOH溶液调节混合液pH10.0,即得锌前驱体溶液。
(3)加入碲粉(0.3mmol),NaBH4(1.8mmol)以及3mL超纯水,立即以插有注射器针头的橡胶塞封口,室温搅拌65min。在此过程中,瓶中产生的氢气通过橡胶塞上的针头排出,并将瓶内原有的空气排出;溶液颜色由深紫红色变为淡粉色,说明反应已经结束,得到NaHTe溶液。
(4)在剧烈搅拌下将新制备的2mL NaHTe溶液迅速注入上述制备的镉前体溶液中,回流25min。在反应液沸腾的状态下注入10mL锌前体溶液,回流15min。然后再次注入10mL锌前体溶液,继续回流15min。停止100℃加热,反应液冰水浴冷却至室温,即得红色荧光CdTe@ZnS QDs分散液。将CdTe@ZnS QDs溶液旋转蒸发浓缩至原体积的1/3,加入3倍体积的异丙醇,2500g离心10min收集沉淀。沉淀物40℃真空干燥,得到砖红色固体,即为红色荧光CdTe@ZnS QDs。
实施例2捕获探针功能化红色荧光量子点(RQDs-CP)的制备
本发明实施例提供一种捕获探针功能化红色荧光量子点(RQDs-CP)的制备方法,所述方法包括以下步骤:
(1)加入实施例1中制备的RQDs(10mg)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC,50mg)、单链DNA结合蛋白(SSB,2nmol)和1mL 4-羟乙基哌嗪乙磺酸(HEPES,25mM)缓冲液(pH 7.5),室温下反应2h。
(2)反应结束后加入1mL无水乙醇,16500g离心3min,得到的红色沉淀重分散在0.5mL Tris-HCl缓冲液(25mM,pH8.0)中,加入0.5mL无水乙醇,混匀后16500g离心3min,弃去上清液。最后,将红色沉淀分散在5mL Tris-HCl缓冲液(25mM,pH 8.0)中,终浓度为2mg/mL,4℃保存备用。
实施例3单链结合蛋白修饰的磁性纳米颗粒(MNPs-SSB)的制备
本发明实施例提供一种单链结合蛋白修饰的磁性纳米颗粒(MNPs-SSB)的制备方法,所述方法包括以下步骤:
(1)首先,用偶联缓冲液(25mM HEPES,pH=7.5,0.1% Tween-20)将羧基化磁性纳米颗粒(MNPs-COOH)洗涤3次,然后重分散于偶联缓冲液中,终浓度为5mg/mL,4℃保存备用。
(2)在2mL离心管中加入50mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)、5μLSSB(10mg/mL)和1mL MNPs-COOH分散液(5mg/mL),混匀后室温反应2h。反应结束后进行磁分离,用1mL偶联缓冲液洗涤3次,重分散于1mL 偶联缓冲液中,浓度为5mg/mL,4℃保存备用。
采用BCA法验证单链结合蛋白(SSB)在MNPs表面的负载量。按照BCA试剂盒说明书规定的方法,将25μL牛血清白蛋白(BSA)标准曲线样品与200μL 新鲜配置的BCA工作液混匀,在37℃下孵育45min后测定562nm吸光度。MNPs-SSB(10mg/mL)也按照同法操作,考虑到磁性纳米颗粒对可见光具有广泛而强烈的吸收,在进行吸光度检测前先用磁铁将磁珠吸至96孔板的一侧,以避免造成信号干扰。
分别对实施例1-3制备的产物进行表征,如图2所示,RQDs的紫外吸收图谱(图2A)在555nm处有一个明显的激子吸收峰,荧光图谱显示最大发射波长位于600nm。图2A的内插图为RQDs分散液分别在可见光与365nm紫外光下的照片,图片显示RQDs在365nm激发光照射下具有明亮的红色荧光。当RQDs与捕获探针(CP)连接后,对比RQDs-CP与RQDs的紫外-可见吸收光谱,结果如图2B所示,RQDs-CP在260nm附近有一个微小的凸起,主要来源于捕获探针的特征吸收,说明了捕获探针已经成功结合在RQDs表面。此外,RQDs与RQDs-CP在555nm处的激子吸收峰并未消失,说明偶联过程中RQDs晶体结构没有发生明显的变化。荧光图谱验证了同样的结果,RQDs-CP最大发射波长从600nm红移至610nm(图2C),在365nm紫外灯下的红色荧光也发生了肉眼可见的变化(图2D)。采用高分辨透射电子显微镜(HRTEM)对RQDs-CP的微观形貌及分散程度进行了表征。如图2E所示,合成的RQDs-CP具有良好的分散性,呈类球形,具有明显清晰的晶格条纹(图2F),平均粒径为3.6±0.5nm,尺寸分布集中(图2G)。采用BCA法验证单链结合蛋白(SSB)在MNPs表面的负载量。结果表明,MNPs-SSB中SSB的负载量为7.81±0.359μg/mg。由于在MNPs-SSB制备时,MNPs与SSB的投料比为100:1,因此SSB的连接效率约为78.1%。上述结果表明SSB已经成功连接在MNPs上,连接率较高,避免SSB的损耗。
实施例4诺西那生钠检测可行性分析
本发明实施例提供一种诺西那生钠检测方法,其中待测样品为梯度浓度的诺西那生钠溶液,具体包括以下步骤:
(1)加入50μL不同浓度的诺西那生钠溶液(0、0.1、1、10、50、100、200nM)或超纯水、20μL的RQDs-CP分散液(0.2mg/mL),室温孵育15min后,加入20μL MNPs-SSB分散液(5mg/mL),在室温下继续孵育15min。
(2)孵育结束后进行磁分离,取80μL上清液于96孔板中,采用多功能酶标仪(SpectraMax M2e)测定610nm荧光强度,荧光光谱扫描范围500~700nm,激发波长为365nm。
捕获探针的本质是一段单链DNA(ssDNA),能够通过碱基互补配对与诺西那生钠结合形成DNA-RNA杂交双链。根据前期的文献报道,单链结合蛋白能够特异性结合ssDNA,而几乎不与双链DNA(dsDNA)、双链RNA(dsRNA)以及DNA-RNA杂交双链结合,单链结合蛋白与RNA的亲和力仅为与DNA的十分之一。因此,在检测体系中,单链结合蛋白功能化磁球(MNPs-SSB)能够特异性地结合RQDs-CP表面的单链捕获探针,上清液中的RQDs-CP减少,荧光强度减弱。
捕获探针的本质是一段单链DNA(ssDNA),能够通过碱基互补配对与诺西那生钠结合形成DNA-RNA杂交双链。根据前期的文献报道,单链结合蛋白能够特异性结合ssDNA,而几乎不与双链DNA(dsDNA)、双链RNA(dsRNA)以及DNA-RNA杂交双链结合,单链结合蛋白与RNA的亲和力仅为与DNA的十分之一。因此,在检测体系中,单链结合蛋白功能化磁球(MNPs-SSB)能够特异性地结合RQDs-CP表面的单链捕获探针,上清液中的RQDs-CP减少,荧光强度减弱。
实施例5检测条件优化
为了提高诺西纳生钠的荧光信号响应及分析效率,本实施例对RQDs-CP的浓度以及RQDs-CP与诺西那生钠孵育温度等实验条件进行了优化。
首先考察了RQDs-CP的浓度,具体步骤如下:
(1)将20μL不同浓度RQDs-CP与50μL诺西那生钠溶液(20nM或0nM)室温下孵育15min,加入20μL MNPs-SSB分散液(5mg/mL),室温下继续孵育15min。
(2)磁分离后取80μL上清液测定610nm荧光强度,激发波长为365nm,将F/F0最大时的RQDs-CP浓度作为最适浓度,其中F与F0分别为20nM与0nM诺西那生钠样品上清液在610nm处的荧光强度。
此外,本实施例对反应温度进行了考察。具体步骤如下:
(1)将20μL RQDs-CP(0.2mg/mL)与50μL诺西那生钠溶液(20nM或0nM)在不同温度(4℃、25℃、37℃)下孵育15min,加入20μL MNPs-SSB分散液(5mg/mL),继续孵育15min。
(2)磁分离后取80μL上清液测定610nm荧光强度。
图3A显示RQDs-CP与不同浓度MNPs-SSB(0、0.5、1、2、3、4、5mg/ml)混合孵育后,上清液在610nm处的荧光强度急剧下降,内插图显示这种变化甚至可以用肉眼观察到。上述结果表明,MNPs-SSB能够在较短的时间内(15min)高效捕获RQDs-CP。当体系中存在诺西那生钠(NC)时,诺西那生钠能够与RQDs-CP互补配对形成RQDs-CP-NS复合物,由于DNA-RNA杂交双链几乎不与单链结合蛋白结合,因此RQDs-CP-NS复合物在经过磁分离后仍然存在于上清液中,导致上清液荧光增强。图3B显示,随着诺西那生钠浓度的升高,磁分离后的上清液在610nm附近的发射峰明显增强,当诺西那生钠浓度达到1μM时,上清液荧光强度与未添加MNPs-SSB时接近。上述结果说明,MNPs-SSB确实能够通过特异性捕获RQDs-CP或RQDs-NS的方式反应样品中的诺西那生钠浓度变化,表明本项目所提出的检测策略是可行性。
为了提高检测荧光响应信号及分析效率,对RQDs-CP的浓度以及孵育温度分别进行了优化。如图3C所示,RQDs-CP浓度为0.2mg/mL时检测信号达到最大值。在进行孵育温度优化时,当孵育温度为25℃时,检测信号达到最大值,升高温度至37℃后检测信号反而下降(3D)。因此,在后续的检测中,RQDs-CP浓度均为0.2mg/mL,RQDs-CP与诺西那生钠孵育温度均为25℃。
实施例6生物样本分析
本发明实施例提供一种诺西纳生钠的检测方法,包括以下步骤:
(1)将空白大鼠血浆用超纯水稀释100倍,得到1%大鼠血浆。将10μL不同浓度诺西那生钠工作溶液)((4000、2000、1000、200、100、20、0nM))加入190μL大鼠血浆中,得到不同浓度的诺西那生钠血浆样品(200、100、50、10、5、1、0nM)。
(2)分别向上述溶液中加入20μL RQDs-CP(0.2mg/mL),25℃孵育15min,磁分离后,取80μL上清液测定610nm荧光强度。
在最佳实验条件下建立诺西那生钠的生物分析标准曲线,结果表明(图4A),水溶液中诺西那生钠在0.1~200nM的范围内,检测信号F/F0与诺西那生钠浓度之间有良好的线性关系(F/F0=0.0114[Nusinersen]+0.6865,R2=0.9959)。血浆生物样品中诺西那生钠在0.1~200nM范围内,检测信号F/F0与诺西那生钠浓度之间有良好的线性关系(F/F0=0.0054[Nusinersen]+0.8540,R2=0.9152)。与以往的报道相比,此方法具有类似的灵敏度,且一个样品的分析检测周期约为30min。将上述构建的方法用于血浆生物样本中的诺西那生钠的定量检测,准确度在91.6%~121.3%范围内,精密度(RSD%)在6.2%~23.3%范围内(表1)。随机将已知浓度的诺西那生钠加入生物样本中制备了5个浓度的生物样品,回收率(%)在84.0%~112.8%范围内,精密度(RSD%)在4.5%~21.4%范围内(表2)。上述结果符合FDA生物样本分析指导原则。本发明为生物样品中诺西那生钠的检测提供了一种更为直接的新型检测策略,且分析周期远远短于以往报道的ASOs药物检测方法,能够快速、准确、灵敏地检测生物样品中诺西那生钠的血药浓度。
表1人血浆中诺西那生钠的精密度与准确度数据(n=6)
conc.:concentration;SD:standard deviation;RSD:relative standarddeviation;n:number of replicates.
表2人血浆中诺西那生钠的提取回收率数据(n=6).
conc.:concentration;SD:standard deviation;RSD:relative standarddeviation;n:number of replicates.
实施例7选择性考察
为了考察所建方法对诺西那生钠检测的特异性,分别考察了NaCl、KCl、Na2SO4、NaH2PO4、Glucose(10mM)、CaCl2、MgCl2(1mM)和还原型谷胱甘肽(GSH)、L-赖氨酸(L-Lys)、L-组氨酸(L-His)(0.1mM)以及碱基突变序列等在相同检测条件下对生物样品中诺西那生钠的干扰。其中,诺西那生钠的浓度为100nM,超纯水作为阴性对照样品,测定610nm荧光强度。
如图4B所示,除还原型谷胱甘肽(GSH)外,其余考察范围内的小分子物质均不会导致检测信号F/F0的明显升高,说明该方法对常见小分子物质具有良好的选择性。由于RQDs-CP与诺西那生钠的结合依赖于二者的碱基互补配对,某些具有相似序列的寡核苷酸可能会在个别碱基错配的情况下与RQDs-CP结合,导致假阳性信号的产生。如图4C所示,在相同浓度下,单碱基(Selective Probe1)、二碱基(Selective Probe 2)、三碱基(SelectiveProbe 3)突变的选择性探针检测信号均弱于诺西那生钠,选择性探针所产生的检测信号可能有两个来源:选择性探针与RQDs-CP的错配结合以及选择性探针与MNPs-SSB的结合,这是因为所用的选择性探针为ssDNA,它们本身就可以与MNPs-SSB发生特异性结合,导致MNPs-SSB捕获到的RQDs-CP减少,因而上清液的荧光强度增加。尽管选择性探针也可以产生检测信号,但其信号均明显低于同浓度的诺西那生钠,说明所提出的方法对于诺西那生钠相似序列也具有较好的选择性。
Claims (7)
1. 一种用于检测反义寡核苷酸类药物的荧光探针,其特征在于,所述荧光探针包括MNPs-SSB和RQDs-CP,所述MNPs-SSB由磁球和修饰在磁球上的单链DNA结合蛋白构成;所述RQDs-CP由红色荧光量子点和修饰在红色荧光量子点上的单链DNA构成;所述单链DNA的序列与待测反义寡核苷酸类药物的序列互补,所述单链DNA结合蛋白可特异性结合所述单链DNA;其中,所述反义寡核苷酸类药物为诺西那生钠,所述单链DNA序列如下: 5’-H2N-(CH2)6-CCA GCA TTA TGA AAG TGA-3’。
2.根据权利要求1所述的用于检测反义寡核苷酸类药物的荧光探针,其特征在于,所述红色荧光量子点为CdTe@ZnS荧光量子点。
3.根据权利要求2所述的用于检测反义寡核苷酸类药物的荧光探针,其特征在于,所述CdTe@ZnS荧光量子点由以下步骤制备所得:
(1)镉源和还原型谷胱甘肽加溶剂溶解,获得镉前体溶液;
(2)锌源和还原型谷胱甘肽加溶剂溶解,获得锌前体溶液;
(3)NaHTe溶液注入镉前体溶液中,回流,在反应沸腾状态下,注入锌前体溶液,继续回流,反应结束,得到CdTe@ZnS荧光量子点。
4.根据权利要求3所述的用于检测反义寡核苷酸类药物的荧光探针,其特征在于,所述RQDs-CP由以下步骤制备所得:
(1)CdTe@ZnS荧光量子点、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和单链DNA结合蛋白在缓冲溶液中反应;
(2)反应结束后,收集红色沉淀,即为RQDs-CP。
5.根据权利要求1所述的用于检测反义寡核苷酸类药物的荧光探针,其特征在于,
所述单链DNA结合蛋白为T4 Gene 32 Protein。
6.根据权利要求5所述的用于检测反义寡核苷酸类药物的荧光探针,其特征在于,所述MNPs-SSB由以下步骤制备所得:
(1)羧基化磁性纳米颗粒洗涤后,分散于偶联缓冲液中,得到羧基化磁性纳米颗粒分散液;
(2)所述羧基化磁性纳米颗粒分散液与1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和单链结合蛋白混合,室温反应,反应结束后,进行磁分离,所得产物即为MNPs-SSB。
7.一种根据权利要求1-6任一所述的荧光探针在制备用于检测反义寡核苷酸类药物试剂中的应用,所述反义寡核苷酸类药物为诺西那生钠。
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