WO2019153137A1 - 硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法 - Google Patents

硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法 Download PDF

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WO2019153137A1
WO2019153137A1 PCT/CN2018/075601 CN2018075601W WO2019153137A1 WO 2019153137 A1 WO2019153137 A1 WO 2019153137A1 CN 2018075601 W CN2018075601 W CN 2018075601W WO 2019153137 A1 WO2019153137 A1 WO 2019153137A1
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rna
pba
dna
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彭伟
钱思宇
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大连理工大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis

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  • the invention belongs to the technical field of nucleic acid detection, and utilizes boronic acid molecular modified nanoparticles (PBA-NPs) to identify and amplify RNA, and can be used for multiple detection platforms, such as surface plasmon resonance (SPR) detection, Fluorescence detection, chromatographic detection, Surface-enhance Raman Scattering (SERS) detection, electrochemical detection, etc.
  • SPR surface plasmon resonance
  • SERS Surface-enhance Raman Scattering
  • Phenylboronic acid is capable of reversible binding to cis diols. By changing the pH, reversible binding and separation of boric acid and glycols can be achieved.
  • Carbohydrates and their derivatives (such as glycoproteins, ribonucleic acids, etc.) play an important role in the physiological activities of the human body. Due to its polyhydroxyl character, some sugars and their derivatives which conform to the cis- ortho-diols can specifically bind to boric acid, and the mono-hydroxy sugars have weak binding ability to boric acid.
  • RNA The basic structural unit of RNA is a ribonucleotide consisting mainly of ribose, a nitrogenous base, and a phosphate group. Ribose contains a bishydroxy structure, so boric acid can specifically bind to RNA sequences.
  • the DNA structural unit is a deoxyribonucleotide consisting mainly of deoxyribose, a nitrogenous base and a phosphate group.
  • Deoxyribose contains only a monohydroxy structure, so the ability of boric acid to bind DNA is weak.
  • SPR sensing technology Due to the low concentration of nucleic acid, small molecular weight, and no signal amplification function, it can not be directly detected by SPR sensing technology, SERS enhancement technology, electrochemical sensing, fluorescence and other technologies.
  • Nanoparticles such as gold nanoparticles and silver nanoparticles, are chemically stable and have good biocompatibility, and are widely used in genetic testing. Modification of nucleic acids or other molecules on the surface of nanoparticles can be achieved by surface self-assembly techniques.
  • RNA In detecting a specific sequence of RNA, a base sequence capable of complementary pairing with the RNA to be tested is usually modified on the surface of the nanoparticle.
  • this method is cumbersome and cannot distinguish between RNA and DNA having the same base sequence.
  • the PBA-NPs system is capable of efficiently binding to RNA for signal labeling and amplification.
  • the advantage of PBA-AuNPs is that they can selectively distinguish RNA and DNA with the same base sequence. It is not necessary to change the PBA-NPs system when testing different base sequences, and it has strong versatility in RNA recognition and detection.
  • the invention provides a novel RNA sequence recognition and enhancement detection technology. While specifically recognizing RNA sequences, it is possible to selectively distinguish between RNA and DNA of the same sequence.
  • the phenylboronic acid molecule containing a thiol group is modified on the surface of the gold nano or silver nanoparticle by a thiol self-assembly method to form a PBA-NPs system.
  • PBA-NPs can rapidly and efficiently separate and signal the RNA and DNA of the same base sequence, thereby eliminating the interference of DNA on RNA detection to some extent.
  • RNA or DNA sequences that are not capable of base complementary pairing are removed by recognition of DNA.
  • both the RNA to be tested and the DNA can be base-paired with the recognition DNA.
  • RNA and DNA having the same base sequence are selectively distinguished by the PBA-NPs system. Because the recognition DNA is capable of specific binding to a specific sequence of RNA. Therefore, the PBA-NPs system can be applied to recognize single-stranded RNAs of different sequences, and is capable of selectively distinguishing single-stranded RNAs and single-stranded DNAs of the same base sequence.
  • the PBA-NPs system can be applied to a variety of nucleic acid detection platforms to selectively distinguish between single-stranded RNA and DNA with the same base sequence.
  • the binding of PBA-NPs to the sensing surface can significantly change the refractive index of the sensor, thereby amplifying the RNA detection signal; further modifying the PBA-NPs based on the SERS detection platform.
  • the probe molecule can realize the amplification of the RNA detection signal; in the electrochemical detection platform, the fixation of the PBA-NPs on the surface of the sensing electrode can change the electrode resistance and realize the RNA detection.
  • gold nanoparticles and silver nanoparticles can effectively quench molecular fluorescence.
  • the present invention can further selectively distinguish RNA and DNA of the same base sequence by PBA-NPs in combination with other fluorescence-related gene detection techniques.
  • Figure 1 is a schematic diagram of the selective discrimination of RNA and DNA by PBA.
  • Figure 2 is a schematic diagram of the selective discrimination of RNA and DNA on the sensing surface of the PBA-NPs system.
  • Figure 3 is a signal response (SPR test) of PBA-NPs to RNA and DNA having the same base sequence.
  • Figure 4 is a selective test (SPR test) of different nucleic acid sequences of the PBA-NPs system.
  • the recognition DNA is immobilized on the sensing surface, and the recognition DNA can be base-paired with the characteristic RNA of the characteristic sequence; then, the RNA of the sensing surface is recognized and amplified by PBA-NPs.
  • Figures 1 and 2 show the principle of selective discrimination of RNA and DNA by the PBA-NPs system in the sensor.
  • a phenylboronic acid molecule containing a mercapto group is modified on the surface of a gold nano or silver nanoparticle by a thiol self-assembly method to form a PBA-NPs system.
  • the recognition DNA is then immobilized on the surface of the sensor chip.
  • the recognition DNA is capable of base pairing with complementary single-stranded RNA or DNA and immobilized on the sensing surface.
  • the recognition DNA is not capable of immobilizing it on the sensing surface.
  • the recognition DNA can bind to complementary single-stranded RNA or DNA and immobilize it on the sensing surface.
  • PBA-NPs By introducing PBA-NPs solution, PBA-NPs can specifically bind to RNA immobilized on the sensing surface to achieve specific recognition and signal amplification of RNA. Since PBA-NPs have strong binding ability to RNA and weak binding ability to DNA, differential detection of single-stranded RNA and DNA of the same sequence can be achieved.
  • the target detection RNA is Let-7a, which belongs to microRNA (miRNA) and plays a regulatory role in human physiological activities.
  • the HS-ssDNA is a recognition surface for DNA, and is subjected to surface self-assembly modification by a thiol group to vaporize a sensing surface on which a gold film is deposited.
  • DNA-1 has the same base sequence as Let-7a
  • RNA-2 is a single base mismatched RNA
  • RNA-3 is a random RNA.
  • the PBA-NPs system is capable of efficiently identifying the specific base sequence of Let-7a to be tested, and is capable of selecting Let-7a and DNA-1 of the same base sequence. Sexual distinction.

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Abstract

硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法，属于核酸检测技术领域。将含有巯基的苯硼酸分子通过硫醇自组装方式修饰在金纳米和银纳米粒子表面，形成PBA-NPs体系。PBA-NPs体系能够有效的与RNA进行结合，进行信号标记及放大。PBA-NPs的最大优势在于能够选择性区分具有相同碱基序列的RNA和DNA，并能够与多种核酸检测技术联用，实现对特定序列RNA的专一性检测。

Description

硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法 技术领域
本发明属于核酸检测技术领域，利用硼酸分子修饰的纳米粒子(PBA-NPs)对RNA进行识别和信号放大，可以用于多个检测平台，如表面等离子体共振(Surface plasmon resonance,SPR)检测、荧光检测、色谱检测、表面增强拉曼检测(Surface-enhance Raman Scattering,SERS)检测、电化学检测等。
背景技术
苯硼酸(Phenylboronic acid,PBA)能够与顺式二醇类物质进行可逆结合。通过改变PH，可以实现硼酸与二醇类物质的可逆性结合与分离。糖类及其衍生物(如糖蛋白、核糖核酸等)在人体的生理活动中起着重要的作用。糖类由于其多羟基特性，部分符合顺式邻位二醇类物质的糖类及其衍生物能够和硼酸进行特异性结合，单羟基结构的糖类与硼酸结合能力很弱。
RNA的基本结构单元为核糖核苷酸，主要由核糖、含氮碱基和磷酸基团组成。核糖含有双羟基结构，因此硼酸可以与RNA序列进行特异性结合。
而DNA结构单元为脱氧核糖核苷酸，主要由脱氧核糖、含氮碱基和磷酸基团组成。脱氧核糖只含有单羟基结构，因此硼酸结合DNA能力弱。
由于核酸浓度比较低、分子量小，并且本身不具有信号放大功能，通常不能被SPR传感技术、SERS增强技术、电化学传感、荧光等技术直接进行检测。
金纳米粒子和银纳米粒子等纳米粒子(Nanoparticles，NPs)具有化学性质稳定且生物相容性好等特点，被广泛应用于基因测试。通过表面自组装技术，可以实现核酸或其他分子在纳米粒子表面的修饰。
在检测特定序列RNA中，通常在纳米粒子表面修饰能够和待测RNA进行 互补配对的碱基序列。然而这种方法过程繁琐，并且不能够对具有相同碱基序列的RNA和DNA进行区分。
PBA-NPs体系能够有效的与RNA进行结合，进行信号标记及放大。PBA-AuNPs的优势在于能够选择性区分具有相同碱基序列的RNA和DNA，测试不同碱基序列时无需改变PBA-NPs体系，并且在RNA识别和检测过程中具有很强的通用性。
发明内容
本发明提供了一种新型的RNA序列识别及增强检测技术。在特异性识别RNA序列的同时，能够对相同序列的RNA和DNA进行选择性区分。
本发明的技术方案：
硼酸-纳米粒子体系对RNA的特异性识别及检测方法，步骤如下：
将含有巯基的苯硼酸分子通过硫醇自组装方式修饰在金纳米或银纳米粒子表面，形成PBA-NPs体系。PBA-NPs能够快速、高效的对相同碱基序列的RNA和DNA进行特异性区分及信号放大，从而在一定程度上排除DNA对RNA检测的干扰。
首先，通过识别DNA去除掉不能够进行碱基互补配对的RNA或DNA序列。然而，当DNA具有与待测RNA相同的碱基序列时，待测RNA和DNA都能够与识别DNA进行碱基互补配对。进一步，通过PBA-NPs体系对具有相同碱基序列的RNA和DNA进行选择性区分。由于识别DNA能够对特定序列的RNA进行专一性结合。因此，PBA-NPs体系能够应用于识别不同序列的单链RNA，并且能够选择性区分相同碱基序列的单链RNA和单链DNA。
本发明的有益效果：
PBA-NPs体系能够应用于多种核酸检测平台，对具有相同碱基序列的单链RNA和DNA进行选择性区分。
在SPR检测平台中，PBA-NPs在传感表面与RNA的结合可以显著改变传感器折射率的变化，从而实现对RNA检测信号的放大；在SERS检测品台，在PBA-NPs基础上进一步修饰拉曼探针分子，可以实现RNA检测信号的放大；在电化学检测平台，PBA-NPs在传感电极表面的固定可以改变电极电阻，实现RNA检测。
在荧光检测领域，因为金纳米粒子和银纳米粒子能够有效的猝灭分子荧光。本发明可以结合其他与荧光相关的基因检测技术，通过PBA-NPs进一步对相同碱基序列的RNA和DNA进行选择性区分。
附图说明
图1是PBA对RNA和DNA进行选择性区分原理图。
图2是PBA-NPs体系在传感表面对RNA和DNA选择性区分原理图。
图3是PBA-NPs对具有相同碱基序列的RNA和DNA的信号响应(SPR测试)。图4是PBA-NPs体系对不同核酸序列的选择性测试(SPR测试)。
具体实施方式
以下结合附图，以SPR检测为例，进一步说明本发明的具体实施方式。
首先，将识别DNA固定在传感表面，识别DNA能与特性序列的待检测RNA进行碱基互补配对；然后，通过PBA-NPs对传感表面的RNA进行识别及信号放大。图1、2为传感器中PBA-NPs体系对RNA和DNA选择性区分原理图。
具体实施步骤：
首先，将含有巯基的苯硼酸分子通过硫醇自组装方式修饰在金纳米或银纳 米粒子表面，形成PBA-NPs体系。
然后，将识别DNA固定在传感芯片表面。识别DNA能够与互补的单链RNA或者DNA进行碱基互补配对，并将其固定在传感表面。当待测核酸序列不能够与识别DNA进行碱基互补配对时，识别DNA不能够将待其固定在传感表面。
进一步，通入一定浓度的待测单链核酸序列溶液。识别DNA可以与互补的单链RNA或DNA结合，并将其固定在传感表面。
通入PBA-NPs溶液，PBA-NPs能够与固定在传感表面的RNA进行特异性结合，从而实现对RNA的特异性识别和信号放大。由于PBA-NPs与RNA结合能力强，与DNA结合能力弱，因此可以实现对相同序列的单链RNA和DNA的区别检测。
在测试实验中，所用的核酸均为单链核酸。目标检测RNA为Let-7a，属于microRNA(miRNA)，在人体生理活动中起调控作用。HS-ssDNA为识别DNA，通过巯基进行表面自组装修饰在蒸镀有金膜的传感表面。DNA-1具有与Let-7a相同的碱基序列，RNA-2为单碱基错配RNA，RNA-3为随机RNA。从附图3、和附图4可以看出，PBA-NPs体系能够对特定碱基序列的待测Let-7a进行有效识别，并能够对相同碱基序列的Let-7a和DNA-1进行选择性区分。
表1测试PBA-NPs体系所用的RNA及DNA序列
Figure PCTCN2018075601-appb-000001

Claims (3)

  1. 一种硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法，其特征在于，步骤如下：
    将含有巯基的硼酸分子通过自组装方式修饰在纳米粒子表面，形成PBA-NPs体系，用于PBA-NPs对具有相同碱基序列的RNA和DNA进行选择性区分。
  2. 根据权利要求1所述的硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法，其特征在于，所述的纳米粒子为金纳米粒子或银纳米粒子。
  3. 根据权利要求1或2所述的硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法，其特征在于，与其他核酸检测方法联用，实现对RNA的特异性检测。
PCT/CN2018/075601 2018-02-07 2018-02-07 硼酸修饰纳米粒子体系对核糖核酸的特异性识别及检测方法 WO2019153137A1 (zh)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2011035195A1 (en) * 2009-09-18 2011-03-24 Nano Terra Inc. Functional nanofibers and methods of making and using the same
WO2013039819A2 (en) * 2011-09-12 2013-03-21 Nanoselect, Inc. Layer-by-layer carbon nanostructure surface functionalization and devices
CN106596676A (zh) * 2016-12-22 2017-04-26 安阳师范学院 一种用于microRNAs检测的电化学方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011035195A1 (en) * 2009-09-18 2011-03-24 Nano Terra Inc. Functional nanofibers and methods of making and using the same
WO2013039819A2 (en) * 2011-09-12 2013-03-21 Nanoselect, Inc. Layer-by-layer carbon nanostructure surface functionalization and devices
CN106596676A (zh) * 2016-12-22 2017-04-26 安阳师范学院 一种用于microRNAs检测的电化学方法

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