阅读说明：本技术 一种银纳米团簇印迹聚合物对肿瘤标志物的靶向检测方法 (Targeting detection method of silver nanocluster imprinted polymer on tumor marker ) 是由 丁伟华 孙斐 顾亚云 李文清 于 2019-10-23 设计创作，主要内容包括：本发明公开了一种银纳米团簇印迹聚合物及其在肿瘤标志物靶向检测中的应用,以设计获得的PSA智能适配体为基础,对其优化改造提高特异性结合能力,并以其为模板合成银纳米团簇,结合印迹聚合物的特异性识别优势制备银纳米团簇印迹聚合物,探究银纳米团簇印迹聚合物对肿瘤组织中PSA的靶向识别应用。本发明有望为提高肿瘤标志物的靶向识别提供新的研究思路,对临床上癌症的靶向诊断和治疗具有重要意义。(The invention discloses a silver nanocluster imprinted polymer and application thereof in tumor marker targeted detection, wherein the PSA intelligent aptamer obtained by design is taken as a basis, the PSA intelligent aptamer is optimized and modified to improve specific binding capacity, the silver nanocluster is taken as a template to synthesize the silver nanocluster, the silver nanocluster imprinted polymer is prepared by combining the specific recognition advantage of the imprinted polymer, and the targeted recognition application of the silver nanocluster imprinted polymer to PSA in tumor tissues is explored. The invention is expected to provide a new research idea for improving the targeted identification of tumor markers, and has important significance for the targeted diagnosis and treatment of cancer in clinic.)

1. A silver nanocluster imprinted polymer characterized by: synthesizing silver nanoclusters in a cytosine region of an intelligent aptamer of a tumor marker, and then imprinting the tumor marker by using the silver nanoclusters as a fluorescence signal response group through a surface imprinting method to obtain a silver nanocluster imprinted polymer with a mesoporous structure;

the tumor marker intelligent aptamer comprises a cytosine region, an i-motif region, a connecting region and an aptamer region.

2. The silver nanocluster imprinted polymer according to claim 1, characterized in that: the tumor marker is a prostate cancer marker PSA.

3. The silver nanocluster imprinted polymer according to claim 2, characterized in that: the intelligent aptamer sequence of the tumor marker PSA is shown as SEQ ID number 1.

4. The use of the silver nanocluster imprinted polymer of claim 1 in the preparation of a tumor marker targeted recognition reagent.

5. Use according to claim 5, characterized in that: the tumor marker is a prostate cancer marker PSA.

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a silver nanocluster imprinted polymer and application thereof in tumor marker targeted detection.

Background

At present, the targeted identification of tumor markers is an important link in the targeted diagnosis and treatment of cancer, and has important significance for reducing the cancer mortality. The traditional target recognition technology is greatly limited in application due to the problem of the source of the biological antibody. The aptamer is widely applied to the field of specificity analysis because of the advantages of easy synthesis, no toxicity, easy modification, lack of immunogenicity and the like. However, since most tumor markers are also expressed in normal tissues, the aptamer can still target the normal tissues, so that it is imperative to improve the targeting of the aptamer to the biomarkers in the tumor tissues. The weak acidity of tumor tissues is an important physiological feature different from that of normal tissues, and in materials responding to acids, DNA i-motif has the ability of conformational spontaneous and rapid transition in acidic environment, and thus has received much attention in DNA nanotechnology. Research shows that the i-motif functionalization can regulate the specific binding of the aptamer and the tumor marker in an acidic environment, but the binding capacity is relatively weak, so that the i-motif functionalized aptamer needs to be further optimized and modified, and the specific identification of the tumor marker is improved.

Disclosure of Invention

The invention aims to provide a silver nanocluster imprinted polymer, which is optimized and modified on the basis of a prostate cancer marker (PSA) intelligent aptamer obtained in the early stage to improve specific binding capacity, and is used as a template to synthesize silver nanoclusters, so that the silver nanocluster imprinted polymer is prepared by combining the specific recognition advantages of the imprinted polymer, and the targeted recognition application of the silver nanocluster imprinted polymer to PSA in tumor tissues is explored.

In order to achieve the above object, the present invention adopts the following technical means:

a silver nanocluster imprinted polymer is characterized in that a silver nanocluster is synthesized in a cytosine region of an intelligent aptamer of a tumor marker, then the silver nanocluster is used as a fluorescence signal response group, and the tumor marker is imprinted by a surface imprinting method to obtain the silver nanocluster imprinted polymer with a mesoporous structure;

the tumor marker intelligent aptamer comprises a cytosine region, an i-motif region, a connecting region and an aptamer region.

Further, the tumor marker is prostate cancer marker PSA.

Further, the intelligent aptamer sequence of the tumor marker PSA is NH2 (CH2)3 CCCCCCC GGGCCC CCT TTT CCC CCA CTA ACT AAT TAA AGC TCG CCA TCA AAT AGC GGT TAG ACC CCCTTT TCC CCC (SEQ ID number 1).

The application of the silver nanocluster imprinted polymer in preparing a tumor marker targeted recognition reagent can be used for improving targeted recognition of a tumor marker.

Further, the tumor marker is prostate cancer marker PSA.

As shown in fig. 1, in an embodiment of the present invention, based on the PSA intelligent aptamer obtained by design, the PSA intelligent aptamer is optimized and modified to improve specific binding capacity, and is used as a template to synthesize silver nanoclusters, and the silver nanocluster imprinted polymer is prepared by combining the specific recognition advantages of the imprinted polymer, and the application of the silver nanocluster imprinted polymer in targeted recognition of PSA in tumor tissue is explored. The invention is expected to provide a new research idea for improving the targeted identification of tumor markers, and has important significance for the targeted diagnosis and treatment of cancer in clinic.

Has the advantages that:

(1) the intelligent PSA aptamer is optimized and modified to obtain the intelligent aptamer with stronger specific binding capacity with PSA under acidic conditions.

(2) And (3) regulating and controlling reaction conditions, and preparing the silver nanoclusters with stable properties by using the intelligent aptamer as a template on the premise of not influencing the specific binding capacity of the intelligent aptamer.

(3) The PSA imprinted silver nanocluster imprinted polymer is prepared based on silver nanoclusters, so that the identification targeting property of the PSA in an acidic environment is improved, and the PSA imprinted silver nanocluster imprinted polymer has the advantages of being good in stability, low in toxicity, high in sensitivity, good in repeatability and the like.

Drawings

Fig. 1 is a schematic diagram illustrating the synthesis and detection of the silver nanocluster imprinted polymer of the present invention.

FIG. 2 is the microcalorimetric electrophoresis assay of the PSA smart aptamer of example 1.

Fig. 3 is a schematic diagram of the synthesis of silver nanoclusters in example 1.

Fig. 4 is a schematic diagram of the synthesis of the silver nanocluster imprinted polymer in example 1.

Fig. 5 is a schematic structural view of the silver nanocluster imprinted polymer in example 1.

Detailed Description

In the early stage, based on weak acid characteristics and i-motif structural characteristics of tumor tissues, the inventor designs and modifies an aptamer of a prostate cancer marker PSA as an example to obtain an i-motif functionalized PSA intelligent aptamer, and adjusts the specific binding capacity of the aptamer by using conformation transition of i-motif in the intelligent aptamer under different pH values.

The existing research shows that i-motif can regulate the specific binding capacity of an aptamer region by influencing the state of a connecting region, and the length of a base sequence of the connecting region has a large influence on the binding capacity of a recognition region, so that the intelligent aptamer is optimized and modified by designing different connecting region sequences. The specific binding capacity of intelligent aptamers with different connecting region sequences and PSA in an acidic environment is explored through a microcalorimetric electrophoresis (MST) technology, and the intelligent aptamers with strong binding capacity are obtained through analysis results. Then based on cytosine and Ag in intelligent aptamer molecules⁺On the basis of not changing the property of the intelligent aptamer, the intelligent aptamer is used as a molecular template, reaction conditions such as temperature, pH value, concentration, reaction time and the like are regulated and controlled, the silver nanocluster with stable property and excellent optical performance is prepared, and the performance and the particle size of the silver nanocluster are characterized. And finally, in order to further improve the chemical stability and the detection sensitivity of the silver nanoclusters, on the premise of not influencing the optical performance of the silver nanoclusters, the silver nanoclusters are taken as fluorescence signal response groups, PSA is imprinted and eluted by a surface imprinting method to obtain a silver nanocluster imprinted polymer with a mesoporous structure, and the performance, the morphology, the structure and the components of the silver nanocluster imprinted polymer are characterized.

In addition, the intelligent aptamer sequence of the prostate cancer marker PSA is changed into the aptamer sequence of other tumor markers, and the targeted identification of other tumor markers can be realized.

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The reagents and the like referred to in this application are commercially available or otherwise publicly available, and are intended to be exemplary only and not exclusive to the present invention. Other suitable tools or biological materials may be substituted, respectively. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.