【摘要】：本論文利用功能性核酸分子將貴金屬和上轉(zhuǎn)換納米材料構(gòu)建成三種類型的納米自組裝生物傳感器,此類傳感器具有獨(dú)特的圓二色性(CD)、表面增強(qiáng)拉曼散射(SERS)或熒光特性�；诹己玫纳锵嗳菪砸约斑m配體與目標(biāo)物的特異性識(shí)別性能,將所構(gòu)建的納米生物傳感器作為光學(xué)活性探針,成功建立了系列高靈敏度、高特異性的細(xì)胞內(nèi)和血清中生物標(biāo)志物檢測(cè)的新原理和新方法。具體內(nèi)容如下:1.以含有核酸適配體的DNA分子為骨架,將不同粒徑的金納米粒子構(gòu)建成一種異質(zhì)二聚體等離子手性傳感器,實(shí)現(xiàn)腫瘤細(xì)胞內(nèi)三磷酸腺苷(ATP)的高靈敏檢測(cè)。在溶液中,異質(zhì)二聚體對(duì)ATP有特異性識(shí)別,使二聚體結(jié)構(gòu)發(fā)生解離,CD信號(hào)對(duì)ATP濃度的變化具有明顯響應(yīng);進(jìn)入細(xì)胞后,該傳感器具有較高的生物相容性和穩(wěn)定性,并能與細(xì)胞中ATP特異性結(jié)合進(jìn)而產(chǎn)生CD信號(hào)的變化。對(duì)細(xì)胞內(nèi)ATP的檢測(cè)靈敏度達(dá)到0.2mmol/L,線性范圍為1.5-4.2 mmol/L。建立了細(xì)胞內(nèi)小分子生物標(biāo)志物的手性傳感檢測(cè)方法。2.基于適配體堿基互補(bǔ)配對(duì)原則構(gòu)建了銀納米粒子三聚體表面增強(qiáng)拉曼傳感器,實(shí)現(xiàn)對(duì)血清中甲胎蛋白(AFP)的超靈敏檢測(cè)。以修飾拉曼信標(biāo)的銀納米粒子作為基底,可顯著提高傳感器的電磁場(chǎng)強(qiáng)度。實(shí)驗(yàn)結(jié)果證明銀納米粒子三聚體具有強(qiáng)烈的拉曼增強(qiáng)效應(yīng)。成功構(gòu)建了SERS傳感檢測(cè)AFP的體系,對(duì)AFP的最低檢測(cè)限(LOD)達(dá)到0.097amol/L,線性范圍為0.2-20 amol/L。該傳感器有望作為一種通用的檢測(cè)平臺(tái)應(yīng)用于其它生物標(biāo)志物的定量檢測(cè)。3.采用DNA驅(qū)動(dòng)的金納米棒二聚體和上轉(zhuǎn)換納米顆粒多級(jí)次衛(wèi)星狀納米結(jié)構(gòu),實(shí)現(xiàn)了腫瘤細(xì)胞內(nèi)microRNA含量和端粒酶活性的同時(shí)檢測(cè)。該傳感器進(jìn)入細(xì)胞后,基于堿基互補(bǔ)配對(duì)和引物延伸原則,利用SERS和熒光信號(hào)分別實(shí)現(xiàn)細(xì)胞中miR-21和端粒酶同時(shí)定量分析。其中,拉曼強(qiáng)度與miR-21濃度之間具有良好的線性關(guān)系,線性范圍是0.021-22.36 amol/ngRNA,LOD達(dá)到0.011 amol/ngRNA;熒光強(qiáng)度與端粒酶活性之間線性范圍是0.6×10-12到31×10-12 IU,LOD達(dá)到3.2×10-13 IU。這方法實(shí)現(xiàn)了活細(xì)胞內(nèi)雙重信號(hào)介導(dǎo)的兩種癌癥標(biāo)志物的同時(shí)檢測(cè)。
[Abstract]:In this paper, three types of nano-self-assembled biosensors were constructed by using functional nucleic acid molecules to synthesize noble metals and up-converted nanomaterials. These sensors have unique circular dichroism (CD), surface-enhanced Raman scattering (SERS) or fluorescence characteristics. Based on the good biocompatibility and the specificity of the aptamer and the target, a series of high sensitivity nanosensors were successfully established by using the constructed nano-biosensors as optical active probes. New principles and methods for the detection of high specific biomarkers in cells and in serum. The details are as follows: 1. Using DNA molecule containing aptamer of nucleic acid as skeleton, a heterogeneous dimer plasma chiral sensor was constructed by using gold nanoparticles with different particle sizes to detect adenosine triphosphate (ATP) in tumor cells with high sensitivity. In the solution, the heterogeneous dimer has specific recognition to ATP, which makes the dissociation CD signal of the dimer structure have obvious response to the change of ATP concentration, and the biosensor has high biocompatibility and stability after entering the cell. And can specifically bind to ATP in cells to produce changes in CD signals. The sensitivity of intracellular ATP was 0.2 mmol / L and the linear range was 1.5-4.2 mmol / L. A chiral sensing method for detection of small molecular biomarkers in cells was established. Based on the principle of aptamer base complementary pairing, a surface-enhanced Raman sensor of silver nanoparticles trimer was constructed to detect alpha-fetoprotein (AFP) in serum. Using silver nanoparticles modified Raman beacons as the substrate, the electric magnetic field intensity of the sensor can be significantly increased. The experimental results show that the silver nanoparticles trimer has a strong Raman enhancement effect. The SERS sensing system for AFP detection is successfully constructed. The minimum detection limit for AFP is 0.097 amol / L, and the linear range is 0.2-20 amol / L. The sensor is expected to be used as a general detection platform for quantitative detection of other biomarkers. DNA driven gold nanorods dimer and up-converted nanocrystalline multistage subsatellite nanostructures were used to detect microRNA content and telomerase activity in tumor cells at the same time. Based on the principle of base complementary pairing and primer extension, the SERS and fluorescence signals were used to realize the simultaneous quantitative analysis of miR-21 and telomerase in the cells. The linear range between Raman intensity and miR-21 concentration was 0.021-22.36 amol / ngRNA-LOD was 0.011 amol / ngRNAs, and the linear range between fluorescence intensity and telomerase activity was 0. 6 脳 10-12 to 31 脳 10-12 IULD was 3. 2 脳 10-13 IU. This method realizes the simultaneous detection of two cancer markers mediated by double signals in living cells.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TP212.3;R446.1

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