基于金纳米杆/PMMA基底SERS特性的高灵敏肿瘤标志物无标记检测
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摘要
高品质贵金属纳米结构基底的制备是应用表面增强拉曼散射(SERS)技术进行高灵敏生物检测的关键。采用改进的Langmuir-Blodgett方法,通过在金纳米杆(Au NRs)溶胶注入乙醇,使得Au NRs迁移至溶胶与甲苯的交界面,并用聚甲基丙烯酸甲酯(PMMA)固定交界面处的Au NRs,形成大面积分布、均匀致密排列的二维畴状Au NRs/PMMA纳米结构薄膜基底。然后,采用等离子体清洗技术处理制备的基底,使得金纳米杆(Au NRs)的表面裸露,以增强基底的SERS特性。实验表明, Au NRs/PMMA基底具有优良的SERS特性,在785 nm波长的激光照射下,增强因子可以达到5.49×10~6。此外,利用制备的Au NRs/PMMA基底,开展前列腺癌症肿瘤标志物——前列腺特异性抗原(PSA)的高灵敏无标记定量检测研究。在PSA的无标记检测过程中,首先对PSA标准溶液和新生牛血清进行SERS光谱的直接检测,得到PSA分别位于823, 1 080, 1 385, 1 586和1 640 cm~(-1)处的主要的拉曼特征峰;其次,通过对PSA标准溶液、临床男性血清样本及女性血清样本的SERS光谱进行测量和分析,筛选出在PSA的SERS光谱中与血清中PSA含量相关的拉曼特征峰,它们是分别位于649, 680以及1 640 cm~(-1)处的拉曼特征峰。进一步,通过对与PSA同属糖蛋白的肿瘤标志物甲胎蛋白(AFP)以及与PSA同源的人腺体激肽释放酶2(hK2)进行SERS光谱检测和分析,发现位于1 640 cm~(-1)处的拉曼特征峰对于PSA具有高的特异性,将其作为临床血清样本中PSA无标记定量检测的具有特异性的拉曼特征峰,并以此为依据,对不同PSA浓度的标准溶液进行检测,得到位于1 640 cm~(-1)处的拉曼特征峰强度与PSA样本溶液中PSA的浓度相关的剂量-响应曲线。最后,开展临床血清样本的应用检测。结果表明,基于Au NRs/PMMA基底的SERS检测结果与化学发光免疫分析(CLIA)方法的检测结果一致,且具有比CLIA更高的检测灵敏度,最低检测极限为0.06 ng·mL~(-1),且无标记检测范围为0.1 mg·mL~(-1)～0.1 ng·mL~(-1)。因此,基于Au NRs/PMMA SERS基底的高灵敏肿瘤标志物无标记检测具有重要应用前景。
Preparation of high-quality noble nanostructure substrate is vital for the application of surface-enhanced Raman scattering(SERS) technology in ultrasensitive bioassay. In our work, based on the improved Langmuir-Blodgett method, the gold nanorods were extracted from colloid to the interface between the colloid and toluene with the help of ethanol, and fixed by polymethyl methacrylate(PMMA), then a uniform and dense array of two-dimensional domain-like nanostructure was formed in a large area. Next, the plasma clean technology was used to treat the fabricated substrate for enhancing its SERS performance due to the exposed surface of the Au NRs. The experimental results showed that the Au NRs/PMMA substrate exhibited the excellent SERS characteristic and its enhancement factor(EF) achieved 5.49×10~6 under irradiating of 785 nm laser. In addition, the highly sensitive label-free quantitative detection of tumor maker, prostate specific antigen(PSA), was developed by using Au NRs/PMMA substrate. In the experiments of label-free detection, the Raman characteristic peaks of the PSA were first acquired by comparing the SERS spectra of the PSA standard solution and new-born battle serum solution, and they were mainly located at 823, 1 080, 1 385, 1 586 and 1 640 cm~(-1). Following, the SERS spectra of PSA standard solution, clinical male serum samples and female serum samples were measured and analyzed to screen the Raman characteristic peaks of PSA associated only with serum PSA levels, and they were located at 649, 680 and 1 640 cm~(-1). Furthermore, the SERS spectra of α-fetoprotein(AFP) belonging to the glycoprotein same with PSA and human kallikrein 2(hK2) homologous with PSA were separately measured as two controls, and the extremely specific Raman characteristic peaks of PSA located at 1640 cm-1 were determined and applied in the detection of clinic serum samples. Subsequently, the does-repose curve was obtained by the relationship of the intensities of the Raman peaks at 1640 cm-1 and the PSA concentrations in the standard solutions. Lastly, the PSA concentrations in the clinical serum samples were detected based on the SERS-based label-free detect proposal. It demonstrated that SERS-based label-free detection not only exhibits a well consistency of test data when compared with that of the chemiluminescent immunoassay(CLIA), but also higher sensitivity, and its limit of detection as low as 0.06 ng·mL~(-1) in the range of 0.1 mg·mL~(-1)～0.1 ng·mL~(-1). Therefore, it reveals that the proposed protocol has a significant application potential for the quantitative detection of tumor marker.
Preparation of high-quality noble nanostructure substrate is vital for the application of surface-enhanced Raman scattering(SERS) technology in ultrasensitive bioassay. In our work, based on the improved Langmuir-Blodgett method, the gold nanorods were extracted from colloid to the interface between the colloid and toluene with the help of ethanol, and fixed by polymethyl methacrylate(PMMA), then a uniform and dense array of two-dimensional domain-like nanostructure was formed in a large area. Next, the plasma clean technology was used to treat the fabricated substrate for enhancing its SERS performance due to the exposed surface of the Au NRs. The experimental results showed that the Au NRs/PMMA substrate exhibited the excellent SERS characteristic and its enhancement factor(EF) achieved 5.49×10~6 under irradiating of 785 nm laser. In addition, the highly sensitive label-free quantitative detection of tumor maker, prostate specific antigen(PSA), was developed by using Au NRs/PMMA substrate. In the experiments of label-free detection, the Raman characteristic peaks of the PSA were first acquired by comparing the SERS spectra of the PSA standard solution and new-born battle serum solution, and they were mainly located at 823, 1 080, 1 385, 1 586 and 1 640 cm~(-1). Following, the SERS spectra of PSA standard solution, clinical male serum samples and female serum samples were measured and analyzed to screen the Raman characteristic peaks of PSA associated only with serum PSA levels, and they were located at 649, 680 and 1 640 cm~(-1). Furthermore, the SERS spectra of α-fetoprotein(AFP) belonging to the glycoprotein same with PSA and human kallikrein 2(hK2) homologous with PSA were separately measured as two controls, and the extremely specific Raman characteristic peaks of PSA located at 1640 cm-1 were determined and applied in the detection of clinic serum samples. Subsequently, the does-repose curve was obtained by the relationship of the intensities of the Raman peaks at 1640 cm-1 and the PSA concentrations in the standard solutions. Lastly, the PSA concentrations in the clinical serum samples were detected based on the SERS-based label-free detect proposal. It demonstrated that SERS-based label-free detection not only exhibits a well consistency of test data when compared with that of the chemiluminescent immunoassay(CLIA), but also higher sensitivity, and its limit of detection as low as 0.06 ng·mL~(-1) in the range of 0.1 mg·mL~(-1)～0.1 ng·mL~(-1). Therefore, it reveals that the proposed protocol has a significant application potential for the quantitative detection of tumor marker.
引文
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[3]Setiawan D,Luttjeboer J,Pouwels K B,et al.JPN.J.Clin.Oncol.,2017,47(3):265.
[4]Skenandore C S,Pineda A,Bahr J M,et al.Domest.Anim.Endoc.,2017,60:61.
[5]Li X G,Meng M,Zheng L,et al.Anal.Chem.,2016,88(17):8556.
[6]Ding S Y,Yi J,Li J F,et al.Nature Reviews Materials,2016,1:1.
[7]Li J F,Zhang Y J,Ding S Y,et al.Chem.Rev.,2017,117(7):5002.
[8]Wang Z Y,Zong S F,Wu L,et al.Chem.Rev.,2017,117(12):7910.
[9]Wang Y,Kang S,Khan A,et al.Sci.Rep.,2016,6:21242.
[10]Wang Y W,Doerksen J D,Kang S,et al.Small,2016,12(40):5612.
[11]Liu Y,Zhou H B,Hu Z W,et al.Biosens.Bioelestro.,2017,94:131.
[12]Lane L A,Qian X M,Nie S M.Chem.Rev.,2015,115(19):10489.
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