多肽的合成和在金屬離子檢測中的應(yīng)用
發(fā)布時間：2018-08-27 12:50
【摘要】：近年來,全球范圍內(nèi)重金屬污染越來越嚴(yán)重,重金屬離子污染已經(jīng)引起了世界各國政府和環(huán)保組織的高度重視,可以說,重金屬污染問題已經(jīng)成為一個全球性環(huán)境問題。所以,對于生命科學(xué)、環(huán)境科學(xué)以及醫(yī)學(xué)來說,發(fā)展一些簡便、高效的檢測重金屬離子的分析方法是極其重要的。多肽作為小分子量的蛋白質(zhì),既有蛋白質(zhì)的一般特性,又具有自身獨特的生理功能,所以多肽的功能引起人們的廣泛關(guān)注。多肽的研究已成為相關(guān)學(xué)科的研究熱點之一,多肽更是憑借著本身的特性和與金屬離子作用的優(yōu)勢成為檢測金屬離子的傳感器中的佼佼者。本文基于金屬離子與多肽的相互作用,設(shè)計、合成了基于多肽的化學(xué)探針,分別研究了它們與金屬離子間的相互作用,探討了它們在金屬離子檢測方面的應(yīng)用。主要內(nèi)容介紹如下:1.特異性檢測Hg~(2+)的多肽比色探針采用Fomc固相合成法合成了比色傳感器多肽Cys-Pro-Gly-Cys-Pro-Gly-Cys-CONH2(P7),應(yīng)用HPLC進(jìn)行了分離純化,并利用質(zhì)譜法進(jìn)行了表征。運(yùn)用紫外可見吸收光譜和圓二色譜等技術(shù)研究了該多肽P7與金屬離子的相互作用。結(jié)果表明,該多肽P7對Hg~(2+)有很強(qiáng)的選擇性,多肽中的半胱氨酸-SH與Hg~(2+)具有特異性的結(jié)合能力。把多肽P7加入到膠體納米金溶液中時,納米金的顏色由紅色變?yōu)樗{(lán)色。當(dāng)將P7和Hg(II)離子的混合溶液加入到膠體納米金溶液中時,納米金的顏色保持不變。但是,當(dāng)將七肽和其他金屬離子的混合溶液加入到膠體納米金溶液中時,納米金的顏色由紅色變?yōu)樗{(lán)色。2.二茂鐵-多肽Fc-CPPCG的合成及電化學(xué)的研究采用Fomc固相合成法合成了二茂鐵-多肽Fc-CPPCG(Fc-P5)。用循環(huán)伏安法(CV)、線性掃描伏安法(LSV)和電化學(xué)阻抗法(EIS)對二茂鐵-多肽Fc-P5修飾的玻碳電極進(jìn)行了電化學(xué)表征,結(jié)果表明該修飾電極具有良好的電化學(xué)性能。研究了該二茂鐵-多肽Fc-P5修飾的玻碳電極對金屬離子的電化學(xué)響應(yīng),即它對金屬離子的選擇性作用。結(jié)果發(fā)現(xiàn),Hg~(2+)使二茂鐵多肽的峰電流和電化學(xué)阻抗都顯著減小,但其他金屬離子對二茂鐵多肽的峰電流和電化學(xué)阻抗幾乎都沒有影響。峰電流和電化學(xué)阻抗的變化均表明,Fc-P5和Hg~(2+)發(fā)生了特異性結(jié)合。峰電流降低可能是由于Hg~(2+)與Fc-P5上Cys殘基發(fā)生反應(yīng),使二茂鐵基團(tuán)的微環(huán)境發(fā)生改變,從而降低了二茂鐵基周圍的電子密度。電化學(xué)阻抗減小則是由于Hg~(2+)與Fc-P5的Cys發(fā)生配位反應(yīng),減少多肽對阻抗液中[Fe(CN)6]3-/4-分子在電極表面的電子傳遞的阻礙作用,促進(jìn)了[Fe(CN)6]3-/4-分子與電極之間的電子傳遞。3.多肽Dansyl-HENRYKW的合成及其對Zn~(2+)的熒光傳感檢測合成了三個丹磺�；揎椀亩嚯腄ansyl-HERYKW(D-P6(1)),Dansyl-HENRKW(D-P6(2))和Dansyl-HENRYKW(D-P7)。通過熒光光譜分別研究了這三個丹磺酰基修飾的多肽與不同金屬離子的相互作用。結(jié)果表明,Dansyl-HERYKW和Dansyl-HENRKW對金屬離子沒有選擇性特異結(jié)合作用,唯有Dansyl-HENRYKW對Zn~(2+)具有特殊的選擇性作用。Zn~(2+)的加入使多肽Dansyl-HENRYKW在515 nm處的熒光強(qiáng)度顯著增強(qiáng)。研究了Dansyl-HENRYKW對Zn~(2+)的熒光傳感性質(zhì)和影響因素,多肽與Zn~(2+)的鍵合比為2:1,結(jié)合常數(shù)為1.8×1011L2×mol-2,檢出限為0.012μmol×L-1。4.多肽CPPC和CPPCGG的合成及其耦合樹脂對汞離子的吸附研究基于多肽與金屬離子的反應(yīng)性,設(shè)計、合成了多肽CPPC(P4)和CPPCGG(P6)及其耦合樹脂(R-P4,R-P6)。運(yùn)用紫外可見吸收光譜,研究了它們與金屬離子的相互作用。結(jié)果表明,P4和P6對Hg~(2+)有很強(qiáng)的選擇性作用。研究了多肽耦合樹脂R-P4和R-P6對Hg~(2+)的吸附作用。原子熒光光譜法測得多肽耦合樹脂R-P4和R-P6對Hg~(2+)的吸附容量分別為96 ng/g和60 ng/g,最佳吸附pH分別為3.0和5.0。
[Abstract]:In recent years, heavy metal pollution has become more and more serious worldwide. Heavy metal ion pollution has attracted the attention of governments and environmental protection organizations all over the world. It can be said that heavy metal pollution has become a global environmental problem. Detection of heavy metal ions is very important. As a small molecular weight protein, polypeptides not only have the general characteristics of proteins, but also have their own unique physiological functions, so the function of polypeptides has attracted widespread attention. In this paper, based on the interaction between metal ions and polypeptides, polypeptide-based chemical probes were designed and synthesized. Their interactions with metal ions were studied and their applications in the detection of metal ions were discussed. The main contents are as follows: 1. Polypeptide colorimetric probe for specific detection of Hg~ (2+) was synthesized by Fomc solid-phase synthesis. The polypeptide Cys-Pro-Gly-Cys-Pro-Gly-Cys-CONH2 (P7) was purified by HPLC and characterized by mass spectrometry. The polypeptide P7 was characterized by UV-Vis absorption spectroscopy and circular dichroism. The results showed that the polypeptide P7 had strong selectivity to Hg~ (2+) and the cysteine-SH in the polypeptide had specific binding ability to Hg~ (2+). When the polypeptide P7 was added to the colloidal nanogold solution, the colour of gold nanoparticles changed from red to blue. When the mixture of P7 and Hg (II) ions was added to the colloidal nanoparticles, the colour of gold nanoparticles changed from red to blue. However, when heptapeptide and other metal ions were mixed into colloidal gold nanoparticles, the color of gold nanoparticles changed from red to blue. 2. Synthesis and electrochemical study of ferrocene-polypeptide Fc-CPPCG Cyclic voltammetry (CV), linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) were used to characterize the ferrocene-polypeptide Fc-P5 modified glassy carbon electrode. The results showed that the modified electrode had good electrochemical performance. The electrochemical response of the ferrocene-polypeptide Fc-P5 modified glassy carbon electrode to metal ions was studied. It was found that Hg~ (2+) significantly reduced the peak current and electrochemical impedance of ferrocene polypeptide, but other metal ions had little effect on peak current and electrochemical impedance of ferrocene polypeptide. The decrease of peak current may be due to the reaction of Hg~ (2+) with Cys residue on Fc-P5, which changes the microenvironment of ferrocene group and reduces the electron density around ferrocene group. The hindrance of electron transfer promotes electron transfer between [Fe(CN)6]3-/4-molecule and electrode. 3. Synthesis of polypeptide Dansyl-HENRYKW and its fluorescence detection of Zn~ (2+) have synthesized three polypeptides, Dansyl-HERYKW (D-P6(1)), Dansyl-HENRKW (D-P6(2)) and Dansyl-HENRYKW (D-P7), respectively. The interaction of these three polypeptides with different metal ions was investigated. The results showed that Dansyl-HERYKW and Dansyl-HENRKW had no selective binding to metal ions, only Dansyl-HENRYKW had special selective effect on Zn~ (2+). The fluorescence intensity of Dansyl-HENRYKW at 515 nm was enhanced by the addition of Zn~ (2+). The fluorescence sensing properties and influencing factors of Dansyl-HENRYKW to Zn~ (2+) were studied. The binding ratio of polypeptide to Zn~ (2+) was 2:1, the binding constant was 1.8 Polypeptides CPPC (P4) and CPPCGG (P6) and their coupling resins (R-P4, R-P6) were synthesized. Their interaction with metal ions was studied by UV-Vis absorption spectroscopy. The results showed that P4 and P6 had strong selectivity for Hg ~ (2+). The adsorption of polypeptide coupling resins R-P4 and R-P6 on Hg ~ (2+) was studied. The adsorption capacities of peptide-coupled resins R-P4 and R-P6 for Hg~ (2+) were 96 ng/g and 60 ng/g, respectively. The optimum adsorption pH was 3.0 and 5.0, respectively.
【學(xué)位授予單位】：聊城大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O629.72;O657.3
本文編號：2207337
[Abstract]:In recent years, heavy metal pollution has become more and more serious worldwide. Heavy metal ion pollution has attracted the attention of governments and environmental protection organizations all over the world. It can be said that heavy metal pollution has become a global environmental problem. Detection of heavy metal ions is very important. As a small molecular weight protein, polypeptides not only have the general characteristics of proteins, but also have their own unique physiological functions, so the function of polypeptides has attracted widespread attention. In this paper, based on the interaction between metal ions and polypeptides, polypeptide-based chemical probes were designed and synthesized. Their interactions with metal ions were studied and their applications in the detection of metal ions were discussed. The main contents are as follows: 1. Polypeptide colorimetric probe for specific detection of Hg~ (2+) was synthesized by Fomc solid-phase synthesis. The polypeptide Cys-Pro-Gly-Cys-Pro-Gly-Cys-CONH2 (P7) was purified by HPLC and characterized by mass spectrometry. The polypeptide P7 was characterized by UV-Vis absorption spectroscopy and circular dichroism. The results showed that the polypeptide P7 had strong selectivity to Hg~ (2+) and the cysteine-SH in the polypeptide had specific binding ability to Hg~ (2+). When the polypeptide P7 was added to the colloidal nanogold solution, the colour of gold nanoparticles changed from red to blue. When the mixture of P7 and Hg (II) ions was added to the colloidal nanoparticles, the colour of gold nanoparticles changed from red to blue. However, when heptapeptide and other metal ions were mixed into colloidal gold nanoparticles, the color of gold nanoparticles changed from red to blue. 2. Synthesis and electrochemical study of ferrocene-polypeptide Fc-CPPCG Cyclic voltammetry (CV), linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) were used to characterize the ferrocene-polypeptide Fc-P5 modified glassy carbon electrode. The results showed that the modified electrode had good electrochemical performance. The electrochemical response of the ferrocene-polypeptide Fc-P5 modified glassy carbon electrode to metal ions was studied. It was found that Hg~ (2+) significantly reduced the peak current and electrochemical impedance of ferrocene polypeptide, but other metal ions had little effect on peak current and electrochemical impedance of ferrocene polypeptide. The decrease of peak current may be due to the reaction of Hg~ (2+) with Cys residue on Fc-P5, which changes the microenvironment of ferrocene group and reduces the electron density around ferrocene group. The hindrance of electron transfer promotes electron transfer between [Fe(CN)6]3-/4-molecule and electrode. 3. Synthesis of polypeptide Dansyl-HENRYKW and its fluorescence detection of Zn~ (2+) have synthesized three polypeptides, Dansyl-HERYKW (D-P6(1)), Dansyl-HENRKW (D-P6(2)) and Dansyl-HENRYKW (D-P7), respectively. The interaction of these three polypeptides with different metal ions was investigated. The results showed that Dansyl-HERYKW and Dansyl-HENRKW had no selective binding to metal ions, only Dansyl-HENRYKW had special selective effect on Zn~ (2+). The fluorescence intensity of Dansyl-HENRYKW at 515 nm was enhanced by the addition of Zn~ (2+). The fluorescence sensing properties and influencing factors of Dansyl-HENRYKW to Zn~ (2+) were studied. The binding ratio of polypeptide to Zn~ (2+) was 2:1, the binding constant was 1.8 Polypeptides CPPC (P4) and CPPCGG (P6) and their coupling resins (R-P4, R-P6) were synthesized. Their interaction with metal ions was studied by UV-Vis absorption spectroscopy. The results showed that P4 and P6 had strong selectivity for Hg ~ (2+). The adsorption of polypeptide coupling resins R-P4 and R-P6 on Hg ~ (2+) was studied. The adsorption capacities of peptide-coupled resins R-P4 and R-P6 for Hg~ (2+) were 96 ng/g and 60 ng/g, respectively. The optimum adsorption pH was 3.0 and 5.0, respectively.
【學(xué)位授予單位】：聊城大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O629.72;O657.3
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