子癇前期關(guān)鍵蛋白電化學(xué)生物傳感器的研究和初步臨床應(yīng)用
發(fā)布時間：2018-09-17 13:27
【摘要】：子癇前期(preeclampsia, PE)作為一種妊娠期特發(fā)疾病,是導(dǎo)致孕產(chǎn)婦和圍產(chǎn)兒發(fā)病率及死亡率上升的主要原因。子癇前期的發(fā)病機(jī)制極為復(fù)雜,盡管多年來對子癇前期發(fā)病機(jī)制的探討一直是產(chǎn)科研究領(lǐng)域研究的熱點和焦點,但迄今為止,仍無一種理論可以全面滿意地闡述其病因。因此,臨床治療只能采取解痙、降壓等對癥治療措施,療效及預(yù)后并不理想。雖然目前關(guān)于子癇前期發(fā)病機(jī)制的基礎(chǔ)研究達(dá)到了前所未有的水平,但仍缺乏準(zhǔn)確、特異有效的臨床預(yù)測指標(biāo),鮮有可單獨用于臨床的標(biāo)志物。另一方面,近年來,生物醫(yī)學(xué)研究正經(jīng)歷從定性到定量、從靜態(tài)到動態(tài)、從分析到綜合的階段,邁向更高層次,對臨床指標(biāo),譬如疾病相關(guān)蛋白質(zhì)的分析檢測也提出了更高更新的要求。發(fā)展簡單、快速、靈敏、準(zhǔn)確、特異,甚至動態(tài)、實時、原位的蛋白質(zhì)分析檢測方法,在蛋白質(zhì)科學(xué)研究和疾病的診斷領(lǐng)域具有十分重要的意義。因此本論文基于蛋白質(zhì)電化學(xué)生物傳感技術(shù),使用新型生物及化學(xué)探針,結(jié)合界面以及納米科學(xué)等相關(guān)領(lǐng)域的新原理新技術(shù),開展蛋白質(zhì)的分子標(biāo)記、分子識別、界面組裝以及信號檢測等方面的研究工作,實現(xiàn)能夠?qū)δ切┰谖磥順O具潛力成為子癇前期發(fā)生發(fā)展預(yù)測評估指標(biāo)的子癇前期關(guān)鍵蛋白,進(jìn)行簡單、快速、靈敏、準(zhǔn)確、特異的檢測分析的愿景。第一部分：基于鋯離子信號放大檢測CREB蛋白磷酸化水平的新方法目的：蛋白質(zhì)的磷酸化修飾是基本的生物學(xué)現(xiàn)象,參與調(diào)控生物體內(nèi)的許多生理過程。CREB (cAMP反應(yīng)元件結(jié)合蛋白)作為一種至關(guān)重要的核轉(zhuǎn)錄因子,它的功能表現(xiàn)在了包括基因轉(zhuǎn)錄調(diào)節(jié)、細(xì)胞凋亡調(diào)控、免疫應(yīng)答等生命活動的諸多方面,而CREB蛋白的生物學(xué)活性正是受其自身磷酸化水平所調(diào)控的。近年來的研究表明,CREB蛋白磷酸化水平的異常表達(dá)與子癇前期等許多疾病的發(fā)生發(fā)展密切相關(guān)。因此本章節(jié)構(gòu)建了一種基于鋯離子信號放大檢測CREB蛋白磷酸化水平的新型電化學(xué)檢測方法,并實現(xiàn)了該檢測手段在臨床實際樣本中的應(yīng)用。方法：以金納米顆粒/DNA/亞甲基藍(lán)納米復(fù)合材料(GNP/DNA/MB)作為信號標(biāo)記,通過Zr4+所介導(dǎo)的納米復(fù)合信標(biāo)對磷酸化位點的特異性標(biāo)記,實現(xiàn)對CREB磷酸化水平的定量檢測。在本工作中,首先將含有CREB蛋白特異性、高親和力結(jié)合位點的捕獲探針DNA序列修飾到金電極的表面,當(dāng)這些探針捕獲有磷酸化的CREB之后,利用Zr4+對磷酸基團(tuán)的分子識別機(jī)制,可以將電極上捕獲的磷酸化CREB蛋白與同樣修飾有磷酸基團(tuán)的GNP/DNA/MB相連接。本方法對純品磷酸化CREB蛋白的檢測限可低至0.25 nM。為了進(jìn)一步驗證該方法在實際生物樣本中的檢測能力,我們選取了南京醫(yī)科大學(xué)第一附屬醫(yī)院產(chǎn)科住院分娩的正常孕婦、輕度子癇前期孕婦、重度子癇前期孕婦各5例,對孕婦胎盤組織中CREB蛋白的磷酸化水平進(jìn)行了檢測分析。結(jié)果：1.條件的優(yōu)化：(1) 500unit/mL為PKA的最優(yōu)濃度條件。(2)120分鐘的孵育時間已經(jīng)可以確保磷酸化CREB蛋白充分地結(jié)合到修飾有捕獲探針的電極表面。(3)Zr4+溶液的濃度達(dá)到0.2mMM時,已經(jīng)足以保證本實驗體系信號的級聯(lián)放大效果。(4)對于GNP/DNA/MB納米復(fù)合信標(biāo)與捕獲有靶標(biāo)蛋白的電極間的孵育時間,60分鐘為最佳。2.通過本方法可獲得CREB蛋白磷酸化水平的飽和增長曲線,并可據(jù)此建立標(biāo)準(zhǔn)工作曲線,用于復(fù)雜樣品磷酸化水平的標(biāo)定。3.在孕婦胎盤組織標(biāo)本中,患有子癇前期的孕婦CREB蛋白磷酸化水平與正常孕婦相比,有所升高。隨著疾病嚴(yán)重程度的增加,CREB磷酸化水平也逐漸升高。結(jié)論：該部分設(shè)計的CREB蛋白磷酸化水平電化學(xué)分析方案展示了理想的靈敏度,以及較高的特異性和良好的重復(fù)性。未來在臨床實際樣本中,在對生理狀態(tài)和病理狀態(tài)下的蛋白質(zhì)磷酸化水平進(jìn)行分析研究方面,具有較為廣闊的應(yīng)用前景。第二部分：基于多肽調(diào)控的蛋白質(zhì)/DNA可逆相互作用檢測STAT 3蛋白活化水平的新方法目的：信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3(signal transducer and activator of transcription 3, STAT 3)參與調(diào)控了細(xì)胞的生長、分化、代謝及凋亡、血管生成等諸多生命活動。近年來的研究表明,STAT3蛋白活化水平的異常不僅與腫瘤的增殖、分化,血管生成,侵襲轉(zhuǎn)移和免疫逃逸等生理功能高度相關(guān),還在子癇前期等一系列妊娠相關(guān)疾病的發(fā)病機(jī)制中發(fā)揮著至關(guān)重要的作用。因此在本章節(jié)中,我們構(gòu)建了一種基于多肽介導(dǎo)的STAT 3蛋白和DNA探針間可逆動態(tài)相互作用從而對STAT 3蛋白活化水平進(jìn)行檢測分析的電化學(xué)分析測試新方法,并將該方法成功運用于實際生物樣本中。方法：本方案借助STAT3蛋白與特異性磷酸化多肽、DNA探針這兩種天然配體間具有強(qiáng)度差異性的相互作用,引入功能化的納米石墨烯復(fù)合信標(biāo)作為信號放大的工具,實現(xiàn)了對STAT3蛋白活化水平的檢測。在本工作中,首先將能夠和STAT 3靶蛋白高特異性、高親和力結(jié)合的末端修飾有磷酸基團(tuán)的捕獲探針雙鏈DNA修飾在金電極表面。當(dāng)電極上DNA探針捕獲活化的STAT 3二聚體后,用DNase I內(nèi)切酶降解電極上過量的未與STAT3結(jié)合的DNA探針,再利用特異性磷酸化多肽來促使靶標(biāo)蛋白STAT 3二聚體的解離,從而使STAT 3與DNA分離。之后,借助Zr4+對磷酸基團(tuán)的分子識別機(jī)制,將磷酸化納米石墨烯復(fù)合信標(biāo)固定到電極表面作為信號放大工具,保證了本方法可以實現(xiàn)對活化的STAT 3蛋白進(jìn)行高靈敏的檢測,檢測限低至0.085 nM。為了進(jìn)一步驗證本方法在實際生物樣本中的檢測能力,我們選取了南京醫(yī)科大學(xué)第一附屬醫(yī)院產(chǎn)科住院分娩的正常孕婦、輕度子癇前期孕婦、重度子癇前期孕婦各5例,對三組孕婦胎盤組織中STAT 3蛋白的活化水平進(jìn)行了檢測分析。結(jié)果：1.條件的優(yōu)化：(1)120分鐘的溫育時間已經(jīng)可以確�；罨腟TAT3蛋白充分地結(jié)合到修飾有捕獲探針的電極表面。(2) DNase I最佳酶切反應(yīng)時間為30分鐘。(3)捕獲有靶標(biāo)蛋白的電極與磷酸化肽間的孵育時間為60分鐘時,可以確保將電極上結(jié)合的STAT 3充分解聚洗脫。(4)對于磷酸化納米石墨烯復(fù)合信標(biāo)與重新釋放了捕獲探針的電極間的孵育時間,60分鐘為最佳。2.在本體系中,STAT 3活化水平隨活化的STAT 3蛋白濃度的增加而增高,到達(dá)一定濃度后飽和。3.在孕婦胎盤組織標(biāo)本中,患有子癇前期的孕婦STAT 3蛋白活化水平與正常孕婦相比,有所降低。隨著疾病嚴(yán)重程度的增加,STAT 3活化水平則逐漸降低。結(jié)論：此部分設(shè)計的STAT 3蛋白活化水平電化學(xué)分析方案表現(xiàn)出理想的靈敏度,較高的特異性和良好的重復(fù)性。未來在臨床復(fù)雜樣本中,在對生理狀態(tài)和病理狀態(tài)下的蛋白質(zhì)活化水平進(jìn)行定量分析研究方面,具有著重要的生物學(xué)意義和廣闊的應(yīng)用前景。第三部分：借助超分子識別對Corin酶進(jìn)行低背景噪音、靈敏檢測的新方法目的：蛋白酶切是蛋白質(zhì)翻譯后修飾的重要手段之一。絲氨酸蛋白酶作為Ⅱ型跨膜嵌合蛋白酶超家族中的重要一員,在限制性蛋白水解過程中起重要作用,參與調(diào)控體內(nèi)激素成熟、細(xì)胞凋亡、血壓調(diào)節(jié)、血液凝固等眾多的生理過程。近年來的研究表明,Corin作為一種新近發(fā)現(xiàn)的Ⅱ型跨膜絲氨酸蛋白酶,其酶活性水平的異常不僅與高血壓、病理性的心肌肥厚、心力衰竭、腫瘤等多種疾病的發(fā)生發(fā)展高度相關(guān),還在子癇前期的發(fā)病機(jī)制中發(fā)揮著至關(guān)重要的作用,極具潛力成為可預(yù)測、評估子癇前期發(fā)生發(fā)展過程的臨床分子標(biāo)志物。因此發(fā)展簡單、快速、靈敏、準(zhǔn)確、特異的蛋白酶活性檢測方法,不僅可以為相關(guān)疾病的病理研究提供新的思路和研究平臺,還在臨床診斷、個體化治療、藥物篩選、靶向治療等應(yīng)用方面具有巨大的潛力。方法：本研究以一段既具備Corin特異性水解底物序列,又具有葫蘆脲識別位點的雙功能多肽作為探針,首先使探針在電極表面形成傳感陣列,在沒有Corin的情況下,電極表面未被切割的多肽探針可以提供葫蘆脲識別位點,利用兩個處于氨基末端的苯丙氨酸可被一個八聚葫蘆脲包裹形成超分子復(fù)合物的特性,通過多肽探針、滾環(huán)擴(kuò)增引物DNA和葫蘆脲間特異性的超分子識別機(jī)制,將引物DNA固定到電極表面并誘導(dǎo)電極界面的滾環(huán)擴(kuò)增反應(yīng)。相反,在有Corin作用下,多肽探針會在特異性位點被水解,帶有葫蘆脲識別位點的多肽片段會從電極表面脫離,電極表面無法形成超分子復(fù)合物,因而引物DNA不能被引入到電極界面,無法誘導(dǎo)滾環(huán)擴(kuò)增反應(yīng)。由于滾環(huán)擴(kuò)增產(chǎn)生的長鏈DNA產(chǎn)物對電極表面有很強(qiáng)的封閉作用,因此,借助于這種封閉作用的有、無可極大地放大有Corin是否存在情況下的信號增量,且信號增量與Corin酶活性的大小呈正相關(guān),因而實現(xiàn)了對Corin酶活性水平低背景信號、高特異性和高靈敏度的檢測。為了進(jìn)一步驗證本方法在實際生物樣本中的適應(yīng)性,我們選取了南京醫(yī)科大學(xué)第一附屬醫(yī)院產(chǎn)科住院分娩的正常孕婦、輕度子癇前期孕婦、重度子癇前期孕婦各5例,對三組孕婦血漿組織中Corin的酶活性水平進(jìn)行了檢測分析。結(jié)果：1.條件的優(yōu)化：(1)16小時的溫育時間已經(jīng)足以保證多肽探針、滾環(huán)擴(kuò)增引物DNA和葫蘆脲間特異性的超分子識別,可確保電極表面超分子復(fù)合物的形成。(2) Corin對電極上修飾的多肽探針最佳酶切反應(yīng)時間為30分鐘。(3)電極上滾環(huán)擴(kuò)增最佳反應(yīng)時間為60分鐘。2.在本體系中,電化學(xué)信號響應(yīng)隨著Corin酶活性水平的升高而增高,到達(dá)一定濃度后信號響應(yīng)飽和。3.在孕婦血漿標(biāo)本中,患有子癇前期的孕婦Corin酶活性水平與正常孕婦相比明顯升高。隨著疾病嚴(yán)重程度的增加,血漿中的Corin酶活性水平也逐步升高。結(jié)論：該部分設(shè)計的Corin酶活電化學(xué)分析方案展示了理想的靈敏度,超低的背景信號,高度的特異性和良好的重復(fù)性。未來在臨床復(fù)雜生物樣本中,在對生理狀態(tài)和病理狀態(tài)下的蛋白酶活性水平進(jìn)行定量分析研究方面,可以提供優(yōu)良的檢測分析手段,具有廣闊的應(yīng)用前景。
[Abstract]:Preeclampsia (PE), as a special disease of pregnancy, is the main cause of increasing morbidity and mortality of pregnant women and perinatal infants. No theory can fully and satisfactorily explain the etiology of preeclampsia. Therefore, clinical treatment can only take symptomatic measures such as spasmolysis, hypotension and so on. The curative effect and prognosis are not ideal. Although the basic research on the pathogenesis of preeclampsia has reached an unprecedented level, there is still a lack of accurate, specific and effective clinical predictive indicators. On the other hand, in recent years, biomedical research is experiencing from qualitative to quantitative, from static to dynamic, from analysis to comprehensive stage, to a higher level. The analysis and detection of clinical indicators, such as disease-related proteins, also put forward higher and newer requirements. Up to now, real-time, in-situ protein analysis and detection methods are of great significance in the field of protein science research and disease diagnosis. Therefore, based on protein electrochemical biosensor technology, this paper uses new biological and chemical probes, and combines new principles and technologies in the fields of interface and nanoscience to develop eggs. Molecular markers, molecular recognition, interfacial assembly and signal detection of white matter have been studied to realize the prospect of simple, rapid, sensitive, accurate and specific detection and analysis of key proteins in preeclampsia that have great potential to be used as predictors of the development of preeclampsia in the future. Ion signal amplification is a new method to detect the phosphorylation level of CREB proteins. Protein phosphorylation is a basic biological phenomenon and involves in regulating many physiological processes in organisms. Recent studies have shown that the abnormal expression of CREB protein phosphorylation is closely related to the occurrence and development of many diseases such as preeclampsia. A novel electrochemical method for detecting the phosphorylation level of CREB protein by zirconium ion amplification was developed, and its application in clinical samples was realized. METHODS: The phosphorylation sites of gold nanoparticles / DNA / methylene blue nanocomposites (GNP / DNA / MB) were detected by Zr4 + mediated nanocomposite beacons. In this work, the DNA sequence of capture probes containing CREB protein-specific and high affinity binding sites was first modified to the surface of gold electrode. After these probes capture phosphorylated CREB, the phosphorylated CREB could be identified by Zr4+ molecular recognition mechanism. The captured phosphorylated CREB protein was linked to GNP/DNA/MB, which was also modified with phosphorylated groups. The detection limit of phosphorylated CREB protein could be as low as 0.25 nM. In order to further verify the detection ability of this method in real biological samples, we selected normal pregnancies of obstetrics in the First Affiliated Hospital of Nanjing Medical University. The phosphorylation level of CREB protein in placenta of pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 500 unit/mL is the optimal concentration of PKA. (2) 120 minutes incubation time can ensure that phosphorylated CREB protein is fully bound to the modified protein. (4) The incubation time between GNP/DNA/MB nanocomposite beacon and the electrode capturing the target protein is the best. 2. The saturated increase of phosphorylation level of CREB protein can be obtained by this method. The phosphorylation level of CREB protein in pregnant women with preeclampsia was higher than that in normal pregnant women. The phosphorylation level of CREB protein in pregnant women with preeclampsia increased gradually with the severity of the disease. The electrochemical assay of phosphorylation level of CREB protein demonstrated ideal sensitivity, high specificity and good repeatability. In the future, it will be widely used in the analysis and research of phosphorylation level of protein in physiological and pathological states in clinical samples. A novel method for detecting STAT 3 protein activation based on protein/DNA reversible interactions regulated by peptides Objective: Signal transducer and activator of transcription 3 (STAT 3) is involved in regulating cell growth, differentiation, metabolism, apoptosis and angiogenesis. Studies have shown that the abnormal activation of STAT3 protein is not only highly correlated with tumor proliferation, differentiation, angiogenesis, invasion, metastasis and immune escape, but also plays an important role in the pathogenesis of preeclampsia and other pregnancy-related diseases. Mediated reversible dynamic interaction between STAT3 protein and DNA probe, a new electrochemical assay method was developed to detect and analyze the activation level of STAT3 protein. The method was successfully applied to practical biological samples. In order to detect the activation level of STAT3 protein, functional graphene nanocomposite beacon was introduced as a signal amplification tool. In this work, the double-stranded DNA, which can bind to STAT3 target protein with high specificity and affinity, was modified into gold electrodes. Pole surface. When DNA probe on the electrode captures the activated STAT 3 dimer, the excessive DNA probe not bound to STAT 3 on the electrode is degraded by DNase I endonuclease, and then the target protein STAT 3 dimer is disintegrated by specific phosphorylated peptides to separate STAT 3 from DNA. The phosphorylated nano-graphene composite beacon was fixed on the electrode surface as a signal amplification tool, which ensured that the method could be used to detect the activated STAT-3 protein with high sensitivity. The detection limit was as low as 0.085 nM. In order to further verify the detection ability of the method in the actual biological samples, we selected the first Nanjing Medical University. The activation levels of STAT 3 protein in placenta of normal pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 120 minutes of incubation time can ensure that the activated STAT 3 protein is fully bound to repair. The optimal digestion time of DNase I was 30 minutes. (3) When the incubation time between the electrode and the phosphopeptides was 60 minutes, the STAT3 bound to the electrode could be fully depolymerized and eluted. (4) The phosphorylated nanographene composite beacon and the capture probe were re-released. In this system, STAT 3 activation level increased with the increase of STAT 3 protein concentration, reached a certain concentration and saturated. 3. In pregnant women placenta tissue samples, preeclampsia pregnant women with STAT 3 protein activation level decreased compared with normal pregnant women. CONCLUSION: The electrochemical assay of STAT 3 protein activation level designed in this part shows ideal sensitivity, high specificity and good reproducibility. Part 3: A new method for detecting Orin enzyme with low background noise by supramolecular recognition. Purpose: Protease digestion is one of the important means for post-translational modification of proteins. Serine proteases are the heavy proteins in type II transmembrane chimeric protease superfamily. In recent years, studies have shown that Corin, as a newly discovered type II transmembrane serine protease, has abnormal enzyme activity levels not only in hypertension but also in pathology. Myocardial hypertrophy, heart failure, tumor and other diseases are highly related to the occurrence and development of preeclampsia, but also play a vital role in the pathogenesis of preeclampsia, has great potential to be predictable, assessment of the occurrence and development of preeclampsia clinical molecular markers. The assay method can not only provide a new idea and research platform for pathological study of related diseases, but also have great potential in clinical diagnosis, individualized treatment, drug screening, targeted therapy and other applications. Peptides are used as probes to form sensor arrays on the electrode surface. Without Corin, the uncut polypeptide probes on the electrode surface can provide cucurbitacin recognition sites. Two phenylalanine at the amino terminal can be encapsulated by an octagourea to form a supramolecular complex. Through the polypeptide probes, the cucurbitacin recognition sites can be rolled. Cyclic amplification primer DNA and cucurbit urea specific supramolecular recognition mechanism, primer DNA is fixed to the electrode surface and induce the roll-around amplification reaction at the electrode interface. Conversely, under the action of Corin, the peptide probe will be hydrolyzed at the specific site, the polypeptide fragment with cucurbit urea recognition site will detach from the electrode surface, and the electrode surface can not. As a result, primer DNA can not be introduced into the electrode interface and can not induce ring-rolling amplification reaction. Because the long-chain DNA products produced by ring-rolling amplification have a strong sealing effect on the electrode surface, whether the signal increment in the presence or absence of Corin can be greatly amplified by means of this sealing effect. In order to further verify the applicability of this method in biological samples, we selected normal pregnant women who delivered in the obstetrics department of the First Affiliated Hospital of Nanjing Medical University. Corin activity in plasma of preeclampsia pregnant women and severe preeclampsia pregnant women were measured and analyzed. Results: 1. The optimum conditions were as follows: (1) 16 hours of incubation time was enough to ensure the specific supramolecular recognition between polypeptide probe, roll-ring amplification primer DNA and cucurbitacin, which could ensure the electrode surface. The formation of supramolecular complexes. (2) The optimal enzyme digestion time of Corin on the electrode modified polypeptide probe was 30 minutes. (3) The optimal reaction time of roll-ring amplification on the electrode was 60 minutes.2. In this system, the electrochemical signal response increased with the increase of Corin enzyme activity, and saturated after reaching a certain concentration. Corin enzyme activity in plasma samples of preeclampsia pregnant women was significantly higher than that of normal pregnant women.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R714.244
，
本文編號：2246058
[Abstract]:Preeclampsia (PE), as a special disease of pregnancy, is the main cause of increasing morbidity and mortality of pregnant women and perinatal infants. No theory can fully and satisfactorily explain the etiology of preeclampsia. Therefore, clinical treatment can only take symptomatic measures such as spasmolysis, hypotension and so on. The curative effect and prognosis are not ideal. Although the basic research on the pathogenesis of preeclampsia has reached an unprecedented level, there is still a lack of accurate, specific and effective clinical predictive indicators. On the other hand, in recent years, biomedical research is experiencing from qualitative to quantitative, from static to dynamic, from analysis to comprehensive stage, to a higher level. The analysis and detection of clinical indicators, such as disease-related proteins, also put forward higher and newer requirements. Up to now, real-time, in-situ protein analysis and detection methods are of great significance in the field of protein science research and disease diagnosis. Therefore, based on protein electrochemical biosensor technology, this paper uses new biological and chemical probes, and combines new principles and technologies in the fields of interface and nanoscience to develop eggs. Molecular markers, molecular recognition, interfacial assembly and signal detection of white matter have been studied to realize the prospect of simple, rapid, sensitive, accurate and specific detection and analysis of key proteins in preeclampsia that have great potential to be used as predictors of the development of preeclampsia in the future. Ion signal amplification is a new method to detect the phosphorylation level of CREB proteins. Protein phosphorylation is a basic biological phenomenon and involves in regulating many physiological processes in organisms. Recent studies have shown that the abnormal expression of CREB protein phosphorylation is closely related to the occurrence and development of many diseases such as preeclampsia. A novel electrochemical method for detecting the phosphorylation level of CREB protein by zirconium ion amplification was developed, and its application in clinical samples was realized. METHODS: The phosphorylation sites of gold nanoparticles / DNA / methylene blue nanocomposites (GNP / DNA / MB) were detected by Zr4 + mediated nanocomposite beacons. In this work, the DNA sequence of capture probes containing CREB protein-specific and high affinity binding sites was first modified to the surface of gold electrode. After these probes capture phosphorylated CREB, the phosphorylated CREB could be identified by Zr4+ molecular recognition mechanism. The captured phosphorylated CREB protein was linked to GNP/DNA/MB, which was also modified with phosphorylated groups. The detection limit of phosphorylated CREB protein could be as low as 0.25 nM. In order to further verify the detection ability of this method in real biological samples, we selected normal pregnancies of obstetrics in the First Affiliated Hospital of Nanjing Medical University. The phosphorylation level of CREB protein in placenta of pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 500 unit/mL is the optimal concentration of PKA. (2) 120 minutes incubation time can ensure that phosphorylated CREB protein is fully bound to the modified protein. (4) The incubation time between GNP/DNA/MB nanocomposite beacon and the electrode capturing the target protein is the best. 2. The saturated increase of phosphorylation level of CREB protein can be obtained by this method. The phosphorylation level of CREB protein in pregnant women with preeclampsia was higher than that in normal pregnant women. The phosphorylation level of CREB protein in pregnant women with preeclampsia increased gradually with the severity of the disease. The electrochemical assay of phosphorylation level of CREB protein demonstrated ideal sensitivity, high specificity and good repeatability. In the future, it will be widely used in the analysis and research of phosphorylation level of protein in physiological and pathological states in clinical samples. A novel method for detecting STAT 3 protein activation based on protein/DNA reversible interactions regulated by peptides Objective: Signal transducer and activator of transcription 3 (STAT 3) is involved in regulating cell growth, differentiation, metabolism, apoptosis and angiogenesis. Studies have shown that the abnormal activation of STAT3 protein is not only highly correlated with tumor proliferation, differentiation, angiogenesis, invasion, metastasis and immune escape, but also plays an important role in the pathogenesis of preeclampsia and other pregnancy-related diseases. Mediated reversible dynamic interaction between STAT3 protein and DNA probe, a new electrochemical assay method was developed to detect and analyze the activation level of STAT3 protein. The method was successfully applied to practical biological samples. In order to detect the activation level of STAT3 protein, functional graphene nanocomposite beacon was introduced as a signal amplification tool. In this work, the double-stranded DNA, which can bind to STAT3 target protein with high specificity and affinity, was modified into gold electrodes. Pole surface. When DNA probe on the electrode captures the activated STAT 3 dimer, the excessive DNA probe not bound to STAT 3 on the electrode is degraded by DNase I endonuclease, and then the target protein STAT 3 dimer is disintegrated by specific phosphorylated peptides to separate STAT 3 from DNA. The phosphorylated nano-graphene composite beacon was fixed on the electrode surface as a signal amplification tool, which ensured that the method could be used to detect the activated STAT-3 protein with high sensitivity. The detection limit was as low as 0.085 nM. In order to further verify the detection ability of the method in the actual biological samples, we selected the first Nanjing Medical University. The activation levels of STAT 3 protein in placenta of normal pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 120 minutes of incubation time can ensure that the activated STAT 3 protein is fully bound to repair. The optimal digestion time of DNase I was 30 minutes. (3) When the incubation time between the electrode and the phosphopeptides was 60 minutes, the STAT3 bound to the electrode could be fully depolymerized and eluted. (4) The phosphorylated nanographene composite beacon and the capture probe were re-released. In this system, STAT 3 activation level increased with the increase of STAT 3 protein concentration, reached a certain concentration and saturated. 3. In pregnant women placenta tissue samples, preeclampsia pregnant women with STAT 3 protein activation level decreased compared with normal pregnant women. CONCLUSION: The electrochemical assay of STAT 3 protein activation level designed in this part shows ideal sensitivity, high specificity and good reproducibility. Part 3: A new method for detecting Orin enzyme with low background noise by supramolecular recognition. Purpose: Protease digestion is one of the important means for post-translational modification of proteins. Serine proteases are the heavy proteins in type II transmembrane chimeric protease superfamily. In recent years, studies have shown that Corin, as a newly discovered type II transmembrane serine protease, has abnormal enzyme activity levels not only in hypertension but also in pathology. Myocardial hypertrophy, heart failure, tumor and other diseases are highly related to the occurrence and development of preeclampsia, but also play a vital role in the pathogenesis of preeclampsia, has great potential to be predictable, assessment of the occurrence and development of preeclampsia clinical molecular markers. The assay method can not only provide a new idea and research platform for pathological study of related diseases, but also have great potential in clinical diagnosis, individualized treatment, drug screening, targeted therapy and other applications. Peptides are used as probes to form sensor arrays on the electrode surface. Without Corin, the uncut polypeptide probes on the electrode surface can provide cucurbitacin recognition sites. Two phenylalanine at the amino terminal can be encapsulated by an octagourea to form a supramolecular complex. Through the polypeptide probes, the cucurbitacin recognition sites can be rolled. Cyclic amplification primer DNA and cucurbit urea specific supramolecular recognition mechanism, primer DNA is fixed to the electrode surface and induce the roll-around amplification reaction at the electrode interface. Conversely, under the action of Corin, the peptide probe will be hydrolyzed at the specific site, the polypeptide fragment with cucurbit urea recognition site will detach from the electrode surface, and the electrode surface can not. As a result, primer DNA can not be introduced into the electrode interface and can not induce ring-rolling amplification reaction. Because the long-chain DNA products produced by ring-rolling amplification have a strong sealing effect on the electrode surface, whether the signal increment in the presence or absence of Corin can be greatly amplified by means of this sealing effect. In order to further verify the applicability of this method in biological samples, we selected normal pregnant women who delivered in the obstetrics department of the First Affiliated Hospital of Nanjing Medical University. Corin activity in plasma of preeclampsia pregnant women and severe preeclampsia pregnant women were measured and analyzed. Results: 1. The optimum conditions were as follows: (1) 16 hours of incubation time was enough to ensure the specific supramolecular recognition between polypeptide probe, roll-ring amplification primer DNA and cucurbitacin, which could ensure the electrode surface. The formation of supramolecular complexes. (2) The optimal enzyme digestion time of Corin on the electrode modified polypeptide probe was 30 minutes. (3) The optimal reaction time of roll-ring amplification on the electrode was 60 minutes.2. In this system, the electrochemical signal response increased with the increase of Corin enzyme activity, and saturated after reaching a certain concentration. Corin enzyme activity in plasma samples of preeclampsia pregnant women was significantly higher than that of normal pregnant women.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R714.244
，
本文編號：2246058
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