發(fā)布時間：2018-06-04 14:57

  本文選題：電化學(xué)多肽傳感器 + 蛋白酶��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：蛋白酶是一種水解蛋白質(zhì)或多肽的水解酶,它與人體代謝過程如細(xì)胞生長與死亡、組織重塑和免疫防御等有關(guān),已作為生物標(biāo)志物應(yīng)用于臨床檢測。建立簡單、靈敏、特異和快速的生物標(biāo)志物檢測方法,對疾病的預(yù)防、診斷和治療具有十分重要的意義。電化學(xué)生物傳感器是將特異性分子識別物質(zhì)例如抗體、酶、適體、多肽等固定在換能器上,以電化學(xué)信號為檢測信號的分析器件,在分析化學(xué)的研究領(lǐng)域中起著越來越重要的地位。將多肽作為分子識別物質(zhì),與抗體和酶相比,它穩(wěn)定、可靠、成本低,并且具有高的親和性,較強(qiáng)的生物活性等優(yōu)點,在蛋白酶生物標(biāo)志物的檢測中得到人們的廣泛關(guān)注。本論文以多肽作為分子識別物質(zhì),致力于設(shè)計不同的信號放大策略,構(gòu)建了一系列操作簡便、成本低廉的電化學(xué)多肽傳感器,實現(xiàn)對前列腺抗原(一種絲氨酸蛋白酶)、基質(zhì)金屬蛋白酶-2、基質(zhì)金屬蛋白酶-7的高靈敏檢測,具體工作如下:1.基于帶正電荷的金納米粒子為信號增強(qiáng)劑檢測前列腺抗原的電化學(xué)多肽傳感器研究大部分的電化學(xué)多肽傳感器是基于目標(biāo)物對標(biāo)記信號標(biāo)簽的多肽進(jìn)行剪切而構(gòu)建的“信號衰減型”(signal-off)傳感器,其靈敏度受到限制,且易產(chǎn)生假陽性信號。當(dāng)前,金屬納米粒子由于可以克服一些酶生物材料固有的不穩(wěn)定性問題,已被廣泛用于信號放大。本工作中,基于正電荷的金納米粒子作為信號增強(qiáng)劑構(gòu)建了一種“信號增強(qiáng)型”(signal-on)的高靈敏的電化學(xué)多肽傳感器。正電荷的金納米粒子修飾在分子識別元件多肽上,它吸附測試底液中帶負(fù)電荷的氧化還原探針鐵氰化鉀([Fe(CN)6]3-/4-),加速了電子間的傳遞,獲得一個較低電化學(xué)阻抗值。在目標(biāo)物前列腺抗原(PSA)存在的條件下,PSA特異性地剪切多肽,從而使得正電荷的金納米粒子離開電極表面,在[Fe(CN)6]3-/4-底液中進(jìn)行電化學(xué)阻抗分析時,得到了一個較大的電化學(xué)阻抗值。通過監(jiān)測電化學(xué)阻抗值的變化,此傳感器實現(xiàn)了對目標(biāo)物PSA的高靈敏檢測。此外,傳感體系是基于目標(biāo)物對多肽的特異性剪切所設(shè)計,具有較高的選擇性和靈敏度,為其他蛋白質(zhì)的分析提供了一個通用性的檢測方法。2.基于核酸外切酶輔助的循環(huán)放大策略檢測基質(zhì)金屬蛋白酶-2的電化學(xué)多肽傳感器研究將目標(biāo)物對肽鏈的剪切直接轉(zhuǎn)化為輸出DNA(output DNA),它可以進(jìn)一步地結(jié)合各種DNA放大技術(shù)實現(xiàn)高效信號放大,提高傳感器的靈敏度。本工作中將多肽剪切事件轉(zhuǎn)換為DNA檢測,并結(jié)合核酸外切酶III(EXO III)輔助目標(biāo)物循環(huán)信號放大策略構(gòu)建了一種超靈敏檢測基質(zhì)金屬蛋白酶-2(MMP-2)的電化學(xué)傳感器。所構(gòu)建的傳感體系可總結(jié)以下幾個優(yōu)點:首先,“信號增強(qiáng)型”(signal-on)傳感器的設(shè)計,在一定程度上減小了由電極表面剝落或者污染導(dǎo)致的假陽性信號的產(chǎn)生;其次,把蛋白質(zhì)分析與DNA放大技術(shù)結(jié)合,有效地提高了傳感器的靈敏度;另外,基于金納米粒子(depAu)良好的導(dǎo)電性和葫蘆脲[7]與電活性物質(zhì)亞甲基藍(lán)(MB)之間強(qiáng)的主客體識別作用,采用CB[7]/depAu修飾的電極作為傳感界面有效地促進(jìn)了電活性物質(zhì)的采集;最后,此方法還可以拓展到其他具有酶剪切活性的蛋白質(zhì)的檢測。3.基于多肽剪切誘導(dǎo)的級聯(lián)信號放大策略檢測基質(zhì)金屬蛋白酶-7的電化學(xué)多肽傳感器研究多肽剪切事件可以直接轉(zhuǎn)化為輸出DNA(output DNA),然而,output DNA通常帶有氨基酸殘基,其位阻效應(yīng)會影響DNA的放大效率。為了解決這個問題,在本工作中,以指數(shù)放大反應(yīng)(EXPAR)作為模型,通過設(shè)計一個兩段式的DNA模板,構(gòu)建了一個高效的DNA放大體系用于高靈敏檢測基質(zhì)金屬蛋白酶-7(MMP-7)�；谶@個兩段式的DNA模板,output DNA作為觸發(fā)劑1(trigger 1)最先引發(fā)了一個低效率的EXPAR,反應(yīng)過程中不僅獲得部分產(chǎn)物DNA(S1),還產(chǎn)生了與output DNA具有相同核苷酸序列的觸發(fā)劑2(trigger 2)。由于trigger 2不包含任何氨基酸殘基,它繼續(xù)誘導(dǎo)了一個額外的,高效的EXPAR放大,迅速地獲得大量的產(chǎn)物S1。產(chǎn)物S1作為催化劑進(jìn)一步誘導(dǎo)了催化發(fā)夾自組裝反應(yīng)(CHA),獲得了級聯(lián)放大的電化學(xué)信號,從而實現(xiàn)了對目標(biāo)物MMP-7的超靈敏檢測,檢測限是0.02 pg·mL-1。本工作中,目標(biāo)物對于多肽的剪切過程,即輸入蛋白轉(zhuǎn)換為output DNA的步驟是在溶液中進(jìn)行的,避免了復(fù)雜的電極制備過程,同時也提高了目標(biāo)物的轉(zhuǎn)換效率。另外,多肽剪切誘導(dǎo)的級聯(lián)放大反應(yīng)包括EXPAR和CHA,它們不僅有較高的放大效率,而且均是等溫反應(yīng)過程,具有簡單,低耗的優(yōu)點,有較好的應(yīng)用前景。
[Abstract]:Protease is a hydrolytic protein or polypeptide hydrolase, which is related to human metabolic processes such as cell growth and death, tissue remodeling and immune defense. It has been used as a biomarker for clinical detection. A simple, sensitive, specific and rapid method for biomarker detection has been established to prevent, diagnose and treat diseases. The electrochemical biosensor is an analytical device which is fixed on the transducer, such as antibodies, enzymes, aptamers, peptides, etc., with electrochemical signals as the detection signal, and plays a more and more important role in the research field of analytical chemistry. It has the advantages of stable, reliable, low cost, high affinity, strong biological activity and so on. People pay much attention to the detection of protease biomarkers. In this paper, polypeptides are used as molecular recognition materials and are devotes to design different signal amplification strategies. A series of simple and inexpensive electrochemical methods have been constructed. Peptide sensors, high sensitive detection of prostatic antigen (a serine protease), matrix metalloproteinase -2, matrix metalloproteinase -7, specific work is as follows: 1. electrochemical polypeptide sensors based on positive charged gold nanoparticles as signal enhancers for detection of prostate antigen most of the electrochemical polypeptide sensors The "signal attenuation" (signal-off) sensor based on the targeting of labeled signal labels is limited and is susceptible to false positive signals. At present, metal nanoparticles have been widely used in signal amplification because they can overcome the inherent instability of some enzyme biomaterials. The positive charged gold nanoparticles are used as signal enhancers to construct a highly sensitive electrochemical polypeptide sensor of "signal enhancement" (signal-on). The positive charged gold nanoparticles are modified on the molecular recognition element polypeptide. It adsorbs the negatively charged oxidation-reduction probe potassium ferricyanide ([Fe (CN) 6]3-/4-) in the test bottom solution. In the presence of the target prostate antigen (PSA), the PSA specific shear peptide, which makes the positive charged gold nanoparticles leave the surface of the electrode and the electrochemical impedance analysis in the [Fe (CN) 6]3-/4- bottom solution, obtains a larger electrochemical impedance value. By monitoring the changes in electrochemical impedance values, the sensor has achieved a high sensitivity detection of the target PSA. Furthermore, the sensing system is designed based on the specific shear of the peptide on the target. It has high selectivity and sensitivity, and provides a universal detection method for the analysis of other proteins based on the nucleic acid exonuclease supplemented. The electrochemical peptide sensor of matrix metalloproteinase -2 was detected by the assisted cyclic amplification strategy. The target substance was transformed directly into the output DNA (output DNA) by the target peptide chain shear. It could be further amplified by various DNA amplification techniques to improve the sensitivity of the sensor. The polypeptide shear event was converted to DNA in this work. An electrochemical sensor for ultra sensitive detection of matrix metalloproteinase -2 (MMP-2) was constructed by combining the nucleic acid exonuclease III (EXO III) assisted target circulation signal amplification strategy. The proposed sensor system can summarize the following advantages: first, the design of "signal enhancement" (signal-on) sensor is reduced to a certain extent. The false positive signals caused by the surface stripping or pollution of the electrode are produced. Secondly, the sensitivity of the sensor is effectively improved by combining the protein analysis with the DNA amplification technology. In addition, the good conductivity of the gold nanoparticles (depAu) and the recognition of the host and guest between the cucurbit [7] and the electroactive methylene blue (MB) are strong. The use of CB[7]/depAu modified electrode as a sensing interface effectively promotes the collection of electroactive substances; finally, this method can also be extended to the detection of other proteins with enzyme shear activity,.3. based on the cascade signal amplification strategy of peptide shear induced detection of the matrix metalloproteinase -7 by the electrochemical polypeptide sensor and the study of the polypeptide scissors The tangent events can be directly converted to the output DNA (output DNA), however, output DNA usually has amino acid residues, and its steric effect affects the amplification efficiency of DNA. In order to solve this problem, in this work, the exponential amplification reaction (EXPAR) is used as a model to construct an efficient DNA amplifier by designing a two segment DNA template. The system is used for highly sensitive detection of matrix metalloproteinase -7 (MMP-7). Based on this two stage DNA template, output DNA as a trigger 1 (trigger 1) first initiate a low efficiency EXPAR. The reaction process not only obtains a partial product DNA (S1), but also produces the trigger agent 2 (trigger 2) with the same nucleotide sequence as output DNA. Ger 2, which does not contain any amino acid residues, continues to induce an extra, efficient EXPAR amplification and rapidly obtain a large number of product S1. products S1 as a catalyst to further induce the catalytic hairpin self assembly reaction (CHA), and obtain a cascade of amplified electrochemical signals. The ultra sensitive detection of the target MMP-7 is realized and the detection limit is achieved. In the 0.02 pg. ML-1. work, the step of the target to the shear process of the polypeptide, that is, the step of the input protein to output DNA, is carried out in the solution, avoiding the complex electrode preparation process and improving the conversion efficiency of the target. In addition, the cascade amplification reaction induced by the polypeptide shear, including EXPAR and CHA, is not only more effective. High magnification efficiency, both of which are isothermal reaction processes, have the advantages of simplicity and low consumption, and have good application prospects.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TP212.3;O657.1

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