本文關鍵詞： 乙醇 膽汁酸 電化學傳感器 妊娠期無癥狀高膽汁酸血癥 妊娠肝內膽汁淤積癥　出處：《重慶醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：建立靈敏、簡單、可用于血液中生物活性物質測定的分析方法是臨床檢驗診斷學學科發(fā)展的重要方向之一。電化學分析法靈敏、快速,且所用儀器簡單、易于微型化,在臨床疾病的輔助診斷和標志物的快速檢測中扮演著重要角色。而液相色譜-質譜聯(lián)用技術具有靈敏度高、結果準確且重現(xiàn)性好等優(yōu)點,可實現(xiàn)血液中已知和未知的多種代謝物同時檢測。鑒于此,本文以氧化/還原型煙酰胺腺嘌呤二核苷酸(NAD+/NADH)為基礎,建立了簡單、實用的血液中乙醇和總膽汁酸(total bile acid,TBA)的電化學分析方法;并采用超高效液相色譜-四級桿飛行時間質譜(UPLC-Triple TOF-MS/MS)技術對妊娠肝內膽汁淤積癥(intrahepatic cholestasis of pregnancy,ICP)和妊娠期無癥狀高膽汁酸血癥(asymptomatic hypercholanaemia of pregnancy,AHP)患者血清膽汁酸代謝輪廓進行全面分析。主要內容包括以下四個部分:1.免修飾絲網(wǎng)印刷碳電極(SPCE)-電化學測定血漿乙醇方法的建立及應用。利用乙醇脫氫酶(ADH)和NAD+特異性催化乙醇生成乙醛和NADH的氧化-還原體系,采用SPCE并結合直接稀釋血液的策略,實現(xiàn)血漿中乙醇的電化學測定。血漿樣本經(jīng)磷酸鹽緩沖液(PBS)稀釋后與ADH和NAD+室溫共反應,再滴加到SPCE表面,采用示差脈沖伏安法(DPV)進行測定。該方法線性良好(0.10~3.20 mg/m L,r=0.9943),靈敏度較高(LOD,40.0mg/m L)。精密度和回收率分別為(RSD,5.1%~9.4%)和(80.1%~103%)。將建立的方法用于20例飲酒自愿者血漿乙醇的測定并與氣相色譜(GC)法測定結果比較,相關性良好(r=0.9311)。該方法簡單、準確,有望用于酒精中毒、酒駕和醉駕等的快速篩查。2.基于雙重氧化的血清TBA超靈敏電化學傳感器的構建及臨床應用。含3α羥基類固醇脫氫酶(3α-HSD)、NAD+和三聯(lián)吡啶釕(Ru(bpy)32+)的PBS溶液滴加到SPCE電極表面并施加恒電位,加入稀釋血樣,3α-HSD特異性催化血清中膽汁酸生成NADH,NADH與被氧化的Ru(bpy)33+反應生成Ru(bpy)32+,Ru(bpy)32+再被氧化為Ru(bpy)33+產(chǎn)生電化學信號,采用計時電流法測定,實現(xiàn)血清TBA超靈敏檢測。該方法測定血清TBA的線性良好(5.0~150.0mmol/L,r=0.9983),靈敏度較高(LOD,0.40mmol/L)。精密度和回收率分別為(RSD,6.4%~11.8%)和(91.4%~108%)。將建立的方法用于正常孕婦(20例)、ICP患者(30例)和AHP患者(22例)血清樣本測定并與酶循環(huán)法測定結果比較,相關性良好(r=0.9372)。與正常孕婦相比,ICP與AHP患者血清TBA均顯著增高。該方法靈敏、快速有望用于床旁檢測。3.AHP與ICP患者血清膽汁酸代謝輪廓分析。利用UPLC-Triple TOF-MS/MS對27例正常孕婦、26例AHP患者和59例ICP患者血清中57種膽汁酸的代謝輪廓進行分析。采用PLS-DA顯示AHP患者的血清膽汁酸代謝輪廓與正常孕婦和ICP孕婦均具有明顯差異,VIP值1的24種膽汁酸,其中甘氨脫氧膽酸(GDCA)、牛磺三羥基膽汁酸的同分異構體4(Ttri-4)、牛磺-ω-鼠膽酸(T-ω-MCA)、甘氨豬膽酸(GHCA)、甘氨三羥基膽汁酸同分異構體3(Gtri-3)這5種膽汁酸在三組間均有顯著差異(P0.05)。采用Logistic回歸篩選出疾病組(AHP和ICP)與正常組的聯(lián)合診斷標志物甘氨膽酸(GCA)、甘氨三羥基膽汁酸同分異構體1(Gtri-1)和�；嵌u基膽汁酸同分異構體1(Tdi-1),ROC曲線下面積(AUC)為0.989,靈敏度98.9%,特異性100%;鑒別AHP與ICP的聯(lián)合診斷標志物石膽酸(LCA)、Gtri-1和鵝脫氧膽酸(CDCA),AUC為0.956,靈敏度92.1%,特異性91.7%。膽汁酸代謝輪廓分析與成分膽汁酸的檢測有助于臨床對AHP的認識,診斷與鑒別診斷。4.芯片-電化學法檢測血清TBA的方法學研究。固相萃取(SPE)預處理的血清樣本經(jīng)聚二甲基硅氧烷(polydimethylsiloxane,PDMS)芯片通道電驅動進樣后電泳到檢測池,血清膽汁酸與檢測池中的3α-HSD、Ru(bpy)32+和NAD+發(fā)生酶促偶聯(lián)-雙重氧化還原反應,生成的Ru(bpy)32+在SPCE電極上被氧化產(chǎn)生電信號,采用計時電流法進行分析。該方法測定血清TBA的線性良好(1.0~100.0mmol/L,r=0.9995),靈敏度較高(LOD,0.10mmol/L)。精密度和回收率分別為(RSD,5.9%~8.9%)和(94.9%~102%)。該方法為后續(xù)建立微流控芯片-電化學檢測膽汁酸代謝輪廓奠定基礎。
[Abstract]:To establish a sensitive, simple and can be used for determination of bioactive substances in the blood is a clinical test for the diagnosis of discipline development one of the important directions. The electrochemical analysis method is sensitive, rapid, and the instrument is simple, easy miniaturization, plays an important role in the rapid detection and diagnosis marker of clinical disease. The liquid chromatography-mass spectrometry technique has high sensitivity, accuracy and repeatability, can achieve a variety of known and unknown metabolites in the blood are detected simultaneously. In view of this, the oxidation of nicotinamide adenine dinucleotide / (NAD+/NADH) as the foundation, established a simple, practical and total bile acid ethanol the blood (total bile, acid, TBA) and electrochemical analysis method; using ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (four UPLC-Triple TOF-MS/MS) on pregnancy with intrahepatic cholestasis (int Rahepatic cholestasis of pregnancy, ICP) and no symptoms of pregnancy hyperbileacidemia (asymptomatic hypercholanaemia of pregnancy, AHP) in patients with serum bile acid metabolic profile were analyzed. The main contents include the following four parts: 1. free modified screen printed carbon electrode (SPCE) - the establishment and application of plasma ethanol electrochemical determination methods. The use of alcohol dehydrogenase (ADH) - and NAD+ specific catalytic oxidation of ethanol to acetaldehyde and the reduction of NADH by SPCE system, combined with the direct blood dilution strategy, electrochemical determination of ethanol in plasma. Plasma samples were extracted with phosphate buffer solution (PBS) were diluted with ADH and NAD+ at room temperature, and then dropped to the SPCE surface. By using differential pulse voltammetry (DPV) were measured. This method has good linearity (0.10~3.20 mg/m, L, r=0.9943), high sensitivity (LOD, 40.0mg/m L). The precision and recovery rate were RSD (, 5.1%~9.4%) and (80.1%~103%). The established method for the measurement of 20 cases of drinking alcohol and plasma volunteers with gas chromatography (GC) method for the determination of results, good correlation (r=0.9311). This method is simple, accurate, is expected to be used for fast screening of.2. alcohol poisoning, drunk driving and drunk driving and other construction and clinical application of serum TBA double oxidation ultra sensitive electrochemical sensor based on containing 3 alpha hydroxysteroid dehydrogenase (3 alpha, NAD+ and -HSD) terpyridyl ruthenium (Ru (bpy) 32+) PBS solution is dripped into the SPCE electrode surface and applying a constant potential, diluted blood samples, 3 alpha -HSD specific catalytic serum bile acid to generate NADH, NADH and Ru (bpy) 33+ oxidation reaction of Ru (bpy) 32+, Ru (bpy) 32+ is oxidized to Ru (bpy) 33+ produce electrochemical signals, measured by chronoamperometry, the serum TBA ultra sensitive detection. The linear determination of serum TBA and this method is good (5.0~150.0mmol/ L, r=0.9983), high sensitivity (LOD, 0.40mmol/L). The precision and recovery rate respectively (RSD, 6.4%~11.8%) and (91.4%~108%). The established method for normal pregnant women (20 cases), ICP patients (30 cases) and AHP patients (22 cases) serum samples were measured and compared with the results of enzyme recycling determination, good correlation (r=0.9372). Compared with the normal pregnant women, ICP and AHP in sera of patients with TBA were significantly increased. The method is sensitive, rapid analysis is expected to be used for bedside detection of.3.AHP and ICP in serum of patients with bile acid metabolic profile. On 27 pregnant women with UPLC-Triple TOF-MS/MS, 26 cases of 57 kinds of metabolic profile in patients with AHP and 59 ICP patients serum bile acids were analyzed. The PLS-DA showed the serum bile acid metabolic profile in patients with AHP and normal pregnant women and pregnant women with ICP has the obvious difference, VIP value of 24 kinds of 1 bile acids, including glycine deoxycholate acid (GDCA), three hydroxy bile acid with sodium 鍒嗗紓鏋勪綋4(Ttri-4),鐗涚：-蠅-榧犺儐閰，

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