化学发光微流动注射分析芯片的研究
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摘要
微流控芯片又称为芯片实验室，是把化学和生物领域中所涉及到的样品制备、反应、分离、检测及细胞培养、分选、裂解等基本操作单元集成或基本集成到一块几平方厘米(或更小)的芯片上，由微通道形成网络，以可控流体贯穿整个系统，用以取代常规化学或生物实验室的各种功能的一种技术平台。其基本特征和最大优势是多种单元技术在整体可控的微小平台上灵活组合、规模集成。微流动注射分析是微流控芯片技术的一个重要分支，是在流动注射分析的基础上发展而来的，具有整体化发展的优势，更加适合于现场分析，已经成为近年来分析科学领域内的一个研究热点。
本论文包括综述和研究报告两部分。综述部分包括三章，即第1-3章，主要涉及微流控芯片的发展概况、微流动的基本理论以及微流动注射分析的现状和发展方向。研究报告部分包括三章，即第4-6章。第4章中采用了双T进样、微阀进样以及改进的不连续微滴进样等几种不同进样方式对食品安全检测和环境分析用的微流动注射分析芯片进行了研究。这几种进样方式的使用大大减少了试剂的消耗体积，提高了测定的灵敏度和检出限。第5章中结合微固相萃取分离技术，通过在化学发光微流动注射芯片上在线富集分离实现了牛奶中β-内酰胺类抗生素的测定。第6章中将微流动注射分析芯片与微透析的取样技术相结合，活体在线测定了兔血中的葡萄糖含量以及金属离子铬在血液中的代谢情况。同时设计了一种可通过精确控制时间来控制进样体积的注射泵，时间控制精度为0.01秒，取样量为纳升至微升级。实验中无须外加采样环，保证每次的进样量为0.4μL，更好地实现了微透析的取样技术与微流动注射芯片的结合。
1．化学发光微流动注射芯片的研究
(1)测定面粉中过氧化苯甲酰的化学发光微流动注射分析芯片
过氧化苯甲酰是面粉中普遍使用的增白剂，它具有漂白和消毒的作用，但面粉中过量添加的过氧化苯甲酰却能引发一些过敏反应和增加致癌的可能性。本文基于PMMA化学发光芯片首次建立了测定面粉中过氧化苯甲酰的方法。芯片上微通道的宽为200μm，深为100μm，微通道的总长为67mm，检测区域面积为5×5mm~2。过氧化苯甲酰能够直接氧化鲁米诺产生化学发光，进样方式采用了两种，分别是双T进样和微阀进样。进样量分别为0.16μL和0.25μL。测定过氧化苯甲酰的线性范围是8×10~(-7)-1×10~(-4)gmL~(-1)，方法的检出限是4×10~(-7)gmL~(-1)。两种进样方式的相对标准偏差分别为3.06％(双T)和2.41％(微阀)。该方法成功应用于面粉中过氧化苯甲酰的测定。
(2)测定自来水中次氯酸根的化学发光微流动注射分析芯片
Cl_2和ClO~-被广泛应用于自来水的消毒剂，然而使用ClO~-作为氯消毒剂会产生对人体有害的氯酸盐和亚氯酸盐。本文基于次氯酸根能够直接氧化鲁米诺产生化学发光的原理，结合微加工技术，建立了微流动注射化学发光检测系统测定自来水中次氯酸根的方法。微芯片是在聚甲基丙烯酸甲酯(PMMA)片(50mm×40mm×5mm)上通过激光雕刻微通道(宽为200μm,深为100μm)并结合双T的进样方式(进样量约为0.3μL)来实现的。该方法测定次氯酸根的线性范围是3.0×10~(-7)-1.0×10~(-4)gmL~(-1)，方法的检出限是1.4×10~(-7)gmL~(-1)。11次平行测定的相对标准偏差为4.5％。方法简单、分析速度快(368 h~(-1))、耗样量少，已成功应用于自来水中次氯酸根的测定。
(3)用于快速测定水样中化学需氧量的化学发光微流动注射分析芯片
化学需氧量(COD)由于代表着水体中有机污染物的多少而成为水质检测的一项重要参数。因此需要低成本、简单的、快速的、试剂消耗量少的测定COD的方法以实现现场分析。本文建立了一种室温下快速测定化学需氧量(COD)的化学发光微流动分析芯片。基于样品在2 molL~(-1)H_2SO_4介质中被K_2Cr_2O_7还原为Cr~(3+)，Cr~(3+)对luminol-H_2O_2化学发光体系具有催化作用来进行测定。芯片材质是聚甲基丙烯酸甲酯，采用的是不连续的微滴进样方式。考察了pH、试剂浓度、通道长度等对发光强度的影响。方法的线性范围为0.27-10 gL~(-1)，检出限为100 mgL~(-1)。该方法成功应用于污水中COD的测定。
(4)用于水样中不同形态的铬的含量测定的化学发光微流动注射分析芯片
建立了一种同时测定水样中Cr(Ⅲ)和Cr(Ⅵ)的化学发光微流控芯片分析系统。基于Cr(Ⅵ)能够被H_2O_2在线还原为Cr(Ⅲ)，Cr(Ⅲ)催化鲁米诺-H_2O_2产生强的化学发光来实现测定。制作微流控芯片的材料为聚甲基丙烯酸甲酯(PMMA)，芯片采用的是微滴进样方式，进样体积为5μL。芯片上微通道的宽为200μm、深为100μm。线性范围分别为5×10~(-8)-1×10~(-5) molL~(-1)(Cr~(3+))和1×10~(-7)-1×10~(-5)molL~(-1)(Cr_2O_7~(2-))，检出限为2×10~(-8)mol L~(-1)(Cr~(3+))和4×10~(-8)mol L~(-1)(Cr_2O_7~(2-))。该法已经成功用于水样中Cr(Ⅲ)和Cr(Ⅵ)的同时测定。
2．微固相萃取分离-化学发光微流动注射芯片的研究
(5)用于牛奶中β-内酰胺类抗生素测定的在线同相萃取化学发光微流动注射分析芯片
β-内酰胺类药物也是牛奶样品中常用的抗生素，而牛奶中的抗生素残留问题已引起广泛的关注。SPE是常用的样品前处理和预富集的分析手段，具有高的选择性、易于操作和试剂消耗低的特点，将在线SPE分离技术用于共存物质干扰的消除是化学发光分析的一种理想的选择。本文建立了一种基于在线固相萃取技术的化学发光微流动注射分析芯片用于牛奶中四种β-内酰胺类抗生素(青霉素、头孢拉定、头孢羟氨苄、头孢氨苄)的测定。基于β-内酰胺类抗生素能够增强luminol-K_3Fe(CN)_6化学发光体系来进行测定。微流动注射分析芯片材料为聚甲基丙烯酸甲酯，其微通道的宽为200μm，深为150μm。C_(18)填装在微通道(长：10mm；宽：1mm；深：500μm)内作为固相萃取装置，微流动注射分析芯片在线完成分析物的萃取和富集，提高了化学发光检测的选择性。样品的检出限分别是青霉素0.5μg mL~(-1)、头孢拉定0.04μg mL~(-1)、头孢羟氨苄0.08μg mL~(-1)和头孢氨苄为0.1μg mL~(-1)。该方法已经成功应用于牛奶样品中β-内酰胺类抗生素的测定。
3．微流动注射分析芯片用于活体在线检测的研究
(6)微透析—微滴进样化学发光微流控芯片测定活体中的葡萄糖
微透析取样技术已成功用于活体分析，具有取样量少的特点，因此与微流动注射分析芯片的结合具有相当的优势。本文建立了在线微透析取样-化学发光微流控芯片测定活体中葡萄糖的方法。化学发光微流控芯片采用的是微滴进样方式，微滴形成于毛细管的尖端，其样品体积为4.5μL。制作微流控芯片的材料为聚甲基丙烯酸甲酯(PMMA)，利用溶胶-凝胶固定化的方法将辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOD)固定在芯片微流通池的内表面上。葡萄糖被GOD氧化为D-葡萄糖酸和H_2O_2，H_2O_2在HRP的存在下氧化鲁米诺产生化学发光。使用微透析探针以20 h~(-1)的频率在兔子血液中采样，成功应用于在线测定兔子血样中的葡萄糖。
(7)微透析取样-化学发光微流动分析芯片活体在线监测血液中Cr(Ⅲ)的代谢
建立了微透析取样-化学发光微流动分析芯片系统用于活体在线监测家兔血液中Cr(Ⅲ)的代谢，基于Cr(Ⅲ)-luminol-H_2O_2体系来进行测定。在聚甲基丙烯酸甲酯的芯片上集成了阴离子交换微柱，芯片上微通道的宽为200μm，深为100μm。插在兔子耳缘静脉内的微透析探针连接的灌流液的流速为5μL min~(-1)，微透析的样品体积为0.4μL，最终实现了兔子血液内Cr(Ⅲ)的代谢活体在位监测。
After the concept of micro total analysis system (uTAS) was introduced by Manz et al. in 1990, the field ofμTAS is growing rapidly. Because miniaturization is currently considered as one of the most important trends in the development of analytical instrumentation, the ultimate purpose ofμTAS is the integration of the entire analytical process on a micro-device. The micro-flow injection system on the chip is part of theμTAS and also has many advantages.
The dissertation is made up of two sections. One is the review ofμTAS and micro-flow injection system on the chip. Another is the research work. In the first part of the research work, the micro-flow injection systems which are used for food analysis and environment analysis are introduced. Different sampling mode, including double-T sampling mode, micro-valve sampling mode and discrete microdroplet sampling mode, is used. With the sampling modes, the reagent consumption is reduced and the sensitivity is improved. In the second part, combining with the solid phase extraction technique, a chemiluminescence (CL) micro-flow system is presented for determination ofβ-lactam antibiotics in milk. In the last part of the research work, microdialysis sampling technique was used on the micro-flow injection system for the determination of glucose and Cr(III) metabolism in blood.
1. Investigation on the chemiluminescence micro-flow injection analysis on a chip
(1) Determination of benzoyl peroxide in flour
Benzoyl peroxide(BP) is a common additive in flour because of its bleaching and sanitizing properties, but excessive benzoyl peroxide in flour could induce allergic reactions, weak cancer causing, Vitamin E and nutrients destruction. A rapid and sensitive chemiluminescence microfluidic chip fabricated in polymethyl methacrylate for determination of benzoyl peroxide in flour is described. The width of the microchannel was 200 urn and the depth was 100μm in the chip (50×40×5 mm). The total length of the microchannel was 67 mm with the detection area of 5×5 mm~2. Benzoyl peroxide can directly oxidize luminol to produce chemiluminescence. Two injectors of double-tee injector and microvalve injector were used. The sampling volume was 0.16μL for the double-tee injector and 0.25μL for the microvalve injector. The linear range of the benzoyl peroxide concentration was 8×10~(-7)-1×10~(-4) g mL~(-1). The detection limit was 4×10~(-7) g mL~(-1) and the R.S.D for eight times is 3.06% for the double-tee injector and 2.41% for the microvalve injector. The proposed method has been successfully applied to the determination of benzoyl peroxide in flour.
(2) Determination of hypochlorite in tap water
A micro-flow injection chemiluminescence detection system fabricated in Polymethyl methacrylate (PMMA) (50mm×40mm×5mm) for determination of hypochlorite in tap water is described, based on the direct chemiluminescence reaction of hypochlorite and luminol. The microchannel on the micro-flow injection chemiluminescence detection system are made by laser ablation (200μm width, 150μm depth) and the sampling volume was about 0.3μL with double-tee mode. The linear range of the hypochlorite concentration was 3.0×10~(-7)-1.0×10~(-4)g/mL. The detection limit is 1.4×10~(-7)g/mlL and the R.S.D for 11 times is 4.5%. The system is easy, fast (sample throughput 368h~(-1)) and reduces reagent consumption. The proposed method has been successfully applied to the determination of hypochlorite in tap water.
(3) Rapid determination of chemical oxygen demand in water
Chemical oxygen demand (COD) is an important parameter for water monitoring because it can represent the organic contaminants in water. A fast, continuous and automatic analysis method for COD determination was needed. A chemiluminescence (CL) micro-flow system for rapid determination of chemical oxygen demand (COD) in water at room temperature is proposed in this paper. In this system, potassium dichromate is reduced to Cr~(3+) in 2 mol L~(-1) H_2SO_4 during chemical oxidation of COD-substances in sample and Cr~(3+) can be measured by the luminol-H_2O_2 CL system. The polymethyl methacrylate micro-flow chip with discrete microdroplet sampling was used here. Effects for COD determination (such as pH, concentrations, the channel length, and interference) were investigated. The linear range for COD determination was 0.27-10 g L~(-1) and the detection limit was 100 mg L~(-1). The proposed method has been successfully applied to the determination of COD in wastewater samples. The datas obtained by the present method were fairly in good agreement with those obtained by the titrimetric method.
(4) Simultaneous determination of Cr (III) and Cr (VI) in water
A sensitive method for the simultaneous determination of chromium (III) (Cr~(3+)) and chromium (VI) (Cr_2O_7~(2-)) using micro-flow chemiluminescence system was developed. The chemiluminescence detection was based on the catalytic effect of Cr (III) on the chemiluminescence reaction of luminol and hydrogen peroxide. Cr (VI) was on-line reduced to Cr (III) with hydrogen peroxide, and then was detected. The polymethyl methacrylate (PMMA) micro-flow system with three plates was used to realize this procedure. The micro-flow system used the discrete sampling with microdroplet, which can be formed at the tip of the capillary with the sampling volume of 5μL. The width of the microchannel was 200μm and the depth was 100μm. The linear ranges were 5×10~(-8)-1×10~(-5)mol L~(-1) for chromium(III) and 1×10~(-7)-1×10~(-5)mol L~(-1) for chromium(VI). The detection limits were 2×10~(-8) mol L~(-1) for chromium (III) and 4×10~(-8)mol L~(-1) for chromium (VI). The proposed method has been successfully applied to the simultaneous determination for chromium (III) and chromium (VI) in water samples.
2. Investigation on the chemiluminescence micro-flow injection analysis on a chip with on-line solid phase extraction
(5) Determination ofβ-Lactam antibiotics in milk
β-Lactam antibiotics are widely used in the treatment of lactating dairy cattle for several infections. Antibiotic residues in milk, besides inhibiting start of cultures in the production of milk products, can provoke allergic reactions in some hypersensitive individuals. On-line introducing in SPE separation technique for interfering element removal is undoubtedly an ideal choice for interference elimination in CL analysis. In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination ofβ-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect ofβ-lactam antibiotics on the luminol-K_3Fe(CN)_6 CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50×40×5mm) with the microchannels of 200μm wide and 150 urn deep. C_(18)-modified silica gel was packed into the microchannel (length: 10mm; wide: 1mm; depth: 500μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5μg mL~(-1) of penicillin, 0.04μg mL~(-1) of cefradine, 0.08μg mL"1 of cefadroxil and 0.1 ug mL"1 of cefalexin. The proposed method was also applied to analyzing theβ-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.
3. Investigation on the chemiluminescence micro-flow injection analysis on a chip by microdialysis sampling
(6) Determination of glucose in vivo
A micro-flow chemiluminescence (CL) system in vivo for glucose determination by the on-line microdialysis sampling is described in this paper. The micro-flow CL system uses discrete sample droplets, which formed at the tip of the capillary with the sampling volume of 4.5μL. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and glucose oxidase (GOD) on the inside surface of the micro-flow cell which was fabricated in polymethyl methacrylate (PMMA). The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H_2O_2, then H_2O_2 oxidizing luminol to produce CL in presence of HRP. The microdialysis probe was utilized for sampling in the rabbit blood; the sample throughput was 20 h~(-1) . The glucose level in blood of the rabbit was on-line monitored with good results.
(7) On-line monitoring of chromium metabolism in vivo
A micro-flow injection chemiluminescence system based on the Cr (III)-luminol-hydrogen peroxide chemiluminescence system for in vivo on-line monitoring of chromium (III) metabolism in blood by microdialysis sampling is described in this paper. It was fabricated in polymethyl methacrylate(PMMA) of two plates and the separation part of anion exchange resin was also integrated on the micro-flow system. The width of the microchannel was 200μm and the depth was 100μm on the plate(50×40×5mm). A microdialysis probe, implanted in the vein of rabbit, was perfused with perfusate at a flow rate of 5μL min~(-1). The dialysate sample volume was about 0.4μL. The metabolism of the chromium (III) in the blood of the rabbit was monitored after administration of the chromium (III) to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.
本论文包括综述和研究报告两部分。综述部分包括三章，即第1-3章，主要涉及微流控芯片的发展概况、微流动的基本理论以及微流动注射分析的现状和发展方向。研究报告部分包括三章，即第4-6章。第4章中采用了双T进样、微阀进样以及改进的不连续微滴进样等几种不同进样方式对食品安全检测和环境分析用的微流动注射分析芯片进行了研究。这几种进样方式的使用大大减少了试剂的消耗体积，提高了测定的灵敏度和检出限。第5章中结合微固相萃取分离技术，通过在化学发光微流动注射芯片上在线富集分离实现了牛奶中β-内酰胺类抗生素的测定。第6章中将微流动注射分析芯片与微透析的取样技术相结合，活体在线测定了兔血中的葡萄糖含量以及金属离子铬在血液中的代谢情况。同时设计了一种可通过精确控制时间来控制进样体积的注射泵，时间控制精度为0.01秒，取样量为纳升至微升级。实验中无须外加采样环，保证每次的进样量为0.4μL，更好地实现了微透析的取样技术与微流动注射芯片的结合。
1．化学发光微流动注射芯片的研究
(1)测定面粉中过氧化苯甲酰的化学发光微流动注射分析芯片
过氧化苯甲酰是面粉中普遍使用的增白剂，它具有漂白和消毒的作用，但面粉中过量添加的过氧化苯甲酰却能引发一些过敏反应和增加致癌的可能性。本文基于PMMA化学发光芯片首次建立了测定面粉中过氧化苯甲酰的方法。芯片上微通道的宽为200μm，深为100μm，微通道的总长为67mm，检测区域面积为5×5mm~2。过氧化苯甲酰能够直接氧化鲁米诺产生化学发光，进样方式采用了两种，分别是双T进样和微阀进样。进样量分别为0.16μL和0.25μL。测定过氧化苯甲酰的线性范围是8×10~(-7)-1×10~(-4)gmL~(-1)，方法的检出限是4×10~(-7)gmL~(-1)。两种进样方式的相对标准偏差分别为3.06％(双T)和2.41％(微阀)。该方法成功应用于面粉中过氧化苯甲酰的测定。
(2)测定自来水中次氯酸根的化学发光微流动注射分析芯片
Cl_2和ClO~-被广泛应用于自来水的消毒剂，然而使用ClO~-作为氯消毒剂会产生对人体有害的氯酸盐和亚氯酸盐。本文基于次氯酸根能够直接氧化鲁米诺产生化学发光的原理，结合微加工技术，建立了微流动注射化学发光检测系统测定自来水中次氯酸根的方法。微芯片是在聚甲基丙烯酸甲酯(PMMA)片(50mm×40mm×5mm)上通过激光雕刻微通道(宽为200μm,深为100μm)并结合双T的进样方式(进样量约为0.3μL)来实现的。该方法测定次氯酸根的线性范围是3.0×10~(-7)-1.0×10~(-4)gmL~(-1)，方法的检出限是1.4×10~(-7)gmL~(-1)。11次平行测定的相对标准偏差为4.5％。方法简单、分析速度快(368 h~(-1))、耗样量少，已成功应用于自来水中次氯酸根的测定。
(3)用于快速测定水样中化学需氧量的化学发光微流动注射分析芯片
化学需氧量(COD)由于代表着水体中有机污染物的多少而成为水质检测的一项重要参数。因此需要低成本、简单的、快速的、试剂消耗量少的测定COD的方法以实现现场分析。本文建立了一种室温下快速测定化学需氧量(COD)的化学发光微流动分析芯片。基于样品在2 molL~(-1)H_2SO_4介质中被K_2Cr_2O_7还原为Cr~(3+)，Cr~(3+)对luminol-H_2O_2化学发光体系具有催化作用来进行测定。芯片材质是聚甲基丙烯酸甲酯，采用的是不连续的微滴进样方式。考察了pH、试剂浓度、通道长度等对发光强度的影响。方法的线性范围为0.27-10 gL~(-1)，检出限为100 mgL~(-1)。该方法成功应用于污水中COD的测定。
(4)用于水样中不同形态的铬的含量测定的化学发光微流动注射分析芯片
建立了一种同时测定水样中Cr(Ⅲ)和Cr(Ⅵ)的化学发光微流控芯片分析系统。基于Cr(Ⅵ)能够被H_2O_2在线还原为Cr(Ⅲ)，Cr(Ⅲ)催化鲁米诺-H_2O_2产生强的化学发光来实现测定。制作微流控芯片的材料为聚甲基丙烯酸甲酯(PMMA)，芯片采用的是微滴进样方式，进样体积为5μL。芯片上微通道的宽为200μm、深为100μm。线性范围分别为5×10~(-8)-1×10~(-5) molL~(-1)(Cr~(3+))和1×10~(-7)-1×10~(-5)molL~(-1)(Cr_2O_7~(2-))，检出限为2×10~(-8)mol L~(-1)(Cr~(3+))和4×10~(-8)mol L~(-1)(Cr_2O_7~(2-))。该法已经成功用于水样中Cr(Ⅲ)和Cr(Ⅵ)的同时测定。
2．微固相萃取分离-化学发光微流动注射芯片的研究
(5)用于牛奶中β-内酰胺类抗生素测定的在线同相萃取化学发光微流动注射分析芯片
β-内酰胺类药物也是牛奶样品中常用的抗生素，而牛奶中的抗生素残留问题已引起广泛的关注。SPE是常用的样品前处理和预富集的分析手段，具有高的选择性、易于操作和试剂消耗低的特点，将在线SPE分离技术用于共存物质干扰的消除是化学发光分析的一种理想的选择。本文建立了一种基于在线固相萃取技术的化学发光微流动注射分析芯片用于牛奶中四种β-内酰胺类抗生素(青霉素、头孢拉定、头孢羟氨苄、头孢氨苄)的测定。基于β-内酰胺类抗生素能够增强luminol-K_3Fe(CN)_6化学发光体系来进行测定。微流动注射分析芯片材料为聚甲基丙烯酸甲酯，其微通道的宽为200μm，深为150μm。C_(18)填装在微通道(长：10mm；宽：1mm；深：500μm)内作为固相萃取装置，微流动注射分析芯片在线完成分析物的萃取和富集，提高了化学发光检测的选择性。样品的检出限分别是青霉素0.5μg mL~(-1)、头孢拉定0.04μg mL~(-1)、头孢羟氨苄0.08μg mL~(-1)和头孢氨苄为0.1μg mL~(-1)。该方法已经成功应用于牛奶样品中β-内酰胺类抗生素的测定。
3．微流动注射分析芯片用于活体在线检测的研究
(6)微透析—微滴进样化学发光微流控芯片测定活体中的葡萄糖
微透析取样技术已成功用于活体分析，具有取样量少的特点，因此与微流动注射分析芯片的结合具有相当的优势。本文建立了在线微透析取样-化学发光微流控芯片测定活体中葡萄糖的方法。化学发光微流控芯片采用的是微滴进样方式，微滴形成于毛细管的尖端，其样品体积为4.5μL。制作微流控芯片的材料为聚甲基丙烯酸甲酯(PMMA)，利用溶胶-凝胶固定化的方法将辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOD)固定在芯片微流通池的内表面上。葡萄糖被GOD氧化为D-葡萄糖酸和H_2O_2，H_2O_2在HRP的存在下氧化鲁米诺产生化学发光。使用微透析探针以20 h~(-1)的频率在兔子血液中采样，成功应用于在线测定兔子血样中的葡萄糖。
(7)微透析取样-化学发光微流动分析芯片活体在线监测血液中Cr(Ⅲ)的代谢
建立了微透析取样-化学发光微流动分析芯片系统用于活体在线监测家兔血液中Cr(Ⅲ)的代谢，基于Cr(Ⅲ)-luminol-H_2O_2体系来进行测定。在聚甲基丙烯酸甲酯的芯片上集成了阴离子交换微柱，芯片上微通道的宽为200μm，深为100μm。插在兔子耳缘静脉内的微透析探针连接的灌流液的流速为5μL min~(-1)，微透析的样品体积为0.4μL，最终实现了兔子血液内Cr(Ⅲ)的代谢活体在位监测。
After the concept of micro total analysis system (uTAS) was introduced by Manz et al. in 1990, the field ofμTAS is growing rapidly. Because miniaturization is currently considered as one of the most important trends in the development of analytical instrumentation, the ultimate purpose ofμTAS is the integration of the entire analytical process on a micro-device. The micro-flow injection system on the chip is part of theμTAS and also has many advantages.
The dissertation is made up of two sections. One is the review ofμTAS and micro-flow injection system on the chip. Another is the research work. In the first part of the research work, the micro-flow injection systems which are used for food analysis and environment analysis are introduced. Different sampling mode, including double-T sampling mode, micro-valve sampling mode and discrete microdroplet sampling mode, is used. With the sampling modes, the reagent consumption is reduced and the sensitivity is improved. In the second part, combining with the solid phase extraction technique, a chemiluminescence (CL) micro-flow system is presented for determination ofβ-lactam antibiotics in milk. In the last part of the research work, microdialysis sampling technique was used on the micro-flow injection system for the determination of glucose and Cr(III) metabolism in blood.
1. Investigation on the chemiluminescence micro-flow injection analysis on a chip
(1) Determination of benzoyl peroxide in flour
Benzoyl peroxide(BP) is a common additive in flour because of its bleaching and sanitizing properties, but excessive benzoyl peroxide in flour could induce allergic reactions, weak cancer causing, Vitamin E and nutrients destruction. A rapid and sensitive chemiluminescence microfluidic chip fabricated in polymethyl methacrylate for determination of benzoyl peroxide in flour is described. The width of the microchannel was 200 urn and the depth was 100μm in the chip (50×40×5 mm). The total length of the microchannel was 67 mm with the detection area of 5×5 mm~2. Benzoyl peroxide can directly oxidize luminol to produce chemiluminescence. Two injectors of double-tee injector and microvalve injector were used. The sampling volume was 0.16μL for the double-tee injector and 0.25μL for the microvalve injector. The linear range of the benzoyl peroxide concentration was 8×10~(-7)-1×10~(-4) g mL~(-1). The detection limit was 4×10~(-7) g mL~(-1) and the R.S.D for eight times is 3.06% for the double-tee injector and 2.41% for the microvalve injector. The proposed method has been successfully applied to the determination of benzoyl peroxide in flour.
(2) Determination of hypochlorite in tap water
A micro-flow injection chemiluminescence detection system fabricated in Polymethyl methacrylate (PMMA) (50mm×40mm×5mm) for determination of hypochlorite in tap water is described, based on the direct chemiluminescence reaction of hypochlorite and luminol. The microchannel on the micro-flow injection chemiluminescence detection system are made by laser ablation (200μm width, 150μm depth) and the sampling volume was about 0.3μL with double-tee mode. The linear range of the hypochlorite concentration was 3.0×10~(-7)-1.0×10~(-4)g/mL. The detection limit is 1.4×10~(-7)g/mlL and the R.S.D for 11 times is 4.5%. The system is easy, fast (sample throughput 368h~(-1)) and reduces reagent consumption. The proposed method has been successfully applied to the determination of hypochlorite in tap water.
(3) Rapid determination of chemical oxygen demand in water
Chemical oxygen demand (COD) is an important parameter for water monitoring because it can represent the organic contaminants in water. A fast, continuous and automatic analysis method for COD determination was needed. A chemiluminescence (CL) micro-flow system for rapid determination of chemical oxygen demand (COD) in water at room temperature is proposed in this paper. In this system, potassium dichromate is reduced to Cr~(3+) in 2 mol L~(-1) H_2SO_4 during chemical oxidation of COD-substances in sample and Cr~(3+) can be measured by the luminol-H_2O_2 CL system. The polymethyl methacrylate micro-flow chip with discrete microdroplet sampling was used here. Effects for COD determination (such as pH, concentrations, the channel length, and interference) were investigated. The linear range for COD determination was 0.27-10 g L~(-1) and the detection limit was 100 mg L~(-1). The proposed method has been successfully applied to the determination of COD in wastewater samples. The datas obtained by the present method were fairly in good agreement with those obtained by the titrimetric method.
(4) Simultaneous determination of Cr (III) and Cr (VI) in water
A sensitive method for the simultaneous determination of chromium (III) (Cr~(3+)) and chromium (VI) (Cr_2O_7~(2-)) using micro-flow chemiluminescence system was developed. The chemiluminescence detection was based on the catalytic effect of Cr (III) on the chemiluminescence reaction of luminol and hydrogen peroxide. Cr (VI) was on-line reduced to Cr (III) with hydrogen peroxide, and then was detected. The polymethyl methacrylate (PMMA) micro-flow system with three plates was used to realize this procedure. The micro-flow system used the discrete sampling with microdroplet, which can be formed at the tip of the capillary with the sampling volume of 5μL. The width of the microchannel was 200μm and the depth was 100μm. The linear ranges were 5×10~(-8)-1×10~(-5)mol L~(-1) for chromium(III) and 1×10~(-7)-1×10~(-5)mol L~(-1) for chromium(VI). The detection limits were 2×10~(-8) mol L~(-1) for chromium (III) and 4×10~(-8)mol L~(-1) for chromium (VI). The proposed method has been successfully applied to the simultaneous determination for chromium (III) and chromium (VI) in water samples.
2. Investigation on the chemiluminescence micro-flow injection analysis on a chip with on-line solid phase extraction
(5) Determination ofβ-Lactam antibiotics in milk
β-Lactam antibiotics are widely used in the treatment of lactating dairy cattle for several infections. Antibiotic residues in milk, besides inhibiting start of cultures in the production of milk products, can provoke allergic reactions in some hypersensitive individuals. On-line introducing in SPE separation technique for interfering element removal is undoubtedly an ideal choice for interference elimination in CL analysis. In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination ofβ-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect ofβ-lactam antibiotics on the luminol-K_3Fe(CN)_6 CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50×40×5mm) with the microchannels of 200μm wide and 150 urn deep. C_(18)-modified silica gel was packed into the microchannel (length: 10mm; wide: 1mm; depth: 500μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5μg mL~(-1) of penicillin, 0.04μg mL~(-1) of cefradine, 0.08μg mL"1 of cefadroxil and 0.1 ug mL"1 of cefalexin. The proposed method was also applied to analyzing theβ-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.
3. Investigation on the chemiluminescence micro-flow injection analysis on a chip by microdialysis sampling
(6) Determination of glucose in vivo
A micro-flow chemiluminescence (CL) system in vivo for glucose determination by the on-line microdialysis sampling is described in this paper. The micro-flow CL system uses discrete sample droplets, which formed at the tip of the capillary with the sampling volume of 4.5μL. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and glucose oxidase (GOD) on the inside surface of the micro-flow cell which was fabricated in polymethyl methacrylate (PMMA). The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H_2O_2, then H_2O_2 oxidizing luminol to produce CL in presence of HRP. The microdialysis probe was utilized for sampling in the rabbit blood; the sample throughput was 20 h~(-1) . The glucose level in blood of the rabbit was on-line monitored with good results.
(7) On-line monitoring of chromium metabolism in vivo
A micro-flow injection chemiluminescence system based on the Cr (III)-luminol-hydrogen peroxide chemiluminescence system for in vivo on-line monitoring of chromium (III) metabolism in blood by microdialysis sampling is described in this paper. It was fabricated in polymethyl methacrylate(PMMA) of two plates and the separation part of anion exchange resin was also integrated on the micro-flow system. The width of the microchannel was 200μm and the depth was 100μm on the plate(50×40×5mm). A microdialysis probe, implanted in the vein of rabbit, was perfused with perfusate at a flow rate of 5μL min~(-1). The dialysate sample volume was about 0.4μL. The metabolism of the chromium (III) in the blood of the rabbit was monitored after administration of the chromium (III) to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.
引文
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106．盖宏伟，於林芬，马银法等，一种基于压力和电动力联用的微流控芯片进样方法：03134125．X，2003．
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109 黄艳贞，方群，李丹妮，连续进样的重力驱动微流控芯片流动分析系统，高等学校化 学学报，2004，25：1628-1631．
110．徐章润，王世立，樊晓峰，王福仁，方肇伦，小型可连续进样微流控芯片分析仪的研制，分析化学，2003，31：1527-1530．
111．吴建刚，岳瑞峰，曾雪锋，刘理天，新型开放式液滴驱动芯片，电子器件，2005，28：699-702．
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17．徐溢，微芯片化学反应器实现酶催化化学发光测定葡萄糖的研究，高等学校化学学报，2003，24：49-51．
18．颜流水，梁宁，罗国安，王义明，王建勇，整体式PDMS电泳芯片快速成型及高灵敏化学发光检测氨基酸，高等学校化学学报，2003，24：1193-1197．
19．苏荣国，林金明，屈锋，高云华，陈志锋，金属离子Cu2+，Co2+，Ni2+的微芯片电泳分离及化学发光检测，化学学报，2003，61：885-888．
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