Micromachined detectors for an enzyme-based FIA

Micromachining techniques were applied to construct biosensor systems. The micromachined biosensors have small size, low production cost, and good reproducibility. We made some detection units for flow injection analysis (FIA). An electrochemical flow cell was fabricated, and both the enzyme immobilized column and electrochemical detector were integrated onto the same chip. A chemiluminescence detector was also fabricated and applied to the determination of glucose and lactic acid contained in human serum and urine.     

Nanomolar Level Amperometric Determination of ATP Through Substrate Recycling in an Enzyme Reactor in a FIA System

Abstract

ATP, adenosine-5′-triphosphate, was determined by recycling in an enzyme reactor with co-immobilized pyruvate kinase and hexokinase in the presence of glucose, NAD⁺, and phosphoenolpyruvate, PEP. Recycling produces glucose-6-phosphate which is converted to an equivalent amount of NADH by glucose-6-phosphate dehydrogenase. The NADH is detected at a graphite flow-through electrode modified with an adsorbed 3,3′-bis(benzo[a]phenoxazin-7-ium, 5-amino-9-(diethylamino))1,4,N,N′-diamidobenzene, BPT. Oxidation of NADH takes place at 0 mM vs Ag/AgCl due to the adsorbed phenoxazine. The amplification factor is directly proportional to the residence time in the reactor and it is increased as the flow rate decreases; it becomes350 at a flow rate of 0.07 ml/min. The amplification factor can be increased further by a controlled stop-time recycling; it became 1200 at a stop-time of 12 min. A theoretical expression for the amplification factor was derived and it shows that the amplification depends o n the residence time and the pseudo-first order rate coefficients of the recycling enzyme systems. The response was linear over more than three decades, from 1 nM to 5 μM ATP. The detection limit, 1 nM ATP was set by cofactor impurities in the reagent rather than by system sensitivity or noise.     

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.     

基于反相有机整体柱的新型蛋白质酶反应器

以牛血清白蛋白(BSA)为标准样品,以鉴定的独立肽段数目、蛋白质鉴定覆盖率和漏切率为考察指标,通过考察酶解缓冲液、反相柱基质的多孔结构、酶解反应时间和整体柱的疏水性对反相蛋白质酶反应器酶解效率的影响,构建了一个基于反相有机整体柱的新型酶反应器,此反应器具有制备简单、通透性好、酶解时间短和蛋白质鉴定覆盖率高等特点。     

牛血红蛋白催化化学发光反应与FIA联用测定人血清中的血糖

基于葡萄糖氧化酶催化葡萄糖氧化并释放过氧化氢,再将其偶合到含鲁米诺的发光体系中;同时采用牛血红蛋白作为发光体系的催化剂,其发光强度在一定范围内与过氧化氢的浓度呈良好的线性关系,据此建立了一种间接测定人体血清中葡萄糖含量的新方法。本法与FIA技术联用,进样频率为112样/小时,线性范围为3.0×10-6~1.0×10-4mol.L-1,检出限可达1.4×10-7mol.L-1。     

测定金鸡纳碱的流动注射化学发光新体系

过氧化氢和高碘酸钠溶液混合能产生弱化学发光,在这一体系中,金鸡纳碱的加入能极大地增强这一化学发光强度,该文对这一化学发光进行了初步的探讨,提出了可能的发光机理,并据此建立了测定金鸡纳碱(奎宁、奎尼丁和辛可宁)的高灵敏度化学发光法;测定奎宁、奎尼丁和辛可宁的线性范围分别为6.0×10-5～1.0×10-2g/L、4.0×10-5～1.0×10-2g/L和1.0×10-3～1.0×10-1g/L,检出限分别为2.1×10-5g/L,1.4×10-5g/L,3.5×10-4g/L;该法适用于奎宁药物的测定.     

固定化酶反应器及其在蛋白质组研究中的应用

综述了近年来国内外酶固定化载体的研究进展,侧重于无机材料和有机聚合物材料上的固定化酶方法;此外,也介绍了固定化胰蛋白酶反应器与分离系统联用在蛋白质样品分析中的应用,并展望了固定化酶反应器的研究方向及其在蛋白质组中的应用前景。     

固定化谷氨酸脱羧酶柱式反应器测定L-谷氨酸

用羧甲基化的N,N’-甲叉双丙烯酰胺交联的烯丙基葡聚糖(简称CM-CADB)凝胶树脂为新型载体,研究了谷氨酸脱羧酶(GDC)在CM-CADB上的固定化与环境的依赖关系.确定了酶固定化最佳条件,研究了固定化GDC的活性与底物浓度、pH、温度的依存性,动态响应特性,热稳定性和寿命,求算了米氏常数.将固定化GDC酶柱与进样系统具CO_2气敏电极的离子活度分析器-计算机数据采集系统匹配,构成酶反应谷氨酸检测装置,测定了固定化GDC酶柱的能斯特线性响应曲线,线性回归方程为y=43.3x+181.6,r为0.9936.     

Development of A Dual Channel Chemiluminescence FIA System for Rapid Measurement of Fish Freshness

Abstract

A dual channel chemiluminescence FIA system for the rapid measurement of the fish freshness index KI $$ where [IMP], [HxR] and [Hx] are, respectively, the inosine 5′-monophosphate, inosine and hypoxanthine contents of fish meat was developed. The system consisted of a pair of chemiluminescence FIAs (CL-FIA); one for the measurement of [IMP] + [HxR] + [Hx], and the other for [HxR] + [Hx]. IMP, HxR or Hx were measured using a reactor comprising immobilized alkaline phosphatase, purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD). The calibration curves for IMP, HxR or Hx were linear from 0.06 μM to 100 μM for a sample volume of 20 μl. The time for a single KI measurement was less than 1 min. This fish freshness FIA system is rapid, convenient and applicable for fish freshness measurements in real fish samples.     

酶膜反应器及其在生物催化领域的应用

酶膜反应器是一种新型的生物催化反应设备,它是反应与分离相耦合的装置,集产品分离、纯化和酶回收利用于一体,具有其它方法不可比拟的优势。在强调资源节约,环境友好和清洁化生产技术的今天,显示一些特别的优点,近年来已成为热门的研究领域。本文主要介绍了酶膜反应器的基本原理、特征及其分类,并根据分类对近年来酶膜反应器在生物催化领域...     

多相酶膜反应器合成生物活性二肽

生物活性肽;多相酶膜反应器合成生物活性二肽     

Micromachined electrochemical T-switches for cell sorting applications

MEMS micro-T-switches actuated via electrochemical bubbles for cell sorting applications in a monolithic chip level are proposed and successfully demonstrated. The electrolysis-bubble actuator, which has the features of low operation temperature and high surface-tension force, is developed to actuate the micro-T-switch sorting structure in our device. The double T-structure design, the T-shape microchannel with the movable micro-T-switch structure located at the junction of the T-shape microchannel, with the electrolysis-bubble actuator makes an active-binary switch function available for cell sorting applications. The room temperature operation and the low voltage required for electrolysis actuation minimize the possibility of cell-damage that happens in the conventional high electric separation instruments, such as flow cytometry. The function of our micro-T-switch chip with a low required actuation voltage of 3.0 approximately 3.5 V is demonstrated by using human hepatoma cells in this paper. The pH-value measurements characterize the pH-value variation and distribution in the actuating chambers and the mainstream microchannels to trace the possible liver-cell injury due to the pH-value variation during electrolysis-actuation operation. The 84.1% cell viability in the sorted human hepatoma cells through our micro-T-switch sorter is observed via the fluorescence assay technique. Furthermore, 70.2% of total injected cells recover in culture after sorting and grow into colonies after micro-T-switch sorting operation. In this paper, we describe the design, microfabrication, and characterization of our micro-T-switch cell-sorting chip. We also report the cell-sorting demonstration and the cell viability results for the mammalian liver cells through our micro-T-switch cell-sorting chip.     

高效液相色谱中固定酶柱后反应器的应用

本文评述了固定酶柱后反应器在高效液相色谱中的应用。阐述了高效液相色谱与固定酶柱后反应器联用的原理,介绍了固定酶柱后反应器的主要类型。评述了固定酶柱后反应器在生化分析和临床分析中的应用。引文43篇。     

Micromachined capillary cross-connector for high-precision fraction collection

A new approach for high-precision fraction collection of double-stranded DNA fragments by capillary electrophoresis coupled to a micromachined plastic capillary cross-connector is presented. The system design integrates four fused-silica capillaries with an acrylic cross-channel connector. The cross-channel structure was introduced to enhance the efficiency of the fraction collection process by electrokinetic manipulations. Following the detection of the sample zone of interest at or slightly upstream of the cross during the separation mode, the potentials were reconfigured to collection mode to direct the selected analyte zone into the corresponding collection vial, while keeping the rest of the sample components virtually stopped within the separation capillary. In this way the spacing between consecutive bands of interest can be physically increased, allowing precise isolation of spatially close sample zones. After collection of the target fraction the separation mode is resumed, and the separation/collection cycle is repeated until all desired sample zones are separated and captured. The capillary cross-connector was fabricated of a transparent acrylic substrate by microdrilling flat end and through channels, matching precisely the O.D. and I.D. of the connected capillary tubing, respectively. This design provided a close to zero dead volume connection assembly for the separation and collection capillaries causing minimal extra band broadening during high-precision micropreparative DNA fractionation.     

Immobilized Enzyme Reactor in On-line LC and Its Application in Drug Screening

This study is to give a brief introduction of immobilized enzyme reactor (IMER) in on-line LC and its application in drug screening. The literature of immobilization techniques, immobilization supports and determination of immobilized enzyme activity were reviewed; the application in the drug screening is briefly introduced. It was found that IMER increased the enzymatic stabilization, strikingly shortens reaction time and can be used to perform fast screening of enzyme inhibitor. IMER has wide fields in drug screening application.     

复合纤维素膜固定化胰蛋白酶反应器及其应用于蛋白质酶解

合成了甲基丙烯酸缩水甘油酯-纤维素复合膜,并以此膜为基质共价键合固定化胰蛋白酶,以N-苯甲酰-L-精氨酰乙酯(BAEE)为底物,应用高效液相色谱系统测定了酶固定化膜柱的催化反应特性。研究结果表明:温度、pH值、离子强度、有机溶剂及蛋白变性剂等都对固定化酶的活力有一定的影响。在最适条件下,固定化胰蛋白酶的活力为17800U/g干膜,蛋白载量为3.6mg/g(≈0.15μmol/g)干膜,活性回收率达到52%.固定化酶表现出较高的使用和储藏稳定性,在40℃下,水解BAEE底物24h活力无显着变化。固定化酶膜柱在4℃冷藏保存100d仍保存90%以上的水解活力。固定化酶反应器被应用于蛋白质酶解的肽谱实验。