含微混合器的微芯片设计和性能研究

微型化分析系统是分析化学的新兴研究领域，研究中设计制作的微芯片，由按层流原理设计制作的交叉分液汇合式微混合器及长反应管道组成。两种荧光染料混合实验可以直观地观测到微混合器的混合效果，而对Ｈｇ＾２＋－若丹明Ｂ的荧光猝灭反应的标准曲线法定量分析表明该设计的微芯片可应用于化学反应过程的定量测试。该微芯片在化学与生化分析和合成等分析检测领域具有巨大的潜在应用价值。     

An integrated on-chip sirtuin assay

A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/μL hSIRT1 was carried out within the serpentine micromixer using a 400 μM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.     

A study on the system of nonaqueous microchip electrophoresis with on‐line peroxyoxalate chemiluminescence detection

This article describes a further development of our previously reported miniaturized analysis system of microchip electrophoresis with on‐line chemiluminescence detection. The system, developed first time for nonaqueous microchip electrophoresis with peroxyoxalate chemiluminescence detection, consists of a suction pressure device for sample or reagent introduction, a constant voltage supplied for electrophoretic separation, an either hydrophilic or hydrophobic porous polymer plug for preventing chemiluminescence reagent flowing upstream and a spiral detection channel for enhancement of both detection sensitivity and reproducibility. Especially, by using organic solvent as BGE medium, the developed system avoided the interface problem between aqueous running buffer and low‐water‐content chemiluminescence solvent in previous reports. The influencing factors on chemiluminescence signal were optimized using rhodamine 6G as model molecule. The system performance was further investigated in the experiment of separation of hydrophilic rhodamine dyes and analysis of hydrophobic polycyclic aromatic hydrocarbon, providing the detection limit (S/N = 3) of 3.5 nmol/L for rhodamine 123, 6.8 nmol/L for rhodamine 6G, and 60 nmol/L for 1‐aminopyrene, respectively. The experimental results showed the system offered a number of benefits, including compact structure, high sensitivity, good reproducibility, and a wide range of application prospect.     

用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

葡萄糖是临床化学诊断以及食品分析中重要的检测项目 ,最常用的测定方法是采用葡萄糖氧化酶(GOD)催化葡萄糖与氧分子间反应 ,生成葡萄糖酸和过氧化氢 [1] ,而对过氧化氢的检测则可采用过氧化酶 (POD)催化鲁米诺的化学发光反应进行 [2 ] .FIA对整个过程的实现是十分有效的方式 ,但由于多采用固定化酶反应器 [3～ 5] 使其在制备及分析上较复杂且费用高 .由 Manz等[6] 提出的微型全分析系统(μ- TAS)在针对不同体系的微量分析及在线监测上均具有突出的优越性 .本文使用的含微混合器的微芯片化学反应器采用μ- TAS设计思想 ,建立了化学发…     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.     

采用甲基丙烯酸丁酯整体微柱芯片系统测定血样中痕量异烟肼

将合成的甲基丙烯酸丁酯(BMA)整体柱与微流控芯片技术结合,在PMMA芯片上以K3Fe(CN)6-NaOH-异烟肼化学发光体系为样品对象,在优化混合发光试剂比例和流速以及选择适合的洗脱液基础之上,实现了BMA整体微柱对异烟肼样品的富集作用,平均富集倍数和回收率分别达到16.8和84.2%,由此建立了流动注射化学发光(FIA-CL)芯片系统测定血液中痕量异烟肼的浓度的方法,可有效地实现异烟肼血药浓度分析的片上预处理和快速测定,检出限低于0.2 mg/L。     

Microfluidic cell disruption system employing a magnetically actuated diaphragm

A microfluidic cell lysis chip equipped with a micromixer and SPE unit was developed and used for quantitative analysis of intracellular proteins. This miniaturized sample preparation system can be employed for any purpose where cell disruption is needed to obtain intracellular constituents for the subsequent analysis. This system comprises a magnetically actuated micromixer to disrupt cells, a hydrophobic valve to manipulate the cell lysate, and a packed porous polymerized monolith chamber for SPE and filtering debris from the cell lysate. Using recombinant Escherichia coli expressing intracellular enhanced green fluorescent protein (EGFP) and lipase as model bacteria, we optimized the cell disruption condition with respect to the lysis buffer composition, mixing time, and the frequency of the diaphragm in the micromixer, which was magnetically actuated by an external magnetic stirrer in the micromixer chamber. The lysed sample prepared under the optimal condition was purified by the packed SPE in the microfluidic chip. At a frequency of 1.96 Hz, the final cell lysis efficiency and relative fluorescence intensity of EGFP after the cell disruption process were greater than 90 and 94%, respectively. Thus, this microfluidic cell disruption chip can be used for the efficient lysis of cells for further analysis of intracellular contents in many applications.     

A high-throughput solid-phase extraction microchip combined with inductively coupled plasma-mass spectrometry for rapid determination of trace heavy metals in natural water

Herein, a hyphenated system combining a high-throughput solid-phase extraction (htSPE) microchip with inductively coupled plasma-mass spectrometry (ICP-MS) for rapid determination of trace heavy metals was developed. Rather than performing multiple analyses in parallel for the enhancement of analytical throughput, we improved the processing speed for individual samples by increasing the operation flow rate during SPE procedures. To this end, an innovative device combining a micromixer and a multi-channeled extraction unit was designed. Furthermore, a programmable valve manifold was used to interface the developed microchip and ICP-MS instrumentation in order to fully automate the system, leading to a dramatic reduction in operation time and human error. Under the optimized operation conditions for the established system, detection limits of 1.64–42.54 ng L⁻¹ for the analyte ions were achieved. Validation procedures demonstrated that the developed method could be satisfactorily applied to the determination of trace heavy metals in natural water. Each analysis could be readily accomplished within just 186 s using the established system. This represents, to the best of our knowledge, an unprecedented speed for the analysis of trace heavy metal ions.     

Analysis of inorganic and small organic ions with the capillary electrophoresis microchip

Capillary electrophoresis microchip devices are receiving considerable attention due to their versatility, portability, and sample handling capabilities. This article is a comprehensive review of the analysis of inorganic and small, charged organic species on microchip platforms. The application of conductivity, amperometry, laser-induced fluorescence, absorbance, and chemiluminescence detection methods are discussed. The potential utilization of these devices for miniaturized analytical systems is described.     

A G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence assay for highly sensitive detection of biotin in flour

Quantitative analysis of biotin in biological fluids, foods, and pharmaceutical is important for diagnosis and treatment of biotin‐related diseases and health maintenance. In this work, a novel G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence (CL) assay method was established for rapid and highly sensitive detection of biotin. This method is based on the specific binding between biotin and streptavidin, the catalytic CL characteristics of G‐quadruplex/hemin DNAzyme to the oxidation–reduction reaction of hydrogen peroxide with luminol, and the on‐line separation function of microchip electrophoresis. Under the optimal experimental conditions, on‐chip biotin analysis was achieved within 1 min. The CL intensity is linearly proportional to the concentration of biotin in the range of 13–630 nM with a detection limit of 6.4 nM. The proposed method has been applied for the detection of biotin in flour, biotin contents in three flour samples are found in the range of 199–223 ng/g with a mean value of 214 ng/g. The recoveries were in the range of 94–103%. With excellent sensitivity and good selectivity, the proposed method could be applied in a wide range of biological fluids, foods, and pharmaceutical analysis.     

Grazing-exit and micro X-ray fluorescence analyses for chemical microchips.

Grazing-exit x-ray fluorescence (GE-XRF) and micro x-ray fluorescence (micro-XRF) methods were applied to chemical microchips as a detection method. Since an energy-dispersive x-ray detector was used, the simultaneous detection of multiple elements was possible. An analyzing region was especially designed on the microchip so that a sample solution could be dried and concentrated in a suitable area corresponding to the size of the primary x-ray beam. Finally, it was confirmed that both analytical methods could be combined well for use with a microchip. In GE-XRF, the background intensity in the XRF spectrum was reduced at grazing-exit angles. In addition, a good relationship between the x-ray fluorescence intensities and the concentrations of standard solutions that were introduced into the microchip was obtained. This indicates that the GE-XRF method is feasible for trace elemental analysis in chemical microchip systems. In micro-XRF, an attempt was made to concentrate and dry the analyte within a small analyzing region. The preliminary results indicated that the micro-XRF method could be applied for the analysis of microchips.     

Development of a surface-reaction system in a nanoliter droplet made by an ink-jet microchip.

A surface-reaction system in a nanoliter water pool using an ink-jet microchip was developed. The reaction system in the nanodroplets formed on a poly(dimethylsiloxane) (PDMS) coated glass slide increased the diffusion-controlled reaction without using a nano-pump, specialized connector or highly sensitive detector. When nanoliter droplets were placed on the PDMS surface with a distance of 100 microm between them by the ink-jet microchip, the repeatabilities of the fluorescence intensity were 2.9% RSD (n = 7). The used ink-jet microchip had 4 different injection ports, and the distance between the ports was 0.995 mm. It was necessary to correct the distance in order to mix or dilute samples in a small droplet. The correction was successfully performed by moving the X-Y stage using inhouse-made software. A linear relationship was obtained between the Resorufin concentrations and the fluorescence intensity. We applied this system to an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin A (IgA), and observed a difference in the fluorescence intensity derived from the amount of IgA (blank, 6.25 ng/mL, 12.5 ng/mL). These results show the usefulness of the open-type micro-analytical systems proposed by us.     

Development of an immune microanalysis system by use of peroxyoxalate chemiluminescence detection.

We developed an immune microanalysis system incorporating chemiluminescence detection, where the peroxyoxalate chemiluminescence (CL) detection using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO)-hydrogen peroxide (H2O2)-fluorescein isothiocianate (FITC) reaction was newly adopted. The analysis system performed the following three processes on a microchip: immune reaction for high selectivity, electrophoresis for formation and transportation of the sample plug, and CL detection. The immune reaction was carried out using an antibody-immobilized glass bead. The glass bead was placed in one of the reservoirs in the microchip along with antigen (analyte) and a known amount of FITC-labeled antigen to set up a competitive immune reaction. The reactant after the immune reaction was fed electrophoretically into the intersection, resulting in a sample plug. The sample plug was then moved into another reservoir containing TDPO-H2O2 acetonitrile solution. At this point, CL detection was performed. The system described here was capable of determining human serum albumin or immunosuppressive acidic protein as a cancer marker in human serum.     

A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection

A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H(2)O(2) system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30 s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0 nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.     

A palm-sized surface plasmon resonance sensor with microchip flow cell

A small-sized surface plasmon resonance (SPR) sensor with a microchip flow cell has been developed for the purpose of enhancing the sensitivity of the SPR detector for low molecular weight compounds. This portable differential SPR detector consisted of an LED, two cylindrical lenses, a round prism, a divided mirror, a CCD, electronics, and a polydimethylsiloxane/gold microchip with two flow paths (10 mm long, 1 mm wide, 20-100 μm deep). 3-Mercaptopropyltrimethoxysilane was used for sealing the microchip. The performance of the on-site orientated SPR detector was estimated using sucrose and IgA. A drastic change in the SPR intensity appeared. The depth of the flow cell was in inverse proportion to the SPR intensity. Compared to a conventional flow cell having the size of 10 mm (L) × 1 mm (W) × 1 mm (D), its sensitivity to 10% sucrose and 0.9 nM IgA increased about 11 and 39 times, respectively. This phenomenon seemed to be due to the increase in the substance on the SPR sensor based on its size effect. These results showed that the application of the microchip sensor for SPR measurement has the possibility for improvement of the SPR intensity for low molecular substances.     

CHARACTERISTICS OF THE CHEMILUMINESCENCE FROM THE BLOOD PLASMA OF A NORMAL HUMAN POPULATION

Oxygen-dependent chemiluminescence was detected from human blood plasma. The intensity of the chemiluminescence increased about three-fold under oxygenation and decreased almost to the background (zero) level under a nitrogen atmosphere. The blood plasma from a sample (n = 100) of donors was tested to determine the variability of several properties of the chemiluminescence in a normal population. No statistically significant difference in blood plasma chemiluminescence between genders was found, but there was a slight increase in luminescence intensity with age. However, the results were found to be dependent on a number of other factors, such as diet, smoking and the length of time between the donor's last meal and the sampling of the blood. Some of the trends in the results coincide with similar trends in the plasma lipoprotein levels and thus support the suggestion that the chemiluminescence arises from the decomposition of lipid hydroperoxides. These factors must all, therefore, be taken into account when using chemiluminescence as an indicator of illness.     

A simple and compact blue diode laser powered excitation source for fluorescence detection in capillary electrochromatographic microchip separation

A simple and compact fluorescence excitation source was prepared using a 405 nm blue laser diode module and characterized in capillary electrochromatographic or capillary electrophoretic microchip separation. An inexpensive blue laser diode module with a tiny focusing lens was simply mounted at the center of an aluminum block on a miniature linear motion guide for heat dissipation and position control. A slit unit has a series of fifteen laser-machined slits with 1 mm space along the direction of the separation channel of the microchip above this unit. The laser beam was focused through a slit with 50 μm width to the separation channel at the position of a desired length. Although the excitation source unit was connected to a simple current controlled power supply, it was stable with 0.1% drift per hour and 1.3% (1σ) fluctuation in intensity. This simple excitation source can be prepared easily with inexpensive minimum optical components and mounted with a microchip on the stage of an ordinary fluorescence microscope for daily separation studies using a CE or CEC microchip. The applicability of the excitation source was evaluated with FITC-amino acid derivative mixtures using a polymer based CEC microchip packed fully with submicron silica beads in its microchannel.     

Recent developments in optical detection methods for microchip separations

This paper summarizes the features and performances of optical detection systems currently applied in order to monitor separations on microchip devices. Fluorescence detection, which delivers very high sensitivity and selectivity, is still the most widely applied method of detection. Instruments utilizing laser-induced fluorescence (LIF) and lamp-based fluorescence along with recent applications of light-emitting diodes (LED) as excitation sources are also covered in this paper. Since chemiluminescence detection can be achieved using extremely simple devices which no longer require light sources and optical components for focusing and collimation, interesting approaches based on this technique are presented, too. Although UV/vis absorbance is a detection method that is commonly used in standard desktop electrophoresis and liquid chromatography instruments, it has not yet reached the same level of popularity for microchip applications. Current applications of UV/vis absorbance detection to microchip separations and innovative approaches that increase sensitivity are described. This article, which contains 85 references, focuses on developments and applications published within the last three years, points out exciting new approaches, and provides future perspectives on this field.     

Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition

A high-performance determination system for alpha-amylase isoenzyme activities in human plasma involving microchip electrophoresis with a plastic chip was developed. The combination of microchip electrophoresis for substrate and hydrolysate separation and an immunoinhibition method for the differentiation of isoenzyme activities using antihuman salivary amylase (S-AMY) mAb allowed the highly selective determination of amylase isoenzyme (S-AMY and pancreatic amylase (P-AMY)) activities even in a complex matrix such as a crude plasma sample. We used 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltohexaose (G6) as a substrate. Amylase in a human plasma sample hydrolyzed APTS-G6 into APTS-maltotriose (G3) and G3, which was measured as the fluorescence intensity of APTS-G3 on microchip electrophoresis. A double logarithm plot revealed a linear relationship between amylase activity and fluorescence intensity in the range of 5-500 U/L of amylase activity (r2=0.9995, p<0.01), and the LOD was 4.38 U/L. Amylase activities in 13 subjects determined by the present method were compared with the results obtained by conventional methods with nitrophenylated oligosaccharides as substrates, respectively. Good correlations were observed for each method on simple linear regression analysis (both p<0.01). The reproducibilities of within-days for total amylase and P-AMY were 2.98-6.27 and 3.83-6.39%, respectively, and these between-days were 2.88-5.66 and 3.64-5.63%, respectively. This system enables us to determine amylase isoenzyme activities in human plasma with high sensitivity and accuracy, and thus will be applicable to clinical diagnosis.     

流动注射化学发光法测定呋塞米

在酸性条件下呋塞米对Ce(Ⅳ)-Na_2SO_3弱化学发光体系具有很强的增敏作用,据此建立了流动注射化学发光法测定呋塞米的新方法.该法测定呋塞米的线性范围为0.01～1.0 mg/L,检出限为4×10~(-3)mg/L(3σ),RSD为2.2%(n=11,ρ=0.2mg/L),回收率为95%～105%.该法已应用于片剂和针剂中呋塞米含量的测定.