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Lab‐on‐a‐Chip Biosensor for Glucose Based on a Packed Immobilized Enzyme Reactor
Abstract
In this work, the development of a packed immobilized enzyme reactor (IMER) and its integration to a capillary electrophoresis microchip is described. The present microchip design differs from others, in the fact that the same design could be used with or without the particles and, just by changing the material used to pack the IMER, different analytes can be detected. The applied procedure involves the separation of the target analyte by capillary electrophoresis (CE), which is then coupled to a post-column IMER that produces H2O2. The H2O2 produced is finally detected downstream at the surface of a working electrode. Glucose was detected above 100 μM by packing particles modified with glucose oxidase at the end of the separation channel. The analytical performance of the microchip-CE has been demonstrated by performing the separation and detection of glucose and noradrenaline. Additions of fructose showed no effect on either the peak position or the peak magnitude of glucose. The microchip-CE-IMER was also used to quantify glucose in carbonated beverages with good agreement with other reports.
... Blanes et al. developed a packed IMER integrated in a PDMS electrophoresis microchip [67]. The working electrode was a gold microwire (25 μm diameter) set at the end of separation channel in a perpendicular position (see Fig. 7). ...
In this book chapter, the advances in the determination of carbohydrates and glycoconjugates by capillary and microchip electrophoresis with electrochemical detection (CE/ME-ED) will be studied, covering from 2002 to 2019. Amperometric detection, capacitively coupled contactless conductivity detection, and enzyme-based amperometric detection as well as electrochemical derivatization schemes will be critically discussed, showing their advantages and limitations. Furthermore, the most important applications in clinical, pharmaceutical, and food fields will also be highlighted. Future outlooks of CE/ME-ED for carbohydrate determination will also be shown, pointing out those strategies that may improve the sensitivity and selectivity of analytical methods.
... Other inherent drawback for the analysis of carbohydrates on ME devices refers to the lack of chromophore or fluorophore groups in these structures, making difficult their monitoring by using UV or LIF detection. Alternatively, electrochemical detectors have emerged as powerful detection modes to monitor electrophoretic separation of these compounds [38][39][40][41][42][43][44]. O'Shea et al. reported a pioneering study showing the use of pulsed amperometric detection (PAD) coupled to a conventional CE system for the determination of several carbohydrates [38]. ...
This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run‐to‐run repeatability (RSD < 1%). The achieved efficiencies ranged from 45,000 ± 6,000 to 70,000 ± 3,000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run‐to‐run repeatability and analytical reliability required for routine and quantitative analysis.
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... Such a device not only is superior to the previous enzymatic silica (~10e30 min) and overnight in-solution approach (>12 h) as reported [39,42,57,58], but also affords higher or comparable performance compared to diverse proteolysis platforms, e.g. microfluidic chips (~2e30 min digestion) [26,27], and capillary columns (~5e45 min digestion) [30,31]. ...
Mass spectrometry (MS)-based proteome profiling is essential for molecular diagnostics in modern biomedical study. To date, sample preparation including protein extraction and proteolysis is still very challenging and lack of efficiency. Recently tips-based sample preparation protocols exhibit strong potentials to achieve the goal of "a proteome in an hour". However, in-tip proteolysis is still rarely reported and far from ideal for dealing with complex bio-samples. In this work, nanoreactors encapsulated micropipette tips were demonstrated as high performance devices for fast (∼minutes) and multiplexing proteolysis to assist the profiling of cancer cells proteome. Nanoporous silica materials with controlled pore size and surface chemistry were prepared as nanoreactors and encapsulated in micropipette tips for efficient in situ proteolysis. The as-constructed device showed desirable sensitivity (LOD of 0.204 ± 0.008 ng/μL and LOQ of 0.937 ± 0.055 ng/μL), selectivity, stability (two months under −20 °C), reusability (at least 10 times), and little memory effect in MS based bottom-up proteomic analysis. It was used for comprehensive protein mapping from cancer cell lines. The number of identified proteins was increased by 18%, 22%, 52%, and 52% dealing with HepG2, F56, MCF7, and HCCLM3 cancer cells, compared to traditional in-solution proteolysis based bottom-up proteomic strategy. With the enhanced performance, our work built a novel, efficient and miniaturized platform for facile proteomic sample preparation, which is promising for advanced biomarkers discovery in biomedical study.
... As a compromise between signal magnitude and selectivity[45,46], the oxidation of hydrogen peroxide was performed at + 650 mV (vs. Ag|AgCl|KCl sat ) and followed by chronoamperometry. ...
... One possibility is the definition of microfluidic structures including steps (Ito et al., 2011;Malecha et al., 2009) or micropillars (Jang and Koh, 2010;Kim et al., 2009) which stop the movement of the particles in the fluid stream at one point of the system. Wires incorporated into microfluidic channels can serve the same purpose (Blanes et al., 2007). The other typical method to retain microparticles is the usage of magnetic particles (Hernandez et al., 2013;Lin et al., 2013;Sheng et al., 2012). ...
Microfluidic biosensing systems with enzyme-based detection have been extensively studied in the last years owing to features such as high specificity, a broad range of analytes and a high degree of automation. This review gives an overview of the most important factors associated with these systems. In the first part, frequently used immobilization protocols such as physisorption and covalent bonding and detection techniques such as amperometry and fluorescence measurements are discussed with respect to effort, lifetime and measurement range. The Michaelis-Menten model describing the kinetics of enzymatic reactions, the role of redox mediators and the limitations of the linear measurement range of enzymatic sensors are introduced. Several possibilities of extending the linear measurement range in microfluidic systems such as diffusion-limiting membranes and the flow injection setup are presented. Regarding the integration of enzymes into microfluidic systems during the fabrication process, the constraints imposed by the biomolecules due to the limited usage of high temperatures and solvents are addressed. In the second part, the most common forms of enzyme integration into microfluidic systems, i.e. in channels and on electrodes, on microparticles, on paper and thread and as injected enzyme solutions, are reviewed, focusing on fabrication, applications and performance.
Copyright © 2015 Elsevier B.V. All rights reserved.
Particle-immobilized enzymes have proven benefits when integrated into biosensors, typically via packed-bed approaches in microfluidic channels. These benefits include dramatically improving sensitivity by increasing the effective surface area to volume ratio and enhancing shelf-life through their thermal stability. However, microfluidic approaches require complex fabrication steps to create weirs or pillars that hold the particles in place and an external pump to control sample flow. In a global trend for affordable diagnostics, there is a need to benefit from the improved performance of particle-based systems while also simplifying the fabrication and readout techniques. Here, we present a new biosensor format, where the bio-functionalized particles are moved through the fluid sample in which they are suspended. We deliver a first study into the main design considerations for this falling particle biosensor, detailing the interdependencies between the kinetics of the enzyme reaction, the mass transport of the substrate to the enzyme on the surface of the particle, and the falling behavior of the settling particles. We detail, through a mathematical model, validated by experimental results, how particle size and enzyme loading are able to influence the outcome measured and establish that this falling particle model does not deviate from the kinetic regime, but that particle size and enzyme loading can be used to tune the signal resolution and deliver simple but highly effective sensors.
The present study describes a simple strategy to integrate electrochemical detection with an assembled microchip-capillary electrophoresis platform. The electrochemical cell was integrated with a microfluidic device consisting of five plastic squares interconnected with fused silica capillaries, forming a four-way injection cross between the separation channel and three side-arms (each of 15 mm in length) acting as buffer/sample reservoirs. The performance of the system was evaluated using electrodes made with either carbon ink, carbon nanotubes, or gold and under different experimental conditions of pH, capillary length, and injection time. Using this system it was possible to separate the neurotransmitters dopamine and cathecol and to quantify phenol from a real sample using a linear calibration curve with a calculated LOD of 0.7 µM. A similar concept was applied to determine glucose, by including a pre-reactor filled with beads modified with glucose oxidase (GOx). The latter system was used to determine glucose in a commercial sample, with a recovery of 95.2 %. Overall, the presented approach represents a simple, inexpensive, and versatile approach to integrate electrochemical detection with CE separations without requiring access to microfabrication facilities.
The sections in this article are
As a result of modern fabrication technology, integrated circuits have become smaller and smaller. This has been achieved while including larger number of components and achieving more and more functions that are called System-on-Chip (SoC). In this paper the patents on SoC and its branches (CoC, NoC, and LoC) have been presented. Chip-on-Chip (CoC) module technology is advantageous because of the density increase. That is, individual semiconductor chips having respective different technology circuits are combined in a stacked structure. A new class of SoC called Network-on-Chips (NoCs) has recently been proposed to solve the architectural and design complexity issues of SoCs. Lab-on-Chip (LoC). For live-cell manipulation, Lab-on-Chip Biosensors, Micro-flow injection analysis system, and Fabrication Methods of Lab-on-Chip have been discussed. Finally, current technology has been discussed and future technology about this subject (can be omitted) is prospected.
Due to its high separation efficiency, short analysis times, low sample volume and simple cleaningup, capillary electrophoresis has become a valuable tool for the research of enzyme inhibitors, such as the screening of potential enzyme inhibitors, the study of inhibitor activities, and the determination of inhibitor type. In this paper, the application of capillary electrophoresis method in the research of inhibitory kinetics of enzyme including the assay method and the application of inhibitory kinetics of enzyme was introduced.
IntroductionSynthesis and Characterization of Biosensor SupportsApplication of Enzyme-Based BiosensorsConclusions
AcknowledgmentsReferences
An electrochemical microfluidic strategy for the separation and enantiomeric detection of D-Methionine (D-Met) and D-Leucine (D-Leu) is presented. These D-amino acids (D-AAs) act as biomarkers involved in relevant diseases caused by Vibrio cholerae. On a single layout microfluidic chip (MC), highly compatible with extremely low biological sample consumption, the strategy allowed the controlled microfluidic D-AA separation and the specific reaction between D-amino acid oxidase (DAAO) and each D-AA biomarker avoiding the use of additives (i.e. cyclodextrins) for enantiomeric separation as well as any covalent immobilization of the enzyme into the wall channels or on the electrode surface such as in the biosensor-based approaches. Hybrid polymer/graphene-based electrodes were end-channel coupled to the microfluidic system to improve the analytical performance. D-Met and D-Leu were successfully detected becoming this proof-of-the-concept a promising principle for the development of point-of-care (POC) devices for in situ screening of V. cholerae related diseases.
In this paper we propose a new image encryption scheme using chaotic maps to protect the distribution of digital images in an efficient and secure way. In order to check the efficiency of proposed method, the new encryption scheme is compared with two current classes of stream ciphers: W7 and Salsa20. So, we performed a series of tests and some comparisons to justify efficiency of image encryption methods. These tests included key space analysis, psychophysical experiments, histogram analysis, information entropy, encryption quality, correlation analysis, differential analysis and sensitivity analysis. Simulation experiment has validated the effectiveness of the proposed system.
A simple and efficient method using enzyme immobilized microfluidic channel as open tubular microreactor was designed for amperometric detection of glucose. The microreactor was composed of a polydimethylsilicone/glass hybrid device with three reservoirs, a cooling cave and a 6 cm capillary with a sampling fracture as microchannel. The microchannel was further modified by thermal polymerization, followed by covalently attaching with glucose oxidase. Through fracture sampling and electrochromatography separation, the production via enzymatic reaction was determinated by Pt electrode at the end of capillary. The linear range for the detection of glucose was 0.05–7.5 mmol·L−1 with detection limit of 23 µmol·L−1. The inter- and intra-chip reproducibilities for determination of 2.5 mmol·L−1 glucose were 98.5% (n=5) and 96.0% (n=5), respectively. With the advantage of flexible assembly, rapid efficiency, good stability and low-cost, this microreactor provided a potential platform for establishing a portable enzyme-based chemical detection system in practical application.
A novel capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) using graphene oxide (GO) as a support was developed by using a simple and reliable immobilization procedure based on layer by layer electrostatic assembly. Using trypsin as a model enzyme, the performance of the fabricated CE-based IMERs was evaluated. Various conditions, including trypsin concentration, trypsin coating time, number of trypsin layers and buffer pH, were investigated and optimized. The Michaelis constant Km (0.24 ± 0.02 mM) and the maximum velocity Vmax (0.32 ± 0.04 mM s(-1)) were determined using the CE-based IMERs, and the values are consistent with those obtained using free trypsin, indicating that enzyme immobilized via the proposed approach does not cause a significant structural change of the enzyme or any reduction of enzyme activity. The presented CE-based IMERs exhibit excellent reproducibility with RSD less than 2.8% over 20 runs, and still remain 79.5% of the initial activity after five days with more than 100 runs. Using the proposed CE-based IMERs, the digestion of angiotensin was completed within 3 min, while quite a number of trypstic peptides were observed for BSA on-line digestion with an incubation time of 30 min. As identified by MS analysis, the online digestion products of BSA using the present CE-based IMER are comparable with those obtained using free trypsin digestion for 12 h incubation. It is indicated that the present immobilization strategy using GO as a support is reliable and practicable for accurate on-line analysis and characterization of peptides and proteins.
The authors described a region-selective immobilization methods of bacteria by using superhydrophobic/superhydrophilic and superhydrophobic/poly(ethylene glycol) (PEG) micropatterns for culture scaffold templates. In the case of superhydrophobic/superhydrophilic micropatterns, the superhydrophobic surface was prepared first by microwave-plasma enhanced chemical vapor deposition (MPECVD) from trimethylmethoxysilane. Then the superhydrophilic regions were fabricated by irradiating the superhydrophobic surface with vuv light through a stencil mask. In the case of the superhydrophobic/PEG micropatterned surfaces, PEG surfaces were fabricated first by chemical reaction of ester groups of p-nitrophenyl PEG with NH2 group of NH2-terminated self assembled monolayer from n-6-hexyl-3-aminopropyltrimethoxysilane. The superhydrophobic regions were fabricated by MPECVD thorough a stencil mask. In this study four bacteria were selected from viewpoint of peptidoglycan cell wall (E. coli versus B. subtilis), extracellular polysaccharide (E.coli versus P. stutzeri, P. aeruginosa), and growth rate (P. stutzeri versus P. aeruginosa). The former micropattern brought discrete adhesions of E. coli and B. subtilis specifically on the hydrophobic regions, Furthermore, using the superhydrophobic/PEG micropattern, adhesion of bacteria expanded for E. coli, B. subtilis, P. stutzeri, and P. aeruginosa. They observed a high bacterial adhesion onto superhydrophobic surfaces and the inhibitive effect of bacterial adhesion on PEG surfaces.
Recent developments in materials, surface modifications, separation schemes, detection systems and associated instrumentation have allowed significant advances in the performance of lab-on-a-chip devices. These devices, also referred to as micro total analysis systems (µTAS), offer great versatility, high throughput, short analysis time, low cost and, more importantly, performance that is comparable to standard bench-top instrumentation. To date, µTAS have demonstrated advantages in a significant number of fields including biochemical, pharmaceutical, military and environmental. Perhaps most importantly, µTAS represent excellent platforms to introduce students to microfabrication and nanotechnology, bridging chemistry with other fields, such as engineering and biology, enabling the integration of various skills and curricular concepts. Considering the advantages of the technology and the potential impact to society, our research program aims to address the need for simpler, more affordable, faster and portable devices to measure biologically active compounds. Specifically, the program is focused on the development and characterization of a series of novel strategies towards the realization of integrated microanalytical devices. One key aspect of our research projects is that the developed analytical strategies must be compatible with each other; therefore, enabling their use in integrated devices. The program combines spectroscopy, surface chemistry, capillary electrophoresis, electrochemical detection and nanomaterials. This article discusses some of the most recent results obtained in two main areas of emphasis: capillary electrophoresis, microchip-capillary electrophoresis, electrochemical detection and interaction of proteins with nanomaterials.
The amperometric glutamate biosensor based on screen-printed electrodes containing carbon nanotubes (CNT), and its integration in a flow injection analysis system, is described herein. The sensor was fabricated by simply adsorbing enzyme glutamate oxidase (GlutOx) on a commercial substrate containing multi-wall CNT. The resulting device displayed excellent electroanalytical properties toward the determination of L-glutamate in a wide linear range (0.01-10 μM) with low detection limit (10 nM, S/N≥3), fast response time (≤5 s), and good operational and long-term stability. The CNT modified screen-printed electrodes have a potential to be of general interest for designing of electrochemical sensors and biosensors.
The importance of analyses of different parameters in food products and monitoring of a production process requires quick
and reliable analytical methods and devices. For this purpose, biosensors can be a suitable option, whereas most of the current
quality control techniques are time consuming, expensive, and unpractical. In this paper, we describe biosensors developed
for analysis of different components present in food samples, namely, glucose, fructose, sucrose, lactose, lactic, malic,
acetic, ascorbic, citric and amino acids, ethanol, glycerol, and triglyceride. Biosensors showed desirable sensitivity, selectivity,
and response time required for various applications. They are often designed to avoid interference from components present
in a complex sample to be analyzed.
KeywordsBiosensors–Food analysis–Enzymes–Analysis
This paper describes a multiplexed enzyme-based assay within a microfluidic device using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles. A microfluidic device was constructed by serially connecting two patterning chambers and a microfilter-integrated detection chamber through a Y-shaped microchannel. Various shapes and sizes of hydrogel microparticles entrapping different enzymes were fabricated in the patterning chamber by photolithography and collected in the detection chamber by pressure-driven flow. Due to the action of the microfilter, hydrogel microparticles were perfectly retained inside the detection chamber without passing through it. The sequential bienzymatic reactions of glucose oxidase (GOX) and peroxidase (POD) or alcohol oxidase (AOX) and POD were successfully investigated using Amplex Red within the hydrogel microparticles. Furthermore, by entrapping GOX and AOX into different shapes of hydrogel microparticles, each enzyme-catalyzed reaction was easily identified, and therefore, simultaneous detection of glucose and ethanol was possible in the concentration range from 1.0 to 10.0 mM without cross-talk by using same fluorescence indicator in a single detection chamber.
Despite multiple orbiter and landed missions to extraterrestrial bodies in the solar system, including Mars and Titan, we still know relatively little about the detailed chemical composition and quantity of organics and biomolecules in those bodies. For chemical analysis on astrobiologically relevant targets such as Mars, Europa, Titan, and Enceladus, instrumentation should be extremely sensitive and capable of analyzing a broad range of organic molecules. Microchip capillary electrophoresis (μCE) with laser-induced fluorescence (LIF) detection provides this required sensitivity and targets a wide range of relevant markers but, to date, has lacked the necessary degree of automation for spaceflight applications. Here we describe a fully integrated microfluidic device capable of performing automated end-to-end analyses of amino acids by μCE with LIF detection. The device integrates an array of pneumatically actuated valves and pumps for autonomous fluidic routing with an electrophoretic channel. Operation of the device, including manipulation of liquids for sample pretreatment and electrophoretic analysis, was performed exclusively via computer control. The device was validated by mixing of laboratory standards and labeling of amino acids with Pacific Blue succinimidyl ester followed by electrophoretic analysis. To our knowledge, this is the first demonstration of completely automated end-to-end μCE analyses on a single, fully integrated microfluidic device.
Miniaturisation is revolutionary to high-throughput proteomics. These technologies have gained much interest in the past decade, as they allow for sensitive parallel analysis of small amounts of biological materials. This review describes the state of the art of proteomics-on-chip, with a particular focus on the fundamental proteomics-on-chip challenges. The important role of bio-interfacial interactions and strategies to control them are presented. Various coating methodologies for on-chip protein/peptide separation are reviewed to provide an overview of the principles of protein-resistant and protein immobilisation coatings, and their effectiveness.
A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.
A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm+ Br-) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm+ Br-) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm+ (CF3SO2)2N-) and poly(ViEtIm+ BF4-). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking content. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days.
Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (μIMER) made of porous silicon with the purpose of investigating the use of such μIMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the μIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the μIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The μIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing μIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the μIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight.
Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6−24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (μ-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The μ-chip IMER provides on-line enzymatic digestion of protein samples (1 μL) within 1−3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50−100%).
Cyclic voltammetric studies of hydrogen peroxide on a polycrystalline gold electrode have permitted to point out that the oxidation of the molecule may occur at two distinct potentials depending on the sample composition and cleanliness and on the redox state of the electrode. At a clean gold surface, the oxidation of hydrogen peroxide gives only one wave at +490 mV (vs. Ag/AgCl) at pH 7.4, besides the gold oxidation wave at +820 mV. Species in solution which adsorb readily on gold (trace impurities originating because of sonication or solution stirring), gold oxide and acidic pH values, inhibit this wave and oxidation of hydrogen peroxide is occurring at a higher potential, i.e., +870 mV in phosphate buffer pH 7.4. Cyclic voltammograms show these two waves, depending on the degree of inhibition of hydrogen peroxide. At oxidized gold electrodes or contaminated by surface active species, oxidation of hydrogen peroxide gives only one wave at +870 mV. From these data it may be inferred that hydroxy species adsorbed on clean gold favor the formation of the first wave. Both waves may be advantageously exploited for quantitative purpose. The latter is not affected by surface active species and permits quantification of hydrogen peroxide down to 20 μM with a detection limit of 4 μM.
A simple and rapid method has been developed for the analysis of four nonsteroidal anti-inflammatory drugs (NSAIDs) in serum using microchip capillary electrophoresis with pulsed amperometric detection. The selected NSAIDs (salicylic acid, acetaminophen, diflunisal, and diclofenac) are among the most commonly used drugs to treat fever, inflammation, and pain. Used above the therapeutic levels, these drugs can cause a wide variety of adverse effects and their fast analysis could have a significant impact in treatment and recovery of the patients. Several conditions, including separation potential, pH, and concentration of the electrolyte solution were studied to optimize the separation and detection. In this study, salicylic acid, acetaminophen, diflunisal, and diclofenac were separated in less than 2 minutes using a 5 mM borate buffer at pH 11.5 and a separation potential of +1200 V. Linear relationships were obtained between the concentration and peak current in the 0.5–15.3 μg/mL range and detection limits around 0.26 μg/mL. After 30 consecutive injections, the stability of both the response and migration time of the analytes showed relative related deviations of less than 4.6% and 1.0%, respectively. The potential of this method was verified by spiking a bovine serum sample with the four NSAIDs and analyzing the recovery ratio.
There is a need to develop analytical methods that are capable of rapidly measuring small biological markers in the field of metabolomics. Among others, carbohydrates play an important role biologically yet are traditionally hard to detect since they have no chromophore or fluorophore. In the present report, the first application of integrated pulsed amperometric detection (iPAD) coupled with microchip electrophoresis to the analysis of glucose, mannose, sucrose, maltose, glucosamine, lactose, maltotriose and galactose is demonstrated. iPAD is an electrochemical detection mode that can be used for direct detection of carbohydrates, amines and sulfur containing compounds. The effect of different solution parameters, including the buffer concentration, pH and the concentration of SDS on both separation and detection response was analyzed. In addition, a comparison study between PAD and iPAD was performed using glucose, glucosamine, sucrose and maltose as model carbohydrates.
A multi-enzyme electrode obtained by a two-step immobilization of the enzymes glucose oxidase, mutarotase and invertase was developed for the determination of sucrose. Glucose oxidase was entrapped in a poly-1,3-diaminobenzene film on a platinum electrode by electrochemical polymerization and a combination of mutarotase and invertase was cross-linked over the electrode via bovine serum albumin and glutaraldehyde. The sucrose concentration was determined from hydrogen peroxide oxidation at +0.7 V vs. Ag/AgCl. This immobilization method minimized interference from ascorbic acid. A second electrode, for glucose only, was constructed containing inactive invertase. This gave an almost identical glucose response to the sucrose sensor and hence could be used for signal subtraction. In this manner, sucrose could be determined in the presence of glucose at higher concentrations. The influence of enzyme content, pH, temperature, and optimum operational conditions for use in a flow injection analysis (FIA) system were determined. When used in FIA, the sensor responded to sucrose in the range 1–300 mmol l−1. The sensor was stable for 15 h of continuous use (150 assays) and retained 70% activity after 30 days. When used to analyze the sucrose and glucose contents in a number of different soft drinks, the sensor showed good agreement with the standard liquid chromatography (LC) method.
The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized. (c) 2005 Elsevier B.V. All rights reserved.
A simple assay of sulfated bile acid (SBA) in urine using flow injection (FI)-spectrophotometry with immobilized enzyme reactors is proposed. The system consists of an injection valve, a switch valve, two immobilized enzyme reactors and a UV–VIS detector with a flow cell. The multi-step enzymatic reactions will occur when an injected sample containing SBA passes through the immobilized enzyme reactors. First, SBA will desulfate under catalysis of immobilized bile acid sulphate sulfatase (BSS), to form 3β-hydroxyl bile acids; the produced 3β-hydroxyl bile acid reacts with nicotinamide adenine dinucleotide (NAD+) under catalysis of co-immobilized 3β-hydroxylsteroid dehydrogenase (3β-HSD), and is converted to the 3-ketosteroid. Meanwhile, β-NAD+ is converted to reduced nicotinamide adenine dinucleotide (NADH). Then by catalysis of immobilized diaphorase, NADH reacts with a novel reagent called “water soluble tetrazolium blue-5” (WST-5) to generate a blue diformazan dye, which is detected at 550 nm.By using FI-spectrophotometry manifold and optimized conditions, we have obtained a linear response for 1–75 μM glycolithocholate sulfate (GLCA-S) with a correlation coefficient of 0.999 and an analytical rate of 15 samples per hour. The R.S.D. was less than 1%. The recoveries (91–108%) of GLCA-S added into urine were satisfactory and the assay correlated well with the manual UBASTEC method. Therefore, it will be applicable for urine tests on patients suffering from hepatobiliary disease.
A new alternative method for bioprocess monitoring based on bienzymatic analytical microreactors integrated in a flow injection analysis (FIA) system is described. Glucose-, alcohol-, lactate-, galactose- and l-amino acid oxidases (GO, AO, LacO, GalO and LAAO) and horseradish peroxidase (HRP) are immobilized on controlled pore glass (CPG) and used for the development of glucose, ethanol, lactate, galactose and amino acid sensors. The analytical methodology is based on HRP catalysed reaction of hydrogen peroxide produced by oxidases with phenol-4-sulfonic acid and 4-aminoantipyrine. The immobilized enzymes are characterized and used for preparation of the packed bed analytical microreactors. Shelf life and operational stability of the microeactors are determined. GO/HRP, AO/HRP and LAAO/HRP microreactors showed excellent shelf life, they could be stored and reused for more than 6 months with no or very little activity loss, while GalO/HRP and LacO/HRP could be stored for shorter periods of time (10–20 days). Operational stability of GO and LacO microreactors was very good: an equivalent to 16,900 FIA injections of 25 μl to a LacO microreactor resulted in loss of half of its activity, immobilized GO was so stable that it was impossible to evaluate enzyme halflife. Immobilized GalO and LAAO lose their operational activity much faster: approximately 1400 and 8000 FIA injections of the respective substrate solution in a FIA set-up resulted in 50% activity loss. The methods with all the described microreactors were successfully validated using off-line samples from S. cerevisiae, E. coli and mesenchymal stem cell cultures with HPLC as the reference method.
A miniaturised analytical system for separating and detecting a range of steroidal oestrogens, based on the coupling of a micromachined capillary electrophoresis chip with glassy-carbon electrode amperometric detector, is described. Factors influencing the on-chip separation utilising the technique micellar electrokinetic chromatography (MEKC) and detection processes are optimised. The addition of modifiers such as organic solvents and surfactants improve separation and resolution of these hydrophobic compounds. Using a borate running buffer (5 mM, pH 11) with 20% methanol and SDS (20 mM) and a separation voltage of 2000 V, baseline resolution is observed for 16-keto-17β-oestradiol, oestriol, 11β-hydroxyoestradiol, oestrone, and β-oestradiol in 420 s with limits of detection 16–84 μM. The implications for on-site environmental analysis are discussed.
A simple and fast method for the simultaneous determination of the antioxidants propyl gallate (PG), octyl gallate (OG), lauryl gallate (LG), and nordihydroguaiaretic acid (NDGA) has been established by using microchip micellar electrokinetic chromatography with pulsed amperometric detection. Under the optimum conditions (30 mM borate buffer, pH 9.7, 30 mM sodium dodecyl sulfate, separation voltage of 1200 V and 5 s injection time) the analytes were baseline separated. Linear relationships were found between the concentration and peak current for all the selected antioxidants. The measured detection limits (S/N ≥ 3) of PG, OG, LG, and NDGA were 2.2, 1.4, 2.3, and 4.6 μM, respectively, which corresponds to 2–6 fmol of analyte. This approach has remarkable advantages with respect to other methodologies involving separations and electrochemical detection including minimal sample consumption, higher analysis speed, lower cost, and portability. Additionally, a highly reproducible signal (migration time and peak current) was obtained for a series of injections (n = 30). In order to demonstrate the capabilities of the method, the determination of antioxidants in a commercial food sample is also presented.
Mushroom tyrosinase was covalently bonded with glutaraldehyde, as activating agent, to aminopropyl-controlled pore glass support by “in situ” immobilization technique. The Schiff's base double bond reduction with sodium cyanoborohydride was made as innovation. Catalytic activity and stability of the chromatographic reactor were evaluated using d,l-3,4-dihydroxyphenylalanine as substrate. The tyrosinase-IMER was characterized by investigation of various parameters influencing the enzymatic activity (pH, temperature, ionic strength and organic solvents). In addition kinetic measurements showed that, by removal of the external diffusional limitation, the enzyme selectivity towards substrate was improved whereas the activity was comparable with respect to that of free enzyme.
The present report describes a new analysis strategy for microchip capillary electrophoresis with pulsed amperometric detection and its application to the determination of glucose. The addition of sodium dodecyl sulfate (SDS) to the mobile phase and detection reservoir stabilized flow rates and enhanced the detection signal for glucose. A higher pH (compared to the running buffer) was used at the waste reservoir in order to improve the detection performance while maintaining good separations. To our knowledge, this is the first report describing the use of post-column pH modification using microchip electrophoresis. Under optimum conditions, a linear relationship between the peak current and the concentration of glucose was found between 10−2–10−5 M, with a limit-of-detection of 1.2 μM. In addition, the separation of glucosamine and glucose was performed at pH 7.1 while the detection was performed at pH 11 to demonstrate the ability to use post-column pH modification.
A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120–200 mesh) CPG was found to give the highest activity (18–20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.
A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25x45x5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3sigma).
Disposable screen-printed, film carbon electrodes (PFCE) were modified with cast-coated Osmium-polyvinylpyrridine-wired horse radish peroxidase gel polymer (Os-gel-HRP) to enable the detection of the reduction at 0 mV of hydrogen peroxide (H2O2) derived from a post-column immobilized enzyme reactor (IMER) containing acetylcholinesterase and choline oxidase. In another series of experiments PFCE were initially modified with cast-coated Os-gel-HRP and then treated with glucose oxidase in bovine serum albumin (BSA) and cross-linked with glutaraldehyde to form a bi-layer glucose-Os-gel-HRP PFCE. This bi-layer glucose-Os-gel-HRP PFCE generated a reduction current at 0 mV to H2O2 derived from the reaction of glucose oxidase and glucose in solution. These enzyme-modified PFCE were housed in a radial flow cell and coupled with cation-exchange liquid chromatographic methods to temporally separate substrates in solution for the determination of acetylcholine (ACh) and choline (Ch) in the first experimental series, or glucose in the second experimental series. These two disposable enzyme-modified PFCE exhibited linear current vs. substrate relations, were durable, being usable for approximately 40 determinations, and were sufficiently sensitive to be employed in biological sampling. Both assays utilized the same HPLC equipment. The limit of detection for ACh was 16 fmol/10 microl and that for glucose was 12 micromol/7.5 microl. ACh and Ch were measured from a microdialysate from the frontal cortex of a rat. Glucose in human urine was determined using the bi-layer glucose oxidase-Os-gel-HRP PFCE.
The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.
This report describes a new "lab-on-a-chip" protocol integrating on-line precolumn biocatalytic reactions of multiple (oxidase and dehydrogenase) enzymes and substrates with effective capillary electrophoresis microseparations and amperometric detection. The operation of the new oxidase/dehydrogenase reaction/separation microchip is illustrated for the simultaneous measurement of glucose and ethanol in connection to the corresponding glucose oxidase and alcohol dehydrogenase reactions, respectively. The enzymatic reactions generate hydrogen peroxide and NADH species that are separated (on the basis of their different charges) and detected amperometrically at the end-column thick-film detector. A driving voltage of 2000 V results in peroxide and NADH migration times of 74 and 230 s, respectively. Operating the gold-coated carbon detector at +1.0 V allows simultaneous anodic detection of both reaction products. Factors influencing the reaction, separation, and detection processes are examined and optimized. The applicability of the new multienzyme assay to wine samples is illustrated.
Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.
Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.
A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.
A continuous amperometric glucose monitoring system is presented. All analytically relevant units are integrated on a microchip (microreactor, control electrode, glucose oxidase based sensor electrode, reference electrode, counter electrode). The scavenging electrochemical microreactor pre-oxidises all interfering compounds enabling a reliable glucose determination. The reliability of the microreactor is demonstrated with the most important antioxidants, ascorbic and uric acids. The glucose sensor system operates at volumetric flow rates of common body interfaces (e.g. microdialysis, microperfusion).
Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol-gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.
A simple method integrating an immobilized enzyme reactor into a microchip electrophoresis device was developed. The enzyme immobilization into a microchip was performed by spotting and drying a drop of dissolved nitrocellulose (NC) on a glass substrate, and adsorbing enzyme on the reconstituted NC membrane. This enzyme-immobilized glass plate was assembled with a polydimethylsiloxane substrate on which the separation channel was fabricated. The advantage of this method is the ability to easily change the position and size of the reactor within the microchip electrophoresis device. A beta-galactosidase reaction was demonstrated with fluorescein di-beta-D-galactopyranoside using this integrated on-column enzyme reactor. A successful electrophoretic separation of its hydrolysis products, i.e., fluorescein mono-beta-D-galactopyranoside (FMG) and fluorescein, was achieved. Enzyme kinetics and inhibition of the beta-galactosidase using FMG and 2-phenylethyl beta-D-thiogalactoside, respectively, were also studied with microchip electrophoresis.
This study presents the use of complementary colorimetric and amperometric techniques to measure the quantity of protein or enzyme immobilised onto a carbon paste electrode modified with a layer of electrodeposited polyaniline. By applying a solution of bovine serum albumin at 0.75 mg/ml, efficient blocking of the electrode from electroactive species in the bulk solution could be achieved. When the horseradish peroxidase was immobilised on the electrode, optimal amperometric responses from hydrogen peroxide reduction were achieved at approximately the same concentration. The mass of enzyme immobilised at this solution concentration was determined by a colorimetric enzyme assay to be equivalent to the formation of a protein monolayer. Under these conditions, amperometric responses from the immobilised layer are maximised and non-specific bulk solution interactions are minimised. At higher immobilised protein concentrations, diminished amperometric responses may be due to inhibited diffusion of hydrogen peroxide to enzyme which is in electronic communication with the electrode surface, or impeded electron transfer.
The self-interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self-interaction chromatography has the potential to provide a quantitative method for determination of protein self-interactions amenable to high-throughput screening. This paper describes the construction and characterization of a microchip separation system for low-pressure self-interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile-phase composition, amount of protein injected, flow rate, and stationary-phase modification. The capacity factors (k') as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B(22)) obtained by static light scattering, showing the ability of the chip to accurately determine protein-protein interactions. A 500-fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein-protein interactions.
This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.
Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.
The separation and detection of underivatized carbohydrates, amino acids, and sulfur-containing antibiotics in an electrophoretic microchip with pulsed amperometric detection (PAD) is described. This report also describes the development of a new chip configuration for microchip electrophoresis with PAD. The configuration consists of a layer of poly(dimethylsiloxane) that contains the microfluidic channels, reservoirs, and a gold microwire, sealed to a second layer of poly(dimethylsiloxane). Example separations of carbohydrates, amino acids, and sulfur-containing antibiotics are shown. The effect of the separation and injection potentials, buffer pH and composition, injection time, and PAD parameters were studied in an effort to optimize separations and detection. Detection limits ranging from 6 fmol (5 microM) for penicillin and ampicillin to 455 fmol (350 microM) for histidine were obtained.
Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.
We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.
Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.
The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.
In the present report, the use of negatively charged surfactants as modifiers of the background electrolyte is reported using poly(dimethylsiloxane) (PDMS) microchips. In particular, the use of anionic surfactants, such as sodium dodecyl sulfate, phosphatidic acid, and deoxycholate, was studied. When surfactants were present in the run buffer, an increase in the electroosmotic flow (EOF) was observed. Two additional effects were also observed: (i) stabilization of the run-to-run EOF, (ii) an improvement in the electrochemical response for several biomolecules. In order to characterize the analysis conditions, the effects of different surfactant, electrolyte, and pH were studied. EOF measurements were performed using either the current monitoring method or by detection of a neutral molecule. The first adsorption/desorption kinetics studies are also reported for different surfactants onto PDMS. The separation of biologically important analytes (glucose, penicillin, phenol, and homovanillic acid) was improved decreasing the analysis time from 200 to 125 s. However, no significant changes in the number of theoretical plates were observed.
This 2-D separation technique can complement MS-based proteomics methods.
The fast separation capability of a novel miniaturized capillary electrophoresis with amperometric detection (CE-AD) system was demonstrated by determining sugar contents in Coke and diet Coke with an estimated separation efficiency of 60,000 TP/m. Factors influencing the separation and detection processes were examined and optimized. The end-capillary 300 microm Cu wire amperometric detector offers favorable signal-to-noise characteristics at a relatively low potential (+0.50 V vs. Ag/AgCl) for detecting sugars. Three sugars (sucrose, glucose, and fructose) have been separated within 330 s in a 8.5 cm length capillary at a separation voltage of 1000 V using a 50 mM NaOH running buffer (pH 12.7). Highly linear response is obtained for the above compounds over the range of 5.0 to 2.0 x 10(2) microg/mL with low detection limit, down to 0.8 microg/mL for glucose (S/N = 3). The injection-to-injection repeatability for analytes in peak current (RSD < 3.6%) and for migration times (RSD < 1.4%) was excellent. The new miniaturized CE-AD system should find a wide range of analytical applications involving assays of carbohydrates as an alternative to conventional CE and micro-CE.
This review summarizes all the research efforts in the last decade (1994-2003) that have been spent to the various application of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography (HPLC). All immobilization procedures including supports, kind of assembly into chromatographic system and methods are described. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. A brief survey of the main applications of IMER both as pre-column, post-column or column in the chemical, pharmaceutical, clinical and commodities fields is also reported.
Microchip immobilized enzyme reactors (microIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two microIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the microIMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of microIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that microIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the microIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The microIMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the microIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The microIMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method.
A one-step procedure for the immobilization of glucose oxidase in fused-silica capillaries and in glass microchips was developed based on enzyme entrapment in a polyacrylamide-based monolithic column. The inner capillary surface was silanized with gamma-methacryloxypropyltrimethoxysilane (gamma-MAPS) to allow covalent binding of the gel to the surface. The composition of the polymer was optimized to prevent the formation of bubbles, allow liquid transportation by electroosmotic flow and to maintain the enzymatic activity. These requirements resulted in the addition of polyethylene glycol and poly(acrylic acid) to the acrylamide mixture. The gel containing the enzyme was formed in situ in the capillaries, respectively, in one channel of the microchip. In the microchip, it was limited to the sample injection channel by accordingly controlled silanization of the inner capillary surface. Glucose was detected via the amperometric determination of hydrogen peroxide. A linear correlation between signals and glucose concentration was observed from 0.05 to 1.1 mM glucose with a correlation coefficient of 0.999. The enzymatic monolithic microreactor showed no loss of activity during 8 h of continuous use and during storage in the running buffer at 4 degrees C for about 2 months. Interferents, such as ascorbic acid, were separated from the analyte electrophoretically, so that glucose could be quantified in diluted juices.
A miniaturized analytical system for separation and detection of three EPA priority phenolic pollutants, based on a poly(dimethylsiloxane)-fabricated capillary electrophoresis microchip and pulsed amperometric detection is described. The approach offers a rapid (less than 2 min), simultaneous measurement of three phenolic pollutants: phenol, 4,6-dinitro-o-cresol and pentachlorophenol. The highly stable response (RSD = 6.1%) observed for repetitive injections (n > 100) reflects the effectiveness of Au working electrode cleaned by pulsed amperometric detection. The effect of solution conditions, separation potential and detection waveform were optimized for both the separation and detection of phenols. Under the optimum conditions (5.0 mM phosphate buffer pH = 12.4, detection potential: 0.7 V, separation potential: 1200 V, injection time: 10 s) the baseline separation of the three selected compounds was achieved. Limits of detection of 2.2 microM (2.8 fmol), 0.9 microM (1.1 fmol), and 1.3 microM (1.6 fmol) were achieved for phenol, 4,6-dinitro-o-cresol and pentachlorophenol, respectively. A local city water sample and two over-the-counter sore-throat medicines were analyzed in order to demonstrate the capabilities of the proposed technique to face real applications.
An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.
In the present chapter, some basic definitions about the photolithography process are explained and then the standard preparation of the silicon wafer, the fabrication of the mold, and the preparation and assembly of poly(dimethylsiloxane) (PDMS)-based microchips are discussed. The purpose of this chapter is to describe the most used techniques for preparation of PDMS microchips. A list of tips is included in order to provide a troubleshooting guide for the most common difficulties found during the fabrication process. Some recent alternative approaches to microfabrication are also discussed.
A rapid and economical method is reported for the preparation of an immobilized enzyme reactor (IMER) using silica-encapsulated equine butyrylcholinesterase (BuChE) as a model system. Peptide-mediated silica formation was used to encapsulate BuChE, directly immobilizing the enzyme within a commercial pre-packed column. The silica/enzyme nanocomposites form and attach simultaneously to the metal affinity column via a histidine-tag on the silica-precipitating peptide. BuChE-IMER columns were integrated to a liquid chromatography system and used as a rapid and reproducible screening method for determining the potency of cholinesterase inhibitors. The IMER preparation method reported herein produces an inert silica-encapsulation matrix with advantages over alternative systems, including ease of preparation, high immobilization efficiency (70-100%) and complete retention of activity during continuous use.
Microreaction technology is an interdisciplinary field combining science and engineering. It has attracted the attention of researchers from different fields for the past few years, resulting in the development of several microreactors. Enzymes are one of the catalysts used in microreactors: they are useful for substance production in an environmentally friendly way and have high potential for analytical applications. However, few enzymatic processes have been commercialized because of problems with stability and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering, particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of enzyme-immobilized microchannel reactors; fundamental techniques for micro enzyme-reactor design and important applications of this multidisciplinary technology in chemical processing are also included in our topics.
Here we reported a novel microfluidic biosensor with an on-column immobilized enzyme microreactor. The fabrication approach of this biosensor is simple and the enzyme microreactors with controlled sizes can be placed at any desired position on the microchip. Taking glucose oxidase (GOx) as an example, electroosmotic flow (EOF) as a driving force and amperometry as a detection method, the performance of biosensors were modulated by changing the length of enzyme reactor from 0.5 cm to 3 cm, and the linear ranges were changed from 0-8.0 mM to 0-30.0 mM with the detection limits from 42 microM to 6.5 microM. The enzyme reactor remained its 65% activity after 23 days storage. It also showed good anti-interference ability and was used to quantify glucose in human serum samples.
In this paper, we describe the separation and detection of six phenolic acids using an electrophoretic microchip with pulsed amperometric detection (PAD). The selected phenolic acids are particularly important because of their biological activity. The analysis of these compounds is typically performed by chromatography or standard CE coupled with a wide variety of detection modes. However, these methods are slow, labor intensive, involve a multistep solvent extraction, require skilled personnel, or use bulky and expensive instrumentation. In contrast, microchip CE offers the possibility of performing simpler, less expensive, and faster analysis. In addition, integrated devices can be custom-fabricated and incorporated with portable computers to perform on-site analysis. In the present report, the effect of the separation potential, buffer pH and composition, injection time and PAD parameters were studied in an effort to optimize both the separation and detection of these phenolic acids. Using the optimized conditions, the analysis can be performed in less than 3 min, with detection limits ranging from 0.73 microM (0.10 microg/mL) for 4-hydroxyphenylacetic acid to 2.12 microM (0.29 microg/mL) for salicylic acid. In order to demonstrate the capabilities of the device, the degradation of a mixture of these acids by two aquatic plants was followed using the optimized conditions.
A miniaturized capillary electrophoretic (CE) microchip device for the simultaneous measurements of lactate and glucose is described. The new microchip bioassay protocol integrates an electrophoretic separation of lactate and glucose, post-column enzymatic reactions of these metabolites with their respective oxidase enzymes, and an amperometric (anodic) detection of enzymatically-liberated hydrogen peroxide at a gold-coated thick-film carbon detector. Factors influencing the response have been examined and optimized, and the analytical performance has been characterized. Applicability of the microchip assay to clinical samples, such as serum and blood, is demonstrated. The microchip protocol obviates cross enzymatic reactions and interferences from major oxidizable constituents common to dual glucose-lactate enzyme electrodes. Such ability to rapidly separate and quantitate lactate and glucose on a small microchip platform should find important clinical and biotechnological applications.