Microfluidics with MALDI analysis for proteomics—A review

Various microfluidic devices have been developed for proteomic analyses and many of these have been designed specifically for mass spectrometry detection. In this review, we present an overview of chip fabrication, microfluidic components, and the interfacing of these devices to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. These devices can be directly coupled to the mass spectrometer for on-line analysis in real-time, or samples can be analyzed on-chip or deposited onto targets for off-line readout. Several approaches for combining microfluidic devices with analytical functions such as sample cleanup, digestion, and separations with MALDI mass spectrometry are discussed.     

Speciation analysis in on-line aerosol mass spectrometry

Ambient aerosol particles affect both the earth's climate and human health. Both effects depend on the chemical composition of the particles including the binding state of specific elements. Modern on-line aerosol mass spectrometry is capable of measuring aerosol composition with high temporal resolution, avoiding artifacts often introduced by classical off-line methods. The two most common types of on-line aerosol mass spectrometers, laser desorption/ionization mass spectrometers (LDI-MS) and thermal desorption electron impact ionization mass spectrometers (TD-EI-MS) provide reliable information on the most common ambient inorganic and organic aerosol species with high temporal resolution. However, for less common aerosol species identification with both types of instrument is frequently associated with large uncertainties. Here, we provide an overview of the element speciation capabilities of current on-line aerosol mass spectrometry for both carbonaceous and non-carbon-containing aerosol species. We describe limitations and other issues for this type of on-line aerosol analysis.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

AgNP/Bi/Nafion‐modified Disposable Electrodes for Sensitive Zn(II), Cd(II), and Pb(II) Detection in Aerosol Samples

A new method for modifying electrodes with Ag nanoparticles (AgNPs) using electrospray deposition for sensitive, selective detection of Zn(II), Cd(II), and Pb(II) in aerosol samples when combined with Bismuth and Nafion coating and square‐wave anodic stripping voltammetry (SWASV) is reported. Carbon stencil‐printed electrodes (CSPEs) fabricated on a polyethylene transparency (PET) sheet were produced for an inexpensive, simple to fabricate, disposable sensor that can be used with the microliter sample volumes for analysis. Sensor performance was improved by modifying the electrode surface with electrospray‐deposited AgNPs. The use of electrospray deposition resulted in more uniform particle dispersion across the electrode surface when compared to drop‐casting. Using AgNP‐modified electrodes combined with Bi and Nafion, experimental detection limits (LODs) of 5.0, 0.5, and 0.1 μg L⁻¹ for Zn(II), Cd(II), and Pb(II), respectively, were achieved. The linear working ranges were 5.0–400.0 μg L⁻¹, 0.5–400.0 μg L⁻¹, and 0.1–500.0 μg L⁻¹ for Zn(II), Cd(II), and Pb(II), respectively. Interference studies showed Cu(II) was the only metal that interfered with this assay but inference could be eliminated with the addition of ferricyanide directly to the sample solution. This electrochemical sensor was applied for the simultaneous determination of Zn(II), Cd(II), and Pb(II) within source particulate matter (PM) samples collected on filters using an aerosol test chamber.     

Particle-into-liquid sampler on-line coupled with solid-phase extraction-liquid chromatography–mass spectrometry for the determination of organic acids in atmospheric aerosols

In the present study, sample collection and preparation were directly integrated with a chromatographic system by coupling a particle-into-liquid sampler for the first time on-line with solid-phase extraction-liquid chromatography–tandem mass spectrometry. Several organic acids, such as adipic, hydroxyglutaric, mandelic, vanillic, cis-pinonic, pinic, azelaic and sebacic, were used in the research. For sample pretreatment and concentration, strong anion exchange material was used in the extraction. Sampling, extraction and analysis conditions were optimized to obtain reliable information about aerosol chemical composition. To evaluate the performance of the on-line coupled system, half of each sample was analysed on-line and the other half was derivatized and analysed off-line by gas chromatography–mass spectrometry. Comparison of the two techniques with use of t-test showed the results to be in an excellent agreement. Limits of detection of studied acids in on-line system were between 0.1 and 0.9 ng. The on-line coupled system is fast and reliable and a promising tool for the real time analysis of organic acids in atmospheric aerosols.     

Combined extraction chromatography and scintillation detection for off-line and on-line monitoring of strontium in aqueous solutions

This paper presents the application of a new sensor materials, whichcombine extraction chromatography and scintillation detection. The resultantsensor embodies the simultaneous dual functionality of selective extractionand radiation detection, which has been applied towards off-line and on-linemonitoring of radiostrontium in aqueous solutions. The sensor materials are(1) a mixture of commercially available Sr-selective extraction chromatographicresin and granular scintillator, (2) bis-4,4'(5')-tert-butylcyclohexano-18-crown-6(DtBuCH18C6) in 1-octanol coated on the surface of a scintillating glass and(3) inert chromatographic resin impregnated with the organic fluors, 2,5 diphenyloxazole,(PPO) and 1-4 bis(4- methyl-5-phenyloxazol-2-yl) benzene, (DM-POPOP) and eitherDtBuCH18C6 in 1-octanol or a proprietary extractant. For off-line measurements,the sensor materials are used in the same manner as the commercially availableextraction chromatographic resin, but rather than eluting the activity fromthe column, the scintillating extraction column is placed in a scintillationvial and counted without the introduction of liquid scintillation cocktail.For on-line measurements, the sensor materials are used in conjunction witha flow-cell scintillation detection system to assess the activity retainedby the extractant in real-time. Characterization of the detection efficiencyand regeneration capability of the extractive scintillator materials is evaluated.     

Cobalt hexacyanoferrate modified multi-walled carbon nanotubes/graphite composite electrode as electrochemical sensor on microfluidic chip

Nanomaterial-based electrochemical sensor has received significant interest. In this work, cobalt hexacyanoferrate modified multi-walled carbon nanotubes/graphite composite electrode was electrochemically prepared and exploited as an amperometric detector for microchip electrophoresis. The prepared sensor displayed rapid and sensitive response towards hydrazine and isoniazid oxidation, which was attributed to synergetic electrocatalytic effect of cobalt hexacyanoferrate and multi-walled carbon nanotubes. The sensitivity enhancement with nearly two orders of magnitude was gained, compared with the bare carbon paste electrode, with the detection limit of 0.91 μM (S/N = 3) for hydrazine. Acceptable repeatability of the microanalysis system was verified by consecutive eleven injections of hydrazine without chip and electrode treatments, the RSDs for peak current and migration time were 3.4% and 2.1%, respectively. Meanwhile, well-shaped electrophoretic peaks were observed, mainly due to fast electron transfer of electroactive species on the modified electrode. The developed microchip-electrochemistry setup was successfully applied to the determination of hydrazine and isoniazid in river water and pharmaceutical preparation, respectively. Several merits of the novel electrochemical sensor coupled with microfluidic platform, such as comparative stability, easy fabrication and high sensitivity, hold great potential for hydrazine compounds assay in the lab-on-a-chip system.     

Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures

In this study, an end-point-based fluorescence assay for soluble epoxide hydrolase (sEH) was transformed into an on-line continuous-flow format. The on-line biochemical detection system (BCD) was coupled on-line to liquid chromatography (LC) to allow mixture analysis. The on-line BCD was based on a flow system wherein sEH activity was detected by competition of analytes with the substrate hydrolysis. The reaction product was measured by fluorescence detection. In parallel to the BCD data, UV and MS data were obtained through post-column splitting of the LC effluent. The buffer system and reagent concentrations were optimized resulting in a stable on-line BCD with a good assay window and good sensitivity (S/N > 60). The potency of known sEH inhibitors (sEHis) obtained by LC–BCD correlates well with published values. The LC–BCD system was applied to test how oxidative microsomal metabolism affects the potency of three sEHis. After incubation with pig liver microsomes, several metabolites of sEHis were characterized by MS, while their individual potencies were measured by BCD. For all compounds tested, active metabolites were observed. The developed method allows for the first time the detection of sEHis in mixtures providing new opportunities in the development of drug candidates.     

Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.     

一种新型超临界流体萃取-高效液相色谱在线联用系统的构建

天然产物研究一直是植物学、化学和药学的重要研究领域．通过从天然产物中寻找生物活性成分和先导物是创制新药的有效途径之一．有效成分的提取是天然产物研究中最基本和最关键的环节．超临界流体萃取（Supercritical fluid extraction,简称SFE）是近年来发展较快的一种新型样品提取技术．超临界CO2作为最常用的萃取剂已被用于天然药物中非极性和弱极性有效成分的提取,尤其是挥发性和热敏性的物质．此外,通过加入适当的添加剂还可有效地萃取极性化合物,和传统的化学方法相比,     

On-line measurement of intramolecular carbon isotope distribution of acetic acid by continuous-flow isotope ratio mass spectrometry

Molecular and intramolecular carbon isotope measurements of acetic acid present in natural environments have been performed by off-line procedures. The off-line method is complicated and time-consuming and requires micromolar to millimolar amounts of sample. This limits geochemical isotopic studies, especially at the intramolecular level, on acetic acid present in natural samples. Here, we examine an on-line measurement of intramolecular carbon isotope distribution of acetic acid using continuous-flow isotope ratio mass spectrometry (CF-IRMS) coupled with an on-line pyrolysis system. This is achieved by measurement of the respective carbon isotope ratios of CH4 and CO2 produced by on-line pyrolysis of acetic acid. Results for authentic standards of pure acetic acid demonstrated the practicality of this on-line method, although the carbon isotope ratio of the methyl group could not be determined directly. The precision of the carbon isotope measurements was 0.4 per thousand (1sigma). The carbon isotope distribution determined by the on-line method was identical to that determined by the conventional off-line method within analytical error. The advantages of the on-line method compared with the conventional off-line method are that it is less laborious, requires less analytical time (less than one hour per sample) and, most importantly, uses smaller sample sizes (ca. 10 nanomole). An application of this on-line method to natural geochemical samples will provide an insight into the geochemical cycle of acetic acid.     

On-line monitoring of the residence time distribution along a kneading block of a twin-screw extruder

An on-line Residence Time Distribution (RTD) measuring set-up that enables the characterisation of twin screw extruders at short axial increments is presented. It uses the light emission of a fluorescent tracer (perylene) to generate concentration versus time curves, which are then used to determine the usual RTD parameters, delay time, mean residence time and variance. The system is initially mounted on a rectangular die coupled to a single screw extruder, on-line and off-line measurements being directly contrasted. Then, the optical probe is positioned at several points along the axis of a modular co-rotating twin screw extruder, on-line and off-line data being again compared. Having gained confidence in the measuring technique, an experimental unit utilizing a transparent barrel is used to characterize the evolution of RTD along a kneading block of a twin screw extruder.     

Determination of Manganese by Cathodic Stripping Voltammetry on a Microfabricated Platinum Thin–film Electrode

In this work, we report on the determination of trace manganese (Mn) using cathodic stripping voltammetry (CSV) using a microfabricated sensor with a Pt thin‐film working electrode. While an essential trace metal for human health, prolonged exposure to Mn tends to gradually impair our neurological system. The potential sources of Mn exposure make it necessary to monitor the concentration in various sample matrices. Previous work by us and others suggested CSV as an effective method for measuring trace Mn. The analytical performance metrics were characterized and optimized, leading to a calculated limit of detection (LOD) of 16.3 nM (0.9 ppb) in pH 5.5, 0.2 M acetate buffer. Further, we successfully validated Mn determination in surface water with ∼90% accuracy and >97% precision as compared with inductively coupled plasma mass spectrometry (ICP‐MS) “gold standard” measurement. Ultimately, with stable, accurate and precise electrochemical performance, this Pt sensor permits rapid monitoring of Mn in environmental samples, and could potentially be used for point‐of‐use measurements if coupled with portable instrumentation.     

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.     

Microfluidic tool based on the antibody-modified paramagnetic particles for detection of 8-hydroxy-2'-deoxyguanosine in urine of prostate cancer patients

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10 μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5 pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.     

Development of an Opto-fluidic Microsystem Dedicated to Chemical Analysis in a Nuclear Environment

Micromachining techniques enable the fabrication of innovative lab-on-a-chip. Following the trend in chemical and biological analysis, the use of microsystems also appears compelling in the nuclear industry. The volume reduction of radioactive solutions is especially attractive in order to reduce the workers radiation exposition in the context of off-line analysis in spent nuclear fuel reprocessing plants. We hence present the development of an opto-fluidic sensor combining microfluidic channels for fluid transportation and integrated optics for detection. With the aim of achieving automated microanalysis with reduced response time the sensor is made compatible with a commercial microfluidic holder. Therefore the chip is connected to computer controlled pumps and electrovalves thanks to capillary tubing. In this paper we emphasis on the fluid handling capacities of the opto-fluidic sensor.     

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.     

On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSⁿ experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase.     

Recent developments in on-line electrochemical stripping analysis--an overview of the last 12 years

This paper presents an overview of the field of electrochemical stripping analysis in flow systems covering developments in the last 12 years (since 1998). The review discusses the flow schemes utilized in stripping analysis, techniques for on-line sample pre-treatment, the main pre-concentration and stripping/detection modes, the most important flow-through cell configurations used and the different types of working electrodes. Finally, applications in inorganic and organic analysis are discussed. Special emphasis is given to different novel approaches developed over the last few years that hold some promise for the future such as the use of the lab-on-a valve (LOV) configuration, microfluidic manifolds, flow-probes for remote sensing, environmentally friendly electrode materials and hyphenation with spectroscopic techniques.     

浅析锡对电感耦合等离子体质谱法测定镉的干扰

在不同的工作条件下,分析了不同Sn(锡)/Cd(镉)比值下Sn对电感耦合等离子体质谱法(ICP-MS)测定Cd的干扰程度,并将经过在线干扰校正和离线干扰校正后的Cd浓度与标准值比较,实验结果表明:当样品中Sn浓度与Cd浓度比值在2以下时,Sn对Cd的干扰很小,Cd的测定值不用经过任何方式校正也比较接近标准值;当Sn浓度与Cd浓度比值在2~30时,采用离线校正和在线校正后的Cd测定值与标准值均比较接近;当Sn浓度与Cd浓度比值大于30时,采用离线校正才能获得更满意的结果。