Shape-anchored porous polymer monoliths for integrated online solid-phase extraction-microchip electrophoresis-electrospray ionization mass spectrometry.

We report a simple protocol for fabrication of shape-anchored porous polymer monoliths (PPMs) for on-chip SPE prior to online microchip electrophoresis (ME) separation and on-chip (ESI/MS). The chip design comprises a standard ME separation channel with simple cross injector and a fully integrated ESI emitter featuring coaxial sheath liquid channel. The monolith zone was prepared in situ at the injection cross by laser-initiated photopolymerization through the microchip cover layer. The use of high-power laser allowed not only maskless patterning of a precisely defined monolith zone, but also faster exposure time (here, 7 min) compared with flood exposure UV lamps. The size of the monolith pattern was defined by the diameter of the laser output (∅500 µm) and the porosity was geared toward high through-flow to allow electrokinetic actuation and thus avoid coupling to external pumps. Placing the monolith at the injection cross enabled firm anchoring based on its cross-shape so that no surface premodification with anchoring linkers was needed. In addition, sample loading and subsequent injection (elution) to the separation channel could be performed similar to standard ME setup. As a result, 15- to 23-fold enrichment factors were obtained already at loading (preconcentration) times as short as 25 s without sacrificing the throughput of ME analysis. The performance of the SPE-ME-ESI/MS chip was repeatable within 3.1% and 11.5% RSD (n = 3) in terms of migration time and peak height, respectively, and linear correlation was observed between the loading time and peak area.

Transmission mode desorption atmospheric pressure photoionization.

RATIONALE: Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that is suitable for the direct analysis of polar and nonpolar compounds from a variety of surfaces. Conventional DAPPI uses reflection geometry, but here transmission mode (TM)-DAPPI is introduced for fast and easy analysis of liquid samples. METHODS: Stainless steel and PEEK meshes were used as sampling support in TM-DAPPI. The sample was applied either in the form of a droplet on the mesh, or by dipping the mesh in the sample solution. Physical parameters affecting the ionization efficiency were optimized for TM-DAPPI. The mesh materials were used to extract compounds from aqueous samples, which were then analyzed by TM-DAPPI. TM-DAPPI and conventional DAPPI were compared. RESULTS: In TM-DAPPI, intense signals for the analytes were achieved with less heating power, and lower nebulizer gas and dopant flow rates than optimally used in conventional DAPPI. Either due to this, or the different sample support material used, a much lower background and improved sensitivity compared to conventional DAPPI was achieved. The analytes could be extracted and concentrated from liquid samples on the mesh material used in TM-DAPPI, which was especially efficient for the nonpolar benzo[a]pyrene. This effect was utilized in the analysis of triacylglycerols from cow milk. CONCLUSIONS: While conventional DAPPI is still the method of choice for solid samples, TM-DAPPI can be utilized as a fast, easily automated method for analyzing liquid samples. The mesh materials can be utilized for extraction of low polarity compounds, such as steroid hormones or PAHs from dilute, aqueous solutions, followed by subsequent analysis by TM-DAPPI.

Microchip technology in mass spectrometry.

Microfabrication of analytical devices is currently of growing interest and many microfabricated instruments have also entered the field of mass spectrometry (MS). Various (atmospheric pressure) ion sources as well as mass analyzers have been developed exploiting microfabrication techniques. The most common approach thus far has been the miniaturization of the electrospray ion source and its integration with various separation and sampling units. Other ionization techniques, mainly atmospheric pressure chemical ionization and photoionization, have also been subject to miniaturization, though they have not attracted as much attention. Likewise, all common types of mass analyzers have been realized by microfabrication and, in most cases, successfully applied to MS analysis in conjunction with on-chip ionization. This review summarizes the latest achievements in the field of microfabricated ion sources and mass analyzers. Representative applications are reviewed focusing on the development of fully microfabricated systems where ion sources or analyzers are integrated with microfluidic separation devices or microfabricated pums and detectors, respectively. Also the main microfabrication methods, with their possibilities and constraints, are briefly discussed together with the most commonly used materials.

Hybrid ceramic polymers: new, nonbiofouling, and optically transparent materials for microfluidics.

A new, commercial hybrid ceramic polymer, Ormocomp, was introduced to the fabrication of microfluidic separation chips using two independent techniques, UV lithography and UV embossing. Both fabrication methods provided Ormocomp chips with stable cathodic electroosmotic flow which enabled examination of the Ormocomp biocompatibility by means of microchip capillary electrophoresis (MCE) and (intrinsic) fluorescence detection. The hydrophobic/hydrophilic properties of Ormocomp were examined by screening its interactions with bovine serum albumin and selected amino acids of varying hydrophobicity. The results show that the ceramic, organic-inorganic polymer structure natively resists biofouling on microchannel walls even so that the Ormocomp microchips can be used in intact protein analysis without prior surface modification. With theoretical separation plates approaching 10(4) m(-1) for intact proteins and 10(6) m(-1) for amino acids and peptides, our results suggest that Ormocomp microchips hold record-breaking performance as microfluidic separation platforms. In addition, Ormocomp was shown to be suitable for optical fluorescence detection even at near-UV range (ex 355 nm) with detection limits at a nanomolar level ( approximately 200 nM) for selected inherently fluorescent pharmaceuticals.

Dynamic coating of SU-8 microfluidic chips with phospholipid disks.

In this work, PEG-stabilized phosphatidylcholine lipid aggregates (disks), mimicking mammalian cell membranes, were introduced as a new biofouling resistant coating for SU-8 polymer microchannels. A rapid and simple method was developed for immobilization of PEGylated phosphatidylcholine disks in microchannels. Microfluidic chips made from SU-8, PDMS, or glass were dynamically coated with the PEGylated disks followed by characterization of their surface chemistry before and after coating. On the basis of the observed changes in EOF and nonspecific protein adsorption, the affinity of the PEGylated disks was shown to be particularly strong toward SU-8. The PEG-lipid coating enabled permanent change in EOF in SU-8 microchannels with an initial value of 4.5 x 10(-8) m(2) V(-1) s(-1), decreasing to 2.1 x 10(-8) m(2) V(-1) s(-1) (immediately after modification), and, eventually, to 1.5 x 10(-8) m(2) V(-1) s(-1) (7 days after modification) for 9 mM sodium borate (pH 10.5) as BGE. As determined by the Wilhelmy plate measurements and microchip-CE analysis of BSA, the PEG-lipid coating also enabled efficient biofouling shield against protein adsorption, similar to that of low amounts of SDS (3.5 mM) or Tween-20 (80 microM) as buffer additives. These results suggest that dynamically attached PEG-lipid aggregates provide stable, biomimicking surface modification that efficiently reduces biofouling on SU-8.

Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates.

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, ß-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.

Electrospray encapsulation of hydrophilic and hydrophobic drugs in poly(L-lactic acid) nanoparticles.

An electrospray method is developed for preparation of beclomethasone-dipropionate- and salbutamol-sulfate-loaded biodegradable poly(L-lactic acid) nanoparticles. Different set-up parameters for electrospraying are examined on particle size, and preparation conditions are optimized for producing spherical-drug-loaded nanoscale particles by controllable processing parameters. Polylactide (PLA)-drug nanoparticles with average diameters of around 200 nm are achieved in a stable cone-jet mode with a flow rate of 4 microL min(-1), polymer concentration of 1%, and ammonium hydroxide content of 0.05%. Morphology and size of the drug-polymer nanoparticles are analyzed by scanning electron microscopy and transmission electron microscopy. Changes in the crystallinity of the PLA polymer and the model drugs are detected by X-ray powder diffraction, and the absence of molecular interactions are confirmed by thermal analyses. The results indicate clearly that electrospraying is a potential method for producing polymeric nanoparticles and for encapsulating both hydrophilic and hydrophobic drugs efficiently into the nanoparticles.

Rapid and sensitive drug metabolism studies by SU-8 microchip capillary electrophoresis-electrospray ionization mass spectrometry.

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface, and an electrospray ionization (ESI) emitter were developed to improve the speed and throughput of metabolism research. Validation of the microchip method was performed using bufuralol 1-hydroxylation via CYP450 enzymes as the model reaction. The metabolite, 1-hydroxybufuralol, was easily separated from the substrate (R(s)=0.5) with very good detection sensitivity (LOD=9.3nM), linearity (range: 50-500nM, r(2)=0.9997), and repeatability (RSD(Area)=10.3%, RSD(Migrationtime)=2.5% at 80nM concentration without internal standard). The kinetic parameters of bufuralol 1-hydroxylation determined by the microchip capillary electrophoresis (CE)-ESI/mass spectrometry (MS) method, were comparable to the values presented in literature as well as to the values determined by in-house liquid chromatography (LC)-UV. In addition to enzyme kinetics, metabolic profiling was demonstrated using authentic urine samples from healthy volunteers after intake of either tramadol or paracetamol. As a result, six metabolites of tramadol and four metabolites of paracetamol, including both phase I oxidation products and phase II conjugation products, were detected and separated from each other within 30-35s. Before analysis, the urine samples were pre-treated with on-chip, on-line liquid-phase microextraction (LPME) and the results were compared to those obtained from urine samples pre-treated with conventional C18 solid-phase extraction (SPE, off-chip cartridges). On the basis of our results, the SU-8 CE-ESI/MS microchips incorporating on-chip sample pre-treatment, injection, separation, and ESI/MS detection were proven as efficient and versatile tools for drug metabolism research.

Implementation of droplet-membrane-droplet liquid-phase microextraction under stagnant conditions for lab-on-a-chip applications.

In the current work, droplet-membrane-droplet liquid-phase microextraction (LPME) under totally stagnant conditions was presented for the first time. Subsequently, implementation of this concept on a microchip was demonstrated as a miniaturized, on-line sample preparation method. The performance level of the lab-on-a-chip system with integrated microextraction, capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection in a single miniaturized device was preliminarily investigated and characterized. Extractions under stagnant conditions were performed from 3.5 to 15 microL sample droplets, through a supported liquid membrane (SLM) sustained in the pores of a small piece of a flat polypropylene membrane, and into 3.5-15 microL of acceptor droplet. The basic model analytes pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from alkaline sample droplets (pH 12), through 1-octanol as SLM, and into acidified acceptor droplets (pH 2) with recoveries ranging between 13 and 66% after 5 min of operation. For the acidic model analytes Bodipy FL C(5) and Oregon Green 488, the pH conditions were reversed, utilizing an acidic sample droplet and an alkaline acceptor droplet, and 1-octanol as SLM. As a result, recoveries for Bodipy FL C(5) and Oregon Green 488 from human urine were 15 and 25%, respectively.

Performance of SU-8 microchips as separation devices and comparison with glass microchips.

Effective analytical performance of native, all-SU-8 separation microdevices is addressed by comparing their performance to commercial glass microdevices in microchip zone electrophoresis accompanied by fluorescence detection. Surface chemistry and optical properties of SU-8 microdevices are also examined. SU-8 was shown to exhibit repeatable electroosmotic properties in a wide variety of buffers, and SU-8 microchannels were successfully utilized in peptide and protein analyses without any modification of the native polymer surface. Selected, fluorescent labeled, biologically active peptides were baseline resolved with migration time repeatability of 2.3-3.6% and plate numbers of 112,900-179,800 m(-1). Addition of SDS (0.1%) or SU-8 developer (1.0%) to the separation buffer also enabled protein analysis by capillary zone electrophoresis. Plate heights of 2.4-5.9 microm were obtained for fluorescent labeled bovine serum albumin. In addition, detection sensitivity through SU-8 microchannels was similar to that through BoroFloat glass, when fluorescence illumination was provided at visible wavelengths higher than 500 nm. On the whole, the analytical performance of SU-8 microchips was very good and fairly comparable to that of commercial glass chips as well as that of traditional capillary electrophoresis and chromatographic methods. Moreover, lithography-based patterning of SU-8 enables straightforward integration of multiple functions on a single chip and favors fully microfabricated lab-on-a-chip systems.

Fabrication of enclosed SU-8 tips for electrospray ionization-mass spectrometry.

We describe a novel electrospray tip design for MS which is fabricated completely out of SU-8 photoepoxy. A three-layer SU-8 fabrication process provides fully enclosed channels and tips. The tip shape and alignment of all SU-8 layers is done lithographically and is therefore very accurate. Fabrication process enables easy integration of additional fluidic functions on the same chip. Separation channels can be made with exactly the same process. Fluidic inlets are made in SU-8 during the fabrication process and no drilling or other postprocessing is needed. Channels have been fabricated and tested in the size range of 10 microm x 10 microm-50 microm x 200 microm. Mass spectrometric performance of the tips has been demonstrated with both pressure-driven flow and EOF. SU-8 microtips have been shown to produce stable electrospray with EOF in a timescale of tens of minutes. With pressure driven flow stable spray is maintained for hours. Taylor cone was shown to be small in volume and well defined even with the largest channel cross section. The spray was also shown to be well directed with our tip design.