Porous polymer monolith assisted electrospray from a glass microdevice

The coupling of a lab-on-a-chip microfluidic device to a nanoelectrospray ionization mass spectrometer has the potential to automate many routine analytical procedures and produce a powerful analytical tool. However, past coupling strategies have relied on complex manufacturing steps including drilling and etching the device to attach a capillary or building a nanospray emitter directly into the device. This study shows that a nanospray emitter can be easily fabricated using a porous polymer monolith (PPM) at the end of a glass microdevice. These devices are able to obtain a stable electrospray at a variety of flow rates (50-500 nL/min) but optimal results are obtained at lower flow rates (50-100 nL/min) compatible with electroosmotic flow processes. The PPM is photo-patterned so that it can be placed in any position within the channel of the device with no dead volume. The porous character and the hydrophobic nature of the PPM both aid in development of a stable electrospray process. Total ion current traces for the constant infusion of leucine-enkephalin and PPG show relative standard errors as low as 4%, and produce mass spectra with good signal-to-noise (S/N 43) from only 2 fmol of material. In addition, multiple experiments in a given day show good repeatability with variability as low as 13%, and the multiple flow paths inherent in the PPM limit sprayer clogging.     

新型纳升级毛细管刻蚀电喷雾质谱接口研究

对普通石英毛细管表面使用氢氟酸刻蚀技术进行刻蚀, 并与商品化鞘流液毛细管电泳-质谱接口(Sheathflow CE-MS interface)结合, 将其改装成一种新型的纳升级电喷雾质谱接口. 玻璃膜接口部分呈多孔结构, 壁厚约10 μm. 以细胞色素c对新型接口加以评价, 样品的流量最低可达到20 nL/min; 在50~500 nL/min流量范围内刻蚀接口具有较高的响应信号. 考察了接触电解质溶液对样品电离的影响; 比较微升级不锈钢接口和新型接口的蛋白质检测结果发现, 在流速为200 nL/min时, 检测灵敏度可以提高3.6倍.     

Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery

Nanospray experiments were performed on an ensemble of drug molecules and their commonly known metabolites to compare performance with conventional electrospray ionization (ESI) and to evaluate equimolar response capabilities. Codeine, dextromethorphan, tolbutamide, phenobarbital, cocaine, and morphine were analyzed along with their well-known metabolites that were formed via hydroxylation, dealkylation, hydrolysis, and glucuronidation. Nanospray exhibited a distinct trend toward equimolar response when flow rate was reduced from 25 nL/min to less than 10 nL/min. A more uniform response between the parent drug and the corresponding metabolites was obtained at flow rates of 10 nL/min or lower. The largest discrepancy was within +/-50% for plasma samples. Nanospray was used as a calibrator for conventional ESI liquid chromatography/tandem mass spectrometry (LC/MS/MS) and normalization factors were applied to the quantitation of an acyl-glucuronide metabolite of a proprietary compound in rat plasma. A nanospray calibration method was developed with the standard curve of the parent drug to generate quantitative results for drug metabolites within +/-20% of that obtained with reference standards and conventional ESI. The nanospray method provides a practical solution for the quantitative estimation of drug metabolites in drug discovery when reference standards are not available.     

An evaluation of the performance of porous polymer monolith assisted electrospray

A novel electrospray interface, which has distinct advantages over conventional pulled silica emitters, has been developed. This novel interface can be easily fabricated by forming a porous polymer monolith (PPM) at the end of a fused-silica capillary that facilitates a stable electrospray over a wide range of flow rates with only a modest increase in back-pressure. A comparison was made between the PPM-assisted electrospray and a commercial nanosprayer in terms of sensitivity, stability and robustness. A PPM-filled electrospray tip produced a day-to-day signal variation of 23% relative standard deviation (RSD) over a 3-day period when spraying a 1.0 microM test peptide solution. Furthermore, three different capillaries fabricated by the same process produced a signal variation of 17% RSD, indicating that the fabrication process shows good reproducibility. The multiple flow paths of the PPM function to split the flow and reduce clogging. Even following the accumulation of debris after prolonged use, a stable spray could still be generated with the PPM-filled capillary while the commercial nanosprayer ceased to function properly. In terms of sensitivity, PPM-assisted electrospray showed an enhancement in sensitivity at infusion flow rates between 100 to 1000 nL/min while commercial nanosprayers performed slightly better at flow rates below 100 nL/min. A sample purification step can be combined with the PPM-assisted sprayer, using the PPM as a stationary phase to desalt and preconcentrate samples prior to mass spectrometric detection.     

Monolithic column plastic microfluidic device for peptide analysis using electrospray from a channel opening on the edge of the device

Electrospray from a channel exit at the edge of a fluorocarbon coated cycloolefin copolymer microfluidic device has been investigated. The fluorocarbon coating facilitated generation of a stable electrospray, thereby enhancing the detectability of electrospray ionization (ESI) mass spectrometry (MS). A microfluidic device of integrated ESI emitters and monolithic liquid chromatography columns has been fabricated on a cycloolefin copolymer chip. The monolithic columns were polymerized in situ using UV irradiation with a photomask to confine the porous polymer monolith to the desired regions of the channels. The monolithic stationary phase was homogenous and well bonded to the channel surfaces, which had been functionalized by graft polymerization. The ESI potential was applied within the channel via a carbon ink line. The performance of this microfluidic device was demonstrated by analysis of a tryptic digest of bovine serum albumin on an ion trap MS instrument.     

A new on-chip ESI nozzle for coupling of MS with microfluidic devices

This paper presents a new on-chip electrospray ionisation (ESI) nozzle, which can be used as an interface for coupling microfluidic devices with mass spectrometric (MS) detection. The nozzle was micromilled in a polymer foil (polymethylmethacrylate (PMMA) 750 microm thick), normally used as a cover for microfluidic chips. The performance of this device was examined in the ESI-MS analysis of the tetrapeptide MRFA (methionine-argenine-phenylalanine-alanine). The spray quality is basically dependent on the inner diameter of the nozzle, beside the part of the organic modifier in the solution to be sprayed. Three different inner nozzle diameters (30, 50, 100 microm) and two different apex angles were investigated. Stable electrospray conditions can be generated with a relative standard deviation less than 10% of the total ion current, and down to a concentration of 0.01 micromol L(-1). The production of this ESI interface is relatively simple for the purpose of a low-cost batch fabrication of miniaturized analytical instruments.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Flow-rate characterization of microfabricated polymer microspray emitters

Microfabricated polymer microspray emitters are characterized in terms of applicable flow rates, temporal and spectral signal-to-noise ratios (SNRs), and solution composition. First, microspray emitters can be operated with 50% methanol/49% water/1% acetic acid from 250 nL/min up to 7 microL/min, with better SNRs above 1 microL/min. Interestingly, even at the lowest flow rates tested, they compare well with nanospray capillaries in terms of mass spectral performances. Secondly, they can be operated with acetonitrile from 10 up to 99% (v/v), with flow rates from 250 nL/min up to 4 microL/min. Even if the mass spectral performances (especially the spectral SNRs) vary with the acetonitrile content, this study validates such microfabricated microspray emitters as interfaces between liquid-phase separations and electrospray mass spectrometers.     

Synchronized dual-polarity electrospray ionization mass spectrometry

This work describes the synchronized dual-polarity (DP) electrospray ionization (ESI) method and demonstrates the first DP ESI mass spectra obtained using two mass spectrometers. Stable double Taylor cones were produced by applying two counter electric voltages with opposite polarities to one electrosprayer. The development of double Taylor cones required higher extraction voltages than conventional ESI, but DP ESI worked effectively at liquid flow rate range three times wider than conventional ESI. Using pure methanol, the emission currents of the two cones were neutralized and no current was drawn from the sprayer. Synchronized DP mass spectra were obtained using electrospray calibrants dissolved in methanol solution of low water content. For bovine insulin with conventional electrospray solution, the gas-assisted electrospray delivered satisfactory sensitivity and stability for routine mass analyses.     

Preparation of Durable Emitter of Electrospray Mass Spectrometry by Covalently Coating the Fused‐Silica Capillary Tip with Carbon‐Nanotube Sol‐Gel Composite Material

A simple approach for the preparation of emitter of electrospray ionization mass spectrometry by covalently coating the fused‐silica capillary tip with the conductive carbon‐nanotube sol‐gel composite material (CNTSCM) is described. The CNTSCM was prepared by dispersing single‐walled carbon nanotubes in the sol composed of a mixture of 3‐glycidoxypropyltrimethoxysilane and 3‐aminopropyltrimethoxysilane, and ethanol. The long‐term stability of the prepared ESI emitters was demonstrated by at least 180 h of continuous use. Signal intensity obtained by the prepared emitter was mass‐flux sensitive when the flow rate was lower than 500 nL/min, while the signal intensity performed a concentration dependence when the flow rate was in the range of 500–800 nL/min. The usefulness of such a prepared emitter was demonstrated by the analysis of various types of samples such as organic small molecular drugs, oligosaccharide, peptide, and protein.     

Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters

Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored because of difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, whereas the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.

Long-lived metallized tips for nanoliter electrospray mass spectrometry

Sheathless electrospray at nL/min flow rates combined with Fourier-transform mass spectrometry has made possible high resolving power (\gr 50,000) mass spectra of subattomole samples of \gr 8 kDa proteins separated by capillary electrophoresis (Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science, 1996, 273, 1199-1202). However, for this new method the mechanical stability of the thin (35 to 100 nm) gold film electrodes has limited tip lifetime to 15 to 30 min. A technique for SiO_x coating of the gold is described that provides a steady ion current (± 10 pA) for 1 to 2 h, even with arcs or interruptions of the electrospray voltage.     

Sheathless capillary electrophoresis-mass spectrometry using a pulsed electrospray ionization source

A sheathless interface has been developed for coupling CE with electrospray IT mass spectrometer. This interface utilized a pulsed ESI source. The use of a pulsed electrospray source allows the use of a sprayer with larger orifice, and thus alleviates the problem of column clogging during conductive coating and CE analysis. A pulsed ESI source operated at 20 Hz and 20% duty cycle was found to produce the optimal signals. For better signals, the maximum ion injection time in the IT mass spectrometer has to be set to a value close to the actual spraying time (10 ms). Using a sprayer with 50 microm od, more stable and enhanced signals were obtained in comparison with continuous CE-ESI-MS under the same flow rate (150 nL/min). The utility of this design is demonstrated with the analysis of synthetic drugs by CE-MS.     

Microfluidic device for capillary electrochromatography-mass spectrometry

A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4-8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement.     

Highly robust stainless steel tips as microelectrospray emitters

Tapered stainless steel spray tips for sheathless microelectrospray ionization (microESI) have been developed. The fabrication procedure for the tapered stainless steel tips was optimized using an electropolishing technique followed by removal of the burr. Using the tip as the microESI emitter, a stable ESI spray was obtained at a flow rate of 20 nL/min. The sensitivity of the microESI system was almost two orders greater than that of the conventional ion spray system. The tip was highly stable, and was successfully used for over 1000 h. Moreover, these stainless steel tips were suitable for use with sheathless capillary electrophoresis/mass spectrometry (CE/MS) and capillary liquid chromatography/mass spectrometry (LC/MS) for routine analysis in proteomic and pharmaceutical applications.     

Microfluidic chips for mass spectrometry‐based proteomics

Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor‐intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high‐throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) are summarized and applications of chip‐based separations and digestion technologies to proteomic analysis are presented. Copyright © 2009 John Wiley & Sons, Ltd.     

Poly(dimethylsiloxane) electrospray devices fabricated with diamond-like carbon-poly(dimethylsiloxane) coated SU-8 masters

This study presents coupling of a poly(dimethylsiloxane) (PDMS) micro-chip with electrospray ionization-mass spectrometry (ESI-MS). Stable electrospray is generated directly from a PDMS micro-channel without pressure assistance. Hydrophobic PDMS aids the formation of a small Taylor cone in the ESI process and facilitates straightforward and low-cost batch production of the ESI-MS chips. PDMS chips were replicated with masters fabricated from SU-8 negative photoresist. A novel coating, an amorphous diamond-like carbon-poly(dimethylsiloxane) hybrid, deposited on the masters by the filtered pulsed plasma arc discharge technique, improved significantly the lifetime of the masters in PDMS replications. PDMS chip fabrication conditions were observed to affect the amount of background peaks in the MS spectra. With an optimized fabrication process (PDMS curing agent/silicone elastomer base ratio of 1/8 (w/w), curing at 70 degree C for 48 h) low background spectra were recorded for the analytes. The performance of PDMS devices was examined in the ESI-MS analysis of some pharmaceutical compounds and amino acids.     

High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry

A new methodology has been developed for high sensitivity electrospray ionization mass spectrometric analyses of phenylthiohydantoin (PTH) amino acid derivatives. Key components of the methodology are the use of a solvent system consisting of methanol/dichloromethane (1:1 v/v) containing 5-mM lithium triflate, a stainless steel electrode having a relatively large surface area, and a microscale electrospray nozzle that provides for stable electrospray at flow rates in the range of 100–500 nL/min. A linear response for the absolute signal intensity of the protonated molecule was observed for a number of derivatives over the concentration range of 50–1000 fmol/µL. For all except the arginine derivative, there was a decrease in the signal intensity with increasing flow rate with 100–300 nL/min being optimum. Collision induced dissociation (CID) product ion spectra were obtained for 21 derivatives including carboxymethyl cysteine and dehydrothreonine. Leucine and isoleucine can be distinquished on the basis of their CID product ion spectra. A subfemtomole detection limit was demonstrated for the phenylalanine PTH derivative in a selected reaction monitoring (SRM) experiment. Samples from an automated Edman microsequencer run have been analyzed using the new technique and compared to results obtained by conventional high-performance liquid chromatography analysis with UV detection. This work demonstrates the feasibility of using mass spectrometry to identify and quantitate the products generated by automated protein microsequencing using standard Edman degradation chemistry.     

Short-capillary electrophoresis with electrochemiluminescence detection using porous etched joint for fast analysis of lidocaine and ofloxacin

This paper describes the properties of a recently developed electrospray emitter coated with a fluorinated polymer of low surface energy. The emitters can produce stable electrospray from solvents of various surface tensions including distilled water with a high surface tension at a flow rate range of micro- to nanoliters without the aid of any nebulizing gas. The electrically non-conductive nature of the tips virtually eliminates electrical discharge and allows stable electrospray in the negative ion mode. The emitters are suitable for hyphenating HPLC to mass spectrometry in both positive and negative ion modes at low flow rates.     

Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry

A novel microfabricated multichannel emitter for electrospray ionization mass spectrometry (ESI-MS) was implemented with polydimethylsiloxane (PDMS) using a soft lithography technique. The emitters are formed as electrospray tips along a thin membrane on the edge of the device with channels of 100 microm x 30 microm dimensions. The electrospray performance of the PDMS emitters for a single channel device and a four channel device interfaced with a time-of-flight mass spectrometer was evaluated for detecting the molecular weight of reference peptides (angiotensin I and bradykinin). The emitters were durable at the flow rate of 1-20 microL min(-1) for more than 30 h of continuous electrospray with limit of detection of 1 microM (S/N 18). This microfabrication method for a PDMS multichannel emitter as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.