Analytical characterization of microfabricated SU-8 emitters for electrospray ionization mass spectrometry

We present a detailed optimization and characterization of the analytical performance of SU-8-based emitters for electrospray ionization mass spectrometry (ESI/MS). The improved SU-8 fabrication process presented here enhances patterning accuracy and reduces the time and cost of fabrication. All emitters are freestanding and enable sample delivery by both pressure-driven and spontaneous flows. The optimized emitter design incorporates a sharp, double-cantilevered tip implemented to the outlet of an SU-8 microchannel and provides highly sensitive ESI/MS detection. Moreover, the optimized design allows the use of relatively large microchannel dimensions (up to 200 x 50 microm(2), w x h) without sacrificing the detection sensitivity. This is advantageous with a view of preventing emitter clogging and enabling reproducible analysis. The measured limits of detection for the optimized emitter design were 1 nM for verapamil and 4 nM for Glu-fibrinopeptide B with good quantitative linearities between 1 nM and 10 microM (R(2) = 0.9998) for verapamil and between 4 nM and 3 microM (R(2) = 0.9992) for Glu-fibrinopeptide B. The measured tip-to-tip repeatability for signal intensity was 14% relative standard deviation (RSD) (n = 3; 5 microM verapamil) and run-to-run repeatability 4-11% RSD (n = 4; 5 microM verapamil) for all individual emitters tested. In addition, long-term stability of < 2% RSD was maintained for timescales of 30 min even under free flow conditions. SU-8 polymer was also shown to be chemically stable against most of the tested electrospray solvents.     

A planar microfabricated nanoelectrospray emitter tip based on a capillary slot

We report on the fabrication and testing of planar nib-like structures for nanoelectrospray ionization-mass spectrometry (nanoESI-MS) applications. The micro-nib structures were fabricated on silicon substrates using the negative photoresist SU-8; they include capillary slots with widths of 8 and 16 microm. A suitable wafer cleaving step made the nib-like structures overhang the edge of a silicon substrate to provide a robust interface for nanoESI-MS applications; this freeing of the nib tip from the wafer surface created a point-like structure that is essential to establish an electrospray. The micro-nib sources were successfully tested on an LCQ Deca XP+ ion trap mass spectrometer using peptide samples at concentrations down to 1 microM. The high voltage was applied using a platinum wire inserted in the sample reservoir upstream to the capillary slot. A Taylor cone was clearly seen at the nib tip. The micro-nibs performed well at voltages as low as 0.8 kV; such performances are state-of-the-art with respect to current micromachined ESI-MS interfaces and are conditions comparable to those used for standard emitter tips. In addition, we clearly observed the influence of the micro-nib slot width on the ionization performances: the narrower the slot, the better the performances.     

A novel nib-like design for microfabricated nanospray tips

We present here novel tips for nanoelectrosray ionization-mass spectrometry (ESI-MS) applications. These ionization sources have a planar geometry in the shape of a nib. Their functioning is based on a principle much akin to that of a fountain pen in that fluids are actuated by capillarity. Once a voltage is applied, an electrospray is formed at the nib tip. The nib fabrication relies on micromachining techniques using the epoxy-based negative photoresist SU-8 and a double exposure photolithographic process. Two types of nib-like sources were fabricated; they were made either conductive by metallization with a nickel layer or non-conductive but hydrophilic by covering them with a SiO(2) layer. In the latter case, the HV was applied via a Pt wire inserted into the reservoir feature of the nib. The nib-like sources were tested on an ion trap mass spectrometer using Gramicidin S samples at concentrations as low as 1 microM and ionization voltages as low as 1.2 kV. We have observed a good overall stability of the spray during the tests with no marked decrease in the signal intensity even under extreme conditions.     

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.     

Miniaturized multichannel electrospray ionization emitters on poly(dimethylsiloxane) microfluidic devices.

A multichannel electrospray ionization (ESI) emitter was fabricated as part of a poly(dimethylsiloxane) (PDMS) microfluidic device using a three-layer photoresist process which also produces a self-alignment system to make a bonding between the top and bottom PDMS parts. The prototype device (2 cm high x 5 cm wide x 5 cm long) had 16-channels (30 microm wide x 50 microm deep) with emitters of 1 mm length and 60 degrees point angle. The PDMS emitter tips enabled interfacing the device to ESI-mass spectrometry; a stable electrospray from the tips was performed with limits of detection under 1 microM for reference peptides (adrenocorticotropic hormone fragment 1-17, angiotensin I and III).     

Open-tubular capillary electrochromatography-mass spectrometry with sheathless nanoflow electrospray ionization for analysis of amino acids and peptides

A novel, rugged sheathless capillary electrochromatography-electrospray ionization (CEC-ESI) device, in which an open-tubular separation capillary and an electrospray tip are integrated with a Nafion tubing junction, is coupled to mass spectrometry (MS) for the analysis of amino acids and peptides. A stable electrospray was generated at nanoflow rates by applying a positive electrical potential at the Nafion membrane junction. To sustain the stable spray, an electroosmotic flow (EOF) to the spray was supported by coating the fused silica capillary with Lupamin, a high-molecular-weight linear positively charged polyvinylamine (PVAm) polymer, which also minimizes analyte adsorption. Electrochromatographic separation of amino acids and peptides was further enhanced by the chromatographic selectivity of Lupamin stationary phase for these molecules. The device was very reliable and reproducible for CEC-ESI-MS analyses of amino acids and peptides for over a hundred injections. The separation and detection behaviors of amino acids and peptides under different conditions including pH, concentration, and composition of mobile phases on Lupamin-coated and uncoated capillaries have been investigated. The relationship between nano electrospray stability and EOF is discussed.     

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.     

A new mass spectrometry electrospray tip obtained via precise mechanical micromachining

A new electrospray tip incorporating a beveled edge has been designed for use in plastic electrophoresis chip/electrospray mass spectrometry. Theoretical hydrodynamic analysis has been conducted to model the analytical sensitivity of the spray tip shape. A larger sample wall, that provides increased hydraulic pressure, is recommended in order to preserve the fluid stability at the tip outlet. A polymer with better hydrophobic characteristics than glass was used for the spray tip in order to restrict moisture accumulation at the spray tip outlet. Experimental results demonstrate that the analytical sensitivity of the proposed scheme is better than that obtained using the flat-head tip. Although a tapered capillary tip is commonly used in electrophoresis chip/electrospray mass spectrometry, the proposed tip offers a similar sensitivity while being more rugged and durable than the conventional tapered capillary tip. The cost of our design is also much lower than conventional spray tips.     

An integrated micropump and electrospray emitter system based on porous silica monoliths

The work presents the design of an integrated system consisting of a high-pressure electroosmotic (EO) micropump and a microporous monolithic emitter, which together generate a stable and robust electrospray. Both the micropump and electrospray emitter are fabricated using a sol-gel process. Upon application of an electric potential of sufficient amplitude (>2 kV), the pump delivers fluids with an electroosmotically induced high pressure (>1 atm). The same potential is also harnessed to electrostatically generate a stable electrospray at the porous emitter. Electrokinetic coupling between pump and spray produces spray features different from sprays pressurized by independent mechanical pumps. Four typical spray modes, each with different drop sizes and charge-to-mass ratios, are observed and have been characterized. Since the monolith is silica-based, this integrated device can be used for a variety of fluids, especially organic solvents, without the swelling and shrinking problems that are commonly encountered for polymer monoliths. The maximum pressure generated by a 100 microm id monolithic pump is 3 atm at an applied voltage of 5 kV. The flow rate can be adjusted in the range of 100 nL/min to 1 microL/min by changing the voltage. For a given applied voltage across the pump and emitter system, it is seen that there exists one unique flow rate for which flow balance is achieved between the delivery of liquid to the emitter by the pump and the liquid ejection from the emitter. Under such a condition, a stable Taylor cone is obtained. The principles that lead to these results are also discussed.     

Characterization of SU-8 for electrokinetic microfluidic applications

The characterization of SU-8 microchannels for electrokinetic microfluidic applications is reported. The electroosmotic (EO) mobility in SU-8 microchannels was determined with respect to pH and ionic strength by the current monitoring method. Extensive electroosmotic flow (EOF), equal to that for glass microchannels, was observed at pH > or =4. The highest EO mobility was detected at pH > or =7 and was of the order of 5.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer. At pH < or =3 the electroosmotic flow was shown to reverse towards the anode and to reach a magnitude of 1.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer (pH 2). Also the zeta-potential on the SU-8 surface was determined, employing lithographically defined SU-8 microparticles for which a similar pH dependence was observed. SU-8 microchannels were shown to perform repeateably from day to day and no aging effects were observed in long-term use.     

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.     

Efficient electrospray ionization from polymer microchannels using integrated hydrophobic membranes

A simple process for realizing stable and reliable electrospray ionization (ESI) tips in polymer microfluidic systems is described. The process is based on the addition of a thin hydrophobic membrane at the microchannel exit to constrain lateral dispersion of the Taylor cone formed during ESI. Using this approach, ESI chips are shown to exhibit well-defined Taylor cones at flow rates as low as 80 nL min(-1) through optical imaging. Furthermore, stable electrospray current has been measured for flow rates as low as 10 nL min(-1) over several hours of continuous operation. Characterization of the electrospray process by optical and electrical monitoring of fabricated ESI chips is reported, together with mass spectrometry validation using myoglobin as a model protein. The novel process offers the potential for low-cost, direct interfacing of disposable polymer microfluidic separation platforms to mass spectrometry.     

Fabrication of low-melting-point alloy microelectrode and monolithic spray tip for integration of glass chip with electrospray ionization mass spectrometry

In this paper, a glass microchip-based emitter with a low-melting-point alloy (LMA) microelectrode and a monolithic tip for electrospray ionization mass spectrometry (ESI-MS) was described. So far, the fabrication of metal microelectrode achieving direct electrical contact in the microchannel of glass chip is still a challenge. A novel fabrication approach for LMA microelectrode in the glass chip was developed to achieve direct electrode-solution electrical contact in the microchannel. An electrode channel and a sample channel were firstly fabricated on a glass chip with a micropore connecting the two channels. The melted LMA was filled into the electrode channel under a pressure of ca. 100 kPa, forming a stable and nicely fitted interface at the micropore between the sample and the electrode channels due to surface tension effect. The melted LMA filled in the electrode channel was then allowed to solidify at room temperature. The channel geometries including the distance between the sample and the electrode channels on the mask and the turning angle of the electrode channel were optimized for fabricating the LMA electrode. In this work, an improved fabrication approach for monolithic emitter tip based on pyramid-shaped tip configuration and stepped grinding method was also developed to fabricate well-defined sharp tips with a smallest tip end size of ca. 15 μm × 50 μm. Two types of emitter tip end including puncher-shaped tip and fork-shaped tip were produced. The emitter with the fork-shaped tip showed better working stability (4.4% RSD, TIC) at nanoliter-scale flow rate of 50 nL/min. The fabrication approaches for the LMA microelectrode and emitter tip are simple and robust, and could be carried out in most of routine laboratories without the need of complicated and expensive instruments. The performance of the emitter was evaluated in the analysis of reserpine, angiotensin II and myoglobin. A continuous experiment over 6 h demonstrated good stability of the present system in long-term analysis.     

A fused silica micro-electrospray tip with an electrically floating metal wire insert to achieve more stable electrospray ionization

A new electrospray tip with a wire insert was tested and compared with the conventional bare fused silica capillary tip. The new tip combined the approach of conventional fused silica spray tips with those containing metal wires. Here, we used a floating wire so that the tips could be prepared and replaced more easily. With the conventional tip, the electrospray process became unstable and the spray current fluctuated significantly in the presence of an air bubble. When the wire-inserted tip was used under the same conditions, much less signal deterioration occurred. The superior performance of this tip over the conventional tip was attributable to its enhanced electric conduction. The new tip has great potential for improving signal stability in LC mass spectrometry.     

Poly(dimethylsiloxane) microchip: microchannel with integrated open electrospray tip

A polymer microchip with an open tip for electrospray mass spectrometry is presented. The tip consists of a groove with parallel walls where a droplet can form at the end surface. A lid covers the whole chip except at the microchannel tip, which is left open. Poly(dimethylsiloxane) (PDMS) microchips were cast using a nickel mould which in turn was replicated from a dry etched silicon wafer. Tips with microchannel widths of around 50 microm could easily be replicated. Since the tip had no cover, the assembly of microchip and cover was simplified. A total ion current variation of 5% during 300 s was achieved for a 1 microM myoglobin solution. The non-complex design of the cover makes it suitable for versatile tests of chip prototypes. The nickel mould was found to be useful for PDMS microstructure fabrication. Also, such a robust mould allows casting electrospray tips in more rigid thermoset materials.     

Electrospray ionization from a gap with adjustable width

In this paper, we present a new concept for electrospray ionization mass spectrometry, where the sample is applied in a gap which is formed between the edges of two triangular-shaped tips. The size of the spray orifice can be changed by varying the gap width. The tips were fabricated from polyethylene terephthalate film with a thickness of 36 microm. To improve the wetting of the gap and sample confinement, the edges of the tips forming the gap were hydrophilized by means of silicon dioxide deposition. Electrospray was performed with gap widths between 1 and 36 microm and flow rates down to 75 nL/min. The gap width could be adjusted in situ during the mass spectrometry experiments and nozzle clogging could be managed by simply widening the gap. Using angiotensin I as analyte, the signal-to-noise ratio increased as the gap width was decreased, and a shift towards higher charge states was observed. The detection limit for angiotensin I was in the low nM range.     

Two-dimensional microfabricated sources for nanoelectrospray

The idea of a novel two-dimensional (2D) nanoelectrospray ionization emitter tip with the shape of a nib is explored here. This novel planar design is studied as an alternative to the needle-like standard emitter tips that suffer from a lack of reproducibility and robustness and from an inherent incompatibility with high-throughput analysis. The composition of the micro-nib sources is analogous to the working of a simple fountain pen, with a liquid reservoir linked to a micro-nib tip from which the sample is electrosprayed via a capillary slot. The micro-nib prototypes described here were fabricated using microtechnology techniques and using the epoxy-based negative photoresist SU-8. The resulting free-standing micro-nib structure was supported by a silicon wafer. We present here two series of such micro-nib sources, the latter series exhibiting improved characteristics such as a 8 micro m source width of the nib tip. They were tested in mass spectrometry experiments on an ion trap mass spectrometer (LCQ Deca XP+, Thermo Finnigan) using standard peptide samples having concentrations down to 1 micro M and with a high voltage (HV) supply around 1 kV for the second series of micro-nib sources. In addition to the stability of the spray, the obtained mass spectra showed the reliability of these sources for peptide analysis; the signal of the spectra was as intense and the signal-to-noise ratio (S/N) as high as that obtained with the use of standard emitter tips.     

Study of SU-8 to make a Ni master-mold: Adhesion, sidewall profile, and removal

For disposable microfluidic devices, easy and inexpensive fabrication is essential. Consequently, replication of microfluidic devices, using injection molding or hot embossing, from a master-mold is widely used. However, the conventional master-mold fabrication technique is unsatisfactory in terms of time and costs. In this regard, direct Ni growth (electroplating) from a back plate is promising when the photoresist is well-defined. Here, we demonstrate the use of SU-8 as a photoresist to define the Ni-growth region. We accomplish this application by focusing on the adhesion, the sidewall profile, and the removal of SU-8: the adhesion is enhanced by controlling the exposure dose, the soft-baking time, and by choosing the adhesion-promoting layer; the sidewall profile is regulated by selecting the intensity of each exposed wavelength, showing an aspect ratio of up to 20.9; and, easy removal is achieved by choosing a proper photoresist-stripper. Using the master-mold fabricated by this method, we test the mechanical stability of the features according to the aspect ratio and length; in the hot embossing process, the features are stable in the aspect ratio of up to 5.8 at a length of 200 microm. In addition, the plastic devices fabricated from this method are applied to the passive stop valves, showing a capillary pressure (-0.2 to -7.2 kPa).     

Control of electroosmotic flow in laser-ablated and chemically modified hot imprinted poly(ethylene terephthalate glycol) microchannels

The fabrication of microchannels in poly(ethylene terephthalate glycol) (PETG) by laser ablation and the hot imprinting method is described. In addition, hot imprinted microchannels were hydrolyzed to yield additional charged organic functional groups on the imprinted surface. The charged groups are carboxylate moieties that were also used as a means for the further reaction of different chemical species on the surface of the PETG microchannels. The microchannels were characterized by fluorescence mapping and electroosmotic flow (EOF) measurements. Experimental results demonstrated that different fabrication and channel treatment protocols resulted in different EOF rates. Laser-ablated channels had similar EOF rates (5.3+/-0.3 x 10(-4) cm(2)/Vs and 5.6+/-0.4 x 10(-4) cm(2)/Vs) to hydrolyzed imprinted channels (5.1+/-0.4 x 10(-4) cm(2)/Vs), which in turn demonstrated a somewhat higher flow rate than imprinted PETG channels that were not hydrolyzed (3.5+/-0.3 x 10(-4) cm(2)/Vs). Laser-ablated channels that had been chemically modified to yield amines displayed an EOF rate of 3.38+/- 0.1 x 10(-4) cm(2)/Vs and hydrolyzed imprinted channels that had been chemically derivatized to yield amines showed an EOF rate of 2.67+/-0.6 cm(2)/Vs. These data demonstrate that surface-bound carboxylate species can be used as a template for further chemical reactions in addition to changing the EOF mobility within microchannels.     

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.