1. Introduction

Miniaturization and integration of electrospray devices has been the subject of intense research and development, due to the ability to generate charged, fine droplets for deposition, sorting, and dispersion of materials. Miniaturized electrospray devices coupled with mass spectrometers offer benefits including minimal sample consumption, fast analysis, and high sensitivity for chemical analysis. Well-fabricated nozzles are crucial to generate disperse ionized electrospray required to obtain reliable mass spectra of analytes. Typically, fused silica capillaries pulled to a narrow tip are used as electrospray sources, which offer simplicity, but are not easily integrated onto microfluidic platforms. Since initial demonstrations of electrospray from microfluidic devices [1, 2], several approaches have been used and are reviewed [3, 4]. In particular, microfabrication techniques have been adopted to form nozzles directly on microfluidic devices [5–9]. Specific examples include high aspect ratio (height to width) nozzles fabricated in silicon [6]; however, silicon substrates are difficult to adapt to microfluidic separation devices that operate using electrokinetic transport. Also, laser ablation techniques have been used to form nozzles in poly(methylmethacrylate) [7] and polyimide [8] substrates. Compared to polymer substrates, glass and silica substrates combine chemical inertness with excellent mechanical, optical, and electrical properties, but obtaining high aspect ratio features on glass using standard microfabrication techniques remains difficult.

Damage by femtosecond lasers is remarkably reproducible and has been widely studied for material processing due to its unique capabilities of high precision and three-dimensional micro- and nanomachining in transparent dielectrics [10–14]. This technique can be enhanced with the presence of water during machining, which efficiently removes ablation debris so that obstruction of features can be avoided [15–26]. Moreover, the machined surfaces are smooth [20, 22], and a number of microfluidic components such as high aspect ratio microholes, microchambers, and nanopores have been successfully fabricated [15–26]. Here, we use water-assisted laser machining to fabricate electrospray nozzles on glass coverslips and assembled microfluidic devices. As seen in Fig. 1, the basic nozzle design consists of a hole through the coverslip and a trough machined around the through-hole to confine the electrospray and prevent liquids from wicking across the glass surface. Of particular interest is the ability to machine nozzles after microfluidic device fabrication, allowing us to place nozzles over any microchannel at any location on the microfluidic device. This circumvents having to align nozzles fabricated in a coverslip over microchannels fabricated in a glass substrate during the bonding step.

 

Fig. 1. Schematic of the laser machining procedure. Using a 1.3 NA objective, the laser is focused through a coverslip onto the glass/water interface on the side distal to the objective. First, a through-hole is drilled through the coverslip, followed by machining of a trough around the through-hole.

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2. Experimental procedure and results

In our initial experiments, we machined nozzles in coverslips (22 mm × 22 mm, Fisherfinest Premium Cover Glass, Fisher Scientific, Inc.) using water-assisted femtosecond laser machining. The laser beam (pulse duration: 400 fs; repetition rate: 1.5 kHz; wavelength: 527 nm; Intralase Corp.) was focused onto a borosilicate coverslip by an oil-immersion objective (numerical aperture (NA): 1.30; working distance: 200 µm; Plan-Neofluar, Carl Zeiss, Inc.) mounted on a Zeiss Axiovert 200 microscope. The sample was translated by a three-dimensional nanomanipulation stage (Nano-Drive, Mad City Labs, Inc.), and the machining process was monitored with a CCD camera (EM-CCD Digital Camera C9100-13, Hamamatsu Photonics K. K.) mounted on the microscope. As depicted in Fig. 1, a hole was drilled through the coverslip, starting from the glass/water interface and moving toward the objective. Bubbles formed in the water droplet during machining helped remove machining debris [17, 25]. The pulse energy was set at 15 nJ/pulse, close to the threshold for optical breakdown, resulting in a smooth wall. For each layer, the substrate was continuously translated in a shrinking circular pattern with a step size of 80 and 300 nm in radial and azimuthal directions, respectively. After the material from a given layer was removed, machining was delayed for 2 s to allow the bubbles to move away from the ablation site and carry machining debris with them. The substrate was then stepped in the vertical direction by 300 nm to machine the next layer. By removing material layer by layer, the hole was drilled through the 150-µm thick coverslip. Next, to complete the nozzle, the laser focus was returned to the top surface of the coverslip to machine the trough surrounding the through-hole. The trough had a larger interaction area with the water droplet than the through-hole, so the ablation debris was much easier to expel, allowing for a higher machining speed. As a result, step sizes were enlarged to 200 nm in the radial direction, 400 nm in the azimuthal, and 400 nm in the vertical. The entire machining process took 15–20 min per nozzle, but this fabrication time could be substantially decreased with a higher repetition rate laser. Figure 2 shows scanning electron microscope (SEM) images of the nozzle, revealing that both the through-hole and trough have smooth surfaces. The through-hole and ring dimensions are similar to those of commercially available electrospray tips (e.g., New Objective, Inc.), and the trough dimensions are similar to microchannels etched in glass substrates. The through-hole, ring, and trough dimensions can be varied independently.

 

Fig. 2. (a). Schematic of the electrospray nozzle with typical dimensions. Scanning electron microscope images of the (a) top and (b) 30° side views of a nozzle machined in a glass coverslip.

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To characterize the electrospray, a coverslip containing a machined nozzle was mounted on a glass microscope slide using a poly(dimethylsiloxane) (PDMS) gasket. We sandblasted an access hole in the glass slide (AEC Air Eraser, Paasche Airbrush Co.) and epoxied a threaded fitting (N-333, Upchurch Scientific, Inc.) to the backside to connect a fused-silica capillary (25 µm i.d., 360 µm o.d., Polymicro Technologies). The capillary was used to couple the electrical potential and pressure-driven flow to the nozzle, which was positioned 3 mm from the inlet on the mass spectrometer. The electrospray experiments were conducted on a Q-TOF Ultima Global mass spectrometer (Waters Corp.) using a scan time of 1 s for all experiments. The capillary was connected to a syringe pump (780100C, Cole-Parmer), which was used to prime the nozzle with a 50/50 water/acetonitrile solution with 0.1% (v/v) formic acid. To induce electrospray, a potential of 3.5 kV relative to the inlet on the mass spectrometer was applied through a tee union connected to the capillary. Electrospray was evaluated for four 30-min periods with pressure-driven flow applied (15 µL/hr) and two 30-min periods without pressure-driven flow. Over these time periods, the electrospray was stable as indicated by monitoring the major ion peaks resulting from unpolymerized residues extracted from the PDMS gasket into the water/acetonitrile solution. These major ions had mass-to-charge ratios (m/z) of 503, 519, 725, and 741. Similar results were obtained using a 50/50 water/methanol solution with 0.1% (v/v) formic acid. During these experiments, the electrospray was not visible using the optics on the mass spectrometer, suggesting the electrospray was very fine and fluid exiting the aperture was not wicking across the coverslip surface.

After demonstrating electrospray with the nozzles fabricated on coverslips, we machined nozzles on fully assembled microfluidic devices. The water-assisted laser machining allows direct fabrication of the nozzles at arbitrary locations over the microchannels of assembled microfluidic devices, circumventing issues with aligning the nozzles over the microchannels prior to device assembly. We first fabricated a microfluidic device with a single microchannel (Fig. 3) using standard fabrication procedures [27]. Microchannels, 20 µm deep and 40 µm wide, were etched into white crown glass substrates (B270, Telic Co.). Holes were sandblasted at the ends of the channel to provide fluid access. No. 1 coverslips (24 mm × 50 mm, VWR, Inc.) were bonded to the B270 substrates by hydrolyzing both pieces in NH₄OH:H₂O₂:H₂O (2:1:2), rinsing thoroughly with water, bringing the substrate and coverslip into contact with each other, and annealing at 350°C for 20 h. Threaded fittings (N-124S, Upchurch Scientific, Inc.) were epoxied over the sandblasted holes to allow pressure and electrical connections to be made.

Because the microfluidic device thickness was > 1 mm, the 200-µm working distance of the 1.3 NA objective used to fabricate the nozzles in Fig. 2 was too short to focus through the microchip and machine nozzles on the surface opposite the objective. Consequently, the setup was reconfigured to machine the nozzle on the surface near the objective, as shown in Fig. 3(b). A temporary coverslip was placed on the near side of the microchip where the nozzle is machined, and an air objective with a 700-µm working distance (0.75 NA, Plan-Neofluar, Carl Zeiss, Inc.) was used instead of the high NA objective. During machining, the microchannel, as well as the gap between the microchip and temporary coverslip (~70 µm), was filled with water. This allowed water-assisted machining on the near side of the microchip, while preventing water and machining debris from contacting the objective.

 

Fig. 3. (a). Schematics of the top and side views of a microfluidic device with an integrated electrospray nozzle. (b). Schematic of the nozzle machining process on a microfluidic device with a temporary coverslip and water layer underneath the microfluidic device.

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Similar to the process used to form the nozzles in the coverslips, the through-hole was machined from the microchannel in the direction of the objective. The procedure was the same as that on the coverslip with step sizes of 80, 300, and 300 nm in the radial, azimuthal, and vertical directions, respectively. The laser energy was 60 nJ/pulse. After the through-hole was completed, we carved the trough in step sizes of 100, 300, and 300 nm in the radial, azimuthal, and vertical directions, respectively. Although the less tightly focused laser beam from the 700-µm working distance objective produced larger machining voxels, we did not change the step sizes significantly for the machining because machining beginning from the front surface is not as efficient as from the rear surface [16]. Single and multiple nozzles were formed over the microchannels, and Fig. 4(a) shows a transmitted light image of a nozzle machined over a microchannel. In addition, these devices were used to electrospray the peptide bradykinin (20 µg/mL) in a 50/50 water/acetonitrile solution with 0.1% (v/v) formic acid. A typical mass spectrum obtained in positive ion mode is shown in Fig. 4(b). The singly charged bradykinin has an m/z of 1060, and the doubly charged species has an m/z of 530. Mass spectra identical to Fig. 4(b) were obtained using a standard capillary electrospray tip (PicoTip Emitter, 15 µm i.d. tip, New Objective, Inc.).

 

Fig. 4. (a). Transmitted light image of an electrospray nozzle fabricated directly over a microchannel in an assembled microfluidic device. The image in (a) is a composite of two images taken at different focal planes. (b) Mass spectrum of bradykinin electrosprayed from the microfluidic device through the nozzle. The most intense peak at m/z = 530 is doubly charged bradykinin.

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3. Summary

We have demonstrated fabrication of high quality electrospray nozzles on glass coverslips and on assembled microfluidic devices using water-assisted femtosecond laser machining. This approach permits integration of electrospray nozzles onto glass microfluidic devices, thus simplifying construction of integrated microfluidic systems which employ mass spectrometric detection.