Technical Note pubs.acs.org/ac
Scanning Electrospray Microscopy with Nanopipets Elizabeth M. Yuill, Wenqing Shi, John Poehlman, and Lane A. Baker* Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, United States S Supporting Information *
ABSTRACT: Electrospray from nanopipets is used to realize scanning electrospray microscopy (SESM). This technique provides an ambient, noncontact method to investigate surface topography with distance dependence of electrospray current as feedback for imaging. SESM approach curves, line scans, and images are reported. Salt deposition on the sample surface from SESM is also characterized.
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n this Technical Note, we make use of the distance dependence of the electrospray process to realize a new form of scanning probe microscopy, which we describe here as scanning electrospray microscopy (SESM). SESM employs a small pipet, similar to that used for scanning ion conductance microscopy (SICM)1 or some modes of scanning electrochemical microscopy (SECM),2−4 as a scanning electrode. Electrospray is generated by application of a suitable potential to an electrolyte solution inside of the pipet, and the magnitude of electrospray current is used for control of the probe−surface distance. As described here, we demonstrate technical considerations for suitable probe control and imaging. We have previously reported the application of nanopipets (capillaries pulled to nanoscale tip dimensions) as electrospray ionization-mass spectrometry (ESI-MS) emitters. For nanopipets, the high S/N and low potentials required to induce electrospray5 (relative to ESI with microscale emitters) prove especially interesting. Because electrospray depends on the electric field between the emitter and collector, the magnitude of ESI current is distance dependent for a constant applied potential. These properties, namely, small tips, low onset potentials, and a distance-dependent current, prove suitable to develop SESM, a new mode of feedback-controlled imaging. Here, in addition to demonstrating imaging with SESM, we use scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) to characterize the SESM process and to demonstrate spatially controlled electrospray deposition at small scales. Deposition as a consequence of this imaging technique provides future opportunities for electrodeposition6−8 and nanofabrication9−11 applications.
Figure 1. (a) Schematic of scanning electrospray microscopy (SESM) setup. (b) Representative approach curves over a gold-coated glass slide at 80, 90, 100, 110, and 120 V with a ∼22 nm inner diameter (i.d.) pipet.
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EXPERIMENTAL SECTION Materials. A solution of 1× phosphate buffered saline (PBS) was prepared with 18 MΩ·cm H2O from a Milli-Q water purification system (EMD Millipore, Billerica, MA) and used as © 2015 American Chemical Society
Received: September 6, 2015 Accepted: October 25, 2015 Published: October 26, 2015 11182
DOI: 10.1021/acs.analchem.5b03399 Anal. Chem. 2015, 87, 11182−11186
Technical Note
Analytical Chemistry
MicroFil needle (World Precision Instruments, Sarasota, FL) and centrifuged before use in experiments. Substrate Preparation. For approach curve experiments, either a gold or ITO-coated glass slide or an agarose gel was prepared as a substrate. Gold-coated glass slides were prepared with a 10 nm chromium adhesion layer, followed by an 80 nm gold layer using a thermal evaporator (BOC Edwards, Auto 306 Vacuum Coater, West Sussex, United Kingdom). To prepare the agarose substrate for both approach curves and for imaging, an agarose solution (5% w/v in 1× PBS) was heated until boiling and cast onto a TEM grid. After gelling, the TEM grid was peeled from the agarose gel, which was prepared for imaging. Agarose gel thickness was approximately 1−2 mm. For experiments, the agarose gel was placed on a gold-coated glass slide, which had a copper contact pad to attach to a grounded electrode. Approach curves were performed over a flat area on the gel, while imaging was performed over the negative of the TEM topographical features. To prepare an insulative feature for imaging, polystyrene microspheres were drop-cast onto agarose gel. Agarose casts were stored in 1× PBS buffer when not in use, to ensure the gel stayed hydrated.
a spray solvent. Agarose solutions (5% w/v) were prepared from molecular grade agarose (Bioline, Taunton, MA) with 1× PBS as the solvent. Ultra fine square 1500 mesh transmission electron microscopy (TEM) grids (G1500HS, Ted Pella, Redding, CA) with 11.5 μm wide holes and 5 μm wide bars were used to prepare a topographical feature for imaging. Polystyrene microspheres (Polyspherex PS-COOH) with an average diameter of 3 μm were obtained from Phosporex, Inc. (Hopkinton, MA) and were used as an insulative topographical feature. SESM Approach and Imaging. A general instrumental setup for scanning electrospray microscopy is shown in Figure 1a (additional experimental details can be found in the Supporting Information). Briefly, nanopipet probes were pulled to inner diameters between 20 and 35 nm. Probe size and integrity were characterized by electron microscopy before and after experiments. Pipets were backfilled with 1× PBS and subsequently mounted in a holder which included a backinserted Pt wire for application of potential. A commercial scanning ion conductance microscope (Park Systems XE-Bio SICM/AFM, Suwon, South Korea) was used to control probe position. An external power supply (Keithley 6487, Cleveland, OH) was used to bias the probe tip at potentials sufficient to result in electrospray. For SESM experiments, at relevant distances, the electrospray current increases (described vide infra) as the probe−surface distance (DPS) decreases. Distance dependence of the current is the opposite case for SICM, where currents decrease as the probe moves toward the surface. To account for this instrumentally, custom electronics were employed to invert the electrospray signal such that it could be fed into the commercial SICM. The current inverter allowed utilization of approach curves, approach−retract scanning (ARS)12 and other instrument protocols available in the SICM software/controller. Substrates examined consisted of either gold or indium tin oxide (ITO)coated glass slides or agarose gels and were connected to ground, while a bias between +80 and +130 V was applied to the pipet electrode to induce electrospray. For approach curves, pipets were approached at a rate of 0.3 μm/s until reaching a current limit of either 25 nA spray current (on gold-coated glass slide) or 15 nA spray current (on ITO-coated glass slide or agarose gel). SESM was used to image topographical features, as well as to deposit salt on the substrate surface for characterization of the SESM process. Approach−retract scanning parameters are seen in Table S1. For salt deposit characterization, fewer pixels were used in comparison to topographical imaging, and 8 × 8 pixels were acquired over a 75 × 75 μm area on a flat, gold-coated glass slide. For all imaging experiments, the probe was first approached to the surface and an appropriate current range was achieved by tuning the applied potential, which varied slightly between pipets. Nanopipet Fabrication and Characterization. A P-2000 CO2 laser puller (Sutter Instrument, Novato, CA) was used to pull quartz capillaries (Q100-70-7.5, Sutter Instrument, Novato, CA) to inner diameters that range from 20 to 35 nm. All capillaries were cleaned with piranha solution (3:1 v/v H2SO4/ 30% H2O2) prior to pulling. (CAUTION: “Piranha” solution reacts violently with organic materials; it must be handled with extreme care.) After fabrication, all pipets were imaged using scanning electron microscopy and scanning transmission electron microscopy (STEM) (Quanta FEG 600F, FEI, Hillsboro, OR). Pipets were backfilled with PBS using a
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RESULTS AND DISCUSSION The basis for SESM comes from the distance dependence of electrospray current. The Pfeifer and Hendricks approximation (eq 1),13 in conjunction with the electric field−distance relationship (eq 2),14 can be used as a starting place to understand the relationship between electrospray current, I (and also electric field, E), with the distance between the probe tip and the collecting (ground) electrode. I = [(4π /ε)3 (9γ )2 ε0 5]1/7 (KE)3/7 (Vf )4/7
(1)
Here, ε is permittivity of solvent, γ is surface tension of solvent, ε0 is permittivity of a vacuum, K is conductivity of solution, Vf is flow rate, and eq 2 is substituted for electric field. E=
AV r ln(4d /r )
(2) 15,16
where A is an empirical constant of 1.499, V is the potential between the emitter tip and grounded substrate, and r is the radius of the emitter. Spraying distance, d, is the distance between the emitter and substrate or the probe−surface distance. The combination of eqs 1 and 2 suggests that, as the probe−surface distance approaches zero, spray current increases rapidly in a nonlinear fashion (eq 3). ⎡ ⎤3/7 V 4/7 I ∝ k⎢ ⎥ (Vf ) ⎣ r ln(4d /r ) ⎦
(3)
Approach curves were measured to characterize the current− distance relationship experimentally (Figure 1b). For example, in a typical approach curve, 100 V was applied to the pipet electrode while the pipet was held over a gold substrate. As the probe approached the surface, the electrospray current between the probe tip and substrate was observed, typically at distances tens of micrometers from the surface. The probe−surface distance was decreased further until either a predetermined distance or a current set point (chosen arbitrarily as 25 nA spray current, Figure 1b) was met. The general shape of the experimental approach curves agrees with a distance-dependent relationship similar to what eq 3 predicts. A plateau region is seen at large DPS values, and electrospray current remains 11183
DOI: 10.1021/acs.analchem.5b03399 Anal. Chem. 2015, 87, 11182−11186
Technical Note
Analytical Chemistry relatively constant as DPS is initially decreased. As DPS decreases further, a sharp increase in spray current is observed. Although the general trend holds, fitting of approach curves with the explicit relationship in eqs 1−3 is not successful. We assume this failure originates from the difference in tip dimensions (micro vs nano) used in the original derivations of equations and in experiments here. We expect that at nanoscale dimensions the influence of parameters, such as fluid viscosity and surface tension,17,18 deviate from the relationship shown. These considerations are outside the scope of this Technical Note and will be detailed in future reports. Multiple approach curves were recorded over a range of potentials (80−120 V) with the same pipet (Figure 1b). Of note, potentials lower than 80 V often did not induce electrospray (presumably due to insufficient electric fields to support electrospray). If no electrospray was induced, an immediate spike in current resulted, which often broke the pipet tip (in the case of gold surfaces), rather than the gradual increase observed for approaches illustrated in Figure 1b. Approach curves generally followed the same trend: a nonlinear, fast rising increase at decreased probe−surface distances. As applied potential increased, higher spray currents were observed at similar probe−surface distances, and the onset of the current rise was recorded further from the surface, as compared to approaches at lower applied potentials. At lower applied potentials, approach curves demonstrated a sharper increase in current with decreased probe−surface distance; an effect we are presently investigating further. Despite similar pipet inner diameters, approach curves varied to some degree for different pipets (Figure S1); however, the overall shape of the approach curve was maintained, which suggests that pipet geometry (and the resulting effects on electric field) might influence the exact current−distance relationship. Due to slight variations between different pipets in the current−distance response, applied potentials were optimized for each pipet prior to imaging but typically fell between 80 and 100 V. Approach and retract curves were also recorded on agarose (Figure S2) and had good agreement with minimal hysteresis, indicative of a robust current−distance relationship (in general, the compliant nature of agarose gels provided a more forgiving surface for repeated approach− retract cycles). Nanopipet dimensions were monitored by SEM and STEM to verify that tips survived approach curves and the electrospray process (e.g., ∼22 nm i.d. before and after approach, Figure S2). To further confirm the feedback mechanism for approach curves was electrospray, control studies were performed (Figure S3) to compare this technique to a similar imaging technique, scanning electrochemical cell microscopy (SECCM), which brings a meniscus at the tip of a pipet into contact with a substrate surface and images via scanning the droplet.19,20 The primary difference between the two techniques is the applied potential (ca. 500 mV for SECCM vs 80−130 V for SESM). To compare techniques, a nanopipet filled with 1× PBS was approached to an ITO-coated glass slide with a potential between 80 and 110 V, retracted a known distance, and then reapproached at 500 mV applied potential. The latter approach curve was characterized by a sudden spike in current when the droplet made contact with the surface. For SESM, the increase in current occurs far from the surface and is gradual. Further, this experiment allows for more accurate estimation of the true DPS from comparison of the two types of approach curves.
The general shape of the approach curves suggests the electrospray currents exhibit distance dependence suitable to serve as a feedback signal for imaging. To realize scanning electrospray microscopy, ARS was utilized as the feedback routine for initial imaging. In ARS mode, the electrospray tip starts at a distance far from the surface and approaches until a current set point is reached. The probe position is recorded, and the probe retracts and is then moved to the next pixel. In essence, each pixel in the image consists of an approach curve, which is then used to determine surface topography in a noncontact fashion.12 As proof-of-concept, SESM was used to image an agarose mold cast from a TEM grid. In Figure 2, an SESM image along
Figure 2. SESM image (a) of an agarose gel replica (negative) of a TEM grid, as compared to an atomic force microscopy (AFM) image (b). Inset in b shows an optical image of the substrate. In c, line scans from SESM and AFM images are compared.
with an atomic force microscopy (AFM) image and an optical image of the mold are shown (Figure 2a, b, b inset, respectively). A 60 × 60 μm area was imaged at 64 × 64 pixels for both SESM and AFM. For SESM, 90 V was applied to the pipet electrode and a set point of 2.5 nA was used for ARS imaging. In the SESM image (Figure 2a), the agarose mold is reproduced with fidelity. Dimensions of the TEM grid (center hole and bar dimensions), which correspond to pillars and spacing between pillars in the agarose mold, are 11.5 and 5 μm, respectively, as compared to 10.7 ± 1.1 μm (n = 9) and 3.9 ± 1.0 μm (n = 9) in the SESM image. SEM images showed the pipet size was ∼25 nm i.d. after imaging, as compared to 22 nm i.d. before experiments, which indicates the pipet remained intact throughout imaging. In Figure 2c, line scans over the images in Figure 2a, b show that SESM measures similar feature depths as AFM (SESM ∼ 1.5 μm, as compared to 2.0 μm for AFM). The choice of applied potential is related to the imaging distance and subsequent resolution. For the current range investigated here, approach curves recorded at the lowest potentials demonstrated the sharpest increase in current with decreasing probe−surface distance (Figure 1b). For data shown in Figure 1b, an x-offset was observed between each approach curve. This arises from the differences in applied potential and 11184
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Analytical Chemistry
0.68 μm, n = 11). In the fast-scan axis, individual spots deposited are not always well resolved, which suggests solvent did not completely evaporate during spray or that spray did not fully turn off when translating in the x-direction to the next pixel location. XPS and XPS mapping were used to characterize deposits left on the sample from SESM (Figures 3c and S5, respectively). In the XPS spectra, peaks for sodium (Na 1s, Na KLL, and Na 2s) appear over the area imaged, and those same peaks are absent in areas not imaged by SESM. Signals from gold are attenuated for XPS on the area imaged by SESM as compared to areas that were not imaged. Since XPS analysis is surface-sensitive, the salt deposits from SESM attenuate the gold signal. Here, salt deposits from 1× PBS are shown; however, the use of alternative solutions may prove SESM as a tool for spatially controlled deposition of biomaterials,6−8 polymers,9 or metals.10,11While risk of clogging of the pipet tip would be problematic, electrospray of macromolecular species from emitters of this size has been demonstrated previously.5 Approach rate and applied potential could serve to control the amount of material deposited and will be studied in future investigations. Insulating features on top of a conductive substrate can also be imaged with SESM. Imaging of polystyrene microspheres on an agarose substrate with a 100 V applied potential produced the topographical image shown in Figure 4. Vertical resolution
the nature of the experiment. Approaches were performed sequentially from lowest to highest applied potential over the same position on the substrate to prevent any effects from surface tilt. Inherent to the electrospray mechanism employed in SESM, salt is deposited on the substrate surface (vide infra). For data shown, subsequent approach curves over the same spot then occur on top of the deposited salt, which may result in a net effect of smaller DPS. Thus, sequential approach curves (such as those shown in Figure 1b) taken at the same position have an increasingly positive x-offset. To illustrate this, approaches that were started at 90 V and increased to 130 V applied potential were recorded (Figure S1a). While the shape of the 80 V approach here is sharper than curves for higher potentials and more similar to the lower potential approaches in Figure 1b, the x-offset is increased, which suggests that a salt buildup on the surface might contribute to x-offset between approaches. Electrospray processes have been used previously for deposition on surfaces.21 Here, salt deposited during imaging demonstrates the possibility of utilizing SESM as a tool for deposition and serves as a method to investigate the process of SESM. To characterize deposited salt, a pipet was used to image a flat gold-coated surface in ARS mode. SEM, XPS, and XPS mapping were then performed for the area imaged by SESM. Deposits were made by spraying 1× PBS solution at 8 × 8 pixel spacing over a 75 × 75 μm area. Salt deposition is observed at each pixel (Figure 3a, b) and was 4.44 ± 0.72 μm in diameter (n = 13 measurements). Image size and the number of pixels gives an expected spacing between deposits of 9.38 μm. Spacing between deposited spots was slightly larger in the y-axis (10.64 ± 0.58 μm, n = 11) than the fast-scan x-axis (9.51 ±
Figure 4. SESM image of 3 μm diameter polystyrene particles on an agarose substrate. Inset, optical image of the same region.
is not representative of the true particle size (seen in attenuation of the particle height, ∼1.4 μm vs the 3 μm mean diameter from the manufacturer), possibly due to the lower conductivity over the particles; however, lateral resolution appears to be consistent with accepted particle size (3.3 ± 0.2 μm in particle diameter, n = 5 measurements). Pipet geometry and size were maintained before and after imaging (Figure S6). These results suggest that, with probe−surface distances that are relatively large (e.g., low current set point, high DPS), the electric field from the conductive agarose gel underneath and surrounding the particles is sufficient to maintain electrospray. Thus, while SESM requires a conductive substrate at present, lateral resolution when imaging insulative features on top of a conductive substrate can be maintained, which may be of significance in future sample analysis. An additional caveat to SESM is the nature of imaging. Deposition of salt can inherently damage the sample; however, for gold substrates and agarose samples used here, the salt could be washed off the surface with no discernible damage to the
Figure 3. (a) Scanning electron microscopy (SEM) image of salt deposits over a 75 × 75 μm area at 8 × 8 pixels from a ∼25 nm i.d. pipet. (b) Zoomed-in image of salt deposits seen in (a). (c) X-ray photoelectron spectroscopy (XPS) spectra show the area off (red) and on (blue) the area imaged in (a). Note: spectra have been offset by 3000 c/s for clarity. 11185
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sample. Additionally, deposition of salts on the surface can be obviated through use of electrospray solutions which do not contain salt (e.g., 70% methanol−water, 0.1% acetic acid).
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CONCLUSIONS In summary, distance-dependent electrospray from nanopipets has been characterized to provide signal for topographic imaging. Approach curves have experimentally demonstrated the relationship between electrospray current and distance between the probe and substrate. Approach−retract scanning mode was utilized to generate an image of both insulative and conductive topographical features. We expect that SESM may find future application in micro/ nano patterning and materials synthesis by electrospinning/ electrospray deposition. SESM as an imaging method also opens up possibilities for collection of concomitant ambient mass spectrometry imaging (MSI) and topographical information. SESM may also be suited for integration with MS in a desorption electrospray ionization (DESI) mass spectrometry imaging format, although additional experimental considerations (e.g., nebulizing gas) are required. Nanopipets would prove much smaller than presently employed DESI emitters and may be useful to lower the spot size of analysis. Sampling from a smaller surface area may also increase spatial resolution, with the caveat that smaller spot sizes may limit the overall MS signal. In addition to possible resolution advantages, SESM holds promise to add dynamic probe-surface distance control and topographic imaging to MSI. We are presently exploring the fundamentals of SESM as an electrochemical imaging platform further, as well as new routes to materials deposition and MSI applications of SESM.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03399. Additional experimental details, approach curves, raw SESM images, XPS maps, and STEM images of pipets after imaging (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (812) 856-1873. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Electronic Instrument Services at Indiana University is gratefully acknowledged for assistance with instrumentation. The authors appreciate XPS and scanning electron microscope use provided by the IU Nanoscale Characterization Facility. Access to XPS was provided by NSF Award DMR MRI1126394. Acknowledgement is given to Park Systems for access to instrumentation. Financial support was provided by Indiana University.
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REFERENCES
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