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Dip-pen nanolithography (DPN) has been extensively used before for patterning
surfaces; however a complete understanding of the ink transport mechanisms is still
lacking. Moreover, quality control of the fabricated structures is a bottleneck in DPN
fabrication, and one aspect of this is the quantification of the ink mass transfer to the
substrate during the lithographic process. There is a demand for measuring the exact
amount of molecules deposited on a surface by lithographic methods, especially for
biological applications. This article demonstrates a quantitative method for measuring
the amount of ink transferred onto the substrate in DPN with phospholipids
by dynamic force spectroscopy. To achieve this, the harmonic oscillation of a
microcantilever in an atomic force microscope is used, obtaining picogram mass
sensitivity in the determination of mass deposition.
1. Introduction
The ability to tailor the chemical composition and structure of a surface at the nanometer length scale is essential for
fabricating novel nanomaterials, understanding underlying
nanoscience and engineering, developing integrated systems
for demanding applications and ultrahighly sensitive biosensors. Nanolithography is the art and science of etching, writing,
or printing at the nanoscopic level, in which the dimensions
of characters are in the order of nanometers. In particular,
scanning probe microscopy (SPM)-based approaches offer
both ultrahigh resolution and in situ imaging capabilities.
Dip-pen nanolithography (DPN), a method for directly
depositing molecules from an ink-coated atomic force microscope (AFM) tip onto a substrate of interest, is a powerful tool
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materials.[15,16] For many applications (e.g., in arraying proteins or other bioactive substances), it is desired to know how
much of an ink was deposited in each lithographic feature.
One approach to quantify the amount of transferred ink is
by subsequent observation with fluorescence microscopy.[17]
The cantilever arrays typically used in DPN offer another
convenient way to address this problem in situ by using the
cantilever itself for mass detection:[1820] generally there are
two kinds of sensing methods utilizing microcantilevers as
a very sensitive chemical sensor or mass sensor; one is to
detect the static bending caused by surface stress due to analyte adsorption onto a flexible cantilever on which a chemically active layer is coated.[21,22] This method is believed to
be very sensitive; however, quantitative evaluation is difficult
because the stress induced by the adsorption is unpredictable.
Furthermore, the drifts of the signal caused by the optical
sensing system make long-time measurements difficult. The
other method is to use resonant frequency changes due to
mass loading;[2224] It seems particularly effective when mass
loading does not cause surface stress. Therefore, we decided
to take the approach of using the harmonic oscillation of a
microcantilever in an AFM to measure the amount of ink
transfer to the substrate in the case of DPN with phospholipids, obtaining a picogram mass sensitivity.
1
2B
k
,
M
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(1)
M
f
f
(2)
Figure 1. Frequency spectra of the cantilever in air before being coated with ink (DOPC+Rhodamine) (black curve), after being coated with ink (red
curve), and after making 50 dots on the surface (green curve). b) The magnified version of the rectangular region marked in (a). The images show
that the resonance frequency of the cantilever increases with decrease of the effective mass of the cantilever and vice versa, as expected.
decay fit to the experimental data points. The area of the dots
has been measured from a fluorescence microscopy image
of the dot patterned substrate using ImageJ.[26] The area of
dots decreases within the first few dots and then stabilizes in
a similar way like the average mass transfer stabilizes after
the first few dots. This is in agreement with the procedure
of bleeding the tips in DPN, where tips are freed from
excessive ink by writing some sacrificial structures before
performing the actual wanted lithographic structure. It is
interesting to note that the measured area is not directly correlated to the amount of transferred ink: although less ink
was transferred to each dot on average in the case of RH =
50% the area per dot is most of the times slightly higher than
in the case of RH = 65%. This reflects the factor that the dot
shape will vary with humidity due to changes in surface tension and contact angle and thus the projected dot area will
not directly translate into the dots mass.
Figure 2. a) Optical micrograph image of the cantilever after being coated with the ink. The tip region is marked with a white circle. b) Fluorescently
labelled dots made on a plasma cleaned cover slip at RH = 50%. White scale bars correspond to 50 m.
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Figure 3. The resonance frequency of the cantilever after writing of each dot at a) RH = 65% and b) RH = 50%. There are two different regimes of
average ink transfer to the substrate marked with a red and green linear fit in the graphs. The average mass transfer per dot corresponding to the
red line is 2.03 0.19 pg for RH = 65% and 1.59 0.10 pg for RH = 50%. For the green lines, the mass transfer is 0.10 0.02 pg for RH = 65% and
0.08 0.02 pg for RH = 50%. The dot area for each dot as measured by fluorescence microscopy is shown for c) RH = 65% and d) RH = 50%.
m A
X2
X1
M2 + M1
(3)
X2
n1
X1
(4)
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Figure 4. Amount of ink transfered to each specific dot for a) RH = 65% and b) RH = 50%. The average mass transferred to the surface corresponding
to the two different regions from Figure 3 has been indicated by the red and the green lines in the graph.
3. Conclusion
We demonstrated a straight forward way of measuring the
mass transfer into single lithographic features during DPN
with phospholipid inks using the change in resonance frequency of a microcantilever and its changing mass. Although
the AFM combined with the commercial DPN setup that we
have used is not as sensitive with regard to frequency shifts
as dedicated AFM setups, we could still achieve sub-picogram
sensitivity by fitting resonance curves and averaging over
several lithographic features. The method has the potential
to be used as a standard method to determine the amount
of deposited material, e.g., during arraying of bioactive substances such as proteins or allergens for immunoassays. It
would be desirable for future DPN setups to enable readout
of the frequency shift for single pens of a pen array before
and after lithography, enabling a quantitative determination
of ink transfer onto the substrate for each individual tip as
well as trying to enhance the stability of the system, so that
the theoretically possible sensitivity limit (0.1 pg; that is,
still one order of magnitude better than the 1 pg obtained
for single-dot analysis) can be achieved. Nevertheless, useful
information on the average amount of active compound in
a single lithographic feature can still be obtained by determining the mass change over several features (e.g., such as the
40 to 50 dots as presented here), which is feasible for quality
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control of immuno- or other protein assays since these usually contain hundreds to thousands of single dot features.
4. Experimental Section
Materials: The DOPC (phase transition temperature, Tm =
16.5 C, molecular weight = 786.113 g/mol), and the fluorophore-labeled
phospholipid
(1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (18:1
lissamine rhodamine/PE), molecular weight = 1319.753 g/mol)
were purchased from Avanti Polar Lipids, Alabaster, AL, USA.
Chloroform (high-performance liquid chromatography (HPLC)
grade) was purchased from Sigma. Ultrapure water with a resistivity of 18.2 Mcm was used. Glass coverslips (18 mm 18 mm)
were purchased from VWR Scientific. Tapping mode cantilevers
were purchased from Budget Sensors.
Experimental Methods: The experiments were carried out using
commercially available instrumentation. Tip coating, DPN writing,
and measuring of the resonance frequency of the cantilever
were carried out using a commercial DPN writer combined with
AFM (DPN 5000, NanoInk Inc., USA, software packages InkCAD
and SPM Cockpit) and the following accessories: Tapping mode
cantilever (from Budget Sensors, aluminum-coated rectangular
cantilever with resonance frequency 300 KHz and spring constant 40 N/m according to manufacturers value) and inkwells of
type IWL-0021-01 (custom-made from NanoInk Inc., USA). The ink
wells are microfluidic chips that allow for depositing the desired
ink dissolved in an appropriate solvent (chloroform) into an ink
reservoir. The ink reservoir is large enough for convenient handling with pipettes and is connected to the actual microwells for
dipping the tips via microchannels (typical width 6 m). The inkwells were filled with a chloroform solution of the phospholipid ink
(1 L, 10 mM, doped with 1 mol% of the dye-labeled lipid). The
solvent was allowed to evaporate for at least 1 h before coating
the tips. The cantilevers were plasma treated with oxygen
(10 sccm, 100 mTorr, 30 W, 5 min) prior to inking. Tips were inked
by placing them in contact with the inkwell and increasing the
humidity to (70 1)% for at least 45 min. Glass substrates were
plasma-treated with oxygen (20 sccm, 100 mTorr, 100 W, 30 s) to
render them hydrophilic. After preparation of the substrates and
coating of the tips with ink, the tip was brought into contact with
the surface for 10 s to write a dot and then retracted 20 m above
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the surface to check the resonance frequency after writing the dot.
This procedure was repeated 40 times to write 40 dots, and the
whole run of 40 dots was performed twice (with re-inking of tip in
between experiments) at two different RH values of (65 1)% and
(50 1)% with a controlled temperature of T = 22 1 C. After
changing the RH inside the glove box, the system was allowed to
equilibrate for approximately an hour to stabilize. The AFM was
operated in noncontact mode (approximately 20 m above the
sample surface) for measuring the resonance frequency of the cantilever before coating the tip with ink, after coating with ink, and
after writing of each particular dot.
Fitting of Resonance Curves: To determine the resonance frequency from the recorded resonance curves, a small in-housedeveloped visual basic program was used. The program reads
in the recorded resonance curves saved by the AFM software
and performs a least-squares fit to determine a Gaussian fit. The
parameters obtained from this first fit are then used as starting
parameters for a second least-squares fit with a Lorentzian function of which the peak frequency is regarded as the resonance
frequency of the cantilever. This two-step approach ensured a
better convergence than directly fitting a Lorentzian function to
the raw data.
Fluorescence Microscopy: This was carried out on an upright
Eclipse 80i fluorescence microscope (Nikon). Patterns were
aligned for imaging using alignment markers scratched onto the
glass surface. Bleaching of fluorophores was minimized (especially
for quantitative measurements) by first focusing on the alignment mark before exposing the patterned area to light, and the
lamp intensity was kept at a minimum. For quantitative measurements of each dot made on the surface as shown in Figure 3, the
fluorescence intensity and area of the dots were measured using
ImageJ.[26]
Supporting Information
Supporting Information is available from the Wiley Online Library
or from the author.
Acknowledgements
The authors want to thank S. Sekula-Neuner, T. Laue, and F.
Brinkmann for their support and useful discussions. S.B. thanks
the Helmholtz Association for nancial support. H.F. acknowledges
support from DFG (TRR 61) and the World Class University program
of the Korean Ministry of Education, Science, and Technology at
Gwangju Institute of Science and Technology. This work was partly
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carried out with the support of the Karlsruhe Nano Micro Facility
(KNMF, www.knmf.kit.edu), a Helmholtz Research Infrastructure at
Karlsruhe Institute of Technology (KIT, www.kit.edu).
This Full Paper is part of the Special Issue dedicated to Chad Mirkin
in celebration of 20 years of inuential research at Northwestern
University.
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