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Single-protein nanomechanical mass spectrometry in real time
M.S. Hanay
1,1
,
S. Kelber
1,2
,
A.K. Naik
1,2
,
D. Chi
1
,
S. Hentz
1,2
,
E.C. Bullard
1
,
E. Colinet
1,2
,
L.
Duraffourg
2
, and
M.L. Roukes
1,*
1
Kavli Nanoscience Institute and Departments of Physics, Applied Physics, and Bioengineering,
California Institute of Technology, MC 149-33, Pasadena, CA, 91125 USA
2
CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
Abstract
Nanoelectromechanical systems (NEMS) resonators can detect mass with exceptional sensitivity.
Previously, mass spectra from several hundred adsorption events were assembled in NEMS-based
mass spectrometry using statistical analysis. Here, we report the first realization of single-
molecule NEMS-based mass spectrometry in real time. As each molecule in the sample adsorbs
upon the NEMS resonator, its mass and the position-of-adsorption are determined by continuously
tracking two driven vibrational modes of the device. We demonstrate the potential of multimode
NEMS-based mass spectrometry by analyzing IgM antibody complexes in real-time. NEMS-MS
is a unique and promising new form of mass spectrometry: it can resolve neutral species, provides
resolving power that increases markedly for very large masses, and allows acquisition of spectra,
molecule-by-molecule, in real-time.
Mass spectrometry (MS) – the identification of species through molecular mass
measurements – is an important analytical tool in chemical and biological research. Since its
first applications to organic compounds more than a half century ago
1,2
, it has assumed an
increasingly dominant role in the life sciences and medicine. It is now arguably the mainstay
of proteomics
3
.
Among recent emerging areas of MS is the elucidation of the structure of complex protein
assemblies
4–7
. Critical to such measurements are spectrometers that are capable of high
resolution in the very large mass range – above several hundred kDa – which is at or beyond
the limit of many conventional MS techniques. Also essential is the development of new,
delicate sample handling methods for molecular ionization/injection, enabling so-called
“native” MS
4,8
, to permit large molecules or molecular assemblies to be transported, intact,
from the fluid phase to the vacuum phase for subsequent analysis. On these new fronts,
NEMS-MS offers significant promise
9–17
. NEMS are sensitive to the inertial mass of neutral
*
Corresponding author: roukes@caltech.edu.
1
These authors contributed equally to this work.
2
Present address: Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore, Karnataka, India
Author Contributions
MLR, AKN, MSH and SK conceived and designed the experiments. MSH, SK and AKN performed the experiments. MSH, SK, AKN
and MLR analyzed the data. MSH, SK, AKN, DC, SH, ECB, EC, LD and MLR contributed materials and analysis tools. MSH, SK,
MLR and AKN wrote the paper.
Competing Financial Interests
The authors declare no competing financial interests.
Additional Information
Supplementary information accompanies this paper at www.nature.com/naturenanotechnology.
Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/.
NIH Public Access
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. 2012 September ; 7(9): 602–608. doi:10.1038/nnano.2012.119.
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particles that accrete upon them; this makes them particularly well suited to studies that
require minimal ionization to avoid structural changes in the protein
4,8
.
We have discussed the principles and ultimate practical limits of NEMS-based mass
detection elsewhere
18
; here we briefly review the salient points. Upon adsorption onto a
NEMS resonator, an
individual
analyte molecule or particle can precipitously downshift the
resonant frequency of each vibrational mode [Supplementary Information]. This is the basis
of the measurement. Theoretical limits to inertial mass resolution from frequency-shift
detection can apparently be as small as the single-Dalton level
17
; indeed, recent endeavors
already report mass resolution at the few hundred Dalton level
15
. However, central to our
present work is that all measurements to date neither measure the mass of
individual
molecules
or nanoparticles, nor can do so in
real time
. This is despite the impressive recent
improvements in mass resolution and the detection of discrete adsorption events
16
. The
reason for this is that the resonant frequency shift induced by analyte adsorption depends
upon both the mass of the analyte and its precise location of adsorption upon the NEMS
resonator.
A way out of this quandary, and first construction of NEMS-MS spectra – albeit, not in real
time – has previously been achieved by employing the known position-dependent mass
responsivity for a doubly-clamped NEMS resonator
16
. In this previous work analytes were
delivered such that they accreted uniformly across the device; this foreknowledge allowed
the deduction of the constituents of simple mixtures after collection of only several hundred
single-molecule adsorption events. (For comparison, conventional mass spectrometry
measurements typically involve measurement of ~10
8
molecules
19
.) The analysis involved
fitting to the statistical ensemble of measured frequency shifts by a rather complex
multidimensional minimization procedure to extract the weights of each constituent, that is,
to deduce the mass spectrum
16,20
. These first results provided a conceptual demonstration of
the potential of NEMS-MS, but the complexity of this process precluded its application to
arbitrarily complex mixtures.
Here we describe an approach that enables direct determination of the mass of
each
arriving
molecule, in
real time
, as it adsorbs upon the NEMS resonator
21
. It is, thereby, directly
applicable to arbitrarily complex mixtures as we shall demonstrate. This approach involves
no assumptions about the sample mixture, and is implemented by simultaneously tracking
the resonant frequency of multiple modes of an individual NEMS resonator and then
resolving the time-correlated, adsorption-induced frequency jumps in several of these
modes. It is known that the resonance frequency of beams and cantilevers is affected both by
the mass of the particle and its landing position. Deconvolution of mass and position is
possible through information from multiple mechanical modes
22–24
. Pairs of these
simultaneous jumps herald a single-molecule adsorption event and are used in the analytical
framework presented here to deduce the nominal values of the mass and position of
adsorption of individual molecules/particles just after adsorption. Beginning with Euler-
Bernoulli beam theory, we have developed a model that includes error analysis. Using the
complete expression for mode shape enables direct determination of the mass and position
uncertainty of each arriving molecule or particle
20,21
. Our analysis provides both a
numerical and a universal graphical approach (Fig. 2) to calculate the mass and position of
the analyte molecule, which arrives randomly in time and position.
Multimode Theory for Single Molecule Mass Measurements
Our approach is briefly summarized here; it is presented more fully in the Supplementary
Information. A point analyte, a single molecule or particle with mass
δ
m
downshifts the
resonant frequency of a nanomechanical resonator with mass
M
in the following way:
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(1)
Here,
f
n
is the resonant frequency of the
n
th
mode and
δ
f
n
is the frequency shift for this
mode. Their ratio,
(
δ
f
n
/f
n
)
, the fractional frequency shift, is proportional to the fractional
mass change,
δ
m/M
. Here,
φ
n
denotes the mode shape for the
n
th
mode, and a denotes the
position-of-adsorption of the molecule upon the beam (normalized to unitary beam length).
The numericalconstant
α
n
depends on the mode number n and is of order unity
(Supplementary Information).
For a symmetric NEMS doubly-clamped beam similar to the one shown in Fig. 1a, resolving
the adsorbate-induced frequency shifts in the first two modes is adequate to determine the
mass of the analyte molecule and its position of adsorption (Supplementary Information).
The mode shapes and the position-dependent responsivities of the first two modes are shown
in Fig. 1c, along with the ratio of these responsivities. The ratio of the responsivities of two
arbitrary modes,
G(a)
φ
n
(a)
2
/
φ
m
(a)
2
, determines whether their simultaneous measurement is
sufficient for real-time mass detection. If
G
is invertible, then a unique value for the
position, and thus the mass of the molecule, can be obtained. Although this condition is not
fulfilled for the first two modes of a doubly-clamped beam (Fig. 1c), analysis can be
restricted to one half of the beam’s length due to the inherent symmetry of such a structure,
and this permits determination of a unique molecular mass and adsorption position relative
to the beam center (Supplementary Information).
In this work, we shall use the first two modes of the NEMS device for mass measurements
of individual protein macromolecules (IgM antibody isoforms) and individual gold
nanoparticles. Each species that physisorbs onto the cooled NEMS device produces a
distinct frequency shift in each of the tracked modes (the fundamental and second mode), as
shown in Fig. 1b (in order to illustrate the changes better, the frequency axes in this plot are
shown as frequency changes from the initial resonance frequencies at t=0). As described
below, these time-correlated frequency shifts are then used to determine both the mass and
position-of-adsorption for
each
of the newly arrived analyte molecules or particles, as well
as their corresponding uncertainties.
Given the aforementioned symmetry of the mode shapes, we restrict our analysis to one half
of the beam,
0<a<0.5
. For this branch, the transformation,
G
, from the fractional-frequency
shift pair,
(
δ
f
1
/f
1
,
δ
f
2
/f
2
)
, to the analyte mass-position pair
(
δ
m/M,a)
is
one-to-one
. Figure 2
graphically represents the transformation of experimentally observed, time-correlated
frequency jumps from the first two modes of a doubly-clamped beam into mass and
position-of-adsorption for each arriving analyte. The real-time experimental data,
i.e.
frequency jumps for the two modes, are represented as fractional-frequency pairs,
(
δ
f
1
/f
1
,
δ
f
2
/
f
2
).
In this representation, this transformation yields analyte position contours that appear as
straight lines passing through the origin, while the deduced mass contours appear as quasi-
elliptical curves.
In a noiseless measurement, each analyte landing on the NEMS would be identified as a
perfectly sharp single point in the
|
δ
f
1
/f
1
|,|
δ
f
2
/f
2
|
plane. However, in practical experiments
the mass and position of the analyte can only be determined up to certain confidence level
that is determined by the frequency instabilities of the two separate, phase-locked modes of
the NEMS resonator. These frequency fluctuations are characterized by their respective
Allan deviations
25
.
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To represent the frequency instability in a single-mode measurement, the induced frequency
shift in each mode is modeled as a random variable with mean value commensurate with the
measured jump, and dispersion identical with that of the frequency noise. For multimode
measurements, the frequency noise statistics for the separate modes are combined into a
joint probability density function (JPDF) representation,
JPDF
δ
f1/f1,
δ
f2/f2
(
δ
f
1
/f
1
,
δ
f
2
/f
2
)
.
Using a bivariate PDF transformation
26
, the
|
δ
f
1
/f
1
|,|
δ
f
2
/f
2
|
plane is mapped onto the (
δ
m/M,
a)
plane and a joint-PDF for mass and position,
JPDF
δ
m, a
(
δ
m, a)
, is calculated
(Supplementary Information).
The JPDF of each analyte in the multimode space describes an elliptically-shaped
distribution, with the length of the principal axes corresponding to the mass and position
uncertainties. This two-dimensional JPDF can be projected onto either the mass or the
position axis to determine the probability distribution of mass or position respectively:
(2)
(3)
These noise-transformation relations can be used to systematically analyze the performance
of NEMS-MS experiments. For example, mass resolution as a function of analyte position-
of-adsorption can be obtained (Supplementary Information, Fig. S3 and S4).
Experimental Technique for NEMS-MS
We utilize the analysis outlined above to perform mass spectrometry on individual IgM
antibody isoforms and gold nanoparticles using two separate and complementary
experimental systems. These systems employ distinct analyte injection/ionization schemes:
electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI). The
experimental setups and their characterization are described in the Supplementary
Information. The results obtained using the systems agree with each other as shown in Fig.
4a.
The NEMS resonators used in the experiments are specifically designed and optimized for
the actuation and detection of the first two modes of a doubly-clamped beam. Details
concerning their fabrication and measurement are presented elsewhere
27,28
and summarized
in the Supplementary Information.
A colorized scanning electron microscope (SEM) image of one representative device used in
the experiments is shown in Fig. 1a. The device is fabricated by CMOS-compatible, top-
down processes designed for very-large-scale integration of NEMS
29
. Electrostatic
actuation is achieved using proximal capacitive gates, and resonator motion is transduced
using symmetric semiconducting piezoresistive strain gauges located near both ends of the
device. Actuation and detection channels of the two modes were combined using high-
frequency electronic components, and a feedback loop
30
was implemented through GPIB
protocol. A schematic of the measurement and actuation circuit, and a summary of the
device characteristics, are provided in the Supplementary Information.
The Allan deviation characterizing the frequency fluctuations of the first NEMS mode was
σ
A
(1)
~8×10
−8
, and of the second mode was
σ
A
(2)
~1×10
−7
at the chosen phase-locked loop
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(PLL) response time of
τ
R
~500 milliseconds unless noted otherwise. For measurements of
the 10nm gold nanoparticles with the ESI system, obtained with a previous generation of
smaller devices, these values were
σ
A
(1)
~3×10
−6
, and
σ
A
(2)
~2×10
−6
, at
τ
R
~ 10 seconds. (A
longer response time was required with the earlier generation of instrumentation used.)
These frequency fluctuations yield a mass resolution of approximately 50kDa and 100kDa
for these two sets of measurements, respectively. With our current, third-generation
instrumentation, not yet deployed for MS, we have demonstrated sub-millisecond time
resolution
31
. The measured noise correlation between the modes was ~0.3 (~0.7 for the first
generation system and devices). Figure 1b shows a snapshot of two-mode PLL data obtained
during electrospray ionization of 10nm gold nanoparticles (Two-mode PLL data from the
IgM run is shown in Fig. S11). Time-correlated, quasi-instantaneous frequency jumps of
different heights in the two modes clearly demonstrate our ability to resolve discrete
adsorption events from individual molecules or nanoparticles accreting onto the NEMS
resonator. These experimentally obtained frequency jumps are subsequently used to
determine the mass and position of each molecule/nanoparticle by mapping the
(
δ
f
1
/f
1
,
δ
f
2
/
f
2
)
pairs onto the
δ
m/M, a
plane as previously described. Figure 3a shows the mass and
position of each adsorbing particle, and their respective uncertainties, for an experiment
where 5nm gold nanoparticles were delivered onto the NEMS mass sensor via MALDI. The
mass spectrum is obtained by integrating the data along the position coordinate. The gold
nanoparticles, as is usual in typical experimental samples, are known to have a large
variance in radius (Sigma Aldrich); this translates in the very large mass spread observed (m
~
r
3
). If the gold nanoparticles were relatively monodisperse, the data would be expected to
appear as relatively narrow bands along the mass axis. This kind of behavior is well
modeled by our Monte-Carlo simulations of 5nm and 10nm gold nanoparticles – if we
assume low size variance and no clustering (Fig. 3b). Note that particles (or molecules) with
smaller masses will produce smaller relative frequency shifts and, in the presence of a fixed
amount of frequency noise, this will appear as a larger position uncertainty. A somewhat
counterintuitive feature of these spectra is the evident decrease in the position uncertainty
for heavier species while the mass uncertainty remains constant. This originates from the
fact that the mass resolution depends on the minimum resolvable frequency shifts – which,
again, remain constant due to the frequency noise, regardless of the magnitude of the actual
shifts from the arriving analytes. On the other hand, position resolution depends on the
minimum resolvable angle in the
(
δ
f
1
/f
1
,
δ
f
2
/f
2
)
plane, and this improves as the magnitude of
the frequency shifts become larger (see Fig. S9). The number of events observed near the
center of the beam is reduced because the second mode has a node at the center, and
therefore particles landing in this region produce jumps below the noise level.
Gold Nanoparticle Measurements
The mass spectra of 10nm gold nanoparticles are shown in Fig. 4a for the data acquired from
the ESI setup (black curve) and from the MALDI setup (blue curve). In order to more easily
compare with the ESI data, the data for the MALDI curve shown here was analyzed with the
same experimental mass resolution that was achieved with the ESI setup for 10nm gold
nanoparticles. Also shown are the best-fit curves for each data set. For the ESI data, the best
fit yielded a diameter of 9.8nm and standard deviation of 2.5nm, while the data using
MALDI yields a diameter of 10.7nm and a standard deviation of 2.8nm. These values are
within the experimental deviation of the vendor specifications for the gold nanoparticles.
Details of our fitting protocols are given in the Supplementary Information.
We complement these 10nm gold nanoparticle measurements with MALDI-based
measurements on 5nm gold nanoparticles. In these experiments two types of MALDI plates
were prepared; each containing 5nm gold nanoparticles, but differing in whether glycerol
was added as a separating agent (Methods). Previous studies have demonstrated that
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clustering effects typically exhibited by metallic nanoparticles can be mitigated by the
addition of various separating agents
32–35
. Figure 4b shows two distinct mass spectra for the
two 5nm gold nanoparticle samples. A clear reduction in gold nanoparticle clustering is
observed with the use of glycerol. To gain further insight into the observed cluster peaks, we
modeled the clustering phenomena using Monte Carlo simulations; the details underlying
our analyses are described in the Supplementary Information. Fig. 4c shows the accumulated
mass spectra, as acquired particle-by-particle, for the 5nm gold nanoparticles
Each event in the current set of measurements provides mass of the adsorbed analyte. This
contrasts with previous measurements
15,16
, wherein each data point was, at best, part of a
statistical ensemble – itself one bit of information convolved with the position dependence
of single-mode NEMS response. Here, as is graphically displayed in Figure 4b and 5a,
spectra can now be built up, particle-by-particle, as each analyte arrives. With this advance it
is now possible to weigh individual molecules in real time, without the need to first collect
an ensemble of identical particles. This enables straightforward analysis of complex
mixtures, as exemplified in Fig. 5 which represents the first time isolated biomolecules have
been weighed by a nanomechanical device.
Human IgM Antibody Measurements
To further demonstrate the utility of NEMS based mass spectrometry for biological species,
we have obtained single-molecule NEMS-MS spectra for human IgM using ESI injection. In
serum, IgM is typically found in macromolecular complexes that are assembled by the
immune system. The presently-known biologically-active isoforms in serum can be
tetrameric, pentameric, hexameric, or dipentameric assemblies of identical ~190kDa
subunits
36–39
. For the prevalent pentamer isoform, an additional small protein (the J chain)
helps link the assemblage and contributes ~15kDa to the total ~960kDa mass of the
complex
40,41
. Our overall mass spectrum, a composite curve accumulated from 74 single
particle spectra, is shown in Fig. 5a. The individual pentameric IgM complex (the highest
intensity peak) is clearly visible at 1.03 ± 0.05 MDa, as is a dimerized pentameric complex
(“dipentamer”) at 2.09 ± 0.05 MDa – their mass ratio very close to two, as expected. These
measured values are very close to the anticipated values 0.96 MDa and 1.92 MDa
41
, which
is remarkable considering the fact that we report mass values without any calibration other
than using the nominal mask dimensions of our mass sensor (Supplementary Information).
The apparent smoothness of the mass spectra for the individual isoforms of Fig 5 arises from
the fact that
each
single-particle/molecule event can be resolved with its own uncertainty
level. Specifically, the mass spectra in Figures 4 and 5 represent information acquired from
sets of 105 and 74 single-particle/molecule adsorption events, respectively. Each of these
events can be represented in the mass-position plane as a continuous probability distribution.
Subsequently, we obtain mass spectra for each event by projecting the individual
distributions onto the mass plane. For each particle/molecule accreted, this yields a smooth
Gaussian-like curve for its mass spectrum, with a width dependent on the specific mass and
position of the particle. The cumulative mass spectra are then Gaussian-like mass
distributions averaged by like IgM isoforms, as in the foreground of Fig 5c, or added overall
to generate a composite spectrum, as in the background grey curve of Fig 5c. Alternatively,
one can report the center of the mass distribution (as done in the inset of Fig. 5b), but then
one loses the unique position and mass uncertainty information for each particle that is
obtained with our uncertainty analysis formalism.
Figure 5b and 5c illustrate the remarkable power of single-molecule NEMS-MS to resolve
spectra. Because the mass of each molecule is individually measured, and using the prior
knowledge of IgM isoformal structure, each molecule can be identified based upon its mass
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as an IgM isoform. In a surprising reversal of the traditional situation, it is now possible to
examine the underlying structure of the composite intensity curve. For instance the apparent
shoulder at 0.82 MDa in the composite intensity curve is seen to arise from the presence of
precisely twelve accreted macromolecular complexes. Each one is a tetramer of IgM that
individually registers on the NEMS mass sensor and was separately measured as part of the
ensemble of 74 molecules collected during this experiment. The legend in Fig. 5c provides
the number of molecules collected for each of the subunit peaks. To identify the different
IgM isoforms with our single-molecule measurements, equidistant thresholds were used
between the expected mass values of adjacent species, shown as gray lines if Fig. 5b. Due to
our mass measurement error, there is a small probability for some events to be misidentified
when the noise level during that particular event happens to exceed the 2
σ
noise threshold
separating two distinct species. We determine the number of potentially misidentified
particles by performing a statistical analysis on the data ensemble (Supplementary
Information). This analysis suggests that less than 7% of the events, that is, only ~5 of the
74 collected molecules, might be misidentified.
Our experiment reveals a sequence of IgM isoforms from trimer (N=3) up to dodecamer
(N=12) within the mass range investigated (excluding the solitary event at 3.6MDa); these
can originate from both physiological and experimental factors. Fragmentation and
nonspecific reassembly of large macromolecular species is expected to occur in ESI
systems
5,42
. However it is known that IgM can also be selectively assembled by the
lymphatic system into pentamer and hexamer complexes as part of an immunologically
driven response to antigens
36,37
. NEMS-MS, unlike conventional MS, does not require the
charging of analytes to achieve its selectivity. The ability to use
neutral injection
methods
that capitalize on the strengths of NEMS-MS will, in future, allow direct determination of
the efficiency of such immunological processes and allow monitoring them in real time and,
for example, in response to potential therapies – without the confounding source of isoforms
from charge-driven fragmentation.
Summary
NEMS-MS systems yield very high resolving powers in the large mass range (>500kDa)
since NEMS mass resolution remains constant over the entire mass range. This is in contrast
with conventional mass spectrometry systems whose resolution degrades with higher mass
especially beyond mega-Dalton range. The present experiments provide the first
experimental validation of real-time NEMS mass spectrometry, and demonstrate the
potential of NEMS mass sensors for performing mass spectrometry on large
macromolecules and nanoparticles with masses deep into the MDa range.
We have demonstrated mass spectrometry using nanomechanical devices wherein the mass
of individual protein macromolecules arriving at the device are measured in real time. The
work clearly demonstrates the utility of NEMS for mass spectrometry of large biomolecules
and, more specifically, for native mass spectrometry. Improving the mass resolution of top-
down fabricated nanomechanical devices by only one or two decades – which is attainable
in the near term – offers exciting prospects for useful applications in bacterial identification,
native mass spectrometry, and structural identification of large macromolecules. Recent
work has dramatically improved the mass resolution of bottom-up fabricated NEMS
devices
13–15
and now offers realistic potential for ultimately creating NEMS-MS
spectrometers with resolution down to a few Daltons. However, much work remains:
bottom-up NEMS devices and approaches have yet to demonstrate mass measurements of
individual molecules, and questions remain about their compatibility with large scale
integration. Ultimately, however, the ability to use VLSI and CMOS-compatible NEMS
with devices providing single Dalton sensitivity will enable the possibility of measuring
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millions of proteins, in real-time, from a small discrete sample – like a single cell – while
retaining single-protein precision over the full range of biological interest.
Methods
Human IgM solution was purchased from Sigma-Aldrich and buffer exchanged to 200mM
aqueous ammonium acetate, with a final antibody concentration of approximately 1 mg/mL .
A nano-electrospray ionization interface with a 20
μ
m Pico-emitter ESI needle (from New
Objective) was employed for IgM measurements. Colloidal gold nanoparticles with nominal
diameters of 5nm (mean diameter = 5.1nm, variance = 19%) and 10nm (mean diameter =
10.7nm, variance = 10%) were purchased from Sigma-Aldrich. For the ESI measurements,
the gold nanoparticle sample was diluted by equal amounts of methanol and introduced into
the ESI needle. The NEMS device was kept at the highest vacuum chamber of a three-stage
differential pumping setup, with a base pressure of 10
−5
Torr before cryo-pumping takes
place. Typical operation temperature for the ESI sample stage was 70 K for the gold
nanoparticle samples and 140 K for the IgM samples. Cooling the NEMS device in both the
ESI and MALDI setups was done to prevent captured particles from rapidly desorbing from
the device surface.
The MALDI sample plates were prepared by washing the stock colloid solutions in water
and, using a centrifuge, concentrating the solutions to ~5×10
14
particles/ml and 1×10
14
particle/ml for the 5 and 10nm gold nanoparticle samples, respectively. For each sample,
38
μ
l of solution was drip-dried onto 3mm diameter spots on a pyrex sample plate. The
glycerol samples were prepared by adding glycerol (Sigma-Aldrich) at 10% concentration to
the gold nanoparticle solution prior to drying on the pyrex sample plate. After drying in air,
the plate was placed inside the vacuum chamber at a distance ~0.5cm from the NEMS
device. A nitrogen laser (model NL 100 from Stanford Research Systems) was focused
through a lens to a spot ~50×100
μ
m
2
that illuminated the backside of the pyrex plate. The
laser was operated at a wavelength of 337nm, 170
μ
J/pulse, 3.5ns pulse width and a
repetition rate of 1Hz. The chamber was maintained at a base pressure of 10
−9
Torr and the
NEMS device stage was cooled to 80K.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank I. Bargatin, E. Myers, M. Shahgholi, I. Kozinsky, M. Matheny, J. Sader, P. Hung, E. Sage and R.
Karabalin for helpful discussions, and C. Marcoux for assistance with device fabrication. We acknowledge critical
support and infrastructure provided for this work by the Kavli Nanoscience Institute at Caltech. This work was
made possible by support from the NIH (grant R01-GM085666-01A1Z), the NSF (MRI grant DBI-0821863), the
Fondation pour la Recherche et l’Enseignement Superieur, an Institut Merieux Research Grant, and a grant the
Partnership University Fund of the French Embassy to the USA. MLR gratefully acknowledges support from an
NIH Director’s Pioneer Award and a Chaire d’Excellence (RTRA) from Fondation Nanosciences. SH and EC
acknowledge partial support from EU CEA Eurotalent Fellowships.
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