Article

pubs.acs.org/jpr

Conductometric Monitoring of ProteinProtein Interactions

Rosanna Spera, Fernanda Festa, Nicola L. Bragazzi, Eugenia Pechkova,, Joshua LaBaer,
and Claudio Nicolini*,,,

Nanoworld Institute, Fondazione EL.B.A. Nicolini, Largo Redaelli 7, 24020, Pradalunga, Bergamo, Italy
Laboratories of Biophysics and Nanobiotechnology, Department of Experimental Medicine, University of Genova, Via Pastore 3,
16132, Genova, Italy

Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, United
States

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Publication Date (Web): November 1, 2013 | doi: 10.1021/pr400445v

ABSTRACT: Conductometric monitoring of proteinprotein and proteinsterol interactions is here proved feasible by
coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays
(NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identication of
binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a
wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the
same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct
interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the
interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.
KEYWORDS: conductometer, nucleic acid programmable protein array, quartz crystal microbalance with dissipation monitoring,
cell free expression system

INTRODUCTION
Protein arrays are rapidly becoming established as a powerful
platform to investigate protein interactions and functions. They
made possible the parallel multiplex screening of thousands of
interactions, encompassing protein-antibody, proteinprotein,
proteinligand or proteinsmall molecules, enzymesubstrate
screening and multianalyte diagnostic assays.1 Moreover,
protein arrays are increasingly generating interest at the
biotechnology levels, especially when coupled to label-free
detecting techniques that do not require the use of reporter
elements (uorescent, luminescent, radiometric, or colorimetric) to facilitate measurements. Label-free techniques can
provide direct and straightforward information on analyte
binding to query molecules typically in the form of mass change
(addition or depletion) from the surface of a sensor substrate2,3
or via changes in a physical bulk property of a sample.4,5
We created a new conductometric biosensor in which the
sensitive biological elements are engineered proteins expressed
in vitro in a self-assembling protein microarray, using NAPPA
technology.6 NAPPA technique relies on the production of
2013 American Chemical Society

proteins directly on the microarray surface using a cell-free in

vitro transcription/translation (IVTT) system7,8 (Figure 1a).
The use of a human-based IVTT system has proven particularly
advantageous since it allows the expression of human proteins
using human machinery and chaperones, increasing the
likelihood of proper folding and post-translational modications. NAPPA, successfully coupled with the human IVTT
system, became the rst high-density protein microarray to
display human proteins expressed in human milieu.9 Typically,
more than 2000 unique features can be spotted in a single glass
slides,7 or more than 8000 features in nanowells.10
The design of NAPPA has been directed to overcome the
limitations of traditional protein microarray technologies.
Among the advantages of NAPPA it is possible to include the
following: minimal manipulation of the proteins; protein
repertoire (cDNA is used as a template for the protein
expression, and the availability of comprehensive cDNA
Received: May 10, 2013
Published: October 9, 2013
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Figure 1. Quartz crystal microbalance with dissipation monitoring (QCM_D) coupled to nucleic acid programmable protein arrays (NAPPA). (a)
Schematic representation of NAPPA technology. In each spot a mixture of plasmid DNA, BSA and anti-GST antibodies is printed and immobilized
on cysteamine coated surface. The antibody is responsible for the capture of the freshly expressed proteins that are tagged, at one of their ends, with
a GST tail. The proteins are translated using an in vitro transcriptiontranslation system (IVTT).2 (b) Nanogravimetric biosensor prototype
scheme. The quartz is positioned in a ow chamber that guarantees the temperature control. Temperature and ux rate are settable trough the
controllers and D factor and frequency values are visible on two displays. It is also possible to record the conductance curves (the frequency and D
factor shifts) in real time (example shown on the right).6

libraries makes it possible to use virtually any cDNA sequence

on the array); protein integrity; and protein stability (proteins
are produced just in time for the assay).
Proteins displayed on NAPPA arrays are properly folded and
active. NAPPA microarray has, in fact, been successfully used
for the study of proteinprotein interaction;7 more than 85%
of the interactions that had been demonstrated biochemically
with puried proteins were detected on the array. Those
interactions occur only between properly folded and active
proteins, demonstrating that the proteins on the array are
properly folded and active. Moreover, it is possible to measure
kinase activity on the array (data not published). Our previous
data shows that proteins displayed on the array can be used for
functional assays up to 24 h after the protein expression.
However it is important to emphasize that NAPPA arrays
printed with the cDNAs of interest can be stored for more than
six months. The expression of the proteins is performed just
when the microarray is needed, and for this reason it is not
necessary to worry about protein stability above the 24 h
window.
NAPPA method, naturally, has also some drawbacks, which
include the following: the potential diusion across spots; the
use of tags for the protein capture, which may impair the
folding and/or activity of the proteins; the presence of plasmid
DNA and the capture antibody in the nal protein microarray,
which may increase the background; and the fact that each
DNA array produces a single protein array.11
Nonetheless, NAPPA microarrays were employed in several
distinct applications including functional assays to detect
proteinprotein interactions7 and biomarker discovery for
breast cancer, 12 arthritis 13 and Pseudomonas aeruginosa
infection.14 Since the proteins on the array are in principle in
a native state, these arrays should allow the parallel detection of
protein activity1517 toward personalized medicine.2 These are
the reasons why we pointed our attentions to NAPPA as
sensing system of our recently developed biosensor.6

Employing NAPPA as sensing system, we are able to develop

a large number of biosensors, simply changing the cDNAs
immobilized on the sensor, without interfering with the sensing
technology.3 One of the main limitations in the development of
a biosensor is, in fact, that the sensing technology is still strictly
dependent to the unique signal transduction properties of the
sensing element and need to be suited to it.
The detection technology we identied as one of the most
promising is the quartz crystal microbalance with dissipation
monitoring (QCM_D) is a useful analytical tool for label-free
and real-time analysis of biological molecule.18 To this aim
NAPPAs was spotted on standard nanogravimetry quartz
crystals (NAPPA-QC). The interaction between immobilized
protein and analyte was monitored by the shift in the resonance
frequency, which is directly proportional to the amount of
molecule immobilized according to the well-known Sauerbrey
equation,18 and the contemporary variations in conductance
and bandwidth, which provides fundamental information about
the viscoelastic properties of the solution and, moreover, the
proper biosensor response.4,1823
The dissipation factor, D, of the crystals oscillation reveals
the lms viscoelasticity, and its measurement can be deduced
from the bandwidth of the conductance curve, 2. It was
demonstrated,19 in fact, that the dissipation factor of the quartz
could be calculated by the following simple equation:
D = 2/f

(1)
19

where f is the peak frequency value.

In the analysis of our results we introduced a further
parameter, the normalized D factor, DN, dened as follows:3,6

D N = 2/Gmax

(2)

where Gmax is the maximum conductance of the conductance

curve. DN is more strictly related to the curve shape, respect D,
and takes into account the conductance variation. Our previous
results3,6 demonstrated that the use of QCM_D as a
conductometric device gives reliable results and allows further
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oscillation. The QCM_D software, QCMAgic-Q5.3.256

(Elbatech srl, Marciana LI, Italy) works in two dierent
modes (not simultaneously): the rst allows to acquire the
conductance curve, and the second allows to acquire
simultaneously the frequency and dissipation factor variation
versus time. Moreover, the software measured also the
normalized D factor.
In order to have a stable control of the temperature, the
experiments were conduced in a temperature chamber. During
proteinprotein interaction analysis the QC was positioned in
a miniature ow-cell produced in house. The ow-cell chamber
volume is 100 L, and it is connected to a BioRad Econo
Gradient Pump, able to pump solution in a ux range of 0.026
mL/min.
Microarrays were produced on standard nanogravimetry
quartz used as highly sensitive transducers. The QC expressing
proteins consisted of 9.5 MHz, AT-cut quartz crystal of 14 mm
blank diameter and 7.5 mm electrode diameter, produced by
ICM (Oklahoma City, OK, USA). The electrode material was
100 Cr and 1000 Au, and the quartz was embedded into
glass-like structures for easy handling.

information about the interactions between antibodyprotein

and proteinprotein, as well as enzymesmall molecules. In
most cases conductometer have been strongly associated with
enzymatic studies, where the ionic strength, and consequently
the conductivity, of a solution between two electrodes changes
as a result of an enzymatic reaction.2426 Although conductometric sensing has not been as extensively implemented as it
could be,27 there are examples of successful development of
these devices for practical application, such as drug detection in
human urine and pollutant detection in environmental
testing.25,28
Even though QCM_D appears to be a promising tool to
study proteinprotein interactions as well as protein
membrane29 and nanoparticlemembrane dynamics,30 relatively few eorts have been made to adapt QCM_D to the
study of proteinprotein interactions.20,3136 To the best of
our knowledge, we coupled for the rst time QCM_D with
NAPPA technology for biomedical applications6 and for
personalized medicine.2
To highlight the extreme versatility of our conductometric
biosensor, we analyzed not only proteinprotein interactions
but also proteinsmall molecule interactions with potentially
useful clinical applications. Moreover multiple proteins on the
same QC have been tested, proving the possibility of analyzing
multiprotein interactions. As proof of principle, applications
with clinical relevance are presented herein, the interaction
between the transcription factors Jun and ATF2 and the
interaction between Cytochrome P540scc and cholesterol.
JunATF2 heterodimers are important for many cellular and
neoplastic processes.37,38 Cytochrome P450scc is a mitochondrial enzyme that catalyzes the cholesterol side chain cleavage
reaction after the hydroxilations at C22 and at C20, the initial
and key step in the regulation of steroid hormone biosynthesis
and steroidogenesis.3942 Cholesterol sensing and monitoring
has a great clinical value and importance, since the association
between elevated plasma levels of cholesterol and cardiovascular diseases is well-known. The response to cholesterol was
measured and compared against traditional technologies,40,43
which employes potentiometric and/or amperometric biosensor (signal is measured as the potential dierence, or the
current intensity, between two electrodes; the signal depends
on the concentration of the analyte in solution).

NAPPA-QC

In Spera et al.,3 we presented our preliminary results

concerning an earlier version of our NAPPA based biosensor;
16 NAPPA spots of 300 m diameter were spotted on the QC
surface, and the functional proteins were synthesized in situ
using a T7-coupled rabbit reticulocyte lysate as an in vitro
transcriptiontranslation (IVTT) system. In Nicolini et al.,6 as
proof of principle of biosensor response to proteinprotein
interaction, we tested the interaction between p53 proteins
immobilized on the NAPPA (after its expression) with a
MDM2 solution.44 The preliminary results presented3,6 had
demonstrated how our prototype of NAPPA based biosensor
gave a valid response to the proteinprotein and drug enzyme
interaction. Moreover we veried the high selectivity and
sensitivity of our biosensor. Here we present the results
obtained employing a further improved version of the biosensor
for the analysis of proteinprotein and proteinsmall molecule
interactions. The NAPPA-QC arrays were printed with 100
spots per QC, to enhance the sensitivity. As IVTT system we
used a human lysate, which guaranteed a higher protein yield
with respect to the rabbit reticulocyte lysate employed in the
preliminary experiments.9 Moreover, the QCM_D software has
been updated and improved, allowing the acquisition of the
conductance curves at higher resolution (namely, the frequency
acquisition interval has been shortened from 120 down to 1
Hz).
Quartzes gold surfaces were coated with cysteamine to allow
the immobilization of the NAPPA printing mix. Briey,
quartzes were washed three times with ethanol, dried with
Argon and incubated overnight at 4 C with 2 mM cysteamine.
Quartzes were then washed three times with ethanol to remove
any unbound cysteamine and dried with argon. Plasmid DNA
coding for GST tagged proteins were transformed into
Escherichia coli, and DNA were puried using the NucleoPrepII
anion exchange resin (Macherey Nagel). NAPPA printing mix
was prepared with 1.4 g/L of DNA, 3.75 g/L of BSA
(Sigma-Aldrich), 5 mM BS3 (Pierce, Rockford, IL, USA) and
66.5 g of polyclonal capture GST antibody (GE Healthcares).
Negative controls, named master mix (hereinafter abbreviated
as MM), were obtained replacing DNA for water in the
printing mix. Samples were incubated at room temperature for

MATERIALS AND METHODS

QCM_D Conductometer

The QCM_D instrument was developed from a basic patent on

QCM biosensor (world patent WO9602830A1 published in
2004 by Nicolini Claudio, Sartore Marco, Troitzky Vladimir
and Adami Manuela) and resulted on a pending application
(patent UIBMGE2012A000080) specic for proteinprotein
interaction study based on QCM_D, Anodic Pourus Allumina
and NAPPA (deposited in 7 October 2012 by Nicolini Claudio,
Bragazzi Nicola, Spera Rosanna and Pechkova Eugenia).
The quartz was connected to an RF gain-phase detector
(Analog Devices, Inc., Norwood, MA, USA) and was driven by
a precision DDS (Analog Devices, Inc., Norwood, MA, USA)
around its resonance frequency, thus acquiring a conductance
versus frequency curve (conductance curve) which shows a
typical Gaussian behavior. The conductance curve peak was at
the actual resonance frequency, while the shape of the curve
indicated how damped the oscillation was, i.e., how the
viscoelastic eects of the surrounding layers aected the
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Figure 2. NAPPA expression for the individual CDK2 (left), P53 (middle) and combined P53 and CDK2 genes (right) on each individual quartz.
The blue curves were acquired before the expression of NAPPA, while the red curves were acquired after the protein expression/capture and washing
process. The curves have been centered to their maximum frequency to better visualize the changing in bandwidths and maximum of conductance.

response to ATF2 on both NAPPA-expressed QCs. It is wellknown that the proto-oncoprotein c-Jun is a major dimerization
partner of ATF2, and c-JunATF2 heterodimers are important
for many cellular and neoplastic processes.37,38 On the contrary,
no interactions are known between ATF2 and CDK2 or p53.

1 h with agitation and then printed on the cysteamine-coated

gold quartz using the Qarray II from Genetix. In order to
enhance the sensitivity, each quartz was printed with 100
identical features of 300 m diameter each, spaced by 350 m
center-to-center. The human cDNAs immobilized on the
NAPPA-QC were CDK2 (Cyclin-Dependent Kinase 2),
CYP11A1 (Cytochrome P450, Family 11, Subfamily A,
Polypeptide 1), Jun, MLH1 (mutL homologue 1), p53, and
polD1 (Polymerase (DNA Directed), Delta 1, Catalytic
Subunit). The size of those proteins ranged from 61 to 150
kDa, including the 27 kDa GST tag.
Gene expression was performed immediately before the
assay, following the protocol described as modied from Festa
et al.9 Briey, in vitro transcription and translation were
performed using HeLa lysate mix (1-Step Human Coupled
IVTT Kit, Thermo Fisher Scientic, Inc.), prepared according
to the manufacturers instructions. The quartz, connected to the
nanogravimeter inside the incubator, was incubated for 1.5 h at
30 C with 40 L of HeLa lysate mix for proteins synthesis, and
then the temperature was decreased to 15 C for a period of 30
min to facilitate the proteins binding on the capture antibody
(anti-GST). After the protein expression and capture, the
quartz was removed from the instrument and washed at room
temperature, in PBS for 3 times. The quartz was then placed in
the ux chamber for proteinprotein interaction analysis. The
protocol described above was followed identically for both
negative control QC (the one with only MM, i.e, all the
NAPPA chemistry except the cDNA) and protein displaying
QC.

Determination of ProteinSmall Molecules Interactions

We analyzed the interaction between CYP11A1 and cholesterol, both in solution and in blood, to acquire information on the
binding kinetics.6 After protein expression and capture
CYP11A1 expressing QC was positioned in the ow chamber
and exposed to a ow of a 50 M cholesterol (Sigma-Aldrich)
solution in 30% sodium cholate (Sigma-Aldrich), at 0.02 mL/
min ow rate, for 10 min at 22 C. We used cytochrome
P450scc (CYP11A1) for the detection of cholesterol (SigmaAldrich) because of its specicity. As negative control we
analyzed the interaction between Clompiramine and polD1 and
MLH1. MLH1 is a protein involved in DNA mismatches repair,
and polD1 is a DNA polymerase. None of these proteins
should interact with Clomipramine.
Mathematical Model

Various kinetic models have been developed to describe

proteinprotein and proteinsmall molecules interactions. For
our data, on the basis of the results of Turon and co-worker,45
we performed mathematical modeling of the proteinprotein
interaction, assuming saturation-like kinetics, that is to say, a
quasi-steady-state condition model. For this purpose, we used
the following experimental equation45 based on an exponential
decay function instead of using deterministic models, focusing
on frequency variation:

Determination of ProteinProtein Interactions

After protein expression and capture, the QCs were washed and
used for the interaction studies as follows: QC displaying Jun
was exposed to a ow of a 33 M ATF2 (Sigma-Aldrich)
solution in PBS (for ow interaction at 0.02 mL/min ow rate)
for 10 min at 22 C; alternatively 60 L of 33 M ATF2
solution in PBS was added on the QC surface (for static
interaction) for 10 min at 22 C.
We also tested the possibility to analyze proteinprotein
interactions in QC displaying multiple proteins. For this aim,
we coprinted cDNA for Jun&CDK2 and Jun&CDK2&p53 on a
single QC, and after the expression, a mixture of these proteins
was displayed on the QC surface. We analyzed the interaction

f = M max (1 e / t )

(3)

where f is the frequency variation (in Hz), Mmax is the

maximum binding value in hertz (corresponding to the
minimum frequency measured, or plateau value), is the
relaxation time (expressed in reciprocal minutes), that is, the
reciprocal of the enzyme protein adsorption/interaction rate,
and t is the time (in minutes).
The sensitivity of our NAPPA based biosensor was
determined by the QC characteristics and the sensitivity of
the nanogravimeter. At the moment, the minimum frequency
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Figure 3. Matching of conductance curves of Jun expressing QC, CDK2 expressing QC and p53 expressing QC after lysate addition.

Figure 4. Conductance curves of MM QC (upper) and of CYP11A1 QC (lower). The curves were collected in dierent steps of NAPPA protocol,
as reported in the legend, and after the addition of cholesterol. In the box the MM+CYP11A1+cholesterol conductance curves acquired with
frequency acquisition steps of 1 Hz.

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Table 1. Main Parameters of QC-NAPPA Displaying No Proteins (MM) or CYP11A1a

MM Conductance Curves
baseline
IVTT addition
90 min IVTT addition
postcapture (30 min)
postwash
CYP11A1 Conductance Curves
baseline
IVTT addition
90 min IVTT addition
postcapture (30 min)
postwash
MM+CYP11A1+cholesterol

f (Hz)b

(Hz)b

Gmax (mS)b

D 103 c

DN (Hz/mS)c

9497485
9493570
9493495
9493120
9489085

2422
4562
4500
4875
6255

0.432
0.370
0.390
0.378
0.046

0.51
0.96
0.95
1.03
1.32

11218
24653
23071
25814
273144

9487615
9481540
9479935
9479790
9475975
9478300

2265
4198
3984
3980
5625
6410

0.440
0.399
0.401
0.400
0.150
0.268

0.48
0.87
0.84
0.84
1.19
1.35

10286
21069
19895
19895
75050
47782

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a
Conductance curves were collected in dierent steps of NAPPA protocol. bf is peak frequency, is the half-width half-maximum, and Gmax is the
max conductance. cD factor and DN = 2/Gmax normalized D factor.

Table 2. Shift of the Main Parameters of MM and CYP11A1 Conductance Curves after Lysate Addition and the Corresponding
Mass of Immobilized Protein on the QC Surface
MM Conductance Curves
IVTT addition
90 min IVTT addition
postcapture (30 min)
CYP11A1 Conductance Curves
IVTT addition
90 min IVTT addition
postcapture (30 min)
MM+CYP11A1+cholesterol

CV (%)a

Db

DN (Hz/mS)b

f (Hz)c

f (Hz)c

m (g)c

m (g)c

3.3
4.1
4.0

0.01
0.07

1582
1161

3915
3990
4365

75
450

17.0
17.3
18.9

0.4
2.0

4.8
4.8
4.6
6.9

0.03
0.03
0.48

1174
1174
26713

6075
7680
7825
9315

1605
1750
3240

26.3
33.3
33.9
40.3

7.0
7.6
14

Coecient of variation of three independent experiments. bD factor and normalized D factor shifts (D and DN) respect the values immediately
after lysate addition cFrequency shifts respect the initial frequency (f) and respect the frequency immediately after lysate addition (f ) and
corresponding molecular masses (m and m).

Figure 2 shows the conductance curves for three NAPPAQCs expressing p53, CDK2 and a mixture of p53 and CDK2
(both cDNAs were coimmobilized in each feature). The blue
curves were acquired before the expression of NAPPA, while
the red curves were acquired after the protein expression/
capture and washing process. The curves have been centered to
their maximum frequency to better visualize the changing in
bandwidths and conductance. These data pointed to a unique
conductance curve shape for each protein and suggested the
possibility to identify the expressed proteins by QCM-D even
when combined on the same expressing QC (Figure 3).
To test the possibility to acquire information on the kinetic
constant of a proteinsmall molecule interaction, we realized a
NAPPA-QC expressing CYP11A1 to be tested for cholesterol
interaction as proof of principle. The P450scc-cholesterol
interaction, in fact, is well characterized, and the results
obtained have been satisfactorily compared with those in
literature.39,4648 In Figure 4 are reported the conductance
curves for the negative control (MM) or CYP11A1 expressing
NAPPA-QC arrays. The conductance curves were acquired in
dierent steps of NAPPA expression process. In particular:
before the beginning of the gene expression process (baseline,
the QC is dry); after the addition of human IVTT lysate at 30
C (IVTT addition), i.e., prior protein expression; after 90
min from the addition of human IVTT lysate, i.e., after protein
expression (90 min IVTT addition); after 120 min from the
addition of human IVTT lysate, i.e., at the end of QC

shift detectable is of 0.05 Hz that corresponds to about 0.3 ng

of detected molecules.3

RESULTS AND DISCUSSION

QCM_D measures were calibrated for frequency and for D
factor shifts. The frequency calibration was performed using
dierent amounts of thaumatin. The calibration curve equation
(obtained with Ordinary Least Squares methods, OLS) is
f = 7.16 231.18m

(4)

with r2 = 0.9986.
The D factor calibration in function of the viscosity was
obtained using fructose samples at dierent concentrations.
The calibration curve equation (obtained with OLS methods)
is
D = 0.831 + 0.286

(5)

with r2 = 0.9990. For both the curves, a good correlation with

the linear slope was found.3,6
We analyzed the conductance curves acquired in NAPPAQCs in dierent steps of the expressing and capturing process.
Coecients of variation (CV) have been computed for each
conductance curve, according to the following equation:

C V = /

(6)

where is the standard deviation and the mean.

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Figure 5. FLOW interaction CYP11A1 with HDL cholesterol in blood (upper panel) and in solution (lower panel).

incubation at 15 C, which is the capture step (postcapture

(30 min)); after the nal washing process with PBS
(postwash).
At the end of the whole expression process, the CYP11A1
expressing QC was positioned in the ow chamber and exposed
to a ow of a 50 M cholesterol solution in 30% sodium
cholate. The conductance curve MM+CYP11A1+cholesterol
(see Figure 4b) was recorded in this condition.
In Table 1 are reported the main parameters of the
conductance curves of Figure 4.
It is evident that the decrease in the frequency (f) is due to
the human IVTT lysate addition. There is also a change in
viscoelastic properties of the quartzes after the human IVTT
lysate addition, leading to a measurable increase of the
bandwidth (). During the incubation, on the contrary, the
frequency and bandwidth variations were minimal. This eect
could be related to two main eects: rst, merely because of the
IVTT lysate addition on the QC surface, when the QC comes

in contact with a solution the frequency decreases depending

upon the viscosity and the density of the solution, and there is a
decrement in damping the resonant oscillation;47 and second,
because of the change of the composition of both QC surface
and IVTT lysate after the gene expression and the protein
synthesis and immobilization. The conductance curves acquired
after PBS washing evidenced the further changes of solution in
contact with the QC.
In Table 2 is reported the shift of the main parameters
normalized against the baseline conductance curve and the
conductance curve acquired after human IVTT lysate (IVTT
addition). It is also reported the mass of molecules
immobilized on the QC surface at the end of immobilization
protocol and after the interaction with cholesterol (m),
estimated through the calibration coecients (eq 5).
To estimate the amount of proteins anchored on the QC
surface after the NAPPA expression, we had to account for the
human IVTT lysate molecules aspecically adsorbed on the
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Figure 6. Conductance curves of Jun and CDK2 expressing QC (upper panel). Conductance curves of Jun, CDK2 and p53 expressing QC (lower
panel). The curves were collected in dierent steps of NAPPA process, as reported in the legends, and after the addition of ATF2 solution.

obtained by subtracting 2 g of molecules aspecically

adsorbed to the 7.6 g of molecules adsorbed in correspondence of postcapture (30 min) curve (see Table 2), while the
amount of cholesterol was 6.4 g, obtained subtracting 5.6 g
of CYP11A1 proteins captured on the array and 2 g of
molecules aspecically adsorbed from the 14 g of molecules
adsorbed in correspondence of MM+CYP11A1+ cholesterol
curve. We veried (data not reported) that the exposure of the
QC to sodium cholate solution aected only the bandwidth of
the curve, considerably increasing the D factor, leaving the

quartz surface.3,22,48 Assuming that on each QC surface there

was the same aspecic adsorption,48 we could estimate it from
reference quartz conductance curves. In particular we
considered the frequency shift between the reference QC
conductance curves acquired immediately after the human
IVTT lysate addition (IVTT addition) and that acquired at
the end of the protein anchorage (90 min IVTT addition);
this value is 450 Hz (see Table 1), which corresponds to 2 g
of molecules aspecically adsorbed (see Table 2). The values of
immobilized CYP11A1 molecules, therefore, was 5.6 g,
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Table 3. Main Parameters of QC-NAPPA Displaying Jun&CDK2a

Jun&CDK2 conductance curves

f (Hz)b

(Hz)b

Gmax (mS)b

D 103 c

DN (Hz/mS)c

IVTT addition
5 min IVTT addition
15 min IVTT addition
postcapture (5 min)
postwash
PBS-ux
MM+Jun&CDK2+ATF2

9482473
9482145
9481600
9481018
9479200
9480764
9481309

3073
2945
2800
2836
2400
2909
2982

0.461
0.457
0.447
0.410
0.261
0.313
0.294

0.65
0.62
0.59
0.60
0.51
0.61
0.63

13336
12885
12528
13822
18391
18577
20284

Conductance curves were collected in dierent steps of NAPPA protocol. bf is peak frequency, is the half-width half-maximum, and Gmax is the
max conductance. cD factor and DN = 2/Gmax normalized D factor.

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resonance frequency unchanged. This result also served as

control to verify the stability of the biosensor.
Our QCM_D instrument gave us the opportunity to monitor
in real-time the trend of D factor and f. In Figure 5 we reported
frequency vs time during the interaction between CYP11A1
and cholesterol, both in solution (as previously reported) and
in blood. Using the eq 3 to t this experimental data in solution
(Figure 5), we obtained a K of about 100 M, a value in good
agreement with the values found in the literature (see, for
example, refs 49 and 50).
In the rst experiments, conductance curves were acquired
every 5 min (data not reported), and it was noticed that 15 min
after IVTT lysate addition at 30 C the position and shape of
the curves did not change until we decreased the temperature
to 15 C. After a few minutes at 15 C, again position and
shape of the curves did not change until the washing step.
Deducing from these results that the protein expression took
place in the rst minutes and that also their capture needed
only few minutes, we decided then to perform the next
experiments, reducing the expression time from 90 to 15 min
(at 30 C) and the capture time from 30 to 5 min (at 15 C).
To test the possibility to analyze multiprotein interactions,
we immobilized on the same QC both Jun and CDK2 cDNAs
and tested the response to ATF2 interaction. c-Jun is a partner
of ATF2 in many cellular processes;38 however, no interactions
are known between CDK2 and ATF2.
The conductance curves of NAPPA-QC expressing
Jun&CDK2 acquired in dierent steps of NAPPA expressing
process are reported in Figure 6a. After protein expression and
capture, the Jun&CDK2 expressing QC was positioned in the
ow chamber and exposed to a ow of 33 M ATF2 solution in
PBS. The conductance curve MM+Jun&CDK2+ATF2
(Figure 6a) was recorded after 3 min of ux. In Table 3 are
reported the main parameters of conductance curves of Figure
6a.
In Table 4 is reported the shift of the main parameters
normalized against the conductance curve acquired immediately after human IVTT lysate addition (IVTT addition).
Through eq 5 we evaluated the mass of immobilized proteins
on the quartz surface at the end of capture protocol and after
the interaction with ATF2 (m).
The amount of molecules captured on the QC 5 min after
protein capture corresponded to about 6 g. A further
frequency decrease was recorded after the washing. This
decrease was not due to further molecules capture on the QC
surface but rather to the change of the solution in contact with
the QC surface, since the frequency is dependent upon the
viscosity and the density of the solution. This eect is mostly
evident considering the shift in D and normalized D factor (see
Table 4). In particular, postwash DN increases about 100

Table 4. Shift of the Main Parameters of Jun&CDK2

Conductance Curves after Lysate Addition and after
ProteinProtein Interaction, and Relative Mass of
Immobilized Proteins on the Quartz Surface
Jun&CDK2
conductance curves

CV
(%)a

Db

DN
(Hz/mS)b

f
(Hz)b

m (g)c

5 min IVTT addition

15 min IVTT addition
postcapture (5 min)
postwash
PBS-ux
MM
+Jun&CDK2+ATF2

5.5
5.7
5.3
4.8
7.3
8.0

0.03
0.06
0.06
0.28
0.21
0.20

455
660
509
45965
11182
9644

456
807
1432
3895
2561
2491

2.0
3.5
6.2
17.0
5.8d
6.1d

a
Coecient of variation. We performed three independent experiments. bD factor, normalized D factor and frequency shifts (D, DN,
and f ) with respect the values immediately after IVTT lysate
addition (see Table 3). cMass of molecules immobilized on the quartz
surface for each step (calculated by eq 5). dThese values were obtained
considering the frequency shifts respect postwash curve, since the
QC was in contact with PBS solution.

times. What is meaningful is the frequency shift after QC

exposure to ATF2 ux. We recorded, in fact, a frequency
increase, rather than a decrease as expected. Evidently the ux,
even if very gentle, caused spoliation of the QC, removing some
captured molecules and making it impossible to assess the
interaction between Jun and ATF2.
In order to overcome the spoliation problem, the interaction
between Jun and AFT2 was tested in a static way, by simply
adding the ATF2 solution on the QC surface. For this
experiment, QC with immobilized cDNA of Jun&CDK2&p53
were expressed for a period of 90 min and captured for 30 min.
After protein expression and capture, the QC was washed with
PBS and incubated for 10 min with 60 L of 33 M ATF2
solution in PBS.
In Table 5 are reported the main parameters of conductance
curves of Figure 6b, and in Table 6 is reported the shift of these
parameters with respect those of the conductance curve
acquired immediately after lysate human IVTT lysate addition
(IVTT addition).
The unique conductance curve shape for each of the three
proteins conrms the possibility to identify the three expressed
proteins by QCM_D even when combined on the same
expressing QC (Figure 2).
The amount of molecules captured on the QC 90 min
postexpression and 30 min postcapture corresponded to about
8 g, not much dierent from the 6 g obtained in the previous
experiments. Again a frequency decrease was recorded after the
washing (because of PBS solution). In contrast to what
previously obtained, the frequency further decreased after QC
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Table 5. Main Parameters of QC-NAPPA Displaying Jun&CDK2&p53a

Jun&CDK2&p53 conductance curves

f (Hz)b

(Hz)b

Gmax (mS)b

D 103 c

DN (Hz/mS)c

baseline
IVTT addition
10 min IVTT addition
80 min IVTT addition
postcapture (30 min)
postwash
MM+Jun&CDK2&p53+ATF2
MM+Jun&CDK2&p53+ATF2 (10 min)

9489250
9483250
9482625
9481750
9481250
9477250
9475125
9475625

2375
4625
4675
4625
4688
5375
6000
6063

0.436
0.347
0.339
0.342
0.300
0.111
0.188
0.187

0.50
0.98
0.99
0.98
0.99
1.13
1.27
1.28

10900
26629
27543
27038
31250
97262
63687
64894

The conductance curves were collected in dierent steps of NAPPA protocol. bf is peak frequency, is the half-width half-maximum, and Gmax is
the max conductance. cD factor and DN = 2/Gmax normalized D factor.
a

the same solution (PBS), and any changes in the frequency is

due to the binding of ATF2. The amount of ATF2 molecules
bound to the QC was 9.2 g.
As negative control, a NAPPA-QC coexpressing polD1 and
MLH1 was tested for Clomipramine interaction. After protein
expression and immobilization and after PBS washing, 60 L of
110 M Clomipramine solution in PBS was spotted on the
polD1&MLH1 expressing QC. The conductance curve MM+
polD1&MLH1+Clomipramine (Figure 7) was recorded 1 min
after Clomipramine solution addition.
In Table 7 are reported the main parameters of conductance
curves of Figure 7 and in Table 8 are reported the shifts of
these parameters with respect to those of IVTT addition
curve.
The amount of molecules captured on the QC 15 min after
the expression and 5 min after the capture corresponded to
about 15 g (Table 8). A frequency decrease was recorded after
the washing (because of PBS solution). After Clomipramine
addition, no signicant frequency shift was recorded.
This result conrms that no interaction occurs between
polD1 or MLH1 and Clomipramine, as expected.
As reported in the literature,5153 CV values are usually very
low, conrming the repeatability of the experiments and the
validity and portability of the technique. In our hands, NAPPAbased QCM_D proved to have an intra-assay overall CV of 5%
(range 3.38.0%).

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Table 6. Shifts of the Main Parameters of Jun&CDK2&p53

Conductance Curves after Lysate Addition and Relative
Mass of Immobilized Protein on the Quartz Surface
Jun&CDK2&p53
conductance curves

CV
(%)a

Db

DN
(Hz/mS)b

f
(Hz)b

m
(g)c

10 min IVTT addition

postexpression (80 min)
postcapture (30 min)
postwash
MM
+Jun&CDK2&p53+ATF2
MM
+Jun&CDK2&p53+ATF2
(10 min)

5.4
6.0
5.5
5.2
6.5

0.01
0.00
0.01
0.15
0.29

914
409
4621
70633
37058

625
1500
2000
6000
8125

2.7
6.5
8.7
26.2
9.2d

8.0

0.30

38265

7625

7.1d

a
Coecient of variation. We performed two dierent experiments. bD
factor, normalized D factor, and frequency shifts (D, DN, and f )
with respect the values immediately after IVTT lysate addition (see
Table 5). cAmount of molecules immobilized on the QC surface
(calculated through eq 5 respect the frequency recorded immediately
after IVTT addition). dThese values were obtained considering the
frequency shifts respect postwash curve, since the QC was in contact
with PBS solution.

exposure to ATF2 ux, suggesting that the interaction between

Jun and ATF2 was favored by the static process. To evaluate
the amount of ATF2 bound to Jun, we considered the
frequency shift between postwash and MM+Jun&CDK2&p53+ATF2 curves, because in both conditions the QC was in

Figure 7. Conductance curves of polD1 and MLH1 expressing QC. The curves were collected in dierent steps of NAPPA process, as reported in
the legend, and for polD1 and MLH1 expressing QC, after the addition of Clomipramine solution, as negative control.
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Table 7. Main Parameters of polD1&MLH1 Conductance Curves Collected in Dierent Steps of NAPPA Expression Protocol

polD1&MLH1 conductance curves

f (Hz)a

(Hz)a

Gmax (mS)a

D 103 b

DN (Hz/mS)b

baseline
IVTT addition
5 min IVTT addition
15 min IVTT addition
postcapture (5 min)
postwash
MM+polD1&MLH1+Clomipramine

9486895
9486439
9486088
9485463
9483000
9484333
9484404

2860
2702
2596
2596
1509
1877
1895

0.450
0.441
0.431
0.393
0.051
0.157
0.170

0.60
0.57
0.55
0.55
0.32
0.40
0.40

12710
12254
12050
13219
58674
23892
22354

f is peak frequency, is the half-width half-maximum, and Gmax is the max conductance. bD factor and DN = 2/Gmax normalized D factor.

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Table 8. Shifts of the Main Parameters of polD1&MLH1 Conductance Curves after Lysate Addition and after ProteinProtein
Interaction, and Relative Amount of Molecules Immobilized on the Quartz Surface
polD1&MLH1 conductance curves

CV (%)a

Db

DN (Hz/mS)b

f (Hz)b

m (g)c

5 min IVTT addition

15 min IVTT addition
postcapture (5 min)
postwash
MM+polD1&MLH1+Clomipramine

4.8
5.3
6.0
5.2
7.1

0.02
0.02
0.25
0.17
0.17

204
965
46420
11638
10100

351
976
3439
2106
2035

1.5
4.3
15.0
9.2
0.3d

Coecient of variation. We performed two dierent experiments. bD factor, normalized D factor, and frequency shifts (D, DN, and f ) with
respect the values immediately after IVTT lysate addition (see Table 7). cAmount of molecules immobilized on the QC surface (calculated through
eq 5 respect the frequency recorded immediately after IVTT addition). dThese values were obtained considering the frequency shifts respect
postwash curve, since the QC was in contact with PBS solution.
a

CONCLUSIONS
We presented the results obtained applying our innovative
conductometer,2 realized by combining NAPPA technology
with QCM_D, to the characterization of proteinprotein and
proteinsterol interactions in a multiparametric way, taking
advantage of the multiple information provided by the analysis
of the conductance curves (i.e., conductance, viscoelasticity and
adsorbed mass). Moreover, through our conductometer we
acquired information on the kinetic constant of enzymatic
interaction.
Results about the sensitivity and selectivity of the original
prototype have been presented in previous papers.3,6,54 The
data here presented have been obtained employing a further
improved version both ow and static of our conductometer.
The main objective of this communication was to establish
two independent proofs of principle by choosing very wellknown pairs of interacting proteins and proteinsteroid:
CYP11A1 and cholesterol, Jun and ATF2.
An interesting implication for potential clinical applications
concerned the possibility to drastically reduce the time of
protein expression and capture under our experimental
conditions. We noticed that 15 min after IVTT lysate addition
peak frequency and bandwidth of the curves did not change;
the same was true, after few minutes at 15 C, for protein
capture. We deduced from these results that the protein
expression took place in the rst minutes and that also their
capture needed only few minutes, and we performed
experiments reducing the expression time and the capture
time. The results presented seem to conrm our hypothesis.
The conductance curves obtained showed that protein
expression and capture and proteinprotein interactions were
successfully performed with few exceptions. To estimate the
amount of molecules aspecically captured on the QC surface
after the NAPPA expression, we employed a reference QC, and
we estimated an amount of 2 g of molecules aspecically
adsorbed. For CYP11A1 quartz, we obtained 5.6 g of
CYP11A1 and 4.4 g of cholesterol immobilized on the quartz.

The QCM_D instrument we used allowed us to monitor in

real-time the trend of D factor and f during the interaction
between CYP11A1 and cholesterol, both in solution and in
blood. Fitting this experimental data, we obtained a K of about
100 M, a value in good agreement with the values found in the
literature.39,55
Finally to verify the possibility to analyze simultaneously
more interactions in as single NAPPA-QC, we immobilized on
the same QC up to three cDNAs, successfully identied all of
them, and subsequently analyzed the response to multiprotein
interactions. Jun&CDK2 and Jun&CDK2&p53 coexpressed in
the same QCs were indeed tested for ATF2 interaction, in ow
and statically. The results revealed that the ow, even if very
gentle, caused a spoliation of the QC surface, removing some
captured molecules and making it impossible to assess the
interaction between Jun and ATF2. On the contrary, the static
process favored the interaction between Jun and ATF2. We
obtained an amount of ATF2 molecules immobilized on the
QC of 9.2 g.
Taken all together, we demonstrated the versatility of the
NAPPA-QC biosensors for the detection of proteinprotein
interactions and proteinsterol interaction. Because of the
simplicity in which new NAPPA-QC biosensors can be
generated, we envision the use of this platform for the
development of biosensors for other applications, including, but
not limited to, proteinsmall molecules, proteinlipids, and
proteinDNA.

AUTHOR INFORMATION

Corresponding Author

*Fax and Tel.: +39-035767215. E-mail: president@

fondazioneelba-nicolini.org.
Notes

The authors declare no competing nancial interest.

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ACKNOWLEDGMENTS
This work was supported by FIRB Nanobiosensors (ITALNANONET RBPR05JH2P_003) of MIUR (Ministero dellIstruzione, Universita e Ricerca) to Claudio Nicolini University of
Genoa, and by a grant Funzionamento by MIUR (Ministero
dellIstruzione, Universita e Ricerca) to Fondazione El.B.A.
Nicolini.

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ABBREVIATIONS
QCM_D, quartz crystal microbalance with dissipation monitoring; NAPPA, nucleic acid programmable protein arrays;
NAPPA-QC, nucleic acid programmable protein arrays printed
on quartz crystal; IVTT, in vitro transcription and translation;
QC, quartz crystal; CDK2, cyclin-dependent kinase 2; MLH1,
MutL homologue 1, colon cancer, nonpolyposis type 2; ATF2,
activating transcription factor 2; CYP11A1, cytochrome P450,
family 11, subfamily A, polypeptide 1; polD1, polymerase
(DNA directed), delta 1, catalytic subunit; NADPH,
nicotinamide adenine dinucleotide phosphate; ACTH, corticotrophin; SF-1, steroidogenic factor type 1; AP-1, activating
protein type 1 isoform; AP-2, activating protein type 2
isoform; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; DDS, direct
digital synthesizer; BSA, bovine serum albumin; BS3, Bis[sulfosuccinimidyl] suberate; MM, master mix; MDM2, mouse
double minute 2 homologue; CV, coecients of variation

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