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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
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
Article
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
(1)
19
D N = 2/Gmax
(2)
Article
NAPPA-QC
QCM_D Conductometer
Article
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.
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
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)
Article
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.
5539
Article
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
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
(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)
C V = /
(6)
Article
Figure 5. FLOW interaction CYP11A1 with HDL cholesterol in blood (upper panel) and in solution (lower panel).
Article
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.
Article
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.
CV
(%)a
Db
DN
(Hz/mS)b
f
(Hz)b
m (g)c
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.
Article
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
CV
(%)a
Db
DN
(Hz/mS)b
f
(Hz)b
m
(g)c
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.
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
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.
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
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.
AUTHOR INFORMATION
Corresponding Author
Article
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.
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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