Abstract

In the last years, the evolution and the advances of the nanobiotechnologies applied to the systematic study of proteins, namely proteomics, both structural and functional, and specifically the development of more sophisticated and largescale protein arrays, have enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details. Here, we present a further advancement of our previously introduced and described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We coupled Quartz Crystal Microbalance with Dissipation factor Monitoring (QCM_D) with piezoelectric inkjet printing technology (namely, the newly developed ActivePipette), which enables to significantly reduce the volume of probe required for genes/proteins arrays. We performed a negative control (with master mix, or MM) and a positive control (MM_p53 plus MDM2). We performed this experiment both in static and in flow, computing the apparent dissociation constant of p53-MDM2 complex (130 nM, in excellent agreement with the published literature). We compared the results obtained with the ActivePipette printing and dispensing technology vs. pin spotting. Without the ActivePipette, after MDM2 addition the shift in frequency (Δf) was 7575 Hz and the corresponding adsorbed mass was 32.9 μg. With the ActivePipette technology, after MDM2 addition Δf was 7740 Hz and the corresponding adsorbed mass was 33.6 μg. With this experiment, we confirmed the sensing potential of our device, being able to discriminate each gene and protein as well as their interactions, showing for each one of them a unique conductance curve. Moreover, we obtained a better yield with the ActivePipette technology.

Keywords

Cell-free expression system, MDM2, Nanoconductometric sensor, NanoProbeAssay, Nucleic Acid Programmable Protein Array (NAPPA), p53, Piezoelectric inkjet printing, Quartz Crystal Microbalance with Dissipation factor Monitoring (QCM_D)

Introduction

In the last decades, the rapidly increasing evolution and the advances of the nanobiotechnologies applied to the systematic study of proteins, namely proteomics, both structural and functional, and specifically the development of more sophisticated and large-scale protein arrays [1], have enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details [2]. Protein arrays are an important proteomics tool, together with mass spectrometry and 2-dimensional gel electrophoresis [3-6]. Moreover, so-called cell-free protein arrays [7, 8] can be coupled with label-free approaches: which offer unique advantages in the study of human proteome [9-11].

In this manuscript, we report and discuss some preliminary results of protein expression of genes related to cancer, coupling Nucleic Acid Programmable Protein Array (NAPPA) with a recently improved nanogravimetric apparatus which exploits the quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies [11, 12] and using a newly developed piezoelectric liquid dispensing technology.

The selected proteins are p53 and MDM2 because of their importance and biological roles, which will be briefly summarized and reviewed in the following paragraphs.

p53, as a nuclear transcription factor and oncosuppressor, plays a major role in the regulation of the cell cycle, DNA repair, and cell death/senescence/apoptosis, responding to DNA damage, hypoxia, oncogene activation and other kinds of stress. It finely tunes survival of proteins in the mitochondria, microRNA processing, and protein translation among the other biological processes in which it is involved [13]. Mouse double minute 2 homolog protein (MDM2) is one of the main p53 negative regulators since it acts as an E3 ubiquitin ligase that catalyzes the ubiquitination of p53 for degradation, recognizing the N-terminal trans-activation domain (TAD) of p53 [14]. Inhibitors that target p53, MDM2 or p53/MDM2 interaction are an important class of anticancer therapeutics [14, 15].

At a molecular level, MDM2 has a deep binding pocket for p53, which is only 18 Å long but is fundamental for the interaction with p53's Trp23, Leu26, and Phe19 (the so-called "three finger pharmacophore" model) [13].

We chose NAPPA since this innovative technology avoids any time-consuming task in the difficult process of obtaining highly purified proteins, relying instead on the production of proteins from high quality super-coiled DNA. For this purpose, complementary DNAs (cDNAs) of selected genes tagged with a C-terminal glutathione S-transferase (GST) are spotted on the microarray surface and expressed using a cell-free transcription/translation system (IVTT, in vitro transcription and translation). The newly expressed protein is captured on the array by an anti-GST antibody that have been co-immobilized with the expression clone on the microarray surface.

The advantages and benefits of NAPPA technologies can be briefly summarized [11]:

(1) The demanding and challenging process of obtaining highly purified proteins is replaced by a single quick step; furthermore, cDNAs and clones are more easily available;

(2) Proteins expressed on the NAPPA arrays are stable, properly folded and biologically, functionally active.

NAPPA microarrays can be useful in biomarkers discovery and for other clinical applications [16], such as biosensor development, especially in the effort of moving towards Personalized Medicine [17-19]. For this task we coupled NAPPA with a new generation of conductometric devices, namely QCM. QCM_D indeed appears a promising tool to study protein-protein interactions especially in the field of oncology, both cellular and molecular.

Inkjet printing confers more advantages: in the field of nanotechnologies, it has been exploited to produce a formulation capable of controlling the release of a drug [20], to better functionalize biosensors [21-24].

To the best of our knowledge, we coupled for the first time QCM_D with NAPPA technology and piezoelectric inkjet printing for biomedical applications. The objective of the present research regards the analysis of protein-protein interaction towards potentially useful clinical applications, namely in the field of cancer studies.

Clinical implications are also envisaged and addressed.

Materials and Methods

QCM_D conductometer

Nanogravimetry makes use of functionalized piezoelectric quartz crystals (QC), which vary their resonance frequency (f) when a mass (m) is adsorbed to or desorbed from their surface. This is well described by the well-known Sauerbrey's equation:

Δf = S ∙ Δm = – 2 ∙ f²/Z_p ∙ Δm

where Δf is the frequency shift, Δm is the change of mass per area, S is the mass sensitivity, f is the resonant frequency, Z_p is the acoustical impedance.

This equation can be expressed also as:

Δf/f₀ = - m/(A ∙ ρ ∙ l)

where f₀ is the fundamental frequency, A is the surface area covered by the adsorbed molecule and ρ and l are the quartz density and thickness, respectively.

Quartz resonators response strictly depends on the biophysical properties of the analyte, such as the viscoelastic coefficient. The dissipation factor (D) of the crystal's oscillation is correlated with the softness of the studied material and its measurement can be computed by taking into account the bandwidth of the conductance curve 2Γ, according to the following equation:

D = 2Γ/f

where f is the peak frequency value.

In our analysis we introduced also a "normalized D factor", D_N, that we defined as the ratio between the half-width halfmaximum (Γ) and the half value of the maximum value of the conductance (G_max) of the measured conductance curves [11]:

D_N = 2Γ/G_max

D_N is more strictly related to the curve shape, reflecting the conductance variation [2, 11].

ActivePipette piezoelectric inkjet technology

For this experiment, we exploited a newly developed technology that enables to significantly reduce the volume of probe required for genes/proteins arrays [25-27]. This technology relies upon the precise, non-contact printing capabilities of a piezoelectric inkjet printer. When compared to pin spotters, it enables to avoid some drawbacks, such as slow printing and dispensing speed, satellite spots, ring-like stains or "coffee mug" effects, inhomogeneous spots, misplaced or even absent spots due to evaporation issues and poorly controlled deposition and confinement, and spot contamination [27, 28]. Moreover, two important advantages of piezoelectric inkjet printing are the possibility of spotting on complex surfaces [27, 29] and using also clinical samples of limited volume and amount, thus showing more biotechnological and biomedical potential and implications.

NAPPA experiments

The QCM_D instrument was developed by Elbatech (Elbatech srl, Marciana – LI, Italy). 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 the viscoelastic effects of the surrounding layers affected the oscillation. The QCM_D software, QCMAgic-Q5.3.256 (Elbatech srl, Marciana – LI, Italy) allows to acquire the conductance curve or the frequency and dissipation factor variation versus time. In order to have a stable control of the temperature, the experiments were conducted in a temperature chamber. 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, USA). The electrode material was 100Å Cr and 1000Å Au and the quartz was embedded into glass-like structures for easy handling [11, 30].

The NAPPA-QC arrays were printed with 100 spots per QC.

Quartzes gold surfaces were coated with cysteamine to allow the immobilization of the NAPPA printing mix. Briefly, quartzes were washed three times with ethanol, dried with Argon and incubated over night at 4°C with 2 mM cysteamine. Quartzes were then washed three times with ethanol to remove any unbound cysteamine and dried with Argon. Plasmids DNA coding for GST tagged proteins were transformed into E. coli and DNA were purified using the NucleoPrepII anion exchange resin (Macherey Nagel). NAPPA printing mix was prepared with 1.4 μg/ul DNA, 3.75 μg/ul BSA (Sigma-Aldrich), 5mM BS3 (Pierce, Rockford, IL, USA) and 66.5 μg 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 1 hour with agitation and then printed on the cysteaminecoated gold quartz using the Qarray II from Genetix. In order to enhance the sensitivity, each quartz was printed with 100 identical features of 300 microns diameter each, spaced by 350 microns center-to-center. The human cDNAs immobilized on the NAPPA-QC were: p53 and MDM2.

Gene expression was performed immediately before the assay, following the protocol described in [11]. Briefly, IVTT was performed using HeLa lysate mix (1-Step Human Coupled IVTT Kit, Thermo Fisher Scientific Inc.), prepared according to the manufacturers' instructions. The quartz, connected to the nanogravimeter inside the incubator, was incubated for 10 min at 30°C with 40 μl of HeLa lysate mix for proteins synthesis and then, the temperature was decreased to 15°C for a period of 5 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 500 mM NaCl PBS for 3 times. 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.

After protein expression, capture, and washing the QCs were used for the interaction studies QC displaying the expressed protein was spotted with 40 μl of MDM2 at 22°C.

Reproducibility of the experiments was assessed computing the coefficient of variation (CV, or σ^*), using the following equation:

σ^* = σ/μ,

where σ is the standard deviation, and μ is the mean.

We performed experiment both in static and in flow. This method enables to compute the apparent dissociation constant, using the following equation [31]:

where f(t) is the frequency at time t, t₀ represents the start of dissociation, f₀ is the frequency at t₀, k_diss is the apparent dissociation rate constant.

Results and Discussion

QCM_D measures were calibrated for frequency and for D factor shifts. The calibration curves equation (obtained with Ordinary Least Squares methods, OLS) are:

Δf= - 7.16 – 231.18 m; with r2= 0.9986,
and:
D = 0.831 + 0.286 η; with r2= 0.9990.

We analyzed the conductance curves acquired in NAPPAQCs in different steps of the expressing and capturing process: after the addition of human IVTT lysate at 30°C ("IVTT addition"), i.e. prior protein expression; after the final washing process with PBS ("Post-wash"); after addition of the substrate.

In Figure 1 are reported the conductance curves of the negative control (MM experiment), while in Figure 2 are shown the conductance curves of quartz carrying p53 gene being expressed and thereafter interacting with MDM2 are reported.

Figure 3 shows the same experiment of Figure 2 conducted in flow (frequency versus time, and D factor versus time).

We computed the apparent dissociation constant of the p53-MDM2 complex 130 nM, which is in excellent agreement with the extant literature [32].

In Tables 1, 2, 3 and 4, the main parameters of the conductance curves of Figure 1 and 2 are reported.

In Table 5, we compared the results obtained with the piezoelectric inkjet printing and dispensing ActivePipette technology with the values yielded without [30]. We can see that without the ActivePipette, after expressing the protein and washing Δf was 4530 Hz and the corresponding adsorbed mass was 19,7 μg, while after MDM2 addition Δf was 7575 Hz and the corresponding adsorbed mass was 32,9 μg.

With the ActivePipette technology, after expressing the protein and washing Δf was 4665 Hz and the corresponding adsorbed mass was 20,3 μg, while after MDM2 addition Δf was 7740 Hz and the corresponding adsorbed mass was 33.6 μg.

Conclusions

In this paper, we successfully introduced an advancement in our previously described NAPPA-based nanoconductometric sensor, coupling it with a newly developed piezoelectric inkjet printing and dispensing technology (namely, the ActivePipette). We performed a negative control (MM) and a positive control (MM_p53 plus MDM2). We confirmed the sensing potential of our device, being able to discriminate each gene and protein as well as their interactions, showing for each one of them a unique conductance curve. Moreover, in this communication, we showed that we obtained a better yield with ActivePipette piezoelectric dispensing technology.

Acknowledgements

This project was supported by grants to FEN (Fondazione Elba Nicolini) and to Professor Claudio Nicolini of the University of Genova, Italy by the FIRB Nanobiosensors (ITALNANONET RBPR05JH2P_003) and by a grant Funzionamento from MIUR (Ministero dell'Istruzione, Università e Ricerca; Italian Ministry for Research and University). The authors are very grateful to Prof. Joshua LaBaer for his precious cooperation at the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ. USA during Professor Nicolini Visting Professorship stage. ActivePipette piezoelectric inkjet dispensing technology was provided by Engineering Arts (Tempe, AZ).

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