|Calcium oxide (CaO) matrices; Carbon dioxide (CO2);
Carbon nanotube composites (CNTs); Laccase; Mass spectrometry
(MS); Nanosensors for biosafety; Nucleic Acid Programmable Protein
Arrays (NAPPA); Phenols and phenolic derivatives and compounds;
Protein synthesis Using Recombinant Elements (PURE) technology;
Quartz crystal microbalance with dissipation monitoring (QCM_D);
|According to the Cartagena Protocol , biosafety (known also
as biosecurity or biocontainment) is defined as a discipline dealing
with the prevention of the loss of the biological integrity and with its
safeguard and preservation from natural and anthropogenic hazards.
|It is definitely a multidisciplinary science, combining the expertise
of different fields, ranging from biomedicine and agriculture, to
chemistry and ecology.
|In order to unravel the key elements of both biomedical and
environmental sciences, it is mandatory to exploit a complex strategy,
which unifies many different technologies [2-7]. Bioinformatics tools
play a role in identifying the most important genes and/or molecules
involved in key biological processes . Our previously described
Leader Genes algorithm [9,10] enables to find the so-called “hub
genes”, that, being highly interconnected, form an important subnetwork
within the complex gene-gene interactions map useful to
design and investigate with the help of DNA analyzer (DNASER) 
ad hoc targeted micro-arrays. The selected genes of high interest can be
subsequently expressed and investigated using label free technologies
[12-14] based on NAPPA with or without the SNAP-tag [15,16] (Figure
1). This new approach overcomes indeed the limits of correlated
fluorescence detection plagued by the background still present after
extensive washes. This is being accomplished in conjunction with an
homogeneous and well defined human cell free expression capable
to achieve the above goals making realistic the ambitious objective
to quantify the regulatory protein networks in humans, a task
being fundamental for nanomedicine and in the fields of biosafety
and natural disasters, through the development of a quartz crystal
microbalance with dissipation monitoring (QCM_D) sensor for health
and environment of new conception [17,18] which can be utilized along with mass spectrometry (MS) and Nucleic Acid Programmable
Protein Arrays (NAPPA)-SNAP arrays [19 ].
|NAPPA arrays may play a key role, being able to detect bacterial
and viral agents. Molecular epidemiology and surveillance have been
found to be useful in monitoring natural disasters, as proven by scholars
after the Katrina hurricane in Louisiana (US) with enterococcosis
, gonorrhea  and other sexually transmitted diseases (STDs),
and after an earthquake in Japan with influenza  and tuberculosis
. Molecular biotechnologies are recommended by international
organisms and health authorities during Public Health emergencies
, and thus the role of NAPPA is emerging.
|A second concern with profound implication on biosafety is the
environment, where nanotechnology did play a role since the onset
with the implementation of effective gas sensors based on the fact
that the conformation of the polymer backbone seems also to affect
the molecular rearrangement occurring during the doping process and
in some cases, due to the sterical hindrance generated by “too close”
substituents to the aromatic ring, is responsible of the spontaneous
undoping process [6,7]. Sensors for the detection and monitoring
of carbon dioxide (CO2) , an important greenhouse gas, can be
extremely helpful in revealing the quality of indoor air and the increased
emissions of gases from fossil fuels and coal combustion, industrial
processes (such as the manipulation of hydrocarbons), and agriculture
associated to deforestation and habitat destruction. This can results in
consequent changes in the atmospheric chemical composition with direct biological effects and negative impact on the Earth’s climate,
such as the global warming issue, considering that fossil fuel CO2
emissions can remain high in the atmosphere for long periods (also up
to tens of thousands of years).
|Moreover, CO2 has been involved in sea and ocean acidification
and other environmental hazards. The instruments generally employed
in these determinations basically consist of infrared (IR) detectors
performing a continuous monitoring and plotting in the site with a
good degree of accuracy, but unfortunately they cannot be used for
extensive mapping work because of the large number of expensive
devices and specialized people that would have to be involved. Longterm
sampling devices such as diffusive sampling techniques are
therefore the cheapest and easiest way. Environment plays also a key
role in shaping human diseases and for this reason ecological factors
cannot be neglected. Climate change, wild urbanization together with
deforestation and globalization are having indeed a tremendous impact
on human health.
|Natural disasters such as hurricanes and earthquakes are
environmental drivers of diseases since they favor the dispersal and
dissemination of vectors and pathogenic microbes. Environmental
drivers are considered both precursors and determinants of diseases,
such as air pollution as a driver of influenza , heat waves as a driver
of West Nile fever, Dengue, Chikungunya and other tropical diseases
, tsunami as a driver for mycobacteriosis , and El Niño Southern
Oscillation (ENSO), the Indian Ocean Dipole (IOD) as determinants of
cholera epidemics . Even though the increase of natural hazards is
only apparent and is considered controversial by some scholars ,
its consequences in term of damages and crop and resources losses are
|Our third goal was finally to build the prototype of an enzymebased
biosensor for biomedical and environmental purposes, in which
the immobilization procedure was carried out via Langmuir-Blodgett
(LB) films. The enzyme implemented in our device  was laccase
, which is a blue oxidase capable of oxidizing phenols and aromatic
amines and their derivatives by reducing molecular oxygen to water
by means of a cluster of copper atoms. Laccase belong to a large group
of the multicopper enzymes, which catalyze the oxidation of highly
diverse and heterogeneous compounds.
|Materials and Methods
|Taking into account all the considerations so far discussed and the
sensors recently introduced , here in this section we summarize the
techniques and procedures utilized in the construction of the abovementioned
|In order to prove the feasibility of the NAPPA arrays [12-16], we
have used a matrix-assisted laser desorption ionization time-of-flight
MS (MALDI-TOF MS) and a model protein system where the known
query proteins immobilized in a blind order were successfully identified
(Figures 2 and 3) . Thus, we have recently shown that is possible
to utilize label free techniques coupled with MS for the detection of
NAPPA slides containing Jun, p53, Cdk2 and CdkN1A genes spotted,
immobilized and later expressed , as shown in Figures 2 and 3.
The scheme of the experimental set up for the MS is shown in Figure
2 below and in reference . In alternative to fluorescent staining
and labeling approaches, the new label free NAPPA method emerging
from the combined utilization of independent and complementary nanotechnological approaches finds applications in analyzing protein
function and protein-protein interaction both in basic and applied
studies ideal for nanomedicine [2,18], namely QCM_D and QCM_Fbased
nanogravimetry [17,18], MS , atomic force microscopy
(AFM)  and anodic porous alumina (APA) . QCM_D,
measured in a temperature chamber where the quartz was positioned
and was monitored at the same time for frequency and dissipation
factor variations, is discussed later in the “Results” section; it basically
detects the normalized dissipation factor of the quartz crystal by means
of the “half-width half-height” principle of its impedance curve. In our
case, the quartz was connected to an RF gain-phase detector (Analog
Devices, Inc., Norwood, MA, USA) and was driven by a precision
Direct Digital Synthesizer (DDS) (Analog Devices, Inc., Norwood, MA,
USA) around its resonance frequency, thus acquiring conductance vs.
frequency curve, which showed a typical Gaussian behavior.
|For what concerns the health prototypes, one biosensor of new
conception was realized coupling the traditional QCM_F and QCM_D
using an innovative protein cell-free expression system named
NAPPA that allowed immobilizing on the quartz surface, as sensing
molecule, any kind of proteins. Standard nanogravimetry exploited
the piezoelectric quartz crystals properties to vary the resonance
frequency, f, when a mass, Δm, was adsorbed to or desorbed from their
surface according to Sauerbrey equation . Quartz resonators used
in fluids are more than mere mass or thickness sensors: sensor response
depends also on viscoelastic properties of the adhered biomaterial,
surface charges of adsorbed molecules and surface roughness. The
modern QCM_D technology utilizing impedance measurement offers
access to the resonance bandwidth in addition to resonance frequency,
which is strictly connected with the viscoelastic properties of the
sample. Building upon the successful use of in vitro NAPPA translated
protein (IVTT), Ramachandran et al. substituted the use of purified
proteins with the use of cDNAs encoding the target proteins at each
feature of the microarray [15,16]. The proteins were translated using
a T7-coupled rabbit Reticulocyte Lysate IVTT system. Mammalian
proteins can be expressed in a mammalian milieu, providing access
to vast collections of cloned cDNAs. The addition of a C-terminal
glutathione S-transferase (GST) tag to each protein enabled its capture
to the array through an antibody to GST printed simultaneously with
the expression plasmid. We manage to couple QCM_D and QCM_F
with NAPPA technology, optimizing the monitoring in real time of the
kinetics of the reaction to obtain information not only on the mass but
also on the viscosity of the sample and sensing the interaction among a
query protein and the expressed protein.
|As a follow-up in cooperation with the New England Biolabs
(NEB) and the Biodesign Institute at Arizona State University (ASU),
we have used an innovative cell-free expression NEB SNAP system
based on bacteria recently proposed  in which all protein species
and concentrations are well defined. The combination of NAPPA and
SNAP  is emerging as a promising tool, since utilizes a complex
mammalian cell free expression system to produce proteins in situ ,
shown in Figure 3. On the whole, considering the abundance of protein
material different from target and query protein, the results are very
encouraging, showing the potential to couple the NAPPA and SNAP
technologies with the MALDI-TOF MS for a label free investigation
of protein samples in combination with sophisticated bioinformatics
tools and software . The chemistry and the algorithms progressively
implemented prove for the first time that MS can characterize proteins
immobilized on NAPPA, pointing that, with further developments, this label-free procedure will prove to be fully effective when compared
to the fluorescence NAPPA (Figure 3, left), that has already seen
significant clinical applications in the last decades [15,16]. To verify the
proper protein expression and capture on SNAP-NAPPA, a preliminary
test has been carried out using fluorescence analysis. The same SNAPNAPPA
samples employed for MS analysis (namely, p53, CDK2,
SH2-Src and SH2-PTPN11) were spotted on microscope glass in a 2x2
spots per box configuration using increasing SNAP concentrations. As
negative control on the gold slides was printed a box only with master
mix (Figure 3, left), while the positive controls mouse IgG and rabbit
IgG were added in a printing mix, instead of DNA.
|Proteins were synthesized by two different IVTT systems, a new
system extracted from human cells (1-Step Human Coupled IVTT,
HCIVTT, Thermo Scientific) and E. coli IVTT. It is known that HCIVTT
performs better than RRLIVTT. The yield of protein synthesized in
HCIVTT is more than 10 times higher than RRL. Moreover, HCIVTT
showed a robust lot-to-lot reproducibility. In immune assays, the
signals of many antigens were detected only in HCIVTT-expressed
arrays, mainly due to the reduction in the background signal and
the increased levels of protein on the array . The protein yields
obtained through the process “Protein synthesis Using Recombinant
Elements” (PURE) system. PURE system  has then been matched
with that obtained with this innovative cell free IVTT system. In Figure
3 are reported the images of three SNAP-NAPPA slides after proteins
expression fluorescence acquired. Two slides were expressed with
HCIVTT and a third with E. coli IVTT; the level of protein displayed
on the array was measured using respectively anti-SNAP antibody or
anti-p53 antibody followed by a Cy3-labeled secondary antibody. The
results obtained not only confirmed the proper protein expression and
capture on the array surface but, moreover, demonstrated that E. coli
IVTT systems ensured a protein yield from 2 to 8 times higher respect
to HCIVTT, considering the higher SNAP concentration. The gain
respect RRL is, therefore, more than twenty times. For the first time
an improved version of NAPPA, that allows for functional proteins
to be synthesized in situ - with a SNAP tag - directly from printed
cDNAs just in time for assay, has been expressed with a novel cellfree
transcription/translation system reconstituted from the purified
components necessary for Escherichia coli translation - the PURE
system - and analyzed both in fluorescence and in a label free manner
by four different mass spectrometry techniques, namely three MALDITOF,
a Voyager, a Bruker Autoflex and a Bruker Ultraflex, and a liquid
chromatography-electrospray ionization MS (LC-ESI-MS/MS). Due
to the high complexity of the system, an ad hoc bioinformatic tool
has been needed and has been developed for their successful analysis.
The contemporary fluorescence analysis of NAPPA, expressed by
means of PURE system, has been performed to confirm the improved
characterization of this new NAPPA-SNAP system .
|Calcium Oxide (CaO)
|An innovative long-term sampling method for the determination
of environmental CO2 accumulation takes advantages of the properties
of CaO to be carbonated by this gas according to the following equation:
| CaO + CO2 ↔Ca CO3 (1)
|For this purpose, we studied here the variation of mass connected
to the carbonation process in order to assess the quantity of gas
absorbed by a fixed and known amount of composite in relation to
the concentration of environmental CO2 . This long-term sampling
method for the determination of environmental CO2 accumulation
takes advantages of the properties of CaO to be carbonated by this gas. We carried out preliminary tests to assess the best concentration of
CaO in the composite, individuated in the CaO/PEG weight ratio of
¼ and we tested the sensing properties of the composite materials via a
nanogravimetric method by using a home-made glass chamber of 340
ml in volume. The home-made chamber (illustrated in Figure 4) was
provided with four input sockets able to arrange up to four quartzes
at the same time, besides inlet and outlet valves to feed and empty the
gas. As transducers, AT-cut quartz crystals were used, with a native
frequency equal to 9.5 MHz, a blank diameter equal to 0.550”, an etched
surface, an electrode diameter equal to 0.295”, with 100 Å Cr and 1000
Å Au as electrode materials (International Crystal Manufacturing
Co, Inc., Oklahoma City, USA). The preliminary experimental data
highlighted that the composite was able to detect selectively CO2 via
a nanogravimetric method by performing the experiments inside an
atmosphere-controlled chamber filled with CO2. Furthermore, the
composite material showed a linear absorption of CO2 as a function of
the gas concentration inside the atmosphere-controlled chamber, thus
paving the way for the possible use of these matrices for applications
in the field of sensor devices for long-term evaluation of accumulated
|The previous reported considerations allowed us to design and
realize a dosimeter for the long-term analysis of the carbon dioxide. We
used the same transducers but they were inserted in a home-made and
ad hoc designed and built Plexiglas measuring chamber, divided into
two parts: the upper one, with a funnel opening, allowed the exchange
of the sensing matrix with the environment and the lower one that
allowed the housing of the transducer.
|The nanogravimetric instrument, used for relating CO2
concentrations with the mass variations, consisted of a base unit,
interfaced to a PC via USB port and able to drive up to four oscillator
units. The base unit embedded the interface circuitry to/from the
USB port, a digital signal controller and a fast programmable logic
device containing an accurate four-channel counter, plus the four
interfaces to the oscillator units. The counter logic was fully parallel,
this meaning that the four input signals were acquired and counted-up
simultaneously at a gate interval selectable from fractions of a second
to 10 seconds. The base unit was powered by means of an external
pluggable +12 V power supply which sustained input AC voltages from
90 to 240 VAC.
|The choice to have the oscillators outside the base unit allows the maximum flexibility when building an experimental set-up. The noise
immunity was preserved and guaranteed by the oscillator design, which
was based on a precision internal reference crystal, used as a time base
comparator for the working quartz crystal. Only the mixed, lower
frequency signal was then transmitted to the base unit by dedicated
twisted pair lines on the cable. The oscillator units were connected to
the base unit by means of standard Ethernet class V cables.
|The system was driven by an extremely user-friendly software
running under MS-WindowsTM. It consisted of a lower level kernel,
which implemented the necessary data transfer between the computer
and the base unit, and of a higher level set of routines, which drove
the user through an easy-to-perform data acquisition and analysis. In
addition to the data acquisition service routine, a useful data display was
implemented by which the user was easily able to retrieve the acquired
signal values. Oscillating frequency versus time measurements can be
performed following the data acquisition in real time on the computer
screen, by means of strip chart plotting.
|SWNTs and MWNTs embedded in POTO
|Synthesis carried out by polymerizing the monomer in the presence
of a dispersion of CNTs is very simple and can be effectively used for
short-term monitoring of gas [6,7]. Polyaniline derivatives have been
deeply studied among conducting polymers in the last decades for
their good electrical properties, easy methods of synthesis and high
|The chemistry of polyanilines is generally more complex with
respect to other conducting polymers, due to their dependence
on both the pH value and the oxidation states, described by three
different forms known as leucoemeraldine base (fully reduced form),
emeraldine base (EB) (50% oxidized form), and pernigraniline base
(fully oxidized form). The most important is the EB form and its
protonation by means of H+ ions, generated from protic acids, issues
the emeraldine salt form, responsible of the strong increment of the
conducting properties [6,7]. This process is reversible and it is possible
for the presence of imine group basic sites located along the conducting
polymer backbone. Nanogravimetric analysis demonstrates that these
materials are suitable for applications as sensors for carbon dioxide
. From the experimental results, the highest peak of oscillation
is obtained in the presence of O-methylaniline. The standardized
synthesis of nanocomposite materials was carried out by oxidative
polymerization under controlled conditions, maintaining the
temperature at 0 ÷ 4°C by means of an ice bath for 24 h. The medium
of reaction was 200 ml of 1 M HCl solution of the monomers. For the
synthesis of the nanocomposites, we dispersed 100 mg CNTs in the
medium of reaction by sonication. We performed the sonication by
means of SONIC 300 VT equipment, setting a 10% power for 1 min
in order to only disperse CNTs without braking processes. We used
a monomer/MWNTs weight ratio of 100/1 and a monomer/oxidant
molar ratio of 4/1. In order to obtain the solvent processable undoped
form, we filtered and subsequently treated the crude materials in the
doped form (emeraldine salt form) with ammonium hydroxide for 2
h. We finally filtered the undoped materials (EB form) and performed
a treatment with methanol and diethyl ether in order to eliminate
the oligomers, followed by the evaporation of the residue solvents by
vacuum. The final products, in the EB forms, were completely soluble
in chloroform. We fabricated the Langmuir monolayers in a LB trough
(MDTcorp., Russia), 240 mm x 100 mm in size and 300 mL in volume,
having a compression speed of 1.67 mm/s (100 cm2/min). The spreading
solutions of nanocomposites were prepared by dissolving 5 mg of
materials in 20 ml of chloroform. The Langmuir-Schaefer (LS) films
|LB multi-layers of Laccase.
|LB thin biofilm of recombinant laccase from Rigidoporus lignosus
(formerly known as Rigidoporus microporus) and the corresponding
sensor were obtained as reported in Bragazzi et al. , using highly
concentrated sample of laccase enzyme.
|The mixed chloroform solution in equimolar proportions had a
concentration of 1 mg/ml. A volume of 50 μl of the mixture was spread
on a Milli-Q water sub-phase and the monolayer was compressed with
movable barriers at a rate of 70 mm/minute. The transfer pressure
to obtain the LB film (Figure 5, above and left) was about 20 mN/m,
at 22°C. The surface morphology and topology of the LB thin film of
laccase was investigated via AFM. The roughness of the film was found
to be 8.22 nm and the compressibility coefficient about 37.5 m/N as
determined from the LB π-A isotherm at the air-water interface. The
enzyme was deposited onto the electrode via LS technique and a
protocol of immobilization overnight was followed; after depositing
the film, the electrodes were kept at 4°C up to a maximum of 16 hours.
The employed electrodes were ruthenium and graphite ones, while the
counter-electrode was of silver. The area of the electrode (Figure 5,
above and center) was about 0.75 mm per 1 mm. The amperometric
technique was used to polarize the electrochemical couple and to
obtain a current discharge related to the amount of the investigated
drug, namely clomipramine, with an EG & G PARC model 263 A
potentiometer, equipped with dedicated in-house software . This
tricylic tertiary amine antidepressant is widely used for the therapy
of depressive and obsessive compulsive disorders (OCD). Because of
its clinical importance many analytical methods have been developed
to monitor its level: above all chromatographic techniques (like gas
chromatography or GC, high performance liquid chromatography or
HPLC), eventually coupled with tandem MS, but all these techniques
are generally expensive, time-consuming and laborious. Moreover,
the AGNP-TDM panel of experts has emphasized the importance of
therapeutic drug monitoring. In our experiment, clomipramine was
added at varying concentrations in the low micromolar range. The
therapeutic dose is from 75 mg/day to 200 mg/day; the pharmacokinetics
is extremely variable among the patients. Generally, the therapeutic
concentration in the human blood of psychiatric patients is usually
in the low micromolar range. The side-effects of the drug, especially
in case of overdose, are seizures, hematological, cardiological and
neurological adverse effects up to the coma (the so-called tricyclic
|Results and Discussion
|The growing concern for biosafety is here approached in three
distinct different biosafety applications with sound nanotechnological
methods, which have proven encouraging and promising, with
profound implications in health and environment.
|Vaccine Design and Biosafety
|A miniature flow-cell (Figure 6, above and left) was designed for
protein-protein interaction analysis via a conductometric QCM_D
sensor . The flow cell chamber volume was 100 μl and it was
connected to a BioRad Econo Gradient Pump, able to pump solution
in a flux range of 0.02 - 6 ml/min. The transducer consisted of 9.5 MHz,
AT-cut quartz crystal of 14 mm blank diameter and 7.5 mm electrode
diameter and the electrode materials were 100 Å Cr and 1000 Å Au. Microarrays were produced on the quartzes previously described and
connected to the nanogravimeter inside the incubator at 30°C for
proteins synthesis and then, the temperature was decreased to 15°C
to facilitate the proteins binding on the spot surface. After wash the
quartz was placed in the flux chamber for protein-protein interaction
analysis  using genes of significant implications (both clinical and
biological) and using 10x10 spots. In order to investigate the biosensor
response to protein-protein interaction, we added a MDM2 solution to
p53 quartz once we had our proteins expressed. In Table 1 are reported
for p53 quartz, before and after protein expression, the values of the
frequency and the half width at half height (Γ) of the resulting QCM_D
curve, along with the values of the variation in conductance YRE (mS).
The ratio named normalized D factor, DN = 2Γ/I, gives information
on the shape of impedance curves and on sample viscosity, while
the decrease in frequency f (Hertz) is related to the amount of p53
molecules being immobilized.
|The resulting Michaelis-Menten constants of the p53-MDM2 as well as of other protein-protein interactions appeared quite compatible
with the literature .
|The results obtained later using a human Lysate and 10x10 spots per
quartz and with the most recent QCM_D configuration and set-up 
suggest that the NAPPA based biosensor monitor with high selectivity
the single protein being expressed even in a mixture of different genes
(Figure 6, above and right) and, from the analysis of the D-factor,
allowed to acquire in real time information on the characteristics of
each single protein being expressed with unique signature and on
the kinetics constant of the reactions. Then, in order to eliminate
the background signal, we do routinely carry out the measurements
starting from the crystal native frequency subtracted of 15 kHz and
using a step of 1.0 Hz for collecting the whole impedance plots at the
optimal resolution and with a considerable number of data points. For
this reason, we have used a dsPIC (Microchip Inc, USA) featuring at
the same time good computational power and sufficient memory space.
This represents a PC-driven prototype in order to establish the proof
of principle. The industrial prototype is being designed and realized,
under a different contract, in order to have a temperature-controlled
ad hoc chamber, hardware and software optimized to increase speed,
computational power and compactness to incorporate hardware and
to produce a friendly stand-alone device.
|To establish some proofs of principle we choose BRIP1, Jun and
ATF2 . JUN encodes the c-Jun protein, which is a well-known
oncogene, being the putative transforming gene of avian sarcoma virus
17. It encodes indeed a protein with a strong similarity to the viral
protein. It is encoded by the locus 1p32-p31 and its alteration leads to
several malignancies. ATF2 gene (chromosome 2q32) encodes a 505
amino-acids long transcription factor, that belongs to the leucine zipper
family of DNA-binding proteins, and is an important component of
many signaling pathways whose alterations are a cause of malignant
transformation (Figure 6, below).
|An interesting implication for potential, clinical applications
concerned the possibility to drastically reduce the time of protein
expression [35,36] and capture under our experimental conditions
(this is true especially for BRIP1). By acquiring conductance curves
each 5 minutes, we noticed that after the first 15 minutes after IVTT
lysate addition at 30°C the position and shape of the curves did not
change anymore, likewise after few minutes at 15°C, for protein
capture, position and shape of the curves did not change anymore. We
deduced from these results that the protein expression took place in
the first minutes and that also their capture needed only few minutes
and therefore we performed experiments reducing the expression time
from 90 minutes to 15 (at 30°C) and the capture time from 30 to 5
minutes (at 15°C). The results presented in references  and 
confirmed our hypothesis. The conductance curves obtained showed
that protein expression and capture and protein-protein interactions
were successfully performed. Applying the calibration coefficients,
we were able to estimate the amount of protein immobilized on the
biosensor surface. To estimate the amount of molecules aspecifically
captured on the QC surface after the NAPPA expression (human
IVTT lysate molecules not specifically adsorbed on the QC surface) we
employed a reference QC (MM QC) and we estimated an amount of 2
μg of molecules aspecifically adsorbed. For BRIP1 quartz we obtained
2.28 μg while for JUN quartz the amount was 1.69 μg.
|Future perspectives of this biosensor regard its clinical introduction
for assessing cancer prognosis and make a personalized diagnosis and/
or to deliver an individualized treatment, and in the context of biosafety
design effective vaccines (Table 2). Only recently protein arrays were used to discover new antigenic determinants for vaccine development.
NAPPA-based sensors could be used for screening the affinity between
the identified proteins and the immunological synapse (CD4, TCR,
MHC complex). Affinity kinetics can be evaluated also using classical
techniques or via AFM and Surface Plasmon Resonance (SPR). In the
right column of the Table 2 are shown the genes that interact with
immunological human synapse (CD4 + TCR + MHC).
|Carbon dioxide monitoring for biosafety
|We performed nanogravimetric acquisitions of frequency vs.
time in order to assess the variation of mass at regular intervals of
time after placing the small plastic cell in a room. We also measured
the mass of the deposited sample at time zero, in order to have the
value of frequency related to the composite before reacting with
environmental CO2. We consequently carried out acquisitions of
frequency vs. time in order to sample the indoor environmental CO2.
Basing on our previous experiments, we were able to calculate the
variation of mass per week and consequently the average quantity of
CO2 in the environment. Experimental data, summarized in Table 1 for
a clear vision, highlighted the variation of frequency in relation to the
quantity of CO2 absorbed by the composite issued a linear absorption,
coherently with the constant human activity of the sampling period.
Specifically, we found (Table 3) the variation of mass in relation to the
quantity of CO2 present in the environment (indoor), indicated the
concentration of indoor CO2 during the sampling period was 10 times
less than the average concentration in the atmosphere, thus indicating
good quality of the air in our laboratory. Since the very good results
obtained from the usage of this long-term sampling device, we are
presently programming new samplings both in indoor spaces affected
by strong human activities and outdoor.
|Phenols detection for biosafety
|Experiments carried out with cyclic voltammetry of our LB
recombinant laccase  highlighted excellent reproducibility
and linearity of the peaks of oxidation and reduction, related to the
presence of the drug in several biological fluids (see Figure 5, below,
and Table 4).
|These results allowed the design and the creation of a prototype
of a biological sensor for antidepressants and to extend its utilization
for biosafety in the phenols detection. The instrument consisted of a
central unit, able to bias the working electrode on which the enzyme
was deposited and to detect the current generated as a result of the
interaction of the enzyme with the analyte containing the substance
of interest (Figure 5). By a multiple selector, the user can choose the fluid or substance to be analyzed and, through the proper calibration
parameters, on the display, the proper drug concentration will be
provided. The instrument  was powered by two 9V batteries, in
order to avoid noise from the main voltage. Using laccase, screenprinted
electrodes technique and a portable device appear to be an
emerging instrument suitable for investigation in biological, medical
and environmental applications. The same enzyme  and the same
proposed device  are presently being planned also in many different
fields, such as in degradation of polyaromatic hydrocarbons, in textile
industry, in food industry, in waste detoxification and finally for
bioremediation and for biodegradation of industrial pullutants .
|It must be stressed that laccase-based sensors can be used not
only for medical purposes but also for environmental and biosafety
purposes, since they enable to detect contaminants, pollutants such as
phenols and pesticides [30,31].
|In summary, our resulting prototypes appear to yield satisfactory
proof of principles in the shown three specific applications to biosafety.
Indeed, in order to measure CO2, we have realized two new devices
based on the use of a sensor technology (such as the nanogravimetric
one) and an array of capture, highly specific for the gas of interest. CaO
may be considered a valid solution to solve the problem of the longterm
detection of carbon dioxide due to its ability to selectively absorb
this gas through a chemical reaction of carbonation. The reaction of
carbonation leads to a substantial variation of the molecular weight and
was therefore taken into account in the manufacture of nanogravimetric
detection devices. The characteristics of the reaction allowed the
construction of a dosimeter for the long-term analysis of the carbon
dioxide, competitive with respect to the devices already available on the
market based on infrared measurements (CO2 reduces the incidence of
infrared radiation on the sensor, then, depending on the concentration
of CO2) or on measurements of the variations of a voltage across a solid
electrolyte depending on the concentration of CO2.
|Moreover, the latest developments in the field of gas sensing devices
make clear that the next challenge is to monitor personal exposure to
environmental pollution , and nanotechnology can address this
issue , as well as biotechnology . This ambitious task can be
indeed accomplished by integrating into a coherent framework and
sensing platform all the type of sensors that have been described in
this paper. Exposure to pollutants in fact activates particular gene
sub-networks and pathways, and genomics-based biosensors may
provide an early detection of human exposure, as well as the individual
response to it. This could pave the way for a real advancement in
shifting the monitoring from a population and large mass-scale to an
|Gas sensing devices find interesting applications also in the
medical field, such as in anesthesiology and in respiratory medicine as
an “electronic nose” for investigating and characterizing the patient’s
exhaled breath [42,43], as well as in medical toxicology and emergency
|For what concerns the NAPPA QCM_D conductance device, the
results presented demonstrated a valid response for the protein-protein
interaction analysis, exploiting the great advantage of this technique
that allowed the real-time, label-free characterization of molecular
binding kinetics to an immobilized receptor . The most challenging
prospective of the innovative biosensors emerging from this technology
is the potential capability to develop a large number of sensors for
molecules of biological and medical interest, simply changing the
cDNA immobilized on the sensor, without changing the technology
of detection. Among avenues being presently explored NAPPA-based
vaccines identification appears to represent an additional promising
future perspective for biosafety in the frame of the new Omicsbased
Public Health. Vaccinology has indeed emerged as a complex
interdisciplinary science, especially because of the contributions of
the new-Omics disciplines. Worthy of notice for future applications
in human biosafety is finally the innovative kind of self-assembling
protein microarray, the NAPPA expressed with the SNAP tag in E. coli
coupled cell-free expression system, useful to develop a standardized
efficient procedure to analyze in large scale the protein occurred on
the array combining MS with bioinformatics . By the coupling
of our newly developed software SpADS  to K-Means Clustering
algorithm with good results both for known and unknown protein
identification, up to 67% correct score, quite better than earlier MS
without SNAP . The results obtained are encouraging even with
the quite low number of MS spectra so far acquired and without the
subtraction of ab initio known MS spectra of recombinant components
of the E. coli lysate (work in process).
|This project was supported by grants to FEN (Fondazione Elba Nicolini) and
to Professor Claudio Nicolini of the University of Genova by the FIRB Italnanonet
(RBPR05JH2P) from MIUR (Ministero dell’Istruzione, Università e Ricerca; Italian
Ministry for Research and University).
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