Laboratories of Biophysics and Nanobiotechnology, Department of Experimental Medicine (DIMES),
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Nanoworld Institute Fondazione EL.B.A. Nicolini, Bergamo 24100, Italy
Elbatech Srl, Marciana, Marciana 57030, Italy; E-Mails: firstname.lastname@example.org (M.A.);
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Abstract: This paper describes the optimal implementation of three newly conceived
sensors for both health and environmental applications, utilizing a wide range of detection
methods and complex nanocomposites. The first one is inorganic and based on matrices of
calcium oxide, the second is based on protein arrays and a third one is based on
Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances
significant to the environment (such as carbon dioxide) and medicine (drug administration,
cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance
with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring
(QCM_D) technologies. The resulting three implemented nanosensors are described here
along with proofs of principle and their corresponding applications.
Keywords: QCM_D; protein-protein interaction; calcium oxide matrices; CO
This paper describes the utilization of a wide range of complex nanocomposites  for optimal
implementation of three newly conceived sensors within the framework of FIRB Nanoitalnet. We used
inorganic nanocomposites, such as matrices of calcium oxide , and biological ones such as Nucleic
Acid Programmable Protein Arrays (NAPPA) [3,4] along with Langmuir-Blodgett (LB) multi-layers
of proteins of primary interest such as laccase [5,6]. Special attention was paid to the detection of
substances significant for both the environment (such as carbon dioxide) and medicine (drugs
and cancer) by means of a wide variety of detection methods (amperometric, conductometric,
The resulting three organic and biological constructed nanosensors, here presented in their final
versions, were optimized on the outcome of their proof of principles studies and applied for three pilot
cases, two for health use and one for the environment. However, all three nanosensors have prominent
medical implications, since it is well known that there is a strong link between environment and health
and that, according to the WHO (World Health Organization), at least 25% of diseases are due to
environmental risk factors.
Sensors for detection of CO
 can be useful to reveal the increased emissions of gases from fossil
destruction. This can result in consequent changes in the chemical composition of the atmosphere with
direct biological effects and negative influence on the Earth’s climate  considering that fossil fuel
instruments generally employed in these determinations basically consist of infrared (IR) detectors
performing a continuous plotting in the site with a good degree of accuracy, but 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. Long-term sampling devices such as diffusive sampling techniques are
the cheapest and easiest way.
An alternative long-term sampling method for the determination of environmental CO
reference , according to the following equation:
order to assess the quantity of gas absorbed by a fixed amount of composite in relation to the
concentration of environmental CO
coupling the traditional quartz crystal microbalance with frequency monitoring (QCM_F)  with the
quartz crystal microbalance with dissipation monitoring (QCM_D) [8,9], and with an innovative
protein cell-free expression system named Nucleic Acid Programmable Protein Arrays
(NAPPA) [10,11], that allowed us to immobilize on the quartz surface, as sensing molecule, any kind
square and functional proteins were synthesized in situ directly from printed cDNAs (complementary
DNAs) just before the assay [4,12]. 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 the Sauerbrey equation:
is the fundamental frequency,
thickness, respectively [7–9]. Quartz resonators used in fluids are more than mere mass or thickness
sensors; sensor response depends also on the viscoelastic properties of the adhered biomaterial, surface
charges of adsorbed molecules and surface roughness. The modern QCM_D technology utilizing
impedance measurement (as in the sensor introduced in references [3,12]) offers access to the
resonance bandwidth in addition to resonance frequency. Bandwidth value is strictly connected with
the viscoelastic properties of the sample [8,9]. Building upon the successful use of
NAPPA- translated protein, Ramachandran
of cDNAs encoding the target proteins at each feature of the microarray . The proteins were
translated using a T7-coupled rabbit reticulocyte lysate
in vitro transcription-translation (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 on the array through an antibody to GST printed simultaneously with the
expression plasmid [10,11]. In the present research, we coupled QCM_D and QCM_F with NAPPA
technology [3,12], optimizing the monitoring in real time of the kinetics of the reaction to obtain
information not only concerning the mass, but also on the viscosity of the sample and sensing the
interaction among a query protein and the expressed protein.
The proteins monitored by the NAPPA-based nanogravimetric biosensor, namely p53 and MDM2,
are of fundamental importance in the molecular mechanisms leading to malignant cell transformation
and cancer; p53 is in fact a 53-kiloDalton phosphoprotein oncosuppressor, encoded by a 20-kilobases
gene situated on the short arm of human chromosome 17 and termed as the “guardian of the genome”
and the “policeman of oncogenes” [13,14]. Mutated, it is involved in up to 70% of human tumors,
being responsible of cell growth arrest, senescence, apoptosis in response to an array of stimuli such as
DNA damages (DSB, or double-strand-breaks), hypoxia, telomeres shortening, cell adhesion,
oncogene activation and other molecular and cellular stresses . MDM2, a p53-specific E3 ubiquitin
ligase, inhibits p53 functions by binding to its
N-terminal transcription-promoting domain, thus having
an oncogenic activity. The MDM2-p53 plays a major role in cancer, being also a molecular therapeutic
target and its monitoring is of crucial importance in cancer diagnosis and treatment .
In this report, our goal was finally to build the prototype of the enzyme-based biosensor for medical
purposes, in which the immobilization procedure was carried out via Langmuir-Blodgett films. The
enzyme implemented in our device  was laccase , which is a blue oxidase capable of oxidizing
phenols and aromatic amines by reducing molecular oxygen to water by means of a full complement of
copper atoms. Laccase belong to a large group of multicopper enzymes, which includes among others
ascorbic acid oxidase and ceruloplasmine. They catalyze the oxidation of diverse compounds such as
o-, p-diphenols, aminophenols, polyphenols, polyamines, lignin, some inorganic ions, aryldiamines,
benzenthiols, and phenothiazines.
Taking into account all the considerations so far discussed and the three sensors recently introduced
in the literature [2,3,5], in this section we summarize the techniques and procedures utilized in the
construction of the three distinct prototypes.
The alternative long-term sampling method for the determination of environmental CO
accumulation takes advantages of the properties of CaO to be carbonated by this gas. We carried out
CaO/PEG weight ratio of 1/4, by studying the variation of mass connected to the carbonation process,
in order to assess the quantity of gas absorbed in relation to the concentration of environmental CO
nanogravimetric method by using a home-made glass chamber of 340 mL in volume . The
home-made chamber 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., ICM, Oklahoma City, OK, USA). The
preliminary experimental data highlighted that the composite was able to selectively detect CO
filled with CO
. Furthermore, the composite material showed a linear absorption of CO
the possible use of these matrices for applications in the field of sensor devices for long-term
evaluation of accumulated environmental CO
. In reference  are illustrated the experimental results
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
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 (Figure 1).
The nanogravimetric instrument used for relating CO
concentrations with mass variations
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 s. 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 (Figure 2).
The choice to have the oscillators outside the base unit allows the maximum flexibility when
design, which was based on a precision internal reference crystal, used as a timebase 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 (see Figure 2 below).
Figure 2. Nanogravimetric base-unit, able to drive up to four oscillating units (Upper).
Oscillators unit connected via Ethernet cable to the base unit (Lower).
The system was driven by an extremely user-friendly software running under MS-Windows
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
versus time measurements can be performed following the data acquisition in real time on
the computer screen, by means of strip chart plotting.
Our system, shown in Figure 3, detects the normalized dissipation factor of the quartz crystal by
means of the “half-width half-height” 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.) around its resonance frequency, thus
chamber that also guaranteed the temperature control. Temperature and flux rate were
settable trough the controllers and D factor and frequency values are visible on two
displays. On connecting the instrument to a PC it is possible to record the impedance
curves, the frequency and D factor shifts in real time.
Temperature and flux controllers
(with inside the quartz)
The curve peak was at the actual resonance frequency while the shape of the curve indicated how
oscillation. In order to have a stable control of the temperature, the experiments were conduced in a
temperature chamber. The experimental set up, illustrated by the scheme in Figure 3, consisted of a
temperature chamber were the quartz was positioned and monitored at the same time for frequency and
dissipation factor variation. We designed a miniature flow-cell (Figure 4) to employ in protein-protein
interaction analysis. 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.
Figure 4. Plexiglas chamber utilized during the flow analysis of protein-protein
interaction: the upper part, with the flow inlet and outlet, allowed the easy insertion of the
biological fluid to be analysed and the lower allowed the proper housing of the quartz.
The transducer consisted of 9.5 MHz AT-cut quartz crystal of 14 mm blank diameter and 7.5 mm
electrode diameter, produced by ICM. The electrode materials were 100 Å Cr and 1,000 Å Au.
Microarrays were produced on the quartzes already described as highly sensitive transducers. Each
quartz was printed with 4 × 4 NAPPA spots of 300 microns diameter, spaced 350 microns
center-to-center , using the given genes and corresponding expressed proteins. The blank quartz was
sent to the Harvard Institute of Proteomics (HIP) for the NAPPA spots printing as described in [3,4];
in order to express proteins we adopted the protocol described in . Gene expression and protein
synthesis took place at 30 °C for about 1.5 h; to prepare rabbit reticulocyte lysate mix (Expression Kit,
Promega, Madison, WI, USA) we mixed 16 μL of TNT buffer, 8 μL of T7 polymerase, 4 μL of Cys,
8 μL of RNaseOUT (Invitrogen, Carlsbad, CA, USA), 160 μL of DEPC water (Ambion, Foster City,
CA, USA), and 200 μL of reticulocyte lysate (TNT® T7 Coupled Reticulocyte Lysate System,
Promega). Sixty μL of IVT lysate mix was added per quartz. The quartz, connected to the
nanogravimeter inside the incubator, was incubated for 1.5 h at 30 °C for proteins synthesis and then,
the temperature was decreased to 15 °C for a period of 0.5 hour to facilitate the proteins binding on the
spot surface. The quartz was subsequently removed from the instrument and washed three times at
room temperature (22 °C) in Milli-Q water. The quartz was then placed in the flux chamber for
protein-protein interaction analysis. The protocol described above was followed identically for both
control quartz (blank quartz) and working quartz. The proofs of principle to verify sensor response to
NAPPA protein expression and immobilization were carried out immobilizing the following genes and
corresponding expressed proteins: CDK2_Human gene, jun_Human gene and p53_Human gene .
As reference, a blank quartz was employed. For protein-protein interaction proof of principle, we
tested the interaction between p53 proteins immobilized on the NAPPA surface (after its expression)
with a MDM2 solution. In parallel experiments (submitted elsewhere as a result of a cooperation with
the Biodesign Institute at the Arizona Institute of Proteomics) several other genes of significant clinical
and biological implication and others configurations (10 × 10 spots) were tested. In particular
CYP11A1_Human genes were immobilized and cholesterol was chosen for interaction analysis and
their comparison with similar cholesterol sensors based on traditional technologies [18,19].
A LB thin film  of recombinant laccase from
concentrated sample of laccase. 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
(>17 MΩ) and the monolayer was compressed with movable barriers at a rate of 70 mm/minute. The
deposition was of Y-type with a dipping rate of 25 mm/min. The drainage rate (in order to remove the
film) was about 3.5 mm/min. The transfer pressure to obtain the LB film (Figure 5) was about
20 mN/m, at 22 °C.
The surface morphology and topology of the LB thin film of laccase was investigated via Atomic Force
Microscopy (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
(Figure 5, Left). The enzyme was deposited onto the electrode via Langmuir-Schaefer (LS) technique 
and a protocol of immobilization overnight was followed; after depositing the film, the electrodes (Figure
5, Right) 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 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 [5–21], with an EG & G
PARC model 263A potentiometer, equipped with dedicated software.
Figure 5. π-A isotherm of the laccase thin film at the air-water interface (Left). A couple
of screen-printed electrodes used as a transducer based on graphite/ruthenium ink and the
counter on silver one (Right).
3. Results and Discussion
We performed nanogravimetric acquisitions of frequency
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 CO
. We consequently carried out acquisitions of frequency
in order to sample the indoor environmental CO
in the room, illustrated in Figure 6.
and consequently the average quantity of CO
in the environment. Experimental data, summarized in
the sampling period. Specifically, we found the variation of mass in relation to the quantity of CO
present in the environment (indoors), indicated the concentration of indoor CO
during the sampling
period was 10 times less than the average concentration in the atmosphere, thus indicating the good
quality of the air in our laboratory.
Since the very good results were 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
absorbed by the
activity of the sampling period.
present in the environment
was found 10 times less than the average
m (g) Concentration of Environmental CO
0.57 ± 0.007
0.72 ± 0.007
0.75 ± 0.007
The QCM_D results were calibrated both for frequency and D factor shifts using fructose [3,4]. We
monitored by QCM-D the viscoelastic behaviour of the quartzes during the NAPPA expression
process, recording the impedance curves in correspondence with the main steps of the expression
process. Moreover we monitored by QCM-F the variation in the mass adsorbed on the surface of the
sensors (corresponding to a decrease of the resonance frequency). Figure 7 Left shows the impedance
curves of p53 quartz at different steps of the expression protocol, as following:
protein synthesis plus 30 min at 15 °C for protein immobilization) and after washing process,
at 22 °C;
In order to investigate the biosensor response to protein-protein interaction the p53 quartz, once
expressed the proteins, was positioned in the flux chamber, connected with the pump, and a MDM2
solution was flowed on the quartz surface: 2 mL of 50 μM MDM2 solution in PBS was injected in the
pump. The corresponding frequency decrease was recorded (Figure 7, Right).
curve), after the protein expression and the washing process (dashed curve) and after
MDM2 addition (dotted curve); (Right) p53 quartz frequency variation in real time; at time
t = 600 s a MDM2 solution (50 μM in PBS) was injected in the flow chamber.
In Table 2 are reported for p53 quartz impedance curves of Figure 7, Left, the values of the
= 2Γ/I, gives information on the shape of
amount of p53 molecules being immobilized.
Peak frequency (
), and normalized
After protein expression and washing
The resulting Michaelis-Menten constants of the p53-MDM2 (calculated from the curve in
Figure 7, Right) interaction appeared quite compatible with the literature.
The results mentioned before obtained using a human lysate and 10 × 10 spots per quartz reported
in a separate communication (pending submission to a different journal) suggest that the NAPPA
based biosensor functioned to monitor with high selectivity the single protein being expressed even in
a mixture of different genes (as shown here in Figure 7, Right) and, from the analysis of D factor,
allowed to acquire in real time information on the characteristics both of single protein being
expressed with unique signature and on the kinetic constant of the reaction.
In order to properly set-up the QCM_D for routine measurements, we are going to immobilize the
NAPPA on the given reference quartzes provided by the manufacturer . Then, in order to eliminate
the background signal, we are going to 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 whole impedance plots
at the necessary resolution and with a considerable number of data points. For this reason, we have
used a dsPIC (Microchip Technology, Inc., Chandler, AZ, USA) featuring at the same time good
computational power and sufficient memory space. This represents a PC-driven prototype to establish
the proof of principle. The industrial prototype is being designed and realized, under a different
contract with significant larger amount of money than here provided by the MIUR under the indicated
FIRB contract, in order to have a temperature-controlled
optimized to increase speed, computational power and compactness to incorporate hardware, in order
to produce a finalized friendly stand-alone device.
3.3. Biological Sensor Based on Langmuir-Blodgett Layers of Laccase
Clomipramine [21,22], a drug belonging to the tricylic tertiary amine antidepressants class, is
widely used for the therapy of depressive and obsessive disorders. Because of its clinical importance
many analytical methods have been developed to monitor its levels, above all chromatographic
techniques (gas chromatography, high performance liquid chromatography), eventually coupled with
tandem mass spectrometry, but all these techniques are 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 micromolar range. The
therapeutic dose is from 75 mg/day to 200 mg/day; the pharmacokinetics among patients is extremely
variable. Generally, the therapeutic concentration in the human blood of psychiatric patients is usually
in the low micromolar range. The side-effects of the drugs, especially in case of overdose, are seizures,
hematological, cardiological and neurological adverse effects up to the coma (tricyclic antidepressant
syndrome, ). Experiments carried out with cyclic voltammetry (see Figure 8) highlighted excellent
reproducibility and linearity of the peaks of oxidation and reduction, related to the presence of the drug
in several biological fluids (in particular in whole blood).
Figure 8. Cyclic voltammetry of laccase-based biosensor for detection of clomipramine at
increasing concentrations in human blood.
These results allowed the design and the creation of a prototype of a biological sensor for
the enzyme was deposited and to detect the current generated as a result of the interaction of the
enzyme with the analyte containing the drug of interest. By a multiple selector, the user can choose the
fluid 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 mains voltage (see Figure 9).
Figure 9. Prototype of laccase sensor jointly constructed with Elbatech Srl, Marciana (LI),
Italy, compact and portable for all fluids indicated in Table 3.
Use of commercial laccase, screen-printed electrodes technique and a portable device appear to
medical applications. The same enzyme and the same proposed device could be used also in many
different fields, such as in degradation of polyaromatic hydrocarbons, in textile industry, in food
industry and in waste detoxification. Laccase-based sensors for detection of clomipramine in breast
milk, saliva and semen were less sensitive than the others, while sensors for the monitoring of the drug
level in urine and blood had better pronounced and separated peaks (as shown in Table 3).
concentrations in different human biological fluids.
Moreover, if we compare these results with our previous findings we can study how changes in
thickness and in number of layers can increase the functionality of a biosensor: the sensitivity of the
biosensor in blood with LB 3-layers was more than the double of the value found here with only one
layer. It is known in fact that highly ordered structured biofilm can increase the sensitivity and the
electron kinetics transfer (difference in the width between the oxidation and reduction peaks).
In summary, our resulting prototypes appear to yield satisfactory proof of principles in the shown
specific health and environmental applications. Indeed, to measure CO
, we have realized a new
capture, highly specific for the gas of interest. Calcium oxide may be considered a valid solution to
solve the problem of the detection of carbon dioxide due to its ability to selectively absorb this gas
through a chemical carbonation reaction. The carbonation reaction leads to a substantial variation of
the molecular weight and was therefore taken into account in the manufacture of gravimetric 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 (CO
reduces the incidence of infrared radiation on the sensor, then,
) or on measurements of the variations of a voltage across 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. A proof of principle was realized immobilizing p53 plasmid, resulting in a
biosensor for MDM2. 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, by simply changing the cDNA immobilized on the sensor, without
changing the detection technology. Among the avenues being presently explored NAPPA-based
vaccines identification appears to represent an additional promising future perspective in the frame of
the new OMICS-based Public Health. Vaccinology has emerged as a complex interdisciplinary
science, especially because of the contributions of the new OMICS disciplines . In addition to
what was anticipated some time ago , only recently were protein arrays used to discover new
antigenic determinants for vaccine development [27,28]. 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 new efforts to
evaluate it via Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR). In the
right column of Table 4 are shown the genes that interact with immunological human synapse
(CD4 + TCR + MHC).
Finally, with the designed and realized amperometric sensor , in order to offer a suitable
instrument for routine medical application, we used only one layer of commercial laccase to fully
characterize clomipramine pharmacokinetics in different biological fluids of relevant medical interest,
namely human blood, saliva, urine, breast-milk, semen and cerebro-spinal fluid or liquor (CSF).
PA0044, PA0807, PA0973 (OprL), PA1080, PA1148, PA1248, PA2300,
PA3407, PA3724, PA3841 (ExoS), PA3931, PA4110, PA4922, PA5369, FlicA,
OprI, OprH2, OprE, OprF, exotoxin A, flagellin
Protein 1 (VC1085), 2 (VC2283), 3 (VC1893), 4 (VC2261), 5 (VC0339, PSD),
6 (VC1494), 7 (VC0556), 8 (VC0975)
VA (V antigen), F1 antigen
PA (protective antigen)
As far as we know, this is the first comprehensive characterization of clomipramine concentration
in different biological fluids of medical interest, since drug monitoring in different biological samples
is very important. Biological fluids were taken from healthy donor volunteers, who gave their
informed consent, and analyzed immediately after being collected. The motivation of studying
electrochemical behavior of clomipramine in different biological fluid of clinical interest was to
provide a comprehensive pharmacokinetic profile. Schimmell
in a breast-fed infant whose mother had taken clomipramine during pregnancy and continued after
giving birth. They found that levels were high following delivery but decreased gradually and were at
the lowest detectable concentration at 35 days, even though breast-feeding continued. Clomipramine
has been used by breast-feeding mothers without adverse effects on the newborn, even if drug
monitoring in human breast milk is of crucial importance. Urine is a biological flood that can be easily
obtained with great acceptability from the patient and can help in rapid assessment and screening in
emergency situations . CSF fluid can be exploited for monitoring the neurochemical changes
during therapy or disease [32,33]. As far as semen is concerned, there were some communications of a
relationship between clomipramine level in sperm and male infertility: clomipramine seems to modify
sperm motility in a significant way, but these findings are controversial and need to be confirmed by
further research. Moreover, clomipramine concentrations in blood, urine and liquor correlate with
patient response state, which is the most important clinical parameter for the assessment of drug
functionality and working.
This work was supported by two “Fondo per gli Investimenti della Ricerca di Base” (FIRB–MIUR)
grants to Claudio Nicolini at University of Genova for Nanosensors (RBPR05JH2P) and by a
“Ministero dell’Istruzione dell’Università e della Ricerca” (MIUR) grant to the Fondazione El.B.A.
Nicolini for “Funzionamento” (DM48527).
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