2012, 12, 17112-17127; doi: 10. 3390/s121217112 sensors

Yüklə 416.5 Kb.
Pdf просмотр
ölçüsü416.5 Kb.

Sensors 201212, 17112-17127; doi:10.3390/s121217112 



ISSN 1424-8220 



Prototypes of Newly Conceived Inorganic and Biological 

Sensors for Health and Environmental Applications 

Claudio Nicolini 


*, Manuela Adami 


, Marco Sartore 


, Nicola Luigi Bragazzi 



Valter Bavastrello 


, Rosanna Spera 


 and Eugenia Pechkova 


Laboratories of Biophysics and Nanobiotechnology, Department of Experimental Medicine (DIMES), 

University of Genoa, Genoa 16132, Italy; E-Mails: nicola@genethics.ethicsoft.it (N.L.B.); 

vbavastrello@gmail.com (V.B.); rspera@ibf.unige.it (R.S.); epechkova@ibf.unige.it (E.P.) 

Nanoworld Institute Fondazione EL.B.A. Nicolini, Bergamo 24100, Italy 

Elbatech Srl, Marciana, Marciana 57030, Italy; E-Mails: adami@elbatech.com (M.A.); 

sartore@elbatech.com (M.S.) 

*  Author to whom correspondence should be addressed; E-Mail: cnicolini@ibf.unige.it;  

Tel.: +39-010-353-38217; Fax: +39-010-353-38215. 

Received: 8 October 2012; in revised form: 7 December 2012 / Accepted: 11 December 2012 /  

Published: 12 December 2012 


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



Langmuir-Blodgett; laccase; amperometry; clomipramine 



Sensors 201212 17113 


1. Introduction 

This paper describes the utilization of a wide range of complex nanocomposites [1] for optimal 

implementation of three newly conceived sensors within the framework of FIRB Nanoitalnet. We used 

inorganic nanocomposites, such as matrices of calcium oxide [2], 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,  

and nanogravimetric).  

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


 [2] can be useful to reveal the increased emissions of gases from fossil 

fuel combustion, industrial processes, and agriculture associated to deforestation and habitat 

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 [2] considering that fossil fuel 



 emissions can remain in the atmosphere for periods of up to tens of thousands of years. The 

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



accumulation takes advantages of the properties of CaO to be carbonated by this gas as shown in 

reference [2], according to the following equation: 

CaO + CO






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 amount of composite in relation to the 

concentration of environmental CO


For what concerns the two health prototypes [3,5], one biosensor of new conception was realized by 

coupling the traditional quartz crystal microbalance with frequency monitoring (QCM_F) [7] 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 

of protein [3]. In the QCM_D sensor [12] each quartz contained 4x4 NAPPA spots of 300-micron 

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 

Sensors 201212 17114 


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, 

A is the gold area and ρ and l are the quartz density and 

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

 in vitro  

NAPPA- translated protein, Ramachandran

 et al. substituted the use of purified proteins with the use 

of cDNAs encoding the target proteins at each feature of the microarray [10]. 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 [15]. 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 [16]. 

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 [5] was laccase [6], 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. 

Sensors 201212 17115 


2. Materials and Methods 

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. 

2.1. Inorganic Sensor Based on Matrices of Calcium Oxide 

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 

preliminary tests to assess the best concentration of CaO in the composite, individuated in the 

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



a fixed amount of composite. 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 [2]. 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


 via a 

nanogravimetric method by performing the experiments inside an atmosphere-controlled chamber 

filled with CO


 [2]. Furthermore, the composite material showed a linear absorption of CO


 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 environmental CO


. In reference [2] are illustrated the experimental results 

obtained from these experiments. 

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 


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 (Figure 1). 

The nanogravimetric instrument used for relating CO


 concentrations with mass variations 

consisted of a base unit, interfaced to a PC via USB port and able to drive up to four oscillator  

units [2]. 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). 

Sensors 201212 17116 


Figure 1. Illustration of the plastic cell containing the quartz for environmental CO



long-term sampling, constructed in cooperation with Elbatech Srl, Marciana, Italy. 


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 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


. 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 


 versus time measurements can be performed following the data acquisition in real time on 

the computer screen, by means of strip chart plotting. 





Quartz for nanogravimetric acquisitions

Sensors 201212 17117 


2.2. Biological Sensor Based on NAPPA and QCM_D Technology 

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 [3]. 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 

acquiring conductance

 vs. frequency curve, which showed a typical Gaussian behavior [3]. 

Figure 3. QCM_D biosensor prototype scheme. The quartz was positioned in a flow 

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. 

D factor


Personal Computer 


Temperature and flux controllers



Temperature/flow chamber           

(with inside the quartz)


D factor


Personal Computer 


Temperature and flux controllers



Temperature/flow chamber           

(with inside the quartz)



The curve peak was at the actual resonance frequency while the shape of the curve indicated how 

damped was the oscillation,

 i.e., how the viscoelastic effects of the surrounding layers affected the 

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. 


Sensors 201212 17118 


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 [4], 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 [10]. 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 [17]. 

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]. 

2.3. Biological Sensor Based on Langmuir-Blodgett Multi-Layers of Laccase 

A LB thin film [20] of recombinant laccase from 

Rigidoporus lignosus (formerly known as 

Rigidoporus microporus), obtained as previously reported [6], was prepared using a highly 

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) [5]. 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 [5] 

Sensors 201212 17119 


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 

3.1. Inorganic Sensor Based on Matrices of Calcium Oxide 

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 CO


. We consequently carried out acquisitions of frequency

 vs. time 

in order to sample the indoor environmental CO


 in the room, illustrated in Figure 6. 

Basing on our previous experiments [2], we were able to calculate the variation of mass 

per week 

and consequently the average quantity of CO


 in the environment. Experimental data, summarized in 

Table 1 for a clear vision, highlighted the variation of frequency in relation to the quantity of CO



absorbed by the composite issued a linear absorption, coherently with the constant human activity of 

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  

and outdoors. 

Sensors 201212 17120 


Figure 6. Variation of frequency in relation to the quantity of CO


 absorbed by the 

composite. Experimental data showed a linear absorption, coherently with the human 

activity of the sampling period.  


















Number of acquisitions











11 Aug

16 Aug

21 Aug

27 Aug


Table 1. Variation of mass in relation to the quantity of CO


 present in the environment 

(indoor). The concentration of indoor CO


 was found 10 times less than the average 

concentration in the atmosphere, thus indicating good quality of the air. 

Sampling Period (days) 

Variation of Mass 

m (g)  Concentration of Environmental CO



0.57 ± 0.007 

48 ± 1 

0.72 ± 0.007 

55 ± 1 

0.75 ± 0.007 

55 ± 1 

3.2. Biological Sensor Based on NAPPA and QCM_D Technology 

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: 


At the beginning of the expression process,

 i.e., before reticulocyte lysate addition; 


120 min after reticulocyte lysate addition (

i.e., after 90 min at 30 °C for gene expression and 

protein synthesis plus 30 min at 15 °C for protein immobilization) and after washing process,  

at 22 °C; 


After the addition of a MDM2 50 μM solution. 

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). 

Sensors 201212 17121 


Figure 7. (Left) p53 quartz impedance curves recorded before the gene expression (solid 

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 

frequency and the half width at half height (Γ), along with the values of the variation in conductance 



 (mS). The ratio named normalized D factor, D


 = 2Γ/I, gives information on the shape of 

impedance curves and on sample viscosity, while the decrease in frequency 

f (Hz) is related to the 

amount of p53 molecules being immobilized. 

Table 2.


Peak frequency (

f), half-width at half-height (Γ), maximum conductance 

increment (



), and normalized 

factor for the impedance curves shown in Figure 7 Left. 

Impedance Curve 

f (Hz) 

Γ (Hz) 







Beginning 9475435 




After protein expression and washing 





After MDM2 addition 





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 [4]. 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 

Sensors 201212 17122 


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 

ad hoc chamber, hardware and software 

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 [23]. 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, [24]). 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 

antidepressants. 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 

Sensors 201212 17123 


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 

provide an emerging instrument suitable for investigation in biological fluids as well as for other 

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). 

Table 3. Peak potentials and sensitivity of Laccase-based biosensor measured at increasing 

concentrations in different human biological fluids. 

Fluid Peak 

(mV)  Sensitivity 

Blood +75; 


191.91 mA/M 

Urine +100; 


138.93 mA/M 

Liquor +100; 


31.70 mA/M 

Breast-milk +100; 


9.80 mA/M 

Saliva +150; 


11.14 mA/M 

Sperm +110; 


19.00 mA/M 

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 

Sensors 201212 17124 


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). 

4. Conclusions 

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 

device 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. 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, 

depending on the concentration of CO


) or on measurements of the variations of a voltage across a 

solid electrolyte depending on the concentration of CO



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. 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 [25]. In addition to 

what was anticipated some time ago [26], 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 [5], 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). 

Sensors 201212 17125 


Table 4. Genes which interact with immunological human synapses. 

Microorganism Antigen 

Pseudomonas aeruginosa 

PA0044, PA0807, PA0973 (OprL), PA1080, PA1148, PA1248, PA2300, 

PA3407, PA3724, PA3841 (ExoS), PA3931, PA4110, PA4922, PA5369, FlicA, 

OprI, OprH2, OprE, OprF, exotoxin A, flagellin 

Vibrio cholerae 

Protein 1 (VC1085), 2 (VC2283), 3 (VC1893), 4 (VC2261), 5 (VC0339, PSD), 

6 (VC1494), 7 (VC0556), 8 (VC0975) 

Yersinia pestis 

VA (V antigen), F1 antigen 

Francisella tularensis 


Bacillus anthracis 

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 

et al. [29,30] studied drug plasma levels 

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 [31]. 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).  




Nicolini, C.; Sivozhelezov, V.; Bavastrello, V.; Bezzerra, T.; Scudieri, D.; Spera, R.;  

Pechkova, E. Matrices for sensors from inorganic, organic, and biological nanocomposites. 

Materials 20114, 1483–1518. 

Sensors 201212 17126 




Bezerra, T.C.T.; Bavastrello, V.; Nicolini, C. Calcium oxide matrices and carbon dioxide sensors. 

Sensors 201212, 5896–5905. 



Nicolini, C.; Bragazzi, N.; Pechkova, E. Nanoproteomics enabling personalized nanomedicine. 

Adv. Drug Deliv. Rev. 201264, 1522–1531. 



Adami, M.; Sallam, S.; Eggenhoffner, R.; Sartore, M.; Hainsworth, E.; LaBaer, J.; Nicolini, C. 

Label-free NAPPA via Nanogravimetry, in Functional Proteomics and Nanotechnology-Based 

Microarrays. In 

Pan Stanford Series on Nanobiotechnology; Nicolini, C., LaBaer, J., Eds.; Pan 

Stanford Publishing Pte Ltd.: Singapore, 2010; Volume 2, Chapter 4, pp. 95–108. 



Bragazzi, N.L.; Pechkova, E.; Scudieri, D.; Bezerra, T.C.T.; Adami, M.; Nicolini, C. 

Recombinant laccase: II. Medical biosensor. 

Crit. Rev. Eukaryot. Gene. Expr201222, 197–203. 



Nicolini, C.; Bruzzese, D.; Cambria, M.T.; Bragazzi, N.L.; Pechkova, E. Recombinant laccase: I. 

Enzyme cloning and characterization. 

J. Cell Biochem. 2012, doi:10.1002/jcb.24397. 



Nicolini, C.; Adami, M.; Dubrovsky, T.; Erokhin, V.; Facci, P.; Paschkevitch, P.; Sartore, M. 

High sensitivity biosensor based on LB technology and on nanogravimetry. 

Sens. Actuators B 

Chem. 199524, 121–128. 



Pax, M.; Rieger, J.; Eibl, R.H.; Thielemann, C.; Johannsmann, D. Measurements of fast 

fluctuations of viscoelastic properties with the quartz crystal microbalance. 

Analyst  2005,  130




Poitras, C.; Tufenkji, N. A QCM_D-based biosensor for 

E. coli O157:H7 highlighting the 

relevance of the dissipation slope as a transduction signal. 

Biosens. Bioelectron.  2009,  24,  




Ramachandran, N.; Hainsworth, E.; Bhullar, B.; Eisenstein, S.; Rosen, B.; Lau, A.Y.;  

Walter, J.C.; LaBaer, J. Self-Assembling protein microarrays. 

Science 2004305, 86–90. 



Nicolini, C.; Pechkova, E. An overview of nanotechnology-based functional proteomics for 

cancer and cell cycle progression. 

Anticancer Res. 201030, 2073–2080. 



Nicolini, C.; Bezerra, T.; Pechkova, E. Protein nanotechnology for new design and development 

of biocrystals and biosensor

sNanomedicine (Lond.) 201278, 1117–1120. 



Efeyan, A.; Serrano, M. p53: Guardian of the Genome and Policeman of the Oncogenes.  

Cell Cycle 20076, 1006–1010. 



Levine, A.J. P53, the cellular gatekeeper for growth and division. 

Cell 199788, 323–331. 



Vousden, K.H.; Lu, X. Live or let die: The cell’s response to p53. 

Nat. Rev. Cancer  2002,  2,  




Shangary, S.; Wang, S. Targeting the MDM2-p53 Interaction for cancer therapy. 

Clin. Cancer Res. 

200814, 5318–5324. 



Spera, R.; LaBaer, J.; Nicolini, C. Mass spectrometry detection of nucleic acid programmable 

protein array

. J. Mass. Spectrom201146, 960–965. 



Shumantseva, V.; De Luca, G.; Bulko, T.; Carrara, S.; Nicolini, C.; Usanov, S.A.; Archakov, A. 

Cholesterol amperometric biosensor based on cytochrome P450scc. 

Biosens. Bioelectron. 2004

19, 971–976. 



Stura, E.; Bruzzese, D.; Valerio, F.; Grasso, V.; Perlo, P.; Nicolini, C. Anodic porous alumina as 

mechanical stability enhancer for LDL-cholesterol sensitive electrodes. 

Biosens. Bioelectron. 

200723, 655–660. 

Sensors 201212 17127 




Nicolini, C.; Pechkova, E. Nanostructured biofilms and biocrystals. 

J. Nanosci. Nanotechnol. 

20066, 2209–2236. 



Danish University Antidepressant Group (DUAG). Clomipramine dose-effect study in patients 

with depression: clinical end points and pharmacokinetics. 

Clin. Pharmacol. Ther.  1999,  66,  




Goodman-Gilman, A. 

The Pharmacological Basis of Therapeutics, 7th eds.; Macmillan:  

New York, NY, USA, 1985; Chapter 17, p. 431. 



Baumann, P.; Hiemke, C.; Ulrich, S.; Eckermann, G.; Gaertner, I.; Gerlach, M.; Kuss, H.;  

Laux, G.; Mueller Oerlinghausen, B.; Rao, M.L.; 

et al. The AGNP-TDM Expert Group 

Consensus Guidelines: therapeutic drug monitoring in psychiatry. 

Pharmacopsychiatry 200437




Stage, K.B.; Bech, P.; Kragh-Sørensen, P. Age-related adverse reactions to Clomipramine.  

Acta Psychiat. Scand2002105, 55–59. 



Nalin, D.R. Evidence based vaccinology. 

Vaccine 200220, 11–12. 



Nicolini, C.; Pechkova, E. Nanoproteomics for nanomedicine.

 Nanomedicine 20105, 677–682. 



Moreno, N.; Chevalier, M.; Ronzon, F.; Manin, C.; Dupuy, M.; Krell, T.; Rieu, J.P. Unbinding 

forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy 

correlate with their dissociation rates determined by surface plasmon resonance. 

J. Mol. Recognit

20116, 1105–1114. 



Mandenius, C.F.; Wang, R.; Aldén, A.; Bergström, G.; Thébault, S.; Lutsch, C.; Ohlson, S. 

Monitoring of influenza virus hemagglutinin in process samples using weak affinity ligands and 

surface plasmon resonance. 

Anal. Chim. Acta 2008623, 66–75. 



Schimmell, M.S.; Katz, E.Z.; Shaag, Y.; Pastuszak, A.; Koren, G. Toxic neonatal effects 

following maternal clomipramine therapy. 

J. Toxicol. Clin. Toxicol. 199129, 479–484. 



Kohen, D. Psychotropic medication and breast-feeding. 

Adv. Psychiatr. Treat. 2005 11, 371–379. 



Melanson, S.E.; Lewandrowski, E.L.; Griggs, D.A.; Flood, J.G. Interpreting tricyclic 

antidepressant measurements in urine in an emergency department setting: comparison of two 

qualitative point-of-care urine tricyclic antidepressant drug immunoassays with quantitative 

serum chromatographic analysis. 

J. Anal. Toxicol. 200731, 270–275. 



Altemus, M.; Swedo, S.E.; Leonard, H.L.; Richter, D.; Rubinow, D.R.; Potter, W.Z.;  

Rapoport, J.L. Changes in cerebrospinal fluid neurochemistry during treatment of 


obsessive-compulsive disorder with clomipramine. 

Arch. Gen. Psychiatry 199451, 794–803. 



Mårtensson, B.; Wägner, A.; Beck, O.; Brodin, K.; Montero, D.; Asberg, M. Effects of 

clomipramine treatment on cerebrospinal fluid monoamine metabolites and platelet 


3H-imipramine binding and serotonin uptake and concentration in major depressive disorder. 


Psychiatr. Scand. 199183, 125–133. 

© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article 

distributed under the terms and conditions of the Creative Commons Attribution license 


Поделитесь с Вашими друзьями:

Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©azkurs.org 2019
rəhbərliyinə müraciət

    Ana səhifə