Study of a Low-cost Sensitive Point-of-Care Testing System using

Screen Printed Biosensors for Early Biomarkers Detection Related to

Alzheimer Disease

Sarah Tonello

, Mauro Serpelloni

, Nicola Francesco Lopomo

, Giulia Abate

Daniela Letizia Uberti

and Emilio Sardini

Department of Information Engineering, University of Brescia, Via Branze, 38 - 25123 Brescia, Italy

Department of Molecular and Translational Medicine, University of Brescia, Via Branze, 38 - 25123 Brescia, Italy

Keywords: Screen Printing, Electrochemical Biosensors, Biomarkers, Alzheimer Disease, Point of Care Testing.

Abstract: Among neurodegenerative diseases, Alzheimer Disease (AD) represents one of the most serious pathology,

for which an early diagnosis is still missing. A peculiar expression of an altered conformational isoform of

p53 protein was reported to be a biomarker able to distinguish AD subjects from healthy population,

quantifiable using a blood-based enzyme-linked immunosorbent assay (ELISA). In order to overcome ELISA

limitations related to reliability and to improve sensitivity, this study aimed to realize a low cost highly

sensitive portable point-of-care (PoC) testing system based on screen printed electrochemical sensors (SPES).

The development of the platform specifically included both the design of the sensing probe and of the

electronic circuit devoted to the conditioning and acquisition of the transduced electric signal. In particular,

silver, carbon and silver-silver chloride were selected respectively to realize conductive tracks, working and

counter electrodes, reference electrode in a three-electrodes configuration focusing on Anodic Stripping

Voltammetry (ASV). The conditioning circuit was designed following the scheme for a common potentiostat,

and produced as a Printed Circuit Board (PCB). Initial testing of the circuit were performed recording changes

in the conductivity of NaCl solution and quantifying electrodes coating with antibodies using Electrochemical

Impedance Spectroscopy (EIS) principle. Preliminary results obtained with saline solution, showed the ability

of the circuit to give the best response corresponding to low changes in NaCl concentration (sensitivity 13

mA/(mg/ml)), suggesting a good sensitivity of the platform. Results from EIS showed the ability of the circuit

to discriminate between different concentrations of antibodies coatings (sensitivity 70 mA/µg). The study is

on-going and after a proper calibration, the circuit is intended to be optimized to quantify unknown

concentration of unfolded p53 in samples of real patients, compared results with the one from ELISA analysis,

aiming to realize a low cost, easy usable and highly precise platform.

1 RESEARCH PROBLEM

The number of people over 65 years is rapidly

increasing; nowadays, in Europe, it represents the

16% of the whole population and this percentage will

reach the 25% within the end of 2030. Ageing usually

conveys several issues, which may include

degenerative or chronic disorders. The impact of

these diseases on the single person and on the whole

society could become huge and difficult to support in

the early future. In this perspective, the actual

researches and innovations are pushing to improve

the understanding of the causes and mechanisms

underlying ageing and the associated diseases, thus to

ensure elderlies with a healthy and active condition.

Among neurodegenerative diseases, Alzheimer

Disease (AD) represents indeed one of the most

investigated and serious pathologies, for which an

early reliable diagnosis is still missing. Therapies

currently available help in fact to alleviate the

symptoms of this disease, but they are not able to

specifically slow down the neurodegenerative

process. AD presents a long pre-symptomatic period,

that could last for 20 years, and which is characterized

by biochemical and molecular events that are able to

foresee the beginning of the disease. The ability to

identify early reliable biomarkers (e.g. proteins) to

effectively diagnose the pathology at an early stage is

one of the actual priority of biomedical research in

term of neurology and geriatrics.

Tonello, S., Serpelloni, M., Lopomo, N., Abate, G., Uberti, D. and Sardini, E.

Study of a Low-cost Sensitive Point-of-Care Testing System using Screen Printed Biosensors for Early Biomarkers Detection Related to Alzheimer Disease.

In Doctoral Consortium (DCBIOSTEC 2016), pages 15-23

Recently, among several studies addressing this

issue, different approaches to identify the specific AD

biomarkers have been established and novel one

discussed. To date, the most advanced and accepted

methods to diagnose AD with high specificity and

sensitivity are represented by enzyme-linked

immunosorbent assay (ELISA) measurements in

cerebrospinal fluid (CSF) and imaging biomarkers,

like volumetric magnetic resonance imaging and

positron emission tomography, evaluating glucose

utilization or ligands binding to amyloid plaque. In

addition to these methods, a great challenge is

represented by the search for novel biomarkers in

CSF and in blood by using modern potent methods,

such as microarrays, mass spectrometry,

bioinformatics, since only a combined analysis of

several biomarkers seems the promising path to help

in defining a patient-specific diagnose in the future

(Thal et al., 2006; Humpel, 2011)

In this perspective, scientific literature recently

reported how a peculiar expression of an altered

conformational isoform of p53 protein in AD patients

was able to distinguish them from healthy subjects

with a sensitivity of 90% and a specificity of 77%

(Buizza et al., 2012; Lanni et al., 2008; Uberti et al.,

2006). On this basis, the levels of p53 unfolded could

be an interesting starting point to a reliable blood-

based ELISA performed with a specific

conformational anti-p53 antibody. Even if ELISA

assay actually represents the gold standard technique

used for the detection and quantification of this p53

biomarker, some limitations are related to its

diffusion. These issues specifically include high costs

of the assay implementation, high operator

dependence, lack of standardization and impossibility

to lower the limit of detection, thus to detect the

biomarker in the early stage of the disease when it

would be more useful for the clinicians.

Biosensors represent the emerging technology

that promises to address this challenge, bringing

promising solutions in term of cost and sample use

reduction, ease of use, high portability and sensitivity

(Yager et al., 2008; Polese et al., 2014; Feng et al.,

2015).

In light of this, the development of a portable

point-of-care (PoC) testing system based on a screen

printed electrochemical sensor (SPES), could

represent an innovative and low cost solution to solve

these problems, improving and bringing to a higher

level of sensitivity the specific biomarkers detection

and quantification, with a strong impact on the

possibility to early diagnose AD (Dhawan et al.,

2015).

2 OUTLINE OF OBJECTIVES

The main objective of this project addresses the

design and development of a low-cost portable point-

of-care testing platform for the detection and sensitive

quantification of the unfolder p53, as biomarker for

early detection of AD.

The development of the platform specifically

includes both the design of the sensing probe, with

particular attention to the choice of the materials and

geometry, and of the electronic circuit devoted to the

conditioning and acquisition of the electric signal.

The electrodes were specifically designed to be easily

implemented by means of screen printing

methodology. In details, the outline of the project

could be organized in the following phases:

1. preliminary characterization of the electrodes, and

evaluation of compatibility of screen printing

materials and substrate with wet lab practices;

2. design and production of the screen printed

electrochemical sensor;

3. design and development of the conditioning

electronical circuit to perform the electronical

measurements, which are directly related to the

concentration of the specific protein on the

biological sample;

4. calibration of the sensors and evaluation of the

circuit;

5. functionalization of the sensors and protein

quantification using Electrochemical Impedance

Spectroscopy (EIS) and Anodic Stripping

Voltammetry (ASV).

6. optimization of the acquisition workflow.

7. validation of the sensor by using primary

antibodies specific for p53 unfolded proteins and

real samples from AD patients, compared to the

ELISA assay.

Each reported phase addresses specific objectives.

3 STATE OF THE ART

One of the most pervasive challenges of the research

in medical diagnosis is related to the ability to detect

a specific pathology in its earliest development

(Jacobs et al., 2014). To achieve this goal, in the last

decades, new rapid, low cost and easily accessible

methodologies have been increasingly investigated,

supported by the interest toward customized medicine

and toward rapid and home accessible diagnostic

results (Chan et al., 2013).

DCBIOSTEC 2016 - Doctoral Consortium on Biomedical Engineering Systems and Technologies

For these reasons, the idea to identify and quantify

specific biomarkers in the early stages of a disease

appears to be really promising (Jr et al., 2010;

Svobodova et al., 2012). Biomarkers represent an

indicator of the biological status, which can give

useful information concerning biological processes

happening in physiological or pathological conditions

and during different medical treatments.

As previously stated, in the specific case of AD,

recent studies related to its diagnosis reported how the

presence of a particular conformation of a specific

protein (p53) could be able to discriminate patients

affected by AD from healthy patients (Uberti et al.,

2006; Lanni et al., 2008; Buizza et al., 2012).

In this perspective, clinicians require technologies

able to identify quickly and with a high sensitivity

specific biological biomarkers related to the disease.

Biosensors integrated in lab on a chip (LoC)

devices could represent promising methods to reduce

time, cost and sample needed to perform the analysis.

(Song et al., 2014).

Thanks to their ability to be functionalized and

customized for the detection of different analytes (e.g.

DNA, proteins), electrochemical biosensors represent

the ideal starting point to realize complete platforms,

by integrating the sensor with the conditioning circuit

needed for the electronical measurements, realizing

portable and self-standing devices, useful for PoC

applications (Yager et al., 2008).

Printed electronics represents a successful tool to

realize low cost and sensitive biosensors for these

specific applications. In particular, screen printing

(also defined thick film printing) is the most used

method to realize this kind of electrochemical

sensors, as it arises in several works in the literature

for the sensitive and specific quantification of

different proteins (Silva et al., 2014; Elshafey et al.,

2013; Yun et al., 2011). Protein detection and

quantification is usually obtained by performing the

same functionalization as used in the multiwells

ELISA. More in details, the working electrode of the

sensor is functionalized with the primary antibodies

specific for the proteins that need to be quantified,

thus allowing the formation of an immunocomplex.

After that, the electrode is washed with the sample

containing the proteins and with a solution containing

a specific secondary antibody. After the

immunocomplex is realized, different methods can be

used to quantify the specific proteins.

A label free method is represented from EIS,

which measures the changes of impedance deriving

from a different electrons exchange between the

functionalized surface of the electrode and a

conductive solution (usually potassium ferricyanide),

depending on the concentration of the recognized

proteins (Silva et al., 2014; Elshafey et al., 2013). For

this specific technique, 2–electrode conformation -

including a working (WE) and a reference electrode

(RE) - or 3- electrode conformation – including WE,

RE and a counter electrode (CE) as reference for the

current – have been used to ensure the correct

acquisition of the proteins concentration, depending

on the level of precision and sensitivity required.

A different technique often used is represented by

voltammetry. In this case, the 3-electrodes

conformation is usually adopted. The potential

between RE and WE is varied arbitrarily - either step

by step or continuously - and the actual current value

between WE and CE is measured as the dependent

variable. As reported in different works, this method

can be performed both with the sensor completely

immersed in a buffer solution (Elshafey et al. 2013)

or by placing a drop of solution which cover the three

electrodes (Escamilla-Gómez et al. 2009). Anodic

stripping voltammetry (ASV) represents a very

sensitive kind of voltammetry technique. Several

groups optimized this measurement technique to

detect both proteins (Escamilla-Gómez et al., 2009)

or DNA sequences (Martínez-Paredes et al., 2010),

with limit of detection in the order on ng/ml.

Using the catalyzing action of Alkaline

Phosphatase (AP), ionic silver is selectively reduced

in its metallic form only where the proteins are

recognized, thus allowing the quantification of

proteins converting the amount of deposited silver

into a proportional peak of current during a stripping

step, varying the potential between WE and RE.

To further improve the specificity and the

sensitivity of this technology, limit of detection lower

than 100 pg/ml have been achieved thanks to the

integration of the SPES with nanostructured

materials. Gold nanoparticles for example or carbon

nanotubes or a combination of the two has been used

to modify the surface of the working electrode

allowed to better recognize antigens and DNA

sequences (Escamilla-Gómez et al., 2009; Kara et al.,

2010; Martínez-Paredes et al., 2010; Jeong et al.,

2013).

4 METHODOLOGY

The experimental activities of the project are divided

into 3 main parts:

- a first part dedicated to the evaluation of the

compatibility of the screen printing materials and the

circuit components with the wet lab practices.

Study of a Low-cost Sensitive Point-of-Care Testing System using Screen Printed Biosensors for Early Biomarkers Detection Related to

Alzheimer Disease

- a second part dedicated to the design and

realization of the sensor and the conditioning circuit.

- a final part dedicated to the calibration of the

platform (sensor and circuit) and to p53 protein

quantification.

4.1 Initial Sensors Characterization

Preliminary characterizations are required to assess

the compatibility of the sensor with wet lab practices

and to evaluate how antibodies can be coated on to

electrode surface in order to select the best materials

for the sensor production.

Alumina is identified as the optimal material used

as substrate for the printing of the working electrodes.

The adhesion of different concentration of antibodies

solutions has to be evaluated, in order to optimize the

best concentration for an efficient functionalization of

the sensor. Two different techniques have been

specifically chosen for the analysis: an electronic

method, EIS, and an optical one, evaluating the light

intensity thanks to fluorescent labels on the secondary

antibody.

EIS measurements are performed in presence of

Potassium Ferricyanide (K

[Fe(CN)

]) in a solution

of KCl. This technique is based on the measurement

of the impedance on a wide range of frequencies (200

Hz – 200 KHz), between a carbon WE and a silver-

silver chloride RE, both immersed in an electrolytic

solution of 5 mM K

[Fe(CN)

] in 1 M KCl.

Different designs (single electrodes and multiwell

electrodes) and materials (alumina, glass and

polystyrene) are evaluated in this first optimization

phase in order to better understand the technology to

be implemented in the final layout of the sensor. For

each of this different design, different protocols are

adapted to obtain reliable and repeatable results.

Impedance measurements are specifically

performed using an impedance analyzer (HP4194A),

compared with the fluorescence measurements

registered using a light intensity quantifier

(Odyssey® Fc Dual-Mode Imaging System from LI-

COR Biosciences).

4.2 Sensors Design and Production

The layout is designed using QCAD software. Each

layer, corresponding to a different conducting

material, is separately designed, in order to produce

the masks required to screen print layer by layer the

final structure of the sensor. A specific care should be

put to realize conductive tracks with a resolution

compatible with the printing performance of the

screen printer employed (A2 Model, Baccini srl,

Italy).

Figure 1: Final SPES layout.

Once the geometry is optimized - with a WE of

8.0 mm of diameter - it is printed on a lucid sheet by

means of inkjet printing, thus to allow the realization

by UV photolithography of the mask required for the

screen printing process.

On a 0.4 mm thick alumina substrate, the 3 layers

are consequently printed: firstly the silver for the

conductive tracks, then carbon for the working

electrode and finally silver-silver chloride for the

reference electrode. In order to allow a better

conduction of the signal, the conductive tracks are

isolated using a conductive spray specific for printed

circuit, leaving the terminal part of the tracks free for

the connection with the conditioning circuit (Fig. 1)

The reproducibility of sensor geometry in the

different printing processes is ensured thanks to a

specific care in performing a standardized protocol

while realizing each sensor. Each sensor is accurately

observed under an optical microscope to evaluate the

homogeneity of the printed layer and the resistance

evaluated with a tester.

4.3 Circuit Design and Production

Parallelly to the realization of the electrochemical

sensor, the conditioning circuit is designed in order to

allow the production of a complete PoC testing

platform.

The design of the circuit is performed following

the scheme of a common potentiostat. Its aim is to

control the potential between the functionalized WE

and the RE, and then to measure the current flowing

between WE and CE.

DCBIOSTEC 2016 - Doctoral Consortium on Biomedical Engineering Systems and Technologies

The electronic schematic is realized with OrCAD

software (OrCAD©- Cadence Design Systems -San

Jose, CA) whereas the design for the Printed Circuit

Board is prepared using OrCAD Layout Plus tool

(OrCAD©- Cadence Design Systems -San Jose, CA).

The final PCB realized using OSH Park - community

printed circuit board (PCB) order (Fig. 2).

Figure 2: Schematic of the conditioning circuit.

Figure 3: Final PCB of the conditioning circuit.

All the SMD electronic components are soldered

and the board inserted in a metallic box to avoid

noises on the signal recording and to improve the

sensor sensitivity and precision.

4.4 Calibration and Measurements

After optimizing the design, the platform is tested

following three different protocols, using before a

saline solution, with different concentrations of NaCl,

then performing EIS with Potassium Ferricyanide and

after that performing ASV functionalizing the WE

with proteins.

4.4.1 NaCL Solution Measurements

The first test of the circuit is performed using a saline

solution, varying the concentration of NaCl in order

to change the conductivity of the solution, and

evaluating the ability of the circuit to quantify these

changes as changes in the current peak flowing

between WE and CE.

In the first test the concentrations of the solution

evaluated are 0.0, 15.0, 22.5, 30.0, 37.5 and 45.0

mg/ml. After that, a second round of concentrations

is tested, to evaluate the linearity of the circuit

response in a range between 0.0 and 10.0 mg/ml, in

particular: 0.0, 1.0, 2.2, 4.0, 5.5, 6.0, 10.4 mg/ml.

Finally, the circuit is evaluated with concentrations

lower than 1.0 mg/ml, in particular: 0.00, 0.44, 0.66,

0.88, 1.00 mg/ml.

In each experiment, drop of 2 ml of saline solution

are dropped on WE, CE and RE, assuring that the

drop stays in place with the help of a mask applied on

top of the sensor.

For this analysis, the input signal is considered as

a triangular wave, with amplitude 300 mV and

frequency 40 mHz, obtained using a pulse generator

(HP8116A pulse/function generator 50 MHz

Hewlett-Packard )

The signal is then acquired using an oscilloscope

(Tektronik TDS 1001B – two channel digital storage

oscilloscope 40 MHz, 500 MS/s)).

Experiments are always performed in triplicate.

All graphical and tabulated data are usually displayed

as mean ± mean standard error.

4.4.2 Antibodies Quantification using EIS

EIS is also applied in order to measure changes of the

current detected between WE and CE to quantify

different concentrations of the primary antibodies

released and adhered on WE surface. In particular,

three antibodies concentrations were considered: 0, 4

and 8 µg/ml.

After an overnight incubation at 4 °C the

measurements were performed in presence of a

conductive electrolytic an solution of 5 mM

K3[Fe(CN)6] in 1 M KCl. Once the functionalization

was performed, a drop of 2 ml was placed in order to

cover WE, RE and CE and allow current flow, and the

electronic measurement were performed, giving a

ramp as signal input, and recording the current

between WE and CE using an oscilloscope. In

particular, a first analysis was performed using

triangular waves four different frequencies (40 mHz,

100 mHz, 200 mHz and 1 Hz) with an amplitude of

300 mV, and then a second one fixing the frequency

to 50 mHz.

Study of a Low-cost Sensitive Point-of-Care Testing System using Screen Printed Biosensors for Early Biomarkers Detection Related to

Alzheimer Disease

4.4.3 Protein Measurements

The protocol followed to quantify protein

concentration, both for the preliminary test using a kit

with human interleukin and for the real samples

containing p53 proteins, is represented by the

following steps:

- sensor wash with Phosphate Buffer Saline (PBS)

- WE coating with optimized primary antibody

concentration (8 µg/ml).

- Overnight incubation at 4°C

- Block with a Bovine Serum Albumin (BSA)

solution.

- 2 hours incubation with desired solution

containing a defined concentration of proteins (in

the calibration phase) or with the sample.

Temperature mantained stable at 25°C

- Block with BSA solution.

- 1 hour incubation with biotin labelled secondary

antibodies.

- Block with BSA solution.

- 30 min incubation with streptavidin labelled

Alkaline Phosphatase.

- Block with BSA solution.

- 20 min incubation with a solution of 3 mM AA-p

e 4 mM AgNO3, protected from light.

Once the functionalization is performed, a drop of 2

ml is placed in order to cover WE, RE and CE and

allow current flow, and the electronic measurement

are performed, giving a ramp as signal input, and

recording the current between WE and CE using an

oscilloscope, in the same way indicated in the

previous paragraph.

5 EXPECTED OUTCOME

The complexities and the heterogeneity associated

with AD, requires high precision and sensitivity in the

reliable detection and quantification of specific

biomarkers, able to allow an early diagnosis of the

disease in the pre-symptomatic phase and to acquire

additional information both from the biological and

from the pathoclinical point of view.

The main expected outcome of this project is the

realization of a self-standing portable point-of-care

testing system, able to support clinicians to diagnose

AD from its earliest stages.

The proposed methodology can be used in a

routine automatized diagnosis technique, specifically

quantifying the unfolded p53 biomarker. Following

the development of the platform, this project

inherently addresses different outcomes, specifically

related to:

1. Optimized calibration of the sensor and

conditioning/acquisition electronics (Fig. 3), thus

to discriminate defined protein concentrations

2. Protein sensitive quantification modifying the

sensor materials (e.g. using gold nanoparticles).

3. Final optimization of the platform to realize a self-

standing point of care

Each step described addressed from one side an

increasing in the sensitivity of the sensor itself and

from the other the optimization of the portable point

of care design, aiming to a low cost, easy usable and

highly precise platform.

Figure 4: Point of care overview.

6 STAGE OF THE RESEARCH

6.1 Initial Sensors Characterization

Regarding the compatibility of the materials and of

the printing process with wet lab practices, alumina

substrate represented the optimal solution. Thanks to

the intrinsic porosity of the material, electrodes

printed in this substrate did not show any variation

when washed with water-based solutions during

functionalization steps. On the contrary, electrodes

printed in glass and polystyrene, because of their low

porosity, did not show an efficient adhesion, with

critical modifications during the functionalization

step, compromising the uniformity of the primary

antibodies coating on the WE and the effective

complex formation with the secondary antibody.

Among the different primary antibody

concentrations evaluated (2.0, 2.6, 3.0, 4.0, 4.8, 6.0,

DCBIOSTEC 2016 - Doctoral Consortium on Biomedical Engineering Systems and Technologies

8.0, 10.0 µg/ml), 8.0 µg/ml was identified as the

optimal one to achieve a homogeneous coating of the

WE.

Figure 5: Image obtained from the optical analysis of WE

coated with different primary antibody concentrations.

Figure 6: Electronical measurements (EIS) of different

antibody concentration coatings.

The fluorescence signal recorded from the

Odyssey showed a fluorescence intensity

proportional to the concentration of primary antibody

coated in the range between 2 and 6 µg/ml. (Fig. 4).

Impedance measurements performed on the same

electrodes, showed results in agreement with what

previously evaluated with optical analysis. More

specifically, the linearity observed for concentration

of 0.0, 6.0, 8.0 µg/ml, could be observed with EIS as

well.

Using electrodes printed on ceramic substrates,

thanks to the good compatibility of the materials with

wet lab practices, results obtained appeared to be

repeatable and reliable for all the electrodes tested.

The impedance module measured for electrodes

coated with 8.0 µg/ml primary antibody solution

resulted to be superior in all the frequencies range

evaluated, compared with the one of blank electrodes,

treated with a buffer solution (mean 15.5 ± 4.6 Ohm

between 2 and 20 kHz; max 30 Ohm at 2 kHz and

minimum of 9 Ohm at 200 kHz). The impedance

module measured with electrodes treated with 6.0

µg/ml showed a trend comprised between the

previous two (Fig. 5). Using electrodes printed on

glass substrates, the average difference between the

impedance module of reference electrodes and

electrodes treated with 8.0 µg/ml of primary antibody

was 7.6 ± 1.1 Ohm, constant in the range between 200

and 2 MHz. Because of the poor adhesion and

compatibility of the glass printing process with wet

lab practices, measurements performed on electrodes

coated with intermediate concentrations showed

results compatible with the non-uniform coating

highlighted from the fluorescence analysis.

6.2 Calibration and Measurements

The present stage of the research, after that the design

and development of the sensor and

conditioning/acquisition circuit has been optimized,

is addressing the calibration of the sensor, firstly with

NaCl solution, and then with human interleukin, in

order to optimize the detection protocol and to

prepare the platform for the following step of p53

proteins detection and quantification.

6.2.1 NaCl Solution Measurements

Results from the evaluation of circuit response to

changes in saline solution conductivity showed a

linear response for the specific ranges of

concentrations evaluated. After evaluating the

linearity using high concentration of NaCl, a

narrower range of concentrations was evaluated in

order to understand if the circuit was able to recognize

small changes in solution conductivity and small

current between WE and CE.

Figure 7: Calibration of SPES with NaCl solution.

Study of a Low-cost Sensitive Point-of-Care Testing System using Screen Printed Biosensors for Early Biomarkers Detection Related to

Alzheimer Disease

Performing the same measures with lower

concentration a particular behaviour could be noticed.

Two different slopes could be observed respectively

for concentration lower and higher than 1.0 mg/ml. In

particular, a higher sensitivity was shown for the

concentration lower than 1.0 mg/mg (13 mA/

(mg/ml)), indicating a higher sensitivity of the sensor

for small changes of conductivity and small currents

(Fig. 6). This behaviour suggested that the range of

concentration in which the circuit was able to give the

best response corresponds to low changes in

concentration, resulting in small ionic currents. On

the contrary, high changes in concentration, causing

high changes in conductivity, were discriminated

with an inferior sensitivity (170 mA/(mg/ml))

because they brings to current which cause the circuit

to saturate, and not to be able to discriminate the

difference.

6.2.2 Antibodies Quantification using EIS

Results from EIS measurements showed a

proportional decreasing of the peak of current flowing

between CE and WE, indicating an increased

impedance of the system due to an increasing

concentration of antibodies coated on WE surfaces

resulting in a reduced electrons exchange between

WE surface and electrolytic solution (Fig 8 and 9).

Figure 8: Difference in CE current measured at different

frequencies, evaluating with EIS different concentration

primary antibodies coatings.

Figure 9: Difference in CE current measured at 50mHz,

evaluating with EIS different concentration primary

antibodies coatings.

The same behaviour was observed at all the

frequencies evaluated. Increasing the concentration

of antibodies coated on the WE resulted in reducing

the differences of currents exchanged at different

frequencies (Fig. 9). As showed in Fig. 9, the

sensitivity of the sensor in detecting the change in

antibodies coating concentration was of 70 mA/µg.

6.2.3 Protein Measurements

The activity actually going on refers to the

implementation of the same protocol using

interleukin protein, of dedicated kit DuoSet®

development system for ELISA, Human CXCL8/IL-

8. The different concentrations of proteins are going

to be recognized with two different techniques:

- using EIS in presence of Potassium Ferricyanide

in KCl solution.

- using ASV as described in the methodologies

section.

This phase is essential for an effective calibration of

the platform, in order to proceed with the

quantification of unknown concentration of p53

proteins.

7 FUTURE OUTLOOKS AND

CONCLUSION

In light of the positive results described, the activity

actually going on refers to the implementation of the

same protocol described in the materials and methods

section using interleukin protein, of dedicated kit

(DuoSet® development system for ELISA, Human

CXCL8/IL-8). Different concentrations of proteins

(order of ng/ml) will be recognized using both the

techniques described (EIS and ASV). This phase is

essential for an effective calibration of the platform,

in order to proceed with the quantification of

unknown concentration of p53 proteins. Specifically

regarding ASV, the same protocol will be adopted to

quantify protein concentrations, both for interleukin

and for p53 proteins. It will be characterized by the

use of immucomplexes of primary antibody-protein-

secondary antibody labelled with Alkaline

Phosphatase, as functionalization of the WE. In this

way, through a selective chemical deposition of

silver, the current flowing between WE and CE will

be proportional to the amount of deposited silver,

which in its turns will be proportional to the

recognized protein. Before proceeding with unknown

proteins concentrations, an accurate calibration of the

biosensor will be performed. After the validation, the

DCBIOSTEC 2016 - Doctoral Consortium on Biomedical Engineering Systems and Technologies

proposed methodology and the platform design will

be optimized in order to be easily accessible for a

routine automatized diagnosis technique in the

clinical environment. From these bases, particular

attention will be then addressed to increase the

sensitivity of the method itself, including both the

introduction of nanostructured materials for the

working electrodes and proper ASV measurements.

All this, with the aim to realize an innovative self-

standing portable point-of-care, a low cost, easy

usable and highly precise platform able to support

clinicians to diagnose AD from its earliest stages.

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Aberrations , Single-Nucleotide Variants , and Tumoral

Heterogeneity by Massively Parallel Sequencing. , 224,

pp.211–224.

Dhawan, A.P. et al., 2015. Current and Future Challenges

in Point-of-Care Technologies: A Paradigm-Shift in

Affordable Global Healthcare With Personalized and

Preventive Medicine. Translational Engineering in

Health and Medicine, IEEE Journal of, 3, pp.1–10.

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Study of a Low-cost Sensitive Point-of-Care Testing System using Screen Printed Biosensors for Early Biomarkers Detection Related to

Alzheimer Disease