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Topic 2. Technologies for Chemical and Biochemical Sensing
An Electrochemical Platform for the Point-Of-Care Diagnosis of
Infectious Diseases
Gloria Longinotti, Gabriel Ybarra, Paulina Lloret, Carlos Moina
UT Nanomateriales, Centro INTI-Procesos Superficiales, Instituto Nacional de Tecnología Industrial, Argentina
Mijal Mass, Mariano Roberti, Matías Lloret, Diego Brengi, Salvador Tropea, Francisco Salomón, Laura Malatto,
Liliana Fraigi
Centro de Micro y Nanoelectrónica del Bicentenario, Instituto Nacional de Tecnología Industrial, Argentina
Luciano Melli, María Eugenia Cortina, Diego Rey Serantes, Juan E. Ugalde, Andrés Ciocchini, Diego J.
Comerci
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Argentina
[email protected]
Summary
In this work, we present a point-of-care platform for the serologic diagnosis of infectious diseases. The
principle of diagnosis can be regarded as an electrochemical enzyme-linked immunoassay. Different
recombinant antigens were immobilized onto magnetic microparticles. After incubation with sera and
horseradish peroxidase labeled conjugated antibodies, the magnetic particles were collected and placed
in electrochemical cells where infected sera were amperometrically detected. All components of the
platform were designed and developed during this project, including the electronic instrumentation and
novel, disposable platform was successfully tested for the diagnosis of electrochemical cells, which allow
eight simultaneous measurements. The foot-and-mouth disease, brucellosis, Chagas disease and
infection by STEC O157.
The concept behind the device
The aim of this project was to develop a point-of-care platform to carry out serologic diagnosis of
infectious diseases with reliability comparable to ELISA tests. In contrast to optical detection,
electrochemical transduction is easier to miniaturize and to integrate with electronic circuits. On the other
hand, the immobilization of antigens onto magnetic microparticles has additional advantages over antigen
immobilization in conventional wells, such as the possibility of using higher loads of antigens and the
resulting shorter incubation times; nanoparticles present additional advantages, such as high surface to
volume ratio and superparamagnetism, and are currently being tested as a replacement of microparticles.
The point-of-care platform presented here is based on the combination of magnetic particle-based indirect
immunoassay with electrochemical detection. A schematic representation of the detection principle is
shown in Fig. 1a. Antigen-coated magnetic particles were incubated with sera, washed using a magnetic
rack and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies. The particles
were magnetically collected and placed onto the surface of an electrode, where the enzymatic activity of
HRP was detected [1].
Electrochemical point-of-care platform
All components of the point-of-care platform were developed in this project. Disposable, acrylic
cartridges with eight electrochemical cells were designed and manufactured with dimensions fitting an 8channel micropipette. Each electrochemical cell contains two carbon electrodes and one Ag│AgCl
reference electrode. The electrodes were screen printed onto 0.5 mm acrylic substrates. The central
working electrodes were designed to be aligned with neodymium magnets so as to concentrate the
magnetic particles. The eight electrochemical cells, each one with a volume of 40 µl, were completed with
an acrylic top piece (Fig. 1b). The electronic instrumentation required to control the electrochemical
system and process the resulting signal was presented elsewhere [2]. The electronic circuits are
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connected to a computer via a USB port (Fig. 1c). A software was developed with the capability of
recording amperometric measurements with potential steps and cyclic voltammetries.
a
Fig. 1:
b
c
(a) Schematic representation of the detection principle. (b) Cartridge with eight electrochemical
cells. (c) Electrochemical platform together with a netbook.
Diagnosis of infectious diseases
Glicoconjugates and recombinant proteins were developed and covalently immobilized onto magnetic
particles. A set of different antigens were used to diagnose four different diseases. These antigens were
validated by other methods as ELISA and particle-based immunoassay coupled to fluorimetric detection
[3]. For the serologic tests, coated particles were incubated with different sera for one hour and rinsed with
0.1% Tween 20 in PBS buffer. Then the microparticles were incubated with anti-Ig conjugate, washed with
0.1% Tween 20 in PBS buffer and finally the electrochemical measurements were carried out. As a proof
of principle, four different infectious diseases were tested: foot-and-mouth disease, Chagas disease,
human and bovine Brucellosis, and infections caused by STEC O157.
The results indicate that immunoassays based on the combination of recombinant antigens immobilized
on magnetic particles with electrochemical detection has a great potential for the development of point-of
care diagnosis of human and animal infectious diseases.
Figure 1: (Right) Diagnosis of human brucellosis using four different serum samples from patients with
Brucellosis. Using the glycoprotein OPS-AcrA it is possible to diagnose patients infected with B.
abortus, B. melitensis and B. suis. (Center) Diagnosis of bovine brucellosis using the glycoprotein
OPS-AcrA as antigen in six animals. The immunoassay was developed with pre and post
infection serum samples. (Left) Diagnosis of Chagas disease. Six positive and three negative samples
were used in this immunoassay.
References
[1] G. Longinotti, P. Lloret, G. Ybarra, C. Moina, L. Hermida, O. Milano, M. Roberti, L. Malatto, L. Fraigi, L., Proc. 6th IberoAmerican Congress on Sensors, 2008.
[2] G. Longinotti, P. Lloret, G. Ybarra, C. Moina, A. Ciocchini, D. Rey Serantes, L. Malatto, M. Roberti, S. Tropea, L. Fraigi, Proc.
nd
32 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2010.
[3] A. E. Ciocchini, D. A. Rey Serantes, L. J. Melli, J. A. Iwashkiw, B. Deodato, J. Wallach, M. F. Feldman, J. E. Ugalde, D. J.
Comerci, PLOS Neglected Tropical Diseases, 2013.
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