Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by cognitive and memory deterioration, which requires an early diagnosis for effectiveness of treatment. Immunosensors based on AD biomarkers stand out as a fast, inexpensive and practical alternative for an early detection before it’s first symptoms. The development of Screen-Printed Electrodes (SPEs) meets a growing demand in the market for applications such as signal transducers in biosensor devices. The screen-printing technology stands out due to its simplicity of elaboration, the possibility of mass production of disposable electrodes and ease in miniaturization for use in portable devices. The objective of this work is to develop a simple and low cost method to manufacture a Screen Printed Carbon Electrode (SPCE) system to detect AD. The microelectrodes were obtained from carbon paste on two substrates: one from High Density Polyethylene (HDPE), and another one from Polyvinyl Chloride (PVC), shown Figure 1. The electrochemical response to adsorption of self-assembled polymer and lipid films was studied by cyclic voltammetry. The HDPE designed in Figure 3, offered a less capacitive and sensitive response to the assembly of the films compared to PVC. The immunosensor for AD diagnosis based on the detection of the IgG anti-Aβ1-40 autoantibody was built on SPCEs using layer by layer technique (LBL), as shown in Figure 4. The first bilayer consists of pure or functionalized Polyethyleneimine (PEI), and the second of liposomes of Dipalmitoyl Phosphatidyl Glycerol (DPPG) immobilizing the Aβ1-40 peptide, repeating this process in the second bilayer. In order to improve their sensory response, PEI functionalized with graphene produced via electrochemical exfoliation of graphite (EEG) was used, in the proportions of 0.6%, 1.2%, 2.4% and 10% (w / w).Cyclic voltammetry was used to evaluate the electrochemical behavior of the SPCE in three cycles, at potentials from -0.6 V to 0.6 V with a support electrolyte containing PBS pH 7.4, Figure 5. Electrochemical Impedance Spectroscopy (EIS) measurements, Figure 6, were evaluated, with frequencies ranging from 0.1 MHz to 0.1 Hz, amplitude 10mV, for pure and modified SPCEs with PEI and their EEG composites. For detection, autoantibody solutions, diluted in PBS, were prepared in concentrations ranging from 1ng / ml to 10 µg / ml. There was an evident decrease in capacitance in the samples of PEI with EEG, as seen in the voltammograms. After the functionalization of PEI, the capacitive currents of the electrodes clearly decreased, possibly due to an increase in the conductivity of the electrodes. On the other hand, the EIS curves for the electrodes with PEI functionalized, despite appearing to have larger modules than those in which it is not functionalized, the inclination of impedance spectra at low frequency presents minor slopes. This is possibly due to the higher permeation of charges in the interstices of the PEI, as observed in the microscopies in Figure 7, associating the capacitive process of these functionalized PEI electrodes capable of carrying more charge than the purely capacitive electrodes with bilayers composed only of PEI. This gave them a significantly higher degree of conductivity. After functionalization with EEG, the graphene particles appear to be dispersed in a disordered way on the polymer, being a possible cause of charge dispersion in pores, increasing their contact area, and justifying greater impedance values. PEI bands, as described in Figure 8, refer to asymmetric stretches of the C-N group at 1045, 1119, 1310 cm-1, 1598 and 1651 cm-1. Last band may be associated both to symmetrical angular deformation in plane for the -NH2 to amides (possibly provided by reaction between EEG carbonyl and PEI amines) and PEI existing amines It was noticed a change in the band at 1651 cm-1 in PEI composites with 0.6%, 2.4% and 10% assigned to amide formation, conferring a suitable dispersion. However, for others EEG contents, amide bands may be masked by amine groups of PEI.Sensorial measurements were better performed at HDPE subtract modified with PEI functionalized with EEG until 1.2% of content. There was an exponential growth trend in the normalized area of the voltammograms, Figure 9, as the Aβ1-40 autoantibody concentrations increased, distinguishing its variation. However, after 1μg / ml the curve stabilized showing the limit of detection (LOD). Figure 1
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Jan 12, 2022
Sanjay S Timilsina + 5
Open Access
