Use of data processing for rapid detection of the prostate-specific antigen biomarker using immunomagnetic sandwich-type sensors

Diagnosis of cancer using electroanalytical methods can be achieved at low cost and in rapid assays, but this may require the combination with data treatment for determining biomarkers in real samples. In this paper, we report an immunomagnetic nanoparticle-based microfluidic sensor (INμ-SPCE) for the amperometric detection of the prostate-specific antigen (PSA) biomarker, the data of which were treated with information visualization methods. The INμ-SPCE consists of eight working electrodes, reference and counter electrodes. On the working electrodes, magnetic nanoparticles with secondary antibodies with the enzyme horseradish peroxidase were immobilized for the indirect detection of PSA in a sandwich-type procedure. Under optimal conditions, the immunosensor could operate within a wide range from 12.5 to 1111 fg·L−1, with a low detection limit of 0.062 fg·L−1. Multidimensional projections combined with feature selection allowed for the distinction of cell lysates with different levels of PSA, in agreement with results from the traditional enzyme-linked immunosorbent assay. The approaches for immunoassays and data processing are generic, and therefore the strategies described here may provide a simple platform for clinical diagnosis of cancers and other types of diseases.


Synthesis of magnetic iron oxide nanoparticles
The Fe3O4 magnetic nanoparticles (MNPs) were synthesized according to the coprecipitation method reported by Ferreira and co-workers [1], with modifications. The MNPs were synthesized using 1.55 g of FeSO4·7H2O and 3.05 g of FeCl3·6H2O, dissolved in 2.5 mL of HCl, followed by dilution in 62.5 mL of distilled water and heating to 60 °C. A hot plate IKA C-MAG model HS 7 (IKA Works, Inc.), connected to an integrated temperature control and a Pt1000 temperature probe, was used to prepare the MNPs. A molar ratio of 1:2 between Fe 2+ and Fe 3+ was used during reaction. A solution with 18.75 g NaOH dissolved in 312.5 mL of H2O in a beaker, heated to 60 °C, was mixed with the previous solution and kept under vigorous stirring. The reaction mixture was maintained at 60 °C for 30 min. The magnetic nanoparticle solution was washed repeatedly with distilled water until it reached pH 7 and was then separated by magnetic decantation. Then, 15 mL of this solution were removed and stored in acetone. The solution was left under N2 atmosphere for 30 min and stored in a refrigerator for further characterization.
The surface modification of Fe3O4 magnetic particles was carried out by using sodium citrate. Initially, the MNP solution was heated to 85 °C under stirring for 30 min. Then, sodium citrate was added in excess to the solution under stirring and at constant temperature for another 30 min. The modified particles were washed several times with distilled water until pH 7 was reached. The resulting functionalized MNPs were decanted off using a neodymium magnet and suspended in acetone. Subsequently, the solution was maintained in N2 atmosphere for 30 min and stored in a refrigerator for later use.

Fabrication of electrodes
The all-plastic carbon electrodes consist of eight working electrodes (WE), a pseudoreference electrode (RE), and a counter electrode (CE), designed with Silhouette Studio v. 2.7.4 software. The electrodes were cut out from an adhesive vinyl sheet using an electronic craft cutter (Silhouette Cameo, Silhouette America, Inc.), and unwanted vinyl was removed with tweezers. The mask was used as a template that was transferred to a polyester sheet (transparency film for printers) with dimensions of 21.0 cm by 29.7 mm.
Then, graphite-based carbon ink (Electrodag 423SS) was transferred carefully and heated at 90 °C for 30 min. The pseudo-reference electrode (Ag/AgCl) (C2051014P10, Gwent Electronic Materials Ltd., UK) was then painted and cured for another 30 min at 60 °C.
Finally, the vinyl mask was removed from the polyester sheet.

Sample preparation
The PSA standard solution was prepared by diluting the PSA antigen standard product, which was obtained from Abcam to different concentrations with calf serum. The healthy and prostate cancer cell linage were obtained from the Cell Bank from Rio de Janeiro/Brazil.

Analysis with ELISA
The ELISA test was performed following the Abcam protocol. An ELISA plate containing a solution of 50 µL samples, PSA standards and an antibody cocktail were added to each well. The ELISA plate was shaken (400 rpm) and incubated for 1 h at room temperature. The wells were washed thrice with PBS/0.005% T20 and 50 µL of TMB substrate were added to each well and incubated for 10 min in the dark. Then, 45 µL of  Figure S1A. Field-emission scanning electron microscopy (FE-SEM) images were obtained using a Hitachi model S-4800. Energy-dispersive X-ray spectroscopy (EDX) was performed using Cu Kα radiation (λ = 1.5418 Å, 40 kV and 30 mA) in the range of 2° ≤ 2θ ≤ 80° with steps of 0.05° at a rate of 0.25° min −1 (Figure S1B,C). Figure S1B shows the SEM images of agglomerates (insert in Figure S1B). The average diameters of the agglomerate (inserts of Figure 1B) are similar to the values reported elsewhere [2][3][4].  [5][6][7]. Figure S1D shows the typical EDX spectra of the Fe3O4 nanoparticles. The samples exhibit very strong characteristic Kα and Kβ peaks of iron in the range from 6.40 to 8.06 keV. These results confirm that the particles consist mostly of iron oxide.
Characteristic citrate peaks of C=O stretching at 1626 cm −1 and of OH from COOH between 3012 and 3600 cm −1 ( Figure S1E) were observed [8]. Polarization-modulated S5 infrared reflection absorption spectroscopy (PM-IRRAS) [8] spectra for the step-by-step monitoring of the electrode surface are shown in Figure S1F.