Aptasensor for diagnosis and monitoring of prostate cancer

Description

/ 18 APTASENSOR FOR DIAGNOSIS AND MONITORING OF PROSTATE CANCER Field of invention

[001] The present invention describes the creation of an aptasensor for the diagnosis and monitoring of prostate cancer through the production of carbon electrodes functionalized with gold nanoparticles (AuNps) and the A4 aptamer. Aptamers exhibit many properties such as ease of selection and synthesis, high binding affinity, specificity, low immunogenicity, and low synthesis cost. The A4 aptamer (SEQ ID N 01) was selected using the 3D Cell-SELEX method and evaluated for its effectiveness in prostate cancer. To allow its binding with gold nanoparticles (AuNps), a thiol group was added to the 5' end of the sequence. Printed carbon graphite electrodes were fabricated using carbon and silver ink, which were pre-conditioned in acid and phosphate buffer. These electrodes were functionalized with AuNps to promote the anchoring of the thiolated aptamer.All stages of platform modification were performed using dripping and washing processes, aiming for binding with prostate tumor cells. State of the art

[002] Prostate cancer (PCa) is the second most frequent malignant neoplasm in men and the fifth leading cause of death worldwide (MUNGOVAN SF, BRATT O, BRAY F, BRAWLEY O, LUCKENBAUGH AN, MUCCI L, MORGAN

[003] TM, CARLSSON SV. Update on Prostate Cancer Epidemiology and Risk Factors-A Systematic Review. European Urology, v. 84, n. 2, p. 191-206, 2023), being considered a heterogeneous disease, varying from very slow and benign evolution to progressive, aggressive, metastatic and fatal, even when treated (PORZYCKI P, CISZKOWICZ E. Modern biomarkers in prostate cancer diagnosis. Central European Journal of Urology, v.73, n.3, p.300-306, 2020). The diagnosis of prostate cancer (PCa) is currently based on the test of Petition 870250013754, dated 20 / 02 / 2025, page 22 / 48 2 / 18 Serum prostate-specific antigen (PSA) and / or abnormal digital rectal examination and histopathological evaluation of prostate biopsies. The main disadvantage of PSA is its lack of specificity (HENDRIKS RJ, VAN OORT IM, SCHALKEN JA. Blood-based and urinary prostate cancer biomarkers: a review and comparison of novel biomarkers for detection and treatment decisions. Prostate Cancer Prostatic Diseases, v. 20, n. 1, p. 12-19, 2017), even so, PSA remains the gold standard screening biomarker for prostate cancer.

[004] Although PSA is a serum biomarker for prostate cancer, laboratory tests have low precision and limited specificity. A positive PSA result for prostate cancer requires confirmation through a tissue biopsy, hence the need to develop new and better tools that can assist in the diagnosis of prostate cancer (CRULHAS BP, KARPIK AE, DELELLA FK, CASTRO GR, PEDROSA VA. Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells. Analytical Bioanalytical Chemistry. 2017; CAMPOS-FERNÁNDEZ E, BARCELOS LS, DE SOUZA AG, GOULART LR, ALONSO-GOULART V. Research landscape of liquid biopsies in prostate cancer. American Journal of Cancer Research, v.9, n.7, p. 1309-1328, 2019).

[005] In this sense, liquid biopsies are considered useful tools for finding specific biomarkers for more accurate diagnosis (CAMPOS-FERNANDEZ E, BARCELOS LS, DE SOUZA AG, GOULART LR, ALONSO-GOULART V. Research landscape of liquid biopsies in prostate cancer. American Journal of Cancer Research, v.9, n.7, p.1309-1328, 2019). Liquid biopsy is a non-invasive analysis of biomarkers in biological fluids such as blood, plasma, urine, cerebrospinal fluid, and saliva to allow the detection and monitoring of cancer with sufficient molecular information compared to that derived from tissue biopsies, being less invasive than surgery, allowing verification of the cancer status in an individual (BORGES, RT, DE ANDRADE, RGM, DA LUZ ARAÚJO, J., MEDEIROS, MDSD, VIANA, Petition 870250013754, dated 20 / 02 / 2025, p. 23 / 48 3 / 18 GM L, BITTENCOURT, RJ, & PEREIRA, RW. What is the level of concordance between liquid biopsy and tissue biopsy as a source of biological material for the detection of mutations in the EGFR gene in patients with lung cancer? Revista de Medicina e Saúde de Brasília, v.8, n.3, p. 349-358, 2020; PANG B, ZHU Y, NI J, THOMPSON J, MALOUF D, BUCCI J, GRAHAM P, LI Y. Extracellular sails: the next generation of biomarkers for liquid biopsy-based prostate cancer diagnosis. Theranostics. 2020). Liquid biopsy represents the tumor microenvironment, providing comprehensive information to track tumor progression and the development of different treatment strategies, as well as monitoring the response to therapy (CROCETTO, FELICE, CIMMINO, AMÉLIA, FERRO, MATTEO E TERRACCIANO, DANIELA. Circulating tumor cells in bladder cancer: a new horizon of liquid biopsy for precision medicine. Journal of Basic and Clinical Physiology and Pharmacology, v. 33, n. 5, p. 525-527, 2021).In addition to other constituents, tumors release circulating tumor cells (CTCs) into the bloodstream, whose expression of surface markers and their quantification have already been researched for the diagnosis of various types of cancer (CROWLEY E, Dl NICOLANTONIO F, LOUPAKIS F, BARDELLI A. Liquid biopsy: monitoring cancer genetics in the blood. Nature Reviews Clinical Oncology, v.10, n. 8, p.472-484, 2013), including prostate cancer (HAPACH LA, MOSIER JA, WANG W, REINHART-KING CA. Engineered models to parse apart the metastatic cascade. NPJ Precision Oncology, v. 3, n. 20, p. 1-8, 2019; CAMPOSFERNÁNDEZ E, BARCELOS LS, DE SOUZA AG, GOULART LR, ALONSOGOULART V. Research landscape of liquid biopsies in prostate cancer. American Journal of Cancer Research, v.9, n.7, p.1309-1328, 2019; DAY KC, LORENZATTI HILES G, KOZMINSKY M, DAWSEY SJ, PAUL A, BROSESLJ, SHAH R, KUNJA LP, HALL C, PALANISAMY N, DAIGNAULT-NEWTON S, EL- SAWY L, WILSON SJ, CHATOU A, CHATOS, KY, KELL, E. D, NAGRATH S, MORGAN T, DAY ML. HER2 and EGFR Overexpression Support Metastatic Progression of Prostate Cancer to Bone. Cancer Research, v. 77, n. 1, p. 74-85,2017).

[006] Diagnosis with circulating biomarkers is related to reduced Petition 870250013754, of 20 / 02 / 2025, p. 24 / 48 4 / 18 of intratumoral heterogeneity, allowing us to overcome the variability of molecular information obtained by tissue analysis, which may depend on the location and accessibility of the tumor. Furthermore, liquid biopsy can be a tool to monitor tumor progression, predicting prognosis, overall survival, and treatment efficacy, suggesting personalized therapy (GEEURICKX E, HENDRIX A. Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics. Molecular Aspects of Medicine, v.72, p.1-17, 2020).

[007] Several studies report the use of aptamers in the development of new approaches in liquid biopsy diagnostic strategies for prostate cancer (LI B, LIU C, PAN W, SHEN J, GUO J, LUO T, FENG J, SITU B, AN T, ZHANG Y, ZHENG L. Facial fluorescent aptasensor using aggregation-induced emission luminogens forexosomal proteins profiling towards liquid biopsy. Extracellular surface proteins - vesicles for early detection and classification of cancers. Aptamers are small oligonucleotides, analogous to monoclonal antibodies, with a three-dimensional structure capable of interacting with any desired target.Recently, aptamers have been studied for the treatment and detection of many diseases, including cancer (CRUZ-HERNÁNDEZ CD, RODRÍGUEZ-MARTÍNEZ G, CORTÉS-RAMÍREZ SA, MORALES-PACHECO M, CRUZ-BURGOS M, LOSADA-GARCÍA A, REYES-GRAJEDA JP, GONZÁLEZ-RAMÍREZ I, GONZÁLEZ-COVARRUBIAS V, CAMACHO-ARROYO I, CERBÓN M, RODRÍGUEZ-DORANTES M. Aptamers as Theragnostic Tools in Prostate Cancer and Treatment. Life (Basel).

[009] The 3D Cell-SELEX technique was used as a new method for selecting Petition 870250013754, dated 20 / 02 / 2025, p. 25 / 48 5 / 18 Specific ligands from PC3 cell lines in 3D cell culture. This original system combined Cell-SELEX, an assay that exploited cell structure to generate specific ligands, and 3D cell culture, a methodology that mimics the in vitro tissue microenvironment (SOUZA AG, MARANGONI K, FUJIMURA PT, ALVES PT, SILVA MJ, BASTOS VA, GOULART LR, GOULART VA. 3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line. Experimental Cell Research, v.341, n.2, p. 147-56, 2016).

[010] The aptamers selected by Cell-SELEX present different advantages in relation to monoclonal antibodies, such as small size, lack of immunogenicity, high stability, ability to recognize the native conformation of target molecules present on the cell surface, and can be easily synthesized and modified (SINGER BS, SHTATLAND T, BROWN D, GOLD L. Libraries for genomic SELEX. Nucleic Acids Res. 1997 Feb 15;25(4):781-6. doi: 10.1093 / nar / 25.4.781. Erratum in: Nucleic Acids Research, v. 25, n. 21, p. 781-786, 1997; WANG Y, LUO Y, BING T, CHEN Z, LU M, ZHANG N, SHANGGUAND, GAO X. DNA aptamer evolved by cell-SELEX for recognition of prostate cancer. PLoS One, v.9, n.6, p.1-10, 2014; CRULHAS BP, KARPIK AE, DELELLA FK, CASTRO GR, PEDROSA VA. Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells. Analytical Bioanalytical Chemistry. 2017), in addition to the lower cost of synthesis.Modifications to RNA aptamers can make them more stable, such as the RNA aptamer A4, which binds specifically to prostate tumor cells and has been modified by adding deoxyribonucleotides to its ends to remove the reactive 2' hydroxyl groups from the ribose at the 5' and 3' ends of the aptamer and to stabilize it, preventing contamination by RNases, thus enabling its use in the diagnosis of prostate cancer (CAMPOS-FERNÁNDEZ E, BARCELOS LS, DE SOUZA AG, GOULART LR, ALONSO-GOULART V. Research landscape of liquid biopsies in prostate cancer. American Journal of Cancer Research, v.9, n.7, p.1309-1328, 2019). Petition 870250013754, dated 20 / 02 / 2025, page 26 / 48 / 18

[011] A major advantage of biosensor diagnostics was the creation of a chemiluminescent biosensor, developed using the different properties of 6-FAM conjugated with the G-rich DNA aptamer in the absence and presence of PSA in the guanine chemiluminescence reaction, which was able to quantify trace levels of PSA in human serum in 30 minutes, without the time-consuming and complicated procedures (e.g., multiple incubations and washes) required by conventional immunoassays operated with expensive and difficult-to-handle antibodies. The detection limit of the chemiluminescent biosensor with a wide linear dynamic range (1.9-125 ng / ml) was 1.0 ng / ml. The excellent correlation (R¼0.985) between the chemiluminescent biosensor and the conventional enzyme immunoassay indicates that the accurate, precise, and rapid chemiluminescent biosensor can be applied as a new method for the early diagnosis of prostate cancer (CHA T, CHO S, KIM YT, LEE JH.Rapid aptasensor capable of simply diagnosing prostate cancer. Biosensors Bioelectronics. 2014).

[012] Other simple single-mask gold triple-interdigitated microelectrode biosensors, taking advantage of a self-assembled monolayer, have already been developed and are effective using an amino-silanization technique for the early detection of a prostate cancer biomarker, prostate-specific antigen (PSA). Unlike most interdigitated electrode biosensors, biorecognition occurs between the interdigitated electrodes, which increases the sensitivity and detection limit of the sensor. Using electrochemical impedance spectroscopy (EIS) in Faradaic mode to quantify PSA antigen, the developed detection platform demonstrates logarithmic PSA detection ranging from 0.5 ng / ml to 5,000 ng / ml, an estimated LOD of up to 0.51 ng / ml in serum, and good sensor reproducibility.The developed detection scheme has proven to have great potential as a portable biosensor for prostate cancer, as well as being a universal platform for biomolecular detection with the versatility to implement nanoparticles and others. Petition 870250013754, dated 20 / 02 / 2025, page 27 / 48 7 / 18 Surface chemicals for various applications (IBAU C, MD ARSHAD MK, GOPINATH SCB, NUZAIHAN M N M, M FATHIL MF, ESTRELA P. Gold interdigitated triple-microelectrodes for label-free prognosticative aptasensing of prostate cancer biomarker in serum. Biosensors Bioelectronics. 2019). Early diagnosis is one of the most important factors in determining cancer prognosis. Sensitive detection and quantification of tumor-specific biomarkers have the potential to significantly improve our diagnostic capabilities.

[013] To highlight the importance of early diagnosis, an actionable nanostructure based on an aptamer was presented, based on an oligonucleotide / gold nanoparticle architecture that selectively disassembles in the presence of the biomarker of interest, its optimization being based on the in silico determination of the interactions of the aptamer nucleotides with the protein of interest. This scheme was demonstrated for the case of Prostate Specific Membrane Antigen (PSMA) and PSMA derived from PSMA-positive exosomes, in which the disassembly of the system was tested by means of diameter and counting rate measurements in dynamic light scattering and by inspection of its plasmonic resonance change, after the addition of PSMA, finding appreciable differences up to the subpicomolar range, making it possible to use competitive sensors with biochemical diagnostic assays that require enzymatic amplification.More generally, this scheme has the potential to be applied to a wide range of pathologies with specific biomarkers identified (MATTEOLI G, LUIN S, BELLUCCI L, NIFOSI R, BELTRAM F, SIGNORE G. Aptamer-based gold nanoparticle aggregates for ultrasensitive amplification-free detection of PSMA. Scientific Reports. 2023).

[014] Another target-mounted and self-calibrated aptasensor based on a core-satellite nanostructure using surface-enhanced Raman spectroscopy (SERS) technology was developed for the sensitive and reliable determination of PSA protein, with a detection limit of 0.38 mg mL⁻¹ and a Petition 870250013754, dated 20 / 02 / 2025, page 28 / 48 8 / 18 dynamic detection range of 10 to 10 mg mL. Furthermore, the proposed approach for PSA detection in patient blood samples was implemented, and the results showed that it is capable of providing comparable detection accuracy associated with a larger dynamic detection range and a lower detection limit, as well as less sample requirement (only 5 pL) compared to the commonly used clinical method. Therefore, this SERS-based aptasensor for PSA detection in human blood samples has promising potential to be an alternative tool for clinical application in the precise screening of prostate cancer (WU Q, CHEN G, QIU S, FENG S, LIN D. A target-triggered and self-calibration aptasensor based on SERS for precise detection of a prostate cancer biomarker in human blood. Nanoscale. 2021).

[015] Another study proposed using self-assembled thiolated single-stranded DNA on electrodes functionalized with multi-walled carbon nanotubes (MWCNTs) modified with gold nanoparticles (AuNPs) to realize a low-cost, label-free electrochemical biosensor. In this sense, the PSA aptamer was immobilized by electrostatic adsorption on the surface of a screen-printed MWCNT / AuNPs electrode. The immobilization process was enhanced due to the presence of Au nanoparticles on the electrode surface. The developed sensor has an extended linear range of 1-100 ng / mL and a very low detection limit of up to 1 pg / mL. Furthermore, the reaction has a binding time of only five minutes on the developed electrodes. Investigations of the biosensor's selectivity against various substances revealed efficient selectivity for PSA detection.With this approach, low-cost biosensors with high sensitivity can be realized, which have a wide linearity range and a low detection limit, which are necessary for the early detection of prostate cancer (ALNAIMI A, AL-HAMRY A, MAKABLEH Y, ADIRAJU A, KANOUN O. Gold Nanoparticles-MWCNT Based Aptasensor for Early Diagnosis of Prostate Cancer. Biosensors (Basel). 2022). Patent BR 10 2020 013625 9 describes new modified aptamers and a system. Petition 870250013754, dated 20 / 02 / 2025, page 29 / 48 9 / 18 immunological aptamer comprising at least one of the aforementioned modified aptamers R4 and / or D4 combined with at least one specific antibody, immunological aptamer panels comprising said system, diagnostic or prognostic method and its uses to identify prostate cancer biomarkers in circulating tumor cells from biological samples. From the analysis of these biomarkers by flow cytometry, it is possible to discriminate patients with prostate cancer, potentially aiding in the diagnosis of prostate cancer. The A4 aptamer with a thiol group at the 5' end of the sequence was synthesized for the construction of the aptasensor through the production of carbon electrodes functionalized with gold nanoparticles (AuNps), allowing the detection of circulating tumor cells from samples of patients with prostate cancer, differing from the proposal used in the cited patent, where an immunological aptamer panel with flow cytometry analyses was used.

[016] The aptasensor developed has a differential advantage over the methodologies currently used in the diagnosis and monitoring of prostate cancer by enabling fast and low-cost results. In addition, its ability to detect circulating prostate tumor cells allows for continuous monitoring of disease progression, facilitating adjustments in treatment and providing a more personalized and effective approach for patients. List of figures

[017] The present invention may be better understood based on the attached figures, where: FIGURE 1: Schematic illustration of the steps in the development process of the aptasensor of the present invention. FIGURE 2: Images obtained by Atomic Force Microscopy (AFM) with 1 layer of AuNP for characterization of the aptasensor. FIGURE 3: Scanning electron microscopy (SEM) images obtained for characterization of AuNPs, where A (electrode), B (electrode with AuNPs) and C (electrode with AuNPs and A4 with thiol) at 1000X magnification for characterization. Petition 870250013754, dated 20 / 02 / 2025, page 30 / 48 10 / 18 of the aptasensor surface. FIGURE 4: SEM images obtained for characterization of AuNPs, where A (electrode), B (electrode with AuNPs) and C (electrode with AuNPs and A4 with thiol) at 50,000X magnification for characterization of the aptasensor surface. FIGURE 5: Images obtained using a transmission electron microscope for characterization of AuNPs. FIGURE 6: Images obtained using a transmission electron microscope for characterization of AuNPs. FIGURE 7: Images obtained with the EVOS Microscope to demonstrate the morphology of PC-3 cells. FIGURE 8: Images obtained with the EVOS Microscope to demonstrate the morphology of PC-3 cells. FIGURE 9: Graphical representation of differential pulse voltammetry. FIGURE 10: Histogram representing the mean and standard deviation between the samples analyzed on the electrodes with the control (PBS), the probe (A4thiol aptamer) and the target (PC-3 cell). Description and detailed information on the components of the invention.

[018] The present invention preferably comprises the production of printed carbon electrodes functionalized with gold nanoparticles (AuNps) to anchor the thiolated A4 aptamer, which may be replaced by graphene or carbon nanotubes for application in a biosensor, which could be used in a new diagnostic platform for prostate cancer. The graphite-printed carbon electrodes were produced with carbon and silver ink, pre-conditioned in 0.5 M sulfuric acid and phosphate buffer. They were then functionalized with AuNps, which may be replaced by metallic nanoparticles such as silver or quantum dots, for affinity anchoring of the thiolated A4 aptamer at the 5' end of the sequence. All modification steps of the new platform were performed by dropping and washing in phosphate buffer. Subsequently, characterization was performed by differential pulse voltammetry, which may be replaced by other techniques, such as voltammetry of Petition 870250013754, dated 20 / 02 / 2025, page 31 / 48 / 18 square wave, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Complementary analyses using atomic force microscopy and scanning electron microscopy were performed at each stage of the aptasensor production process. Transmission electron microscopy was also used to characterize the AuNPs. The obtained aptasensor is promising for the rapid detection and monitoring of prostate cancer, since aptamer A4 (SEQ ID N 01) was previously selected and tested as a ligand for prostate tumor cells using the 3D CellSELEX technique; therefore, this aptasensor can identify the presence of circulating prostate tumor cells by liquid biopsy (blood samples), as shown in FIGURE 1. Platform characterization Complementary analyses using atomic force microscopy and scanning electron microscopy were performed at each stage of the aptasensor production process. After immobilization of the thiolated A4 aptamer (SEQ ID N 01) in the electrode working area, bonded to the AuNPs by affinity, a modification occurred on the electrode surface prior to this bonding, which was confirmed by atomic force microscopy and scanning electron microscopy, as shown in FIGURES 2, 3, and 4, respectively. The images generated by transmission electron microscopy revealed the presence of spherical nanoparticles, with an estimated average diameter of 50 nm to 70 nm, which can be visualized in FIGURES 5 and 6. Cell Culture

[019] PC3 cells are a human prostate cancer cell line derived from a bone metastasis of a patient with grade IV prostate adenocarcinoma, exhibit epithelial morphology as shown in FIGURES 7 AND 8, and are characterized by high metastatic potential and used in the investigation of androgen-independent cancer progression. PC3 cells are normally cultured in a medium of Petition 870250013754, dated 20 / 02 / 2025, page 32 / 48 / 18 specialized growth, frequently supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillin and streptomycin) at 37°C with 5% CO2, requiring adherence to a cell culture flask or plate, with regular passages when they reach ideal confluence. PC3 cells lack the functional p53 protein due to a mutation in the TP53 gene, which contributes to their aggressive cancer phenotype and resistance to certain chemotherapeutic agents. The doubling time of PC3 cells varies according to culture conditions, typically between 30 and 48 hours. During thawing, the vials are agitated in a water bath at 37°C until only a small clump of ice remains. After dilution with RPMI medium, the cells are centrifuged to remove the freezing medium and resuspended in growth medium for culture.The initiation of a new PC-3 culture requires a density of 3 x 10⁴ cells / cm². For subcultures, a lower density of 1 x 10⁴ cells / cm² is maintained. For subculture of adherent cells, these are washed with PBS and treated with trypsin. After detachment, the cells are collected by centrifugation, resuspended in RPMI culture medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin), and seeded into new flasks with growth medium. To obtain the cells that will be used as targets in the aptasensor, it is necessary to trypsinize, prepare the cells with trypan blue for viability analysis and cell counting under an EVOS microscope, and resuspend them in PBS to use them as targets for binding the A4 aptamer with a thiol group at the 5' end of the sequence. Electrochemical Characterization

[020] Differential pulse voltammetry (DPV) is a sensitive electrochemical technique used to study the redox properties of substances, providing high resolution in the detection of analytes. To perform differential pulse voltammetry, one begins with the preparation of the sample and the electrolyte solution, choosing a suitable medium for the analysis and dissolving the analyte, if necessary. The electrolyte solution can be, for example, KCl, NaCl or H2SO4, Petition 870250013754, dated 20 / 02 / 2025, page 33 / 48 / 18 depending on the type of redox reaction expected. The analyte concentration must be adjusted according to the experimental conditions. Next, the electrochemical system is assembled, consisting of the working electrode (usually glassy carbon or platinum), the reference electrode (such as Ag / AgCl or SCE), and the auxiliary electrode (usually platinum or graphite), all connected to a potentiostat or other measuring system. The potentiostat is configured to apply a differential pulse wave, where parameters such as the potential range (initial and final), the sweep step, the pulse amplitude, and the pulse frequency are defined. During the measurement, the potentiostat applies a continuous potential to the working electrode and then drives a series of pulses, while recording the current generated at each potential change.The current peak shown in the graph (FIGURE 9) corresponds to the oxidation or reduction reaction of the analyte under study. The peak intensity is directly related to the analyte concentration in the solution, while the potential at which the peak occurs reflects the analyte's redox potential. Data analysis allows for the identification of the analyte (FIGURE 10) and the determination of its concentration. Differential pulse voltammetry is widely used due to its high sensitivity and resolution, being essential for the analysis of substances at low concentrations, and is applied in various fields such as analytical chemistry, biochemistry, and the development of electrochemical sensors. Preparation examples

[021] The data set out below serve to illustrate the implementation of the present invention. Example of preparation 1: Characterization of the platform by atomic force microscopy.

[022] The atomic force microscope (AFM) is a high-precision device used to examine surfaces on a nanometer scale, allowing the observation of structures with atomic resolution. It works through an extremely sharp tip, mounted on a flexible cantilever, which interacts with the surface of the sample. The tip is moved over the sample with the aid of Petition 870250013754, dated 20 / 02 / 2025, page 34 / 48 / 18 of piezoelectric systems, which control their movement with precision. When the tip approaches the surface, atomic interaction forces, such as Van der Waals or repulsive forces, cause the cantilever to deform, and this displacement is detected by an optical system. A laser beam reflects off the back of the cantilever, and the position of the reflected beam is recorded by a detector, allowing the tip's movements to be measured with very high precision. The collected data are used to construct a 3D image of the sample surface, revealing its topography and other microscopic characteristics. For this characterization, 5 pl of AuNPs were dropped onto the electrode's working area, and then it was placed in an oven at 60°C to dry. Immediately afterwards, 5 pl of aptamer A4 with the thiol group at the 5' end (SEQ ID N 01) was added in a humid chamber for 1 hour.Several samples were analyzed in triplicate, the first consisting of the electrode alone, the second the electrode with AuNPs, and the third the electrode with AuNPs and A4-thiol. Preparation example 2: Platform characterization by scanning electron microscopy.

[023] Scanning electron microscopy (SEM) is a technique that uses an electron beam to observe the surface of a sample with very high resolution, allowing detailed analyses of its topography and composition. The process begins with sample preparation, which, to be analyzed, often needs to be coated with a thin layer of conductive material, such as gold or carbon, especially if it is non-conductive, in order to prevent the accumulation of electrical charge during the analysis. In addition, the sample is placed in a vacuum chamber, as the electrons need to travel without being scattered by air molecules, which would occur in environments with atmospheric pressure. Once inside the chamber, the sample is bombarded by an electron beam generated by an emission chamber, usually composed of a tungsten filament or a field emission source (FEG), which emits electrons when heated or when activated by an electric field.The generated electrons are accelerated by an electric field and directed by electromagnetic lenses. Petition 870250013754, dated 20 / 02 / 2025, page 35 / 48 / 18 to form a very thin electron beam, which is focused on the sample. This beam is then systematically scanned over the sample surface in a line-by-line pattern, allowing data to be collected from each point on the sample. When the electron beam hits the sample surface, it interacts with the sample atoms, generating various types of signals, the most important for SEM being secondary electrons and backscattered electrons. Secondary electrons are emitted from the outermost layer of the sample and are used to create images of its topography. The intensity of these electrons depends on the surface structure of the sample, providing a detailed image of its characteristics.Backscattered electrons are those reflected back from the sample and provide information about its chemical composition, as the number of backscattered electrons varies with the atomic number of the elements present. These signals emitted by the sample are detected by specialized sensors, such as the secondary electron (SE) detector for topographic imaging and the backscattered electron (BSE) detector for compositional analysis. The signals are then converted into an electronic signal, which is processed and digitized to create a detailed image of the sample surface. The resulting image is displayed on a monitor and can be analyzed to study characteristics such as roughness, defects, structure, and even chemical properties.Scanning electron microscopy is a powerful tool for obtaining detailed images and data of surfaces at the nanometer level, offering high resolution and the ability to analyze both the morphology and chemical composition of samples, significantly contributing to the understanding of their behaviors and properties. For this characterization, 5 µl of AuNPs were dropped onto the electrode's working area, and then it was placed in an oven at 60°C to dry. Immediately afterwards, 5 µl of aptamer A4 with the thiol group at the 5' end was added in a humid chamber for 1 hour. Several samples were analyzed in triplicate, the first consisting of the electrode alone, the second the electrode with the AuNPs, and the third the electrode with the AuNPs and the... Petition 870250013754, dated 20 / 02 / 2025, page 36 / 48 / 18 A4-thiol. Example of preparation 3: Characterization of AuNPs by transmission electron microscopy (TEM)

[024] The samples to be visualized were mounted on 200 mesh Formvar-coated grids, suitable for transmission electron microscopy, on which the gold nanoparticles from the chemical synthesis were retained. The samples in solution were initially gently homogenized. Next, a volume of 10 pL was used for deposition on the grids, allowing the sample to stand for 3 minutes. Using the tip of a piece of filter paper, the excess liquid was lightly touched to the edge of the grid so that it was absorbed by the paper, and it was kept at room temperature for 8 hours to dry. With the aid of steel tweezers, specific for transmission electron microscopy, the grid was transferred to the sample holder and visualization was carried out using an appropriate voltage acceleration of 80 kV and decreasing the beam intensity to reduce the diameter of the electron beam.After selecting the area to be recorded, a slow scan was performed during image acquisition, resulting in higher quality images with a greater number of pixels. Preparation example 4: Analysis of the shape and size of AuNPs.

[025] Transmission electron microscopy micrographs were analyzed using the ImageJ image processing program. When applying the images to the software, the average diameter of the nanomaterial was measured, which was between 50 nm and 70 nm, with the measurement of the diameter of gold nanoparticles. This analysis allows the characterization of the size and morphology of the gold nanoparticles, which presented a spherical shape. Preparation example 5: Cell Culture

[026] The cultivation of PC-3 cells, a cell line derived from human prostate cancer, requires special care to ensure their proper growth and maintenance. The first step is to prepare the culture environment, including stabilizing the incubator at 37°C with 5% CO2. The medium of Petition 870250013754, dated 20 / 02 / 2025, page 37 / 48 / 18 The most common culture medium for PC-3 cells is RPMI 1640, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). This medium should be warmed to 37°C before use. If the cells are cryopreserved, thawing should be done quickly by placing the vial in a water bath at 37°C. After thawing, the cells are transferred to a 15 mL tube of warm medium and centrifuged at 1000 rpm for 5 minutes to remove the cryoprotectant. After centrifugation, the cells are resuspended in fresh medium and seeded onto the culture plate, generally at a density of 1x10⁴ cells / mL. Once cultured, the cells must be kept under constant monitoring. The culture medium needs to be changed after 24 hours to remove DMSO residue and provide fresh nutrients.Cell growth should be observed with an EVOS microscope, and subculture is performed when cells reach approximately 80% to 90% confluence. For subculture, cells are dissociated with trypsin, incubated at 37°C for 2-4 minutes, and then resuspended in fresh medium. The dissociated cells are subcultured into new flasks. Cell counting should be performed to ensure adequate cell density, using methods such as a hemocytometer or automated counters. Furthermore, continuous monitoring is essential to detect any signs of contamination, such as bacteria, fungi, or mycoplasma, that could compromise the culture. If cells need to be stored long-term, they can be cryopreserved in liquid nitrogen.To do this, it is necessary to prepare a cryoprotective solution with 10% DMSO and 90% fetal bovine serum medium, and freezing should be done slowly, first at 4°C for 2 hours, followed by -80°C overnight and then in liquid nitrogen. To obtain the cells for electrochemical characterization, after centrifugation, they were resuspended in PBS and 5 µl of the cell solution were added to the working surface of the electrode. Example of preparation: Electrochemical characterization

[027] Electrochemical characterization was performed after printing the Petition 870250013754, dated 20 / 02 / 2025, page 38 / 48 / 18 in-house electrodes, their preconditioning, and the construction of the platform with gold nanoparticles (AuNPs), probe immobilization (A4-thiol aptamer), and detection with the target (PC-3 cells) using differential pulse voltammetry (DPV). The graphite carbon printed electrodes were produced with carbon and silver ink, preconditioned in 0.5 M sulfuric acid and phosphate buffer. They were then functionalized with AuNPs for affinity anchoring of the thiolated A4 aptamer at the 5' end of the sequence. All modification steps of the new platform were performed by dripping and washing. Subsequently, characterization was performed by differential pulse voltammetry. For this characterization, 5 pl of AuNPs were dripped onto the electrode's working area, and then it was placed in an oven at 60°C to dry.Immediately after, 5 µl of aptamer A4 with the thiol group at the 5' end was added to a humid chamber for 1 hour and washed with phosphate buffer. Finally, 5 µl of PBS was added to the control group and 5 µl of PC-3 cells resuspended in PBS. All experiments were performed in triplicate. Petition 870250013754, dated 20 / 02 / 2025, pp. 39 / 48

Claims

CLAIMS 1) APTASENSOR FOR THE DIAGNOSIS AND MONITORING OF PROSTATE CANCER characterized by being constructed with printed bioelectrodes modified with nanoparticles and thiolated aptamer A4 at the 5' end (SEQ. ID N 01). 2) APTASENSOR according to claim 1, characterized in that the printed bioelectrodes are printed carbon electrodes functionalized with nanoparticles, which may be replaced by graphene or carbon nanotubes. 3) APTASENSOR according to claim 1, characterized in that the nanoparticles are gold nanoparticles (AuNps) which can be replaced by metallic nanoparticles such as silver or quantum dots. 4) APTASENSOR according to claim 1, characterized by using differential pulse voltammetry as an electrochemical technique for identifying circulating prostate tumor cells, which can be replaced by other techniques such as square wave voltammetry, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Petition 870250077390, dated 08 / 31 / 2025, page 6 / 6

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