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INTRODUCTION The fruit industry employs millions of hectares for production; of these, a significant percentage uses only female or hermaphrodite plants. The selection is carried out by morphological analysis until blooming, which represents an inefficient use of resources. Typically, well-equipped laboratories and trained personnel are required to perform DNA analysis and give valid results1,2. Devices capable of generating this information within a short timespan could contribute to a fast and effective selection of productive plants. Nonetheless, it is necessary to have high sensitivity to differentiate between samples. The objective of this work is the development of an electrochemical and an optical detection system, with high sensitivity and repeatability. With the aim to incorporate the best option in a fully integrated device that ensures an easy-to-use product, safeguarding the correct use by non-trained operators and reducing the time to obtain a result. MATERIALS AND METHODS To evaluate the detection systems a random sequence of DNA (5’-TAGTCGTCAATCCTCCGCTT) was used (50 – 540 ng/µL). The electrochemical detection design is composed by three electrodes (WE-Au, AE-Au, RE-Ag|AgCl) and 0.5 M Ru(NH3)6Cl3 as a DNA intercalating agent3 in 40 mM TAE buffer. Three electrochemical techniques (SWV, DPV and CV) were compared (Bio Logic potentiostat). The optical detection system is composed of a phototransistor (Max. Sens. @ 550 nm), a filter (520 nm) and a LED (Max. WL peak @ 455 nm) to detect the fluorescent DNA intercalator SYBRgreen. Different concentrations of fluorescein (0.005 – 10 µM) were used to evaluate the system capabilities. The system was evaluated with the DNA sequence and SYBRgreen, then compared with a fluorescence microscope (Nikon Eclipse Ti-U). Finally, both systems were tested with plant DNA samples. Genomic material was extracted (CTAB protocol4) form 6 plants (3 female/3 hermaphrodite), amplified by PCR (Thermal cycler miniPCR Amplyus) and analyzed by electrophoresis. RESULTS With the electrochemical detection system, we observed a proportional decrease in the Ru(NH3)6Cl3 signal as the DNA concentration increases, as expected. The data analysis demonstrated that SWV was the most sensitive technique with a R2=0.980, LOD 30.968 ng/µL and LOQ 103.226 ng/µL. Results showed that female plants present an average current of 1.774 μA while hermaphrodite plants had 1.694 μA, allowing us to differentiate hermaphrodite plants accurately with a 90 % confidence interval. On the other hand, in the optical detection system with a R2=0.9568, LOD 2.12 ng/µL and LOQ 7.06 ng/µL, we observed an increase in the signal as de DNA concentration increases. When the optical analysis was compared with the fluorescence microscope results, we observed a similar trend in the signals with a R2=0.995, that validate the functionality of our system. However, the difference between plant samples was detected with only the 70 % confidence interval, female samples showed an average signal of 381 mV while hermaphrodite plants had 352.56 mV. CONCLUSIONS Plant DNA detection was evaluated through two different systems. The optical detection system showed more sensitivity with synthetic samples. However, in the evaluation of plant samples, the electrochemical system is less susceptible to interferences. Therefore, we can differentiate between female and hermaphrodite plants with a 90 % confidence interval. In this way, it represents the best option to evaluate a lab-on-a-chip device. REFERENCES Barrantes-Santamaría W, Loría-Quirós C, Gómez-Alpízar L. Evaluation of two-sex determining systems in papaya plants (Carica papaya) Pococí hybrid. Agron Mesoamerican. 2019;30(2):437-446. doi:10.15517/am.v30i1.34916 Cornelis S. FORENSIC LAB-ON-A-CHIP DNA ANALYSIS. 2019. Li LY, Jia HN, Yu HJ, et al. Synthesis, characterization, and DNA-binding studies of ruthenium complexes [Ru(tpy)(ptn)] 2 + and Ru(dmtpy)(ptn)] 2 +. J Inorg Biochem. 2012;113:31-39. doi:10.1016/j.jinorgbio.2012.03.008 Biosciences. CTAB extraction solution for genomic DNA extraction. Biosciences. 2016:3-7. Figure 1

At present, research and development of a large number of new devices for the detection and quantification of biomedical analytes incorporated into miniaturized platforms of high interest such as organ-on-a-chip (OCC), lab-on-a-chip (LOC), and implantable devices. Together, these systems are characterized by taking continuous measurements for long periods (hours, days, weeks) of the products and/or by-products generated by these platforms[1, 2]. Based on the above, it is essential to detect the failure mechanisms of the systems developed to make improvements in the operational stability of these devices, which could generate biosensing instruments with a longer life span. In this work, the stability of hydrogels was evaluated for the detection of glucose, made up of the branched polyethyleneimine/glucose oxidase (BPEI/GOx) system using ethylene glycol diglycidyl ether (EGDGE) and glutaraldehyde (GA) as crosslinking agents. The stability of these systems was evaluated by light, fluorescence, and electrochemical microscopy (SECM). First, the hydrogels formed were deposited by drop-casting on glass surfaces, gold, and a combination of both (polycrystalline gold chips deposited on glass substrates). The surfaces were treated with different cleaning methodologies and the impact they had on the adhesion of the hydrogels was subsequently evaluated. For their part, the hydrogels were subjected to different conditions such as exposure to aqueous media and potential disturbances to measure their adhesion to the surfaces described. The results showed that, when cleaning the surfaces with the RCA process for 15 minutes, both the hydrogels with EGDGE and GA show better adhesion. Likewise, both hydrogels showed better adherence on gold surfaces compared to glass ones. This can be attributed to electrostatic interactions between the surface and the amino groups present in BPEI and GOx. Subsequently, the spatial arrangement of the BPEI and GOx of both systems was evaluated by fluorescence microscopy and SECM. For this, the GOx was labeled with the fluorophore fluorescein-5-isothiocyanate (FITC) and the BPEI with tetramethylrhodamine-5-Isothiocyanate (5-TRITC). The hydrogels were irradiated with blue, green, and UV light. In the fluorescence images of the controls, unlabeled hydrogels, it was observed that when cross-linked with GA, there was an intense emission of fluorescence at the three wavelengths evaluated. According to the literature, this fluorescence can be attributed to the mechanism proposed by Liu et al.[3] which is based on the formation of Schiff bases from the reaction between aldehydes and amino acid residues. The above-described mechanism was used to confirm the crosslinking reaction between BPEI and enzyme with the crosslinking agent. On the other hand, in hydrogels with EGDGE, heterogeneous structures are seen, in which the distribution of each of the labeled components can be observed. Which facilitated the measurement of stability to time. The spatial arrangement of the enzyme and its stability to time was also evaluated by SECM using the generation of the substrate/collection at the tip (SG/TC) method. A 15 µm Pt UME was used, applying a potential of 0.45 V vs Ag│AgCl to follow the oxidation of H2O2 generated by the enzymatic reaction. As a final step, the concentration of EGDGE and GA was varied to observe the behavior of the hydrogels for the electrochemical detection of different concentrations of glucose in 0.1 M phosphate buffer pH 7.4, following the oxidation of H2O2. Calibration curves were made by chronoamperometry of solutions with concentrations of 0-50 mM of glucose. For EGDGE, the concentration of the solutions ranged from 7.5 to 20%. The results obtained showed that the variation in the concentration of the crosslinker does not affect the diffusion of glucose or H2O2 through the hydrogel and therefore the currents obtained are very similar. In the case of GA, the concentration of the solutions ranged from 4.5 to 0.45%. In this system, it was observed that the concentration of crosslinker significantly affects the diffusion of glucose and/or H2O2 through the hydrogel, reflecting on the currents obtained. In this way, it was determined that the GA concentration of 0.45% allows efficient diffusion through the hydrogel, allowing an increase in the current as of the glucose concentration increases. References Lopez GA, Estevez M-C, Soler M, Lechuga LM (2017) Recent advances in nanoplasmonic biosensors: applications and lab-on-a-chip integration. Nanophotonics 6:123–136. https://doi.org/doi:10.1515/nanoph-2016-0101 Rebelo R, Barbosa AI, Correlo VM, Reis RL (2021) An Outlook on Implantable Biosensors for Personalized Medicine. Engineering. https://doi.org/https://doi.org/10.1016/j.eng.2021.08.010 Liu SG, Li N, Ling Y, et al (2016) pH-Mediated Fluorescent Polymer Particles and Gel from Hyperbranched Polyethylenimine and the Mechanism of Intrinsic Fluorescence. Langmuir 32:1881–1889. https://doi.org/10.1021/acs.langmuir.6b00201

Thiol-based self-assembled monolayers (SAMs) have been widely characterized on gold surfaces, with the desorption potentials of these SAMs being of special interest for nanotechnology and biological applications. However, the study and determination of desorption potentials of thiol SAMs on platinum surfaces has been limited by the hydrogen evolution reaction (HER) catalyzed by the metal, which dominates response signals when studying the system through conventional electrochemical techniques. Through the coupling of electrochemical techniques with fluorescence microscopy, the problem of the HER signal can be avoided in the study of desorption potentials for the platinum-thiol monolayer systems. By using fluorescent thiols, as well as monocrystalline platinum spheres, the desorption potential for different crystallographic orientations and thiol length combinations can be determined. The results obtained show that the desorption of Bodipy C10SH thiols from platinum surfaces occur at more positive potentials in [111] crystallographic orientations, and at more negative potentials at [100] ones. On the other hand, no notable difference was found in the desorption order of Bodipy C16SH from these two orientations. However, the use of fluorescence microscopy for the solution of this problem poses other challenges: the time the thiols need to separate enough from the metallic substrate to not be affected by quenching, as well as the facts that thiols fluoresce less as they dilute and the existing spectral overlap for the molecules, adds difficulties to the establishment of the desorption potentials. Through COMSOL Multiphysics simulations, we try to simulate the systems hereby proposed of platinum with thiol-based SAMs. Using them, the desorption potentials, as well as the monolayer coverage under specific conditions, can be determined. We expect that, with further development of the model, more specific values for these variables can be determined. Figure 1