Development of a Lab on a Chip Prototype for Sex Detection in Plants

Abstract

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, waiting from 10 to 16 weeks 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 results [1-2]. Devices capable of generating this information within a short timespan could contribute to a fast and effective selection of productive plants, that translates in resource savings and an increase in production. In this work, the implementation of an alternative DNA analysis in a single chip is evaluated. The objective is the development of a fully integrated device which incorporates all sequential steps to evaluate vegetal material, that ensure an easy to use product, safeguarding the correct use by non-trained operators and reducing the time to obtain a result. This will cover a chip design able to take the sample, contain the extraction and amplification of genetic material, and the evaluation of two different detection systems (optical and electrochemical). MATERIALS AND METHODS The chip was fabricated in a 3 mm thick acrylic PMMA with a CNC micromilling equipment. Each chip is composed of four cavities (15 to 30 µL) and connected by channels (width 300 – 100 µm, height 150 µm). After the addition of the reactants, are sealed with adhesive double-sided tape and a 1 mm thick PDMS membrane. The electrochemical detection design is composed by three electrodes (WE-Au, AE-Au, RE-Ag|AgCl), 0.5 M Ru(NH3)6Cl3 as a DNA intercalating agent [3] in 40 mM TAE buffer. A random squence of DNA (5’-TAGTCGTCAATCCTCCGCTT) (50 – 540 ng/µL) was tested to evaluate the efficiency of the system by three electrochemical techniques (SWV, DPV and CV) in a BioLogic potentiostat. On the other hand, the optical detection system is composed of a phototransistor (Max. sensitivity @ 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. Then, the system was evaluated with the same DNA sequence and compared with a fluorescence microscope (Nikon Eclipse Ti-u). RESULTS In the electrochemical detection system, we observed a proportional decrease in the Ru(NH3)6Cl3 signal as the DNA concentration increase. The data showed that square wave voltammetry was the most sensitive technique (R2=0.9372, LOD 158.01 ng/µL and LOQ 722.39 ng/µL), although the three were adequate for detection. In the optical detection system, we observed a similar tendency in the signals (LOD 111.50 ng/µl) compared to the microscope results (LOD 94.16 ng/µL) showing a R2=0.982, validating the functionality of our system. Current results show that the optical system is more sensitive and could let us differentiate smaller concentrations of DNA. CONCLUSIONS In this work a lab on a chip prototype was designed, fabricated and tested. The chips are disposable and need minimal human intervention after the sampling. It’s capable of performing DNA detection through two different systems, however, the optical detection system showed more sensitivity. Nevertheless, the evaluation of vegetal material is needed to further validations. REFERENCES [1] Walter Barrantes, Carlos Lorìa, Luis Gòmez, “Evaluation of two-sex determining systems in papaya plants (Carica papaya) Pococì hybrid”, Experimental Station Fabio Baudrit, Costa Rica University, Costa Rica, 2019.[2] Cornelis Senne, “Forensic Lab on a Chip DNA analysis”, Laboratory of Pharmaceutical Biotechnology, Department of life Science Technologies, Imec, Ghent University. Belgium, 2019.[3] Lü-Ying Li, Hai-Na Jia, Hui-Juan Yu, Ke-jie Du, “Synthesis characterization, and DNA binding studies of ruthenium complexes [Ru(tpy)(ptn)]2+ and Ru(dmtpy)(ptn)]2+. KEYWORDS: microfluidics, electrochemical detection, optical detection, fluorescent dye, DNA. Figure 1