Disposable 3D Printed Electrode for the Electrochemical Detection of Delta-9-Tetrahydrocannabinol in Aqueous Solution and 11-Nor-9-Carboxy-Tetrahydrocannabinol in Saliva

Currently, marijuana or cannabis is the most consumed drug worldwide, and is also the one with the highest number of seizures, with 354 tons seized in Brazil in 2017. The increase in its consumption has been called attention, not only by the authorities, due to accidents caused when driving under the influence of the drug, but also by companies that carry out toxicological analyzes on their employees. For this, rapid tests, such as colorimetric tests, are generally used to analyze the urine of users. The need for agile and proper identification of drugs of abuse has encouraged the scientific community to improve and to develop new methodologies. Electrochemical methods appear as a cheap, portable and easy to use alternative. The use of biodegradable materials for the development of disposable electrodes has become paramount today. The use of 3D printers emerges as an alternative for the manufacture of these electrodes and the use of saliva instead of urine, has also been a priority because it is a less invasive sample and difficult to adulterate. In this work, a three dimensional device was created in an innovative way containing the three electrodes, working, counter and reference, in conductive material. The device was then used to detect the active principle present in cannabis, delta-9-tetrahydrocannabinol, in aqueous solution, and its metabolite, 11-nor-9-carboxy-THC, in samples of real saliva, both through cyclic voltammetry. The positive results show that the proposed device can be used, as well as those already found on the market, for the identification of drug use and in the forensic field. 522 Brazilian Journal of Forensic Sciences, Medical Law and Bioethics 9(4):521-533 (2020) E. N. Oiye et al.


Introduction
The history of mankind has been accompanied by the use of psychoactive substances, either for medicinal purposes or as an element of religious rites.
Currently, the term drug, in its more restricted use, refers to illicit drugs in general, among which are cocaine, crack, LSD, ecstasy and marijuana, which is present in almost all related events with drug trafficking 1,2 .
Marijuana, or cannabis, has the main psychoactive substance Δ9-THC (delta 9-THC, short for delta-9-tetrahydrocannabinol). Its main forms of presentation are as herbs (in one piece or crushed) or resins (hashish), whose concentration of Δ9-THC is higher. It can also be found in the form of a cigarette, ready to use, whose consumption can cause euphoria and hallucinations [3][4][5][6] .
According to the World Drug Report of 2019, cannabis trafficking is concentrated in the Americas, accounting for 60% of global seizures, with 38% being concentrated in South America 7  In addition to the analysis of the seized marijuana, there is also the need for the analysis of biological fluids, such as urine and saliva, to detect its metabolites, in order to confirm its consumption 9 . The highest concentrations of Δ9-THC and its main metabolite, 11-nor-9-carboxy-THC (also known as 11-COOH-THC), can be found in biological fluids, as plasma and oral fluid, between 3 and 6 hours after consumption, but its stay in the human system for several days [9][10][11] .
The use of saliva for the verification of drug use allows a less invasive sample collection and increases the difficulty of adulterating the sample 12-14 , being progressively more prioritized by government authorities, in cases of traffic accidents under the influence of such substances 15 , in monitoring of drug addicts 16 , as a clinical toxicology, to control the use of psychotropic substances in employees 17 , and even in sports, with anti-doping tests 18 .
Improving police officers' working conditions to identify the use of narcotics is crucial for driver control. Thus, the development of simple and specific drug detection methods, which can confirm at the scene of the occurrence if the illegal substance was used, is a goal to be pursued to provide tools that facilitate the investigative work of the police.
Most of the preliminary tests used today, for field analysis, employ immunoassays, in which color changes are obtained for certain substances. These tests are easy to produce, cheap, fast and portable, but are mostly applied to urine 14,16,19 .
Despite the practicality and low cost of colorimetric analysis, electrochemical sensors are a more versatile alternative 20,21 , bringing together characteristics such as: 1) light and portable; 2) low cost; 3) have excellent reproducibility with high sensitivity and precision; and 4) it does not require the presence of a professional specialized in its use or instruments of complicated operation 21 .
Electrochemical techniques such as amperometric, potentiometric and voltammetric techniques have been frequently used in the last two decades for the detection and quantification of samples of forensic and pharmacological interest [22][23][24][25][26] .
The literature involving voltammetric determination of Δ9-THC comprehends the use of electrodes with and without chemical modification. There are methodologies for its detection when in seized samples [26][27][28][29][30] , and in saliva 31,32 . However, it should be noted that in the literature only a few studies with artificial saliva have been reported for the electrochemical detection of Δ9-THC 33 .
When it comes to the development of fast, portable and low-cost chemical analysis devices, it is worth mentioning the use of disposable electrodes made in a 3D printer. These printers, initially created to supply needs in the field of engineering, have gained notoriety in other areas due to their speed of manufacture and the reduced use of raw materials, making it interesting for researchers and industry, especially due to its low cost and large-scale manufacturing [34][35][36] .
Despite the advantages of using electrochemical techniques, even considering this new possibility of using polymer as an electrode, there are few studies in the literature, involving the analysis of substances of forensic interest in biological matrix 37 .
Taking this into account, the present work proposes a methodology for the detection of ∆9-THC, in aqueous solution, and its metabolite, 11-COOH-THC, in saliva, using a polymeric material device (polylactic acid) printed on a 3D printer, containing the three electrodes, reference, auxiliary and working, and using the cyclic voltammetry methodology for voltammetric analyzes.

Three-dimensional printed electrode (3DE)
The electrode design was developed using the Inventor (Autodesk) software and printed using a GTMax printer (model A1), with two extruder (0.45 mm nozzle size) at a temperature of 190 °C. The brown PLA filament was used as base and the black conductive PLA filament was used to draw the reference, counter and working electrodes. The printing established through the Simplify3D software.
The electrode design was based on the screen-printed electrodes already found on the market, maintaining the circular shape for the working electrode, while the reference and counter electrodes had their shape modified, Figure 1. After printing, the reference electrode was covered with a layer of silver ink, thus being considered a pseudo reference electrode. Also, to prevent the analytical solution from coming into contact with the electrical contacts, a barrier, in the form of a step, was added to the shape of the electrode.

Standard solutions and samples
For the analysis of ∆9-THC, the solutions were prepared in aqueous medium of It should be noted that the analysis of the "sample blank" was also carried out.
This blank is the saliva sample diluted in the solvent used to prepare the analyte solution, in this case, methanol (Merck, HPLC grade).
Deionized water was used to prepare all aqueous solutions.

Voltammetric analysis
Voltammetric analyzes were performed on µAutolab III (Metrohm), using NOVA 1.11 software. The cyclic voltammetry was performed for the analysis of ∆9-THC and its metabolite 11-COOH-THC, with the objective of detecting it, that is, it focused only on the presence of voltammetric response for the analyte.

Results and discussion
As already mentioned, the scientific police and some companies have as priority only the identification of drug use, and not the quantification of the dosage administered by the user. Devices found on the market, such as immunochromatographic tests, have this purpose, that is, they do not have quantitative information for analysis 14,19 .
Since the highest concentrations of ∆9-THC are found between 1 and 2 hours after the use of marijuana, it is necessary to take this time into account when using saliva as a sample medium. There is an important economic approach for the device, once it reduces in 75% the cost of a screen printed electrode when compared to a commercial one.
The registered patent, PI 1104489-6, describes an oral fluid analysis methodology for detecting drug abuse, including ∆9-THC. It is a micropotentiostat designated with more than 2 working electrodes whose purpose is the simultaneous detection of one or more drugs, using disposable electrodes. However, the results are not presented with the voltammogram records. The apparatus only confirms or not the presence of a drug 38 .

∆9-THC analysis
After the 3DE has been printed and the silver ink has completely dried, it was Previous studies report that the presence of the phenolic group in the Δ9-THC structure results in the observed anodic response 26,29 . The reaction involved is represented in Figure 3.   The analysis of ∆9-THC added in saliva did not present any peak, so the application of the sensor is effective just for seized samples. The advantage is the low cost system applied in aqueous solution with low cost reagents.

Analysis of 11-COOH-THC
Since interest is the indication for the use of marijuana, the detection of its metabolite is essential. After a few hours of cannabis use, the concentration of its metabolite in saliva, 11-COOH-THC, is already high enough to be detected. 9 To simulate a real sample, the use of real saliva fortified with the standard solution of the 11-COOH-THC was necessary to reproduce the condition of a marijuana user as much as possible. We opted for real saliva instead of artificial saliva to give more veracity to the experiment.
The same experimental conditions performed in the detection of Δ9-THC were then used to detect its metabolite. It is possible to observe the appearance of an oxidative peak again, but now in a region of different potential, approximately 0.55 V ( Figure 4). The structural difference between the two analytes is only in the carboxylic group, as shown in the Figure 5 below. Therefore, the oxidative reaction of the metabolite, observed in voltammetry, should probably occur in the phenolic group, just as it does for Δ9-THC. The difference in peak potential could be explained due to this structural difference in the molecule.
The difference of approximately 0.45 V between the peak potentials for both analytes can also indicate that the proposed methodology is able to identify them when present in the same sample.

Conclusions
The results presented showed that it is possible to build, in a simple and practical way, an analytical device with PLA material in a 3D printing, being biodegradable and low cost. Still, as an innovation, the device was built containing the working, reference and counter electrodes, format not yet found in the literature for this type of material.
With the proposed device, it was possible to apply it for the detection of Δ9-THC in aqueous media, that can be used for analysis of seized samples, and also for the analysis of its metabolite, 11-COOH-THC, in real saliva samples, being possible to indicate the cannabis use. Those results demonstrate that the proposed device and methodology can be used for the same goal as immunoassays for forensic purposes.