(E-193) 3D Printed Catheter Platform for Minimally Invasive, Continuous, Real-time Monitoring of Pharmacokinetics in Mice in vivo

As the administration of pharmaceuticals continues to increase, the ability to accurately and continuously monitor analytes in vivo has never been more important. To meet this need, increasingly sophisticated methods of detection, from optical to electrochemical sensors, in biological mediums ranging from blood to cerebrospinal fluid have emerged within the past several decades (Rong et al., 2017). One such method gaining attention is the use of electrochemical, aptamer-based sensors (E-ABs), which utilize the highly specific and reversible binding-induced conformational changes between aptamers and their targets for the real-time detection of a diverse array of compounds, such as antibiotics or chemotherapeutics (Shaver et al., 2022). Already, E-ABs have been used to accurately detect concentrations of doxorubicin, vancomycin, and tobramycin in vivo (Arroyo-Currás et al. 2017, Wu et al., 2022). 

Despite their continuous, sub-second resolution and multi hour stability, current in vivo E-ABs are difficult to fabricate and require complex surgical implantation, making testing of E-ABs in vivo a time-consuming and unreliable process (Figure 1). Herein, we report an open-source, novel method for the manufacturing of E-ABs using stereolithography (SLA) for minimally invasive, continuous monitoring of vancomycin in the jugular vein of mice. Specifically, our device consists of a two-part system; A catheter that is inserted intravenously using a 22G catheter needle, and an insert which houses the electrodes and is slipped into the catheter (Figure 2). 

E-ABs detect the local concentration of a target by analyzing the rate of electron transfer (eT) between the redox reporter attached to the distal end of the surface-bound aptamer – here Methylene blue (MB) – and the gold surface of the working electrode. The rate of exchange depends on the distance between the redox reporter and the surface. Upon binding to the target, the aptamer experiences a conformational change that shifts MB closer to the surface, therefore increasing the rate of eT between the reporter and surface. This change is reflected by a shift in current under the interrogation of square wave voltammetry (SQV), and proportional to the concentration of analyte. Using this principle, the concentration of target molecules may be approximated (Shaver et al. 2022). 

3D Printing and Curing Procedure:

All 3D printing was done using a Formlabs 3B+ printer using Formlabs High Temp V2 resin. 

The pre-cured catheter is washed in isopropanol for 10 minutes. A 22G needle is passed through the catheter to ensure all excess resin is removed. Then, the catheter is cured at 60ºC for 60 minutes.

The two halves of the pre-cured insert mold are washed in isopropanol for 20 minutes. The molds are then briefly rinsed with soap and water to remove any excess resin. The molds are then sonicated for 10 minutes. Afterwards, the molds are cured in deionized water at 60ºC for 60 minutes. Finally, the molds are left to cure outside of water at 60ºC for 30 minutes.  

Silicone Mold Procedure: 

A 1:1 ratio of silicone rubber mix is poured in the resin molds, then cured for 4 hours. After removal from the resin molds, a 20 μL pipette tip is inserted into the groove of the mold to act as an air vent. A 50 μm gold wire is passed through the insert groove, then both halves are sandwiched together using binder clips. Resin is pipetted at the entrance of the mold, then left for 15 minutes. Complete filling of resin is indicated by an accumulation of resin in the vent hole. The resin is then cured inside of the silicone mold at 60ºC for 30 minutes. Afterwards it is removed from the mold, then cured for at 60ºC for an additional 30 minutes. 

A 3D printed catheter with an outer diameter (OD) of 1.30 mm, inner diameter (ID) of 0.80 mm, and height of 33 mm was achieved (Figure 5). In vivo tests were not performed; however, our device is similar in size to industry-standard intravenous catheters and utilizes biocompatible resin, suggesting successful insertion in mice. 

A 3D printed insert was unachievable with our available 3D printer. Although a hollow cylinder of sub-millimeter size could be realized with more advanced printers, such printers are costly and not readily accessible. Instead, using a molding process, an electrode-embedded, resin-poured insert was fabricated from a negative silicone mold that was cured using a 3D printed positive template (Figure 5). The insert is 0.80 mm in diameter and is 30 mm in height, with an 8 mm long stopper. 

Although only the working electrode was cured inside of the resin, our results show a promising first step that may be translated to an insert with 3 electrodes. Further work will focus on incorporating the counter and reference electrodes into the device and simplifying the overall procedure, allowing for simple and accessible in vivo E-AB sensor fabrication.

Rong, G., Corrie, S. R., & Clark, H. A. (2017). In vivo biosensing: Progress and perspectives. ACS Sensors, 2(3), 327–338. https://doi.org/10.1021/acssensors.6b00834  

Shaver, A., Mahlum, J. D., Scida, K., Johnston, M. L., Aller Pellitero, M., Wu, Y., Carr, G. V., & Arroyo-Currás, N. (2022). Optimization of vancomycin aptamer sequence length increases the sensitivity of electrochemical, aptamer-based sensors in vivo. ACS Sensors, 7(12), 3895–3905. https://doi.org/10.1021/acssensors.2c01910  

Arroyo-Currás, N., Somerson, J., Vieira, P. A., Ploense, K. L., Kippin, T. E., & Plaxco, K. W. (2017). Real-time measurement of small molecules directly in awake, ambulatory animals. Proceedings of the National Academy of Sciences, 114(4), 645–650. https://doi.org/10.1073/pnas.1613458114  

Wu, Y., Tehrani, F., Teymourian, H., Mack, J., Shaver, A., Reynoso, M., Kavner, J., Huang, N., Furmidge, A., Duvvuri, A., Nie, Y., Laffel, L. M., Doyle, F. J., Patti, M.-E., Dassau, E., Wang, J., & Arroyo-Currás, N. (2022). Microneedle aptamer-based sensors for continuous, real-time therapeutic drug monitoring. Analytical Chemistry, 94(23), 8335–8345. https://doi.org/10.1021/acs.analchem.2c00829  

Dauphin-Ducharme, P., Ploense, K. L., Arroyo-Curras, N., Kippin, T. E., & Plaxco, K. W. (2022). Electrochemical Aptamer-Based Sensors: A Platform Approach to High-Frequency Molecular Monitoring In Situ in the Living Body. In Biomedical Engineering Technologies (Vol. 1, Ser. Methods in Molecular Biology, pp. 479–492). essay, Springer US.