1. Introduction
Multidisciplinary research in cell biology and bioengineering over the past two decades has resulted in the development of highly effective in-vitro cell culture platforms that allow the representation of micro-environmental signals (1). As a result, increasingly sophisticated systems that can better represent the essential characteristics of cellular microenvironments are replacing traditional in-vitro models in cell biology systems (2, 3). In this context, the concept of organ-on-a-chip (OOC) has been developed and has been rapidly popularized in the last decade as it can effectively mimic the structure and functional response of human organs (4-7). As such, OOC systems are often considered a substitute for animal-testing studies in cell biology as animal-based pharmaceutical studies often fail to anticipate human pathophysiology (8, 9). However, OOC cell culture analysis is mainly performed using conventional instruments, such as optical measurement methods employing microscopy in conjunction with different staining techniques and the collection of supernatants and cellular samples (10, 11). These conventional methods are labor-intensive, necessitate manual sample collection from the microfluidic system, require high working volumes, and are susceptible to system disruption. Label-free, continuous real-time monitoring of cell functionality is an important technical problem encountered in OOC development.
Several review articles have advocated for the integration of functional tools and sensors within the OOC device for performing real-time cell monitoring. In this regard, numerous methods enabling real-time monitoring without impairing the functionality of OOCs have been proposed in recent years (12-17). High-cost microsensors have also been incorporated (18). Zhang et al. recently developed an automated in-situ monitoring platform for the biophysical (pH, oxygen, and temperature) and biochemical parameters of liver-on-a-chip (LOC) and heart-on-a-chip model systems (19). Sensors have been placed on the device’s top, bottom, and cell culture regions without impairing the device’s performance (18, 20-22). To realize optical imaging, impedance monitoring, and metabolite sensing of live kidney cells, Curto et al. employed organic electrochemical transistors along with microfluidics (23). These sensor components were installed at the system’s glass bottom cover by using traditional microtechnology methods and the multistep lithography process in clean rooms. Such sensor integration methods pose challenges in fabrication as requirements such as the use of functionally acceptable materials, low production costs, and simplicity and speed of manufacturing must be considered.
Three-dimensional (3D) printing is a simple, quick, low-cost, and adaptable printing process that is becoming increasingly popular as a manufacturing strategy in several research domains (24-26). Fused deposit molding (FDM)-based 3D printing is suitable for fabricating integrated electrochemical sensors as it offers greater options in terms of material selection and increased flexibility in designing and fabricating sensors in terms of size and geometry. The 3D printing technology is based on open-source software, which ensures design portability between 3D systems and enables quick prototyping, high fabrication speed, low operational cost, high precision, and uniformity (minimal batch-to-batch variation). Low-cost conductive plastic 3D-printed materials have been used for electrode fabrication for electrochemical sensing of ascorbic acid, picric acid, catechol, dopamine, Zn (II), and Pb (II) (27-31). However, plastic electrodes have been rarely used as sensing platforms integrated with the OOC system.
We established a 3D-printed conductive polylactic acid (PLA) (CP)-based three-electrode sensing system to serve as an integrated amperometric glucose biosensor for the LOC platform. Glucose is an important regulatory parameter influencing cell growth and functions; in addition, it is a clinical indicator of diabetes. The designed glucose biosensor comprises three electrodes: a working electrode (WE) that reacts directly with the solution, a reference electrode (RE) for comparison with the WE, and a counter electrode (CE) for completing the electrical circuit. Generally, electrodes such as Ag/AgCl and platinum are used because the RE should have little current flow, and the CE should not react with the electrolyte. However, manufacturing these electrodes is time-consuming and expensive. In addition, all three electrodes must be integrated into a single material for realizing simple integrated devices. Although in a previous study, glucose detection has been performed based on the catalytic activity of glucose oxidase (GOx) absorbed from the surface of Nafion-coated CP electrodes (32), we further took the liberty to investigate the sensitivity of the existing three-electrode sensing system by coating an additional layer of multi-walled carbon nanotube (MWCNT). MWCNTs have been extensively studied for their excellent electrical properties (33, 34). Chemically modifying the electrode surface by using carbon nanotubes (CNTs) increases the activity of the electrodes when reacting with physiologically active species such as hydrogen peroxide, hydrazine, dopamine, cholesterol, and NADH (35). The direct adsorption of large biomolecules onto the surface of immobilized MWCNT may help achieve a direct electrical connection between support electrodes and the active site of redox enzymes, thus augmenting the sensitivity of three-electrode biosensors. Integrated glucose biosensors can be assembled onto the LOC platform containing three holders, allowing the flexibility of easy placement and removal of the three-electrode system as per the experimental requirements. We developed a liver in-vitro model by using a coaxial 3D extrusion bioprinter using hepatocellular carcinoma (HepG2) as the cell source. Therefore, in this study, we established a LOC platform with integrated CP biosensors capable of measuring glucose through the coating of Nafion/MWCNT/GOx.