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.