Electrokinetic Manipulation of Biological Cells towards Biotechnology Applications.

The presented study demonstrates the capability of the template-based electrokinetic assembly (TEA) and guidance to manipulate and capture individual biological cells within a microfluidic platform. Specifically, dielectrophoretic (DEP) focusing of K-562 cells towards lithographically-defined "wells" on the microelectrodes and positioning singles cells withing these "wells" was demonstrated. K-562 lymphoblast cells, are widely used in immunology research. The DEP guidance, particularly involving positive DEP (pDEP), enables the controlled guidance and positioning of conductive and dielectric particles, including biological cells, opening new directions for the accurate and efficient microassembly of biological entities, which is crucial for single cell analysis and other applications in biotechnology. The investigation explores the use of glassy carbon and gold as electrode materials. It was established previously that undiluted physiological buffer is unsuitable for inducing positive DEP (pDEP); therefore, the change of media into a lower ionic concentration is necessary. After pDEP was observed, the cells are resubmerged in the Iscove's modified Dulbecco's medium (IMEM), a cell culturing media, and incubated. A dead/alive staining assay was performed on the cells to determine their survival in the diluted buffer for the period required to capture them. The staining assay confirmed the cells' survival after being immersed in the diluted biological buffer necessary for electrokinetic handling. The results indicate the promise of the proposed electrokinetic bio-sorting technology for applications in tissue engineering, lab-on-a-chip devices, and organ-on-a-chip models, as well as contributing to the advancement of single cell analysis.

Nitrogen-functionalized graphene quantum dot incorporated GelMA microgels as fluorescent 3D-tissue Constructs.

Biopolymer microgels present many opportunities in biomedicine and tissue engineering. To understand their in vivo behavior in therapeutic interventions, long-term monitoring is critical, which is usually achieved by incorporating fluorescent materials within the hydrogel matrix. Current research is limited due to issues concerning the biocompatibility and instability of the conventional fluorescent species, which also tend to adversely affect the bio-functionality of the hydrogels. Here, we introduce a microfluidic-based approach to generate nitrogen-functionalized graphene quantum dot (NGQD) incorporated gelatin methacryloyl (GelMA) hydrogel microspheres, capable of long-term monitoring while preserving or enhancing the other favorable features of 3D cell encapsulation. A multilayer droplet-based microfluidic device was designed and fabricated to make monodisperse NGQD-loaded GelMA hydrogel microspheres encapsulating skeletal muscle cells (C2C12). Control over the sizes of microspheres could be achieved by tuning the flow rates in the microfluidic device. Skeletal muscle cells encapsulated in these microgels exhibited high cell viability from day 1 (82.9 ± 6.50%) to day 10 (92.1 ± 3.90%). The NGQD-loaded GelMA microgels encapsulating the cells demonstrated higher metabolic activity compared to the GelMA microgels. Presence of sarcomeric α-actin was verified by immunofluorescence staining on day 10. A fluorescence signal was observed from the NGQD-loaded microgels during the entire period of the study. The investigation reveals the advantages of integrating NGQDs in microgels for non-invasive imaging and monitoring of cell-laden microspheres and presents new opportunities for future therapeutic applications.

Dissolvable Calcium Alginate Microfibers Produced via Immersed Microfluidic Spinning.

Fabrication of micro- and nanofibers are critical for a wide range of applications from microelectronics to biotechnology. Alginate microfibers with diameters of tens to hundreds of microns play an important role in tissue engineering and fibers of these diameters are impossible to fabricate via electrospinning and can only be produced via fluidic spinning. Typically, microfluidic spinning based on photopolymerization produces fibers that are not easily dissolvable, while fluidic spinning with chemical cross-linking employs complex setups of microfabricated chips or coaxial needles, aimed at precise control of the fiber diameter; however, fluidic spinning introduces significant cost and complexity to the microfluidic setup. We demonstrate immersed microfluidic spinning where a calcium alginate microfiber is produced via displacement of alginate solution through a single needle that is immersed in a cross-linking bath of calcium chloride solution. The resulting diameter of the fiber is characterized and the fiber diameter and topology of the deposited fiber is related to the concentration of the alginate solution (2 wt%, 4 wt%, and 6 wt%), needle gauge (30 g, 25 g, and 20 g), and the volumetric flow rate of the alginate solution (1 mL/min, 2 mL/min, and 2.7 mL/min). The resulting fiber diameter is smaller than the internal diameter of the needle and this dependence is explained by the continuity of the flow and increased rate of fall of the liquid jet upon its issuing from the needle. The fiber diameter (demonstrated diameter of fibers range from 100 microns to 1 mm) depends weakly on the volumetric flow rate and depends strongly on the needle diameter. It also seems that for a smaller needle size, a greater concentration of alginate results in smaller diameter fibers and that this trend is not evident as the needle diameter is increased. In terms of topology of the deposited fiber, the higher wt% alginate fiber produces larger loops, while smaller wt% alginate solution yields a denser topology of the overlaid fiber loops. These fibers can be dissolved in DMEM/EDTA/DSC solution in 20-30 min (depending on the fiber diameter), leaving behind the hollow channels in the hydrogel matrix. We believe that the demonstrated simple setup of the immersed microfluidic spinning of the calcium alginate microfibers will be useful for creating tissue constructs, including the vascularized tissue implants.

Integrating Bio-Sensing Array with Blood Plasma Separation on a Centrifugal Platform.

Numerous immunoassays have been successfully integrated on disc-based centrifugal platforms (CDs) over the last 20 years. These CD devices can be used as portable point-of-care (POC) platforms with sample-to-answer capabilities where bodily fluids such as whole blood can be used as samples directly without pre-processing. In order to use whole blood as a sample on CDs, centrifugation is used to separate red blood cells from plasma on CDs. There are several techniques for using specific fluidic patterns in the centrifugal fluidic network, such as reciprocation, that enhances the sensitivity of the immunoassays, including those using microarray antigen membranes. Present work demonstrates, for the first time, simultaneous integration of blood plasma separation (BPS) and reciprocation on the CD platform. The integrated design allows plasma that is separated from the red blood cells in a sedimentation chamber to flow into the reciprocation chamber via a narrow connecting channel of 0.5 mm × 0.5 mm cross-section. Due to the small cross-section of the connecting channel, there is no inflow of the red blood cell into the reciprocation chamber during subsequent fluidic operations of the CD. While no inflow of the red blood cells into the reciprocation chamber was observed, the conditions of 20 g jerk acceleration were also simulated in ANSYS finite element analysis software, and it was found that the CD design that was used is capable of retaining red blood cells in the sedimentation chamber. Experimentally, the isolation of red blood cells in the sedimentation chamber was confirmed using the ImageJ image processor to detect the visible color-based separation of the plasma from the blood. A fluorescent analyte testing on the bio-sensing array of the presented novel integrated design and on the standard reciprocation design CD was conducted for 7 min of reciprocation in each case. The test analyte was Europium Streptavidin Polystyrene analyte (10-3 mg/mL) and the microarray consisted of Biotin bovine serum albumin (BSA) dots. The fluorescent signals for the standard and integrated designs were nearly identical (within the margin of error) for the first several minutes of reciprocation, but the fluorescent signal for the integrated design was significantly higher when the reciprocation time was increased to 7 min.

Microvalves for Applications in Centrifugal Microfluidics.

Centrifugal microfluidic platforms (CDs) have opened new possibilities for inexpensive point-of-care (POC) diagnostics. They are now widely used in applications requiring polymerase chain reaction steps, blood plasma separation, serial dilutions, and many other diagnostic processes. CD microfluidic devices allow a variety of complex processes to transfer onto the small disc platform that previously were carried out by individual expensive laboratory equipment requiring trained personnel. The portability, ease of operation, integration, and robustness of the CD fluidic platforms requires simple, reliable, and scalable designs to control the flow of fluids. Valves play a vital role in opening/closing of microfluidic channels to enable a precise control of the flow of fluids on a centrifugal platform. Valving systems are also critical in isolating chambers from the rest of a fluidic network at required times, in effectively directing the reagents to the target location, in serial dilutions, and in integration of multiple other processes on a single CD. In this paper, we review the various available fluidic valving systems, discuss their working principles, and evaluate their compatibility with CD fluidic platforms. We categorize the presented valving systems into either "active", "passive", or "hybrid"-based on their actuation mechanism that can be mechanical, thermal, hydrophobic/hydrophilic, solubility-based, phase-change, and others. Important topics such as their actuation mechanism, governing physics, variability of performance, necessary disc spin rate for valve actuation, valve response time, and other parameters are discussed. The applicability of some types of valves for specialized functions such as reagent storage, flow control, and other applications is summarized.

Electrified lab on disc systems: A comprehensive review on electrokinetic applications.

Many advanced microfluidic Lab-on-disc (LOD) devices require an on-board power supply for powering active components. LODs with an on-board electrical power supply are called electrified-LODs (eLODs) and are the subject of the present review. This survey comprises two main parts. First, we discuss the different means of delivering electrical energy to a spinning disc including slip-ring, wireless power transmission, and on-board power supply. In the second part, we focus on utilizing electrical power on eLODs for three electrokinetic microfluidic processes: electrophoresis, electroosmotic flow, and dielectrophoresis. Electrokinetic phenomena enable propulsion, separation, and manipulation of different fluids and various types of microparticles/cells. We summarize the theoretical and experimental results for all three electrokinetic phenomena enacted on centrifugal platforms. While extensive numerical modeling and experimental research are available for electrokinetics on stationary platforms, there is a noticeable lack of development in this area when executed on rotating platforms. The review concludes by comparing the strengths and weaknesses of different electrokinetic techniques implemented on centrifugal platforms, and additionally, the most promising applications of electrokinetic-assisted eLOD devices are singled out.

Elastic membrane enabled inward pumping for liquid manipulation on a centrifugal microfluidic platform.

Nowadays, centrifugal microfluidic platforms are finding wider acceptance for implementing point-of-care assays due to the simplicity of the controls, the versatility of the fluidic operations, and the ability to create a self-enclosed system, thus minimizing the risk of contamination for either the sample or surroundings. Despite these advantages, one of the inherent weaknesses of CD microfluidics is that all the sequential fluidic chambers and channels must be positioned radially since the centrifugal force acts from the center of the disk outward. Implementation of schemes where the liquid can be rerouted from the disk periphery to the disk center would significantly increase the utility of CD platforms and increase the rational utilization of the real estate on the disk. The present study outlines a novel utilization of elastic membranes covering fluidic chambers to implement inward pumping whereby the fluid is returned from the disk periphery to the center of the disk. When the disk revolves at an angular velocity of 3600 rpm, liquid enters the chamber covered by the elastic membrane. This membrane is deflected upward by liquid, storing energy like a compressed spring. When the angular velocity of the disk is reduced to 180 rpm and thus the centrifugal force is diminished, the elastic membrane pushes the liquid from the chamber inward, closer to the center of the disk. There are two channels leading from the elastic membrane-covered reservoir-one channel has a higher fluidic resistance and the other (wider) has a lower fluidic resistance. The geometry of these two channels determines the fluidic path inward (toward the center of the disk). Most of the liquid travels through the recirculating channel with lower resistance. We demonstrated an inward pumping efficiency in the range of 78%-89%. Elastic membrane-driven inward pumping was demonstrated for the application of enhanced fluid mixing. Additionally, to demonstrate the utility of the proposed pumping mechanism for multi-step assays on the disk, we implemented and tested a disk design that combines plasma separation and inward pumping.

Artificial Intelligence Algorithms Enable Automated Characterization of the Positive and Negative Dielectrophoretic Ranges of Applied Frequency.

The present work describes the phenomenological approach to automatically determine the frequency range for positive and negative dielectrophoresis (DEP)-an electrokinetic force that can be used for massively parallel micro- and nano-assembly. An experimental setup consists of the microfabricated chip with gold microelectrode array connected to a function generator capable of digitally controlling an AC signal of 1 V (peak-to-peak) and of various frequencies in the range between 10 kHz and 1 MHz. The suspension of latex microbeads (3-µm diameter) is either attracted or repelled from the microelectrodes under the influence of DEP force as a function of the applied frequency. The video of the bead movement is captured via a digital camera attached to the microscope. The OpenCV software package is used to digitally analyze the images and identify the beads. Positions of the identified beads are compared for successive frames via Artificial Intelligence (AI) algorithm that determines the cloud behavior of the microbeads and algorithmically determines if the beads experience attraction or repulsion from the electrodes. Based on the determined behavior of the beads, algorithm will either increase or decrease the applied frequency and implement the digital command of the function generator that is controlled by the computer. Thus, the operation of the study platform is fully automated. The AI-guided platform has determined that positive DEP (pDEP) is active below 500 kHz frequency, negative DEP (nDEP) is evidenced above 1 MHz frequency and the crossover frequency is between 500 kHz and 1 MHz. These results are in line with previously published experimentally determined frequency-dependent DEP behavior of the latex microbeads. The phenomenological approach assisted by live AI-guided feedback loop described in the present study will assist the active manipulation of the system towards the desired phenomenological outcome such as, for example, collection of the particles at the electrodes, even if, due to the complexity and plurality of the interactive forces, model-based predictions are not available.

Capillary Flow-Driven and Magnetically Actuated Multi-Use Wax Valves for Controlled Sealing and Releasing of Fluids on Centrifugal Microfluidic Platforms.

Compact disc (CD)-based centrifugal microfluidics is an increasingly popular choice for academic and commercial applications as it enables a portable platform for biological and chemical assays. By rationally designing microfluidic conduits and programming the disc's rotational speeds and accelerations, one can reliably control propulsion, metering, and valving operations. Valves that either stop fluid flow or allow it to proceed are critical components of a CD platform. Among the valves on a CD, wax valves that liquify at elevated temperatures to open channels and that solidify at room temperature to close them have been previously implemented on CD platforms. However, typical wax valves on the CD fluidic platforms can be actuated only once (to open or to close) and require complex fabrication steps. Here, we present two new multiple-use wax valve designs, driven by capillary or magnetic forces. One wax valve design utilizes a combination of capillary-driven flow of molten wax and centrifugal force to toggle between open and closed configurations. The phase change of the wax is enabled by heat application (e.g., a 500-mW laser). The second wax valve design employs a magnet to move a molten ferroparticle-laden wax in and out of a channel to enable reversible operation. A multi-phase numerical simulation study of the capillary-driven wax valve was carried out and compared with experimental results. The capillary wax valve parameters including response time, angle made by the sidewall of the wax reservoir with the direction of a valve channel, wax solidification time, minimum spin rate of the CD for opening a valve, and the time for melting a wax plug are measured and analyzed theoretically. Additionally, the motion of the molten wax in a valve channel is compared to its theoretical capillary advance with respect to time and are found to be within 18.75% of the error margin.

Guided Healing of Damaged Microelectrodes via Electrokinetic Assembly of Conductive Carbon Nanotube Bridges.

The subject of healing and repair of damaged microelectrodes has become of particular interest as the use of integrated circuits, energy storage technologies, and sensors within modern devices has increased. As the dimensions of the electrodes shrink together with miniaturization of all the elements in modern electronic devices, there is a greater risk of mechanical-, thermal-, or chemical-induced fracture of the electrodes. In this research, a novel method of electrode healing using electrokinetically assembled carbon nanotube (CNT) bridges is presented. Utilizing the previously described step-wise CNT deposition process, conductive bridges were assembled across ever-larger electrode gaps, with the width of electrode gaps ranging from 20 microns to well over 170 microns. This work represents a significant milestone since the longest electrically conductive CNT bridge previously reported had a length of 75 microns. To secure the created conductive CNT bridges, they are fixed with a layer of electrodeposited polypyrrole (a conductive polymer). The resistance of the resulting CNT bridges, and its dependence on the size of the electrode gap, is evaluated and explained. Connecting electrodes via conductive CNT bridges can find many applications from nanoelectronics to neuroscience and tissue engineering.

Guided Electrokinetic Assembly of Polystyrene Microbeads onto Photopatterned Carbon Electrode Arrays.

Assembly of microdevices from constituent parts currently relies on slow serial steps via direct assembly processes such as pick-and-place operations. Template Electrokinetic Assembly (TEA), a guided, noncontact assembly process, is presented in this work as a promising alternative to serial assembly processes. To characterize the process and its implementation of electrokinetic, dielectrophoretic, and electro-osmotic phenomena, we conducted studies to examine the assembly of polymer microparticles at specific locations on glassy carbon interdigitated electrode arrays (IDEAs). The IDEAs are coated with a layer of lithographically patterned resist, so that when an AC electric field is applied to the IDEA, microparticles suspended in the aqueous solution are attracted to the open regions of the electrodes not covered by photoresist. Interplay between AC electro-osmosis and dielectrophoretic forces guides 1 and 5 µm diameter polystyrene beads to assemble in regions, or "wells", uncovered by photoresist atop the electrodes. It was discovered that AC electro-osmosis under an applied frequency of 1 kHz is sufficient to effectively agglomerate 1 µm beads in the wells, whereas a stepwise process involving the application of a 1 MHz signal, followed by a 1 kHz signal, is required for the positioning of 5 µm beads, which are mainly affected by dielectrophoretic forces. Permanent entrapment of the microparticles is then demonstrated via the electropolymerization process of the conducting polymer polypyrrole.

Step-Wise Deposition Process for Dielectrophoretic Formation of Conductive 50-Micron-Long Carbon Nanotube Bridges.

Carbon Nanotube (CNT) agglomerates can be aligned along field lines between adjacent electrodes to form conductive bridges. This study discusses the step-wise process of dielectrophoretic deposition of CNTs to form conducting bridges between adjacent electrodes. For the first time, the creation of conductive CNT bridges spanning lengths over 50 microns is demonstrated. The CNT bridges are permanently secured using electrodeposition of the conducting polymer polypyrrole. Morphologies of the CNT bridges formed within a frequency range of 1 kHz and 10 MHz are explored and explained as a consequence of interplay between dielectrophoretic and electroosmotic forces. Postdeposition heat treatment increases the conductivity of CNT bridges, likely due to solvent evaporation and resulting surface tension inducing better contact between CNTs.

Fabrication of a Lab-on-Chip Device Using Material Extrusion (3D Printing) and Demonstration via Malaria-Ab ELISA.

Additive manufacturing, such as fused deposition modeling (FDM), has been increasingly employed to produce microfluidic platforms due to ease of use, wide distribution of affordable 3D printers and relatively inexpensive materials for printing. In this work, we discuss fabrication and testing of an FDM-printed fully automated colorimetric enzyme-linked immunosorbent assay (ELISA) designed to detect malaria. The detection platform consists of a disposable 3D-printed fluidic cartridge (with elastomeric silicone domes on top of reagent-storage reservoirs) and a nondisposable frame with servomotors and electronic controls such as an Arduino board and a rechargeable battery. The system is controlled by a novel interface where a music file (so-called "song") is sent to the Arduino board, where the onboard program converts the set of frequencies into action of individual servomotors to rotate their arms a certain amount, thus depressing specific elastomeric domes atop reagent reservoirs and displacing the specific reagents into the detection wells, where bioassay steps are executed. Another of the distinguished characteristics of the demonstrated system is its ability to aspirate the fluid from the detection wells into the waste reservoir. Therefore, the demonstrated automated platform has the ability to execute even the most complex multi-step assays where dilution and multiple washes are required. Optimization of 3D-printer settings and ways to control leakages typical of FDM-printed fluidic systems are also discussed.

Fabrication of a Malaria-Ab ELISA Bioassay Platform with Utilization of Syringe-Based and 3D Printed Assay Automation.

We report on the fabrication of a syringe-based platform for automation of a colorimetric malaria-Ab assay. We assembled this platform from inexpensive disposable plastic syringes, plastic tubing, easily-obtainable servomotors, and an Arduino microcontroller chip, which allowed for system automation. The automated system can also be fabricated using stereolithography (SLA) to print elastomeric reservoirs (used instead of syringes), while platform framework, including rack and gears, can be printed with fused deposition modeling (FDM). We report on the optimization of FDM and SLA print parameters, as well as post-production processes. A malaria-Ab colorimetric test was successfully run on the automated platform, with most of the assay reagents dispensed from syringes. Wash solution was dispensed from an SLA-printed elastomeric reservoir to demonstrate the feasibility of both syringe and elastomeric reservoir-based approaches. We tested the platform using a commercially available malaria-Ab colorimetric assay originally designed for spectroscopic plate readers. Unaided visual inspection of the assay solution color change was sufficient for qualitative detection of positive and negative samples. A smart phone application can also be used for quantitative measurement of the assay color change.

Lab-on-a-CD: A Fully Integrated Molecular Diagnostic System.

The field of centrifugal microfluidics has experienced tremendous growth during the past 15 years, especially in applications such as lab-on-a-disc (LoD) diagnostics. The strength of LoD systems lies in its potential for development into fully integrated sample-to-answer analysis systems. This review highlights the technologies necessary to develop the next generation of these systems. In addition to outlining valving and other fluid-handling operations, we discuss the recent advances and future outlook in four categories of LoD processes: reagent storage, sample preparation, nucleic acid amplification, and analyte detection strategies.

Design and implementation of fluidic micro-pulleys for flow control on centrifugal microfluidic platforms.

Microfluidic discs have been employed in a variety of applications for chemical analyses and biological diagnostics. These platforms offer a sophisticated fluidic toolbox, necessary to perform processes that involve sample preparation, purification, analysis, and detection. However, one of the weaknesses of such systems is the uni-directional movement of fluid from the disc center to its periphery due to the uni-directionality of the propelling centrifugal force. Here we demonstrate a mechanism for fluid movement from the periphery of a hydrophobic disc toward its center that does not rely on the energy supplied by any peripheral equipment. This method utilizes a ventless fluidic network that connects a column of working fluid to a sample fluid. As the working fluid is pushed by the centrifugal force to move toward the periphery of the disc, the sample fluid is pulled up toward the center of the disc analogous to a physical pulley where two weights are connected by a rope passed through a block. The ventless network is analogous to the rope in the pulley. As the working fluid descends, it creates a negative pressure that pulls the sample fluid up. The sample and working fluids do not come into direct contact and it allows the freedom to select a working fluid with physical properties markedly different from those of the sample. This article provides a demonstration of the "micro-pulley" on a disc, discusses underlying physical phenomena, provides design guidelines for fabrication of micro-pulleys on discs, and outlines a vision for future micro-pulley applications.

PPyDEP: a new approach to microparticle manipulation employing polymer-based electrodes.

In this work, a novel approach to 3-dimensional (3D) electrode fabrication, based on electrodeposited polypyrrole (PPy), for dielectrophoresis (DEP) is described. 3D PPy electrodes with post and cage geometries were grown over planar interdigitated electrodes. Computational modelling and experimental work were carried out to assess the performance of the proposed electrode geometries. It was found that these new electrode geometries enhanced the dielectrophoretic trapping efficiency for polystyrene beads by exhibiting larger variations of the electric field and by affecting a larger volume of the fluid sample than planar electrodes. Applications of this work include, but are not limited to, environmental monitoring, food safety control, clinical analysis, and clean energy production.

Theoretical development and critical analysis of burst frequency equations for passive valves on centrifugal microfluidic platforms.

This paper presents a theoretical development and critical analysis of the burst frequency equations for capillary valves on a microfluidic compact disc (CD) platform. This analysis includes background on passive capillary valves and the governing models/equations that have been developed to date. The implicit assumptions and limitations of these models are discussed. The fluid meniscus dynamics before bursting is broken up into a multi-stage model and a more accurate version of the burst frequency equation for the capillary valves is proposed. The modified equations are used to evaluate the effects of various CD design parameters such as the hydraulic diameter, the height to width aspect ratio, and the opening wedge angle of the channel on the burst pressure.

Present technology and future trends in point-of-care microfluidic diagnostics.

This work reviews present technologies and developing trends in Point-of-Care (POC) microfluidic diagnostics platforms. First, various fluidics technologies such as pressure-driven flows, capillary flows, electromagnetically driven flows, centrifugal fluidics, acoustically driven flows, and droplet fluidics are categorized. Then three broad categories of POC microfluidic testing devices are considered: lateral flow devices, desktop and handheld POC diagnostic platforms, and emergent molecular diagnostic POC systems. Such evolving trends as miniaturization, multiplexing, networking, new more sensitive detection schemes, and the importance of sample processing are discussed. It is concluded that POC microfluidic diagnostics has a potential to improve patient treatment outcome and bring substantial savings in overall healthcare costs.

Diffusion-free mediator based miniature biofuel cell anode fabricated on a carbon-MEMS electrode.

We report on the functionalization of a micropatterned carbon electrode fabricated using the carbon-MEMS process for its use as a miniature diffusion-free glucose oxidase anode. Carbon-MEMS based electrodes offer precise manufacturing control on both the micro- and nanoscale and possess higher electron conductivity than redox hydrogels. However, the process involves pyrolysis in a reducing environment that renders the electrode surface less reactive and introduction of a high density of functional groups becomes challenging. Our functionalization strategy involves the electrochemical oxidation of amine linkers onto the electrode. This strategy works well with both aliphatic and aryl linkers and uses stable compounds. The anode is designed to operate through mediated electron transfer between 2,5-dihydroxybenzaldehyde (DHB) based redox mediator and glucose oxidase enzyme. The electrode was first functionalized with ethylene diamine (EDA) to serve as a linker for the redox mediator. The redox mediator was then grafted through reductive amination, and attachment was confirmed through cyclic voltammetry. The enzyme immobilization was carried out through either adsorption or attachment, and their efficiency was compared. For enzyme attachment, the DHB attached electrode was functionalized again through electro-oxidation of aminobenzoic acid (ABA) linker. The ABA functionalization resulted in reduction of the DHB redox current, perhaps due to increased steric hindrance on the electrode surface, but the mediator function was preserved. Enzyme attachment was then carried out through a coupling reaction between the free carboxyl group on the ABA linker and the amine side chains on the enzyme. The enzyme incubation for both adsorption and attachment was done either through a dry spotting method or wet spotting method. The dry spotting method calls for the evaporation of enzyme droplet to form a thin film before sealing the electrode environment, to increase the effective concentration of the enzyme on the electrode surface during incubation. The electrodes were finally protected with a gelatin based hydrogel film. The anode half-cell was tested using cyclic voltammetry in deoxygenated phosphate buffer saline solution pH 7.4 to minimize oxygen interference and to simulate the pH environment of the body. The electrodes that yielded the highest anodic current were prepared by enzyme attachment method with dry spotting incubation. A polarization response was generated for this anodic half-cell and exhibits operation close to maximum efficiency that is limited by the mass transport of glucose to the electrode.