A cost-effective and facile technique for realizing fabric based microfluidic channels using beeswax and PVC stencils.

Microfluidic channels fabricated over fabrics or papers have the potential to find substantial application in the next generation of wearable healthcare monitoring systems. The present work focuses on the fabrication procedures that can be used to obtain practically realizable fabric-based microfluidic channels (µFADs) utilizing patterning masks and wax, unlike conventional printing techniques. In this study, comparative analysis was used to differentiate channels obtained using different masking tools for channel patterning as well as different wax materials as hydrophobic barriers. Drawbacks of the conventional tape and candle wax technique were noted and a novel approach was used to create microfluidic channels through a facile and simple masking technique using PVC clear sheets as channel stencils and beeswax as the channel barriers. The resulting fabric based microfluidic channels with varying widths as well as complex microchannel, microwell, and micromixer designs were investigated and a minimum channel width resolution of 500 µm was successfully obtained over cotton based fabrics. Thereafter, the PVC clear sheet-beeswax based microwells were successfully tested to confine various organic and inorganic samples indicating vivid applicability of the technique. Finally, the microwells were used to make a simple and facile colorimetric assay for glucose detection and demonstrated effective detection of glucose levels from 10 mM to 50 mM with significant color variation using potassium iodide as the coloring agent. The above findings clearly suggest the potential of this alternative technique for making low-cost and practically realizable fabric based diagnostic devices (µFADs) in contrast to the other approaches that are currently in use.

Integrated high-throughput drug screening microfluidic system for comprehensive ocular toxicity assessment.

Traditional experimental methodologies suffer from a few limitations in the toxicological evaluation of the preservatives added to eye drops. In this study, we overcame these limitations by using a microfluidic device. We developed a microfluidic system featuring a gradient concentration generator for preservative dosage control with microvalves and micropumps, automatically regulated by a programmable Arduino board. This system facilitated the simultaneous toxicological evaluation of human corneal epithelial cells against eight different concentrations of preservatives, allowing for quadruplicate experiments in a single run. In our study, the IC50 values for healthy eyes and those affected with dry eyes syndrome showed an approximately twofold difference. This variation is likely attributable to the duration for which the preservative remained in contact with corneal cells before being washed off by the medium, suggesting the significance of exposure time in the cytotoxic effect of preservatives. Our microfluidic system, automated by Arduino, simulated healthy and dry eye environments to study benzalkonium chloride toxicity and revealed significant differences in cell viability, with IC50 values of 0.0033% for healthy eyes and 0.0017% for dry eyes. In summary, we implemented the pinch-to-zoom feature of an electronic tablet in our microfluidic system, offering innovative alternatives for eye research.

A miniaturized wash-free microfluidic assay for electrical impedance-based assessment of red blood cell-mediated microvascular occlusion.

The production of HbS - an abnormal hemoglobin (Hb) - in sickle cell disease (SCD) results in poorly deformable red blood cells (RBCs) that are prone to microcapillary occlusion, causing tissue ischemia and organ damage. Novel treatments, including gene therapy, may reduce SCD morbidity, but methods to functionally evaluate RBCs remain limited. Previously, we presented the microfluidic impedance red cell assay (MIRCA) for rapid assessment of RBC deformability, employing electrical impedance-based readout to measure RBC occlusion of progressively narrowing micropillar openings. We describe herein the design, development, validation, and clinical utility of the next-generation MIRCA assay, featuring enhanced portability, rapidity, and usability. It incorporates a miniaturized impedance analyzer and features a simplified wash-free operation that yields an occlusion index (OI) within 15 min as a new metric for RBC occlusion. We show a correlation between OI and percent fetal hemoglobin (%HbF), other laboratory biomarkers of RBC hemolysis, and SCD severity. To demonstrate the assay's versatility, we tested RBC samples from treatment-naïve SCD patients in Uganda that yielded OI levels similar to those from hydroxyurea (HU)-treated patients in the U.S., highlighting the role of %HbF in protecting against microcapillary occlusion independent of other pharmacological effects. The MIRCA assay could also identify a subset of HU-treated patients with high occlusion risks, suggesting that they may require treatment adjustments including a second-line therapy to improve their outcomes. This work demonstrates the potential of the MIRCA assay for accelerated evaluation of RBC health, function, and therapeutic effect in an ex vivo model of the microcapillary networks.

High-resolution low-cost LCD 3D printing for microfluidics and organ-on-a-chip devices.

The fabrication of microfluidic devices has progressed from cleanroom manufacturing to replica molding in polymers, and more recently to direct manufacturing by subtractive (e.g., laser machining) and additive (e.g., 3D printing) techniques, notably digital light processing (DLP) photopolymerization. However, many methods require technical expertise and DLP 3D printers remain expensive at a cost ∼15-30 K USD with ∼8 M pixels that are 25-40 µm in size. Here, we introduce (i) the use of low-cost (∼150-600 USD) liquid crystal display (LCD) photopolymerization 3D printing with ∼8-58 M pixels that are 18-35 µm in size for direct microfluidic device fabrication, and (ii) a poly(ethylene glycol) diacrylate-based ink developed for LCD 3D printing (PLInk). We optimized PLInk for high resolution, fast 3D printing and biocompatibility while considering the illumination inhomogeneity and low power density of LCD 3D printers. We made lateral features as small as 75 µm, 22 µm-thick embedded membranes, and circular channels with a 110 µm radius. We 3D printed microfluidic devices previously manufactured by other methods, including an embedded 3D micromixer, a membrane microvalve, and an autonomous capillaric circuit (CC) deployed for interferon-γ detection with excellent performance (limit of detection: 12 pg mL-1, CV: 6.8%). We made PLInk-based organ-on-a-chip devices in 384-well plate format and produced 3420 individual devices within an 8 h print run. We used the devices to co-culture two spheroids separated by a vascular barrier over 5 days and observed endothelial sprouting, cellular reorganization, and migration. LCD 3D printing together with tailored inks pave the way for democratizing access to high-resolution manufacturing of ready-to-use microfluidic and organ-on-a-chip devices by anyone, anywhere.

Vasculature-on-chip for Assessment of Bioresorbable Scaffolds and Endothelial Barrier Integrity.

ABSTRACT: Endothelial cells adhere to one another through junctional structures formed by intercellular adhesion molecules. These intercellular proteins regulate barrier function in response to the hemodynamic shear rate and enable the selective passage of solutes and fluids across the endothelium. After endovascular device implantation, the endothelial barrier is compromised and becomes discontinuous, which increases permeability, allowing transmigration of leukocytes and lipoproteins and leading to the accumulation of lipid-laden foamy macrophages in the subendothelial space. Drug-coated bioresorbable vascular scaffold implants have been associated with unexpected thrombotic complications, which were not predicted in animals because of dissimilarities in endothelial regeneration and realignment between animals and humans. The development of a microengineered, microfluidics-based system of patterned channels lined with human endothelial and smooth muscle cells perfused with blood allows for the evaluation of endothelial function and barrier integrity. This review highlights the translational potential of vasculature-on-chip, which recreates the microphysiological milieu to evaluate the impact of drug-eluting bioresorbable vascular scaffolds on endothelial barrier integrity and to characterize polymer biodegradation behavior and drug release kinetic profiles over time.

Triple Emulsion-Based Rapid Microfluidic Production of Core-Shell Hydrogel Microspheres for Programmable Biomolecular Conjugation.

We report a simple and rapid microfluidic approach to produce core-shell hydrogel microspheres in a single step. We exploit triple emulsion drops with sacrificial oil layers that separate two prepolymer phases, forming poly(ethylene glycol)-based core-shell microspheres via photopolymerization followed by spontaneous removal of the oil layer. Our technique enables the production of monodisperse core-shell microspheres with varying dimensions of each compartment by independently and precisely controlled flow rates. This leads to stable and uniform incorporation of functional moieties in the core compartment with negligible cross-contamination into the shell layer. Selective conjugation of biomolecules is enabled through a rapid bioorthogonal reaction with functional groups in the core compartment with minimal non-specific adsorption. Finally, in-depth protein conjugation kinetics studies using microspheres with varying shell porosities highlight the capability to provide tunable size-selective diffusion barriers by simple tuning of prepolymer compositions for the shell layer. Combined, these results illustrate a significant step forward for programmable high-throughput fabrication of multifunctional hydrogel microspheres, which possess substantial potential in a large array of biomedical and biochemical applications.

An inexpensive, high-throughput µPAD assay of microbial growth rate and motility on solid surfaces using Saccharomyces cerevisiae and Escherichia coli as model organisms.

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (µPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed µPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed µPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the µPAD design for channel width (3 mm), agar volume (780 µL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed µPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the µPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and µPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, µPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.

Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation.

Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 µL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes.

A single-view field filter device for rare tumor cell filtration and enumeration.

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 µL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.

Emerging design strategies for constructing multiplex lateral flow test strip sensors.

Conventional lateral flow test strip (LFTS) sensors are insufficiently accurate and reliable due to their single-target detection with limited sample information in a single test. The increasing demand for the simultaneous determination of multiple analytes has recently been accelerating the rapid development of high-throughput and multiplexed LFTS sensing technologies. In this contribution, we systematically summarize the recent achievements on the design, development, and application of multiplexed LFTS sensors for improved rapid on-site diagnostics. The discussion focuses on emerging design strategies to increase multiplexing capacity for enhancing analytical efficiency and precision. As a proof-of-concept, several typical examples are presented. The advantages and disadvantages of such approaches are critically analyzed. Finally, we briefly discuss the current challenges and future perspectives.

Leukocyte adhesion to P-selectin and the inhibitory role of Crizanlizumab in sickle cell disease: A standardized microfluidic assessment.

Upregulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD). Crizanlizumab (ADAKVEO®), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been shown to decrease the frequency of VOCs in clinical trials. However, the lack of reliable in vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to evaluating and improving the therapeutic treatment in SCD. Here, we present a standardized microfluidic BioChip whole blood adhesion assay to assess leukocyte adhesion to P-selectin under physiologic flow conditions. Our results demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to Crizanlizumab. Importantly, treatment with Crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes. Taken together, our results suggest that the microfluidic BioChip system is a promising in vitro assay with which to screen patients, monitor treatment response, and guide current and emerging anti-adhesive therapies in SCD.

Post-exercise Response of Arterial Parameters for Arterial Health Assessment Using a Microfluidic Tactile Sensor and Vibration-Model-Based Analysis: A Proof-of-Concept Study.

OBJECTIVE: Arterial stiffness and endothelial function are two established surrogate markers of subclinical atherosclerosis and are quantified by three arterial parameters: elasticity, viscosity and radius of the arterial wall. Yet, the current methods for their assessment are unsuitable for routine use. Post-exercise response of the cardiovascular (CV) system serves as a more sensitive detection of subclinical arterial abnormalities that are not apparent at-rest. The objective of this study is to propose a novel method that can measure post-exercise response of arterial parameters and is also suitable for routine use. APPROACH: A microfluidic tactile sensor with a location-insensitive configuration was used for arterial pulse signal measurements on six asymptomatic male subjects, offering measurement reliability, ease use by a layperson, and affordability. By treating the arterial pulse signal as a vibration signal of the arterial wall, vibration-model-based analysis of only one measured pulse signal with no calibration was conducted for simultaneous estimation of three arterial parameters. Exercise-intensity-normalized percent changes in arterial parameters were utilized to remove the influence of variation in exercise intensity on post-exercise response, and then their measured values were compared for difference in post-exercise response between the subjects. MAIN RESULTS: One subject who was obese, on subject who had insomnia, and the oldest subject in the study demonstrated differences in post-exercise response at the radial artery (RA), as compared with the three subjects free of those three factors. Despite a lack of statistical significance, the observed difference at the RA between subjects was supported by (i) their consistency with the related findings in the literature, and (ii) their consistency with the measured values at the carotid artery (CA) and superficial temporal artery (STA) and the anatomical difference between the three arteries. SIGNIFICANCE: The proposed method has the potential of offering an affordable and convenient diagnosis tool for routine arterial health assessment.

A pharmaceutical industry perspective on microphysiological kidney systems for evaluation of safety for new therapies.

The human kidney contains approximately one million nephrons. As the functional unit of the kidney, the nephron affords an opportunity to approximate the kidney at a microphysiological scale. Recent emergence of physiologically accurate human tissue models has radically advanced the possibilities of mimicking organ biology and multi-organ combinations in vitro. Anatomically, the nephron is one of the most complex, sequentially integrated microfluidic units in the body making the miniaturized microfluidic systems excellent candidates for capturing the kidney biology in vitro. While these models are promising, there are a number of considerations for practical implementation into a drug development paradigm. Opportunities for pharmaceutical industry applications of new MPS models often start with drug safety testing. As such, the intent of this article is to focus on safety and ADME applications. This article reviews biological functions of the kidney and options for characterizing known roles in nephrotoxicity. The concept of "context-of-use" is introduced as a framework for describing and verifying the specific features of an MPS platform for use in drug development. Overall, we present a perspective on key attributes of microphysiological kidney models, which the pharmaceutical industry could leverage to improve confident safety and ADME evaluations of experimental therapies.

Microphysiological systems for ADME-related applications: current status and recommendations for system development and characterization.

Over the last decade, progress has been made on the development of microphysiological systems (MPS) for absorption, distribution, metabolism, and excretion (ADME) applications. Central to this progress has been proof of concept data generated by academic and industrial institutions followed by broader characterization studies, which provide evidence for scalability and applicability to drug discovery and development. In this review, we describe some of the advances made for specific tissue MPS and outline the desired functionality for such systems, which are likely to make them applicable for practical use in the pharmaceutical industry. Single organ MPS platforms will be valuable for modelling tissue-specific functions. However, dynamic organ crosstalk, especially in the context of disease or toxicity, can only be obtained with the use of inter-linked MPS models which will enable scientists to address questions at the intersection of pharmacokinetics (PK) and efficacy, or PK and toxicity. In the future, successful application of MPS platforms that closely mimic human physiology may ultimately reduce the need for animal models to predict ADME outcomes and decrease the overall risk and cost associated with drug development.

Low-cost multi-core inertial microfluidic centrifuge for high-throughput cell concentration.

We developed a low-cost multi-core inertial microfluidic centrifuge (IM-centrifuge) to achieve a continuous-flow cell/particle concentration at a throughput of up to 20 mL/min. To lower the cost of our IM-centrifuge, we clamped a disposable multilayer film-based inertial microfluidic (MFIM) chip with two reusable plastic housings. The key MFIM chip was fabricated in low-cost materials by stacking different polymer-film channel layers and double-sided tape. To increase processing throughput, multiplexing spiral inertial microfluidic channels were integrated within an all-in-one MFIM chip, and a novel sample distribution strategy was employed to equally distribute the sample into each channel layer. Then, we characterized the focusing performance in the MFIM chip over a wide flow-rate range. The experimental results showed that our IM-centrifuge was able to focus various-sized particles/cells to achieve volume reduction. The sample distribution strategy also effectively ensured identical focusing and concentration performances in different cores. Finally, our IM-centrifuge was successfully applied to concentrate microalgae cells with irregular shapes and highly polydisperse sizes. Thus, our IM-centrifuge holds the potential to be employed as a low-cost, high-throughput centrifuge for disposable use in low-resource settings.

Strong rotating flow in stationary droplets in low power budget using wire electrode configuration.

We explore a simple strategy of generating strong rotating flow in a stationary surface-droplet, using an intricate interplay of local electrical and thermal fields. Wire electrodes are employed to generate on-spot heating without necessitating any elaborate micro-fabrication, which causes strong local gradients in electrical properties to induce mobile charges into the droplet. Applying a low voltage (∼10 V), strong rotational velocity of the order of mm/s can be achieved in the system, within the standard operating ranges of operating and geometrical parameters. Further, altering the diameter of the electrode, vortices can be tuned locally or globally in low power budget, without incurring any droplet oscillations. These results may turn out to be of immense consequence in enhancing micromixing in a plethora of droplet based applications ranging from thermal management to medical diagnostics to be potentially employed in resource-limited settings.

Reliable and reusable whole polypropylene plastic microfluidic devices for a rapid, low-cost antimicrobial susceptibility test.

Using an antimicrobial susceptibility test (AST) as an example, this work demonstrates a practical method to fabricate microfluidic chips entirely from polypropylene (PP) and the benefits for potential commercial use. Primarily caused by the misuse and abuse of antibiotics, antimicrobial resistance (AMR) is a major threat to modern medicine. The AST is a promising technique to help with the optimal use of antibiotics for reducing AMR. However, current phenotypic ASTs suffer from long turnaround time, while genotypic ASTs suffer from low reliability, and both are unaffordable for routine use. New microfluidics based AST methods are rapid but still unreliable as well as costly due to the PDMS chip material. Herein, we demonstrate a convenient method to fabricate whole PP microfluidic chips with high resolution and fidelity. Unlike PDMS chips, the whole PP chips showed better reliability due to their inertness; they are solvent-compatible and can be conveniently reused and recycled, which largely decreases the cost, and are environmentally friendly. We specially designed 3D chambers that allow for quick cell loading without valving/liquid exchange; this new hydrodynamic design satisfies the shear stress requirement for on-chip bacterial culture, which, compared to reported designs for similar purposes, allows for a simpler, more rapid, and high-throughput operation. Our system allows for reliable tracking of individual cells and acquisition of AST results within 1-3 hours, which is among the group of fastest phenotypic methods. The PP chips are more reliable and affordable than PDMS chips, providing a practical solution to improve current culture-based AST and benefiting the fight against AMR through helping doctors prescribe effective, narrow-spectrum antibiotics; they will also be broadly useful for other applications wherein a reliable, solvent-resistant, anti-fouling, and affordable microfluidic chip is needed.

A rapid and low-cost fabrication and integration scheme to render 3D microfluidic architectures for wearable biofluid sampling, manipulation, and sensing.

The large-scale deployment of wearable bioanalytical devices for general population longitudinal monitoring necessitates rapid and high throughput manufacturing-amenable fabrication schemes that render disposable, low-cost, and mechanically flexible microfluidic modules capable of performing a variety of bioanalytical operations within a compact footprint. The spatial constraints of previously reported wearable bioanalytical devices (with microfluidic operations confined to 2D), their lack of biofluid manipulation capability, and the complex and low-throughput nature of their fabrication process inherently limit the diversity and frequency of end-point assessments and prevent their deployment at large scale. Here, we devise a simple, scalable, and low-cost "CAD-to-3D Device" fabrication and integration scheme, which renders 3D and complex microfluidic architectures capable of performing biofluid sampling, manipulation, and sensing. The devised scheme is based on laser-cutting of tape-based substrates, which can be programmed at the software-level to rapidly define microfluidic features such as a biofluid collection interface, microchannels, and VIAs (vertical interconnect access), followed by the vertical assembly of pre-patterned layers to realize the final device. To inform the utility of our fabrication scheme, we demonstrated three representative devices to perform sweat collection (with visualizable secretion profile), sample filtration, and simultaneous biofluid actuation and sensing (using a sandwiched-interface). Our devised scheme can be adapted for the fabrication and manufacturing of current and future wearable bioanalytical devices, which in turn will catalyze the large-scale production and deployment of such devices for general population health monitoring.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

A microfluidic based biosensor for rapid detection of Salmonella in food products.

An impedance based microfluidic biosensor for simultaneous and rapid detection of Salmonella serotypes B and D in ready-to-eat (RTE) Turkey matrix has been presented. Detection of Salmonella at a concentration as low as 300 cells/ml with a total detection time of 1 hour has been achieved. The sensor has two sensing regions, with each formed from one interdigitated electrode array (IDE array) consisting of 50 finger pairs. First, Salmonella antibody type B and D were prepared and delivered to the sensor to functionalize each sensing region without causing any cross contamination. Then the RTE Turkey samples spiked with Salmonella types B and D were introduced into the biosensor via the antigen inlet. The response signal resulted from the binding between Salmonella and its specific antibody demonstrated the sensor's ability to detect a single type of pathogen, and multiple pathogens simultaneously. In addition, the biosensor's selectivity was tested using non-specific binding of E. coli O157 and E. coli DH5 Alpha while the IDE array was coated with the Salmonella antibody. The results also showed the sensor is capable to differentiate low concentration of live Salmonella cells from high concentration of dead Salmonella cells, and high concentration of E. coli cells. A detailed study on antibody immobilization that includes antibody concentration, antibody coating time (0.5-3 hours) and use of cross-linker has been performed. The study showed that Salmonella antibody to Salmonella antigen is not a factor of antibody concentration after electrodes were saturated with antibody, while the optimal coating time was found to be 1.5 hours, and the use of cross-linker has improved the signal response by 45-60%.