Portable Microfluidic Devices Research Articles

Highly efficient cell-microbead encapsulation using dielectrophoresis-assisted dual-nanowell array.

Recent advancements in micro/nanofabrication techniques have led to the development of portable devices for high-throughput single-cell analysis through the isolation of individual target cells, which are then paired with functionalized microbeads. Compared with commercially available benchtop instruments, portable microfluidic devices can be more widely and cost-effectively adopted in single-cell transcriptome and proteome analysis. The sample utilization and cell pairing rate (∼33%) of current stochastic-based cell-bead pairing approaches are fundamentally limited by Poisson statistics. Despite versatile technologies having been proposed to reduce randomness during the cell-bead pairing process in order to statistically beat the Poisson limit, improvement of the overall pairing rate of a single cell to a single bead is typically based on increased operational complexity and extra instability. In this article, we present a dielectrophoresis (DEP)-assisted dual-nanowell array (ddNA) device, which employs an innovative microstructure design and operating process that decouples the bead- and cell-loading processes. Our ddNA design contains thousands of subnanoliter microwell pairs specifically tailored to fit both beads and cells. Interdigitated electrodes (IDEs) are placed below the microwell structure to introduce a DEP force on cells, yielding high single-cell capture and pairing rates. Experimental results with human embryonic kidney cells confirmed the suitability and reproducibility of our design. We achieved a single-bead capture rate of >97% and a cell-bead pairing rate of >75%. We anticipate that our device will enhance the application of single-cell analysis in practical clinical use and academic research.

Recent Achievements in Electrochemical and Optical Nucleic Acids Based Detection of Metal Ions.

Recently nucleic acids gained considerable attention as selective receptors of metal ions. This is because of the possibility of adjusting their sequences in new aptamers selection, as well as the convenience of elaborating new detection mechanisms. Such a flexibility allows for easy utilization of newly emerging nanomaterials for the development of detection devices. This, in turn, can significantly increase, e.g., analytical signal intensity, both optical and electrochemical, and the same can allow for obtaining exceptionally low detection limits and fast biosensor responses. All these properties, together with low power consumption, make nucleic acids biosensors perfect candidates as detection elements of fully automatic portable microfluidic devices. This review provides current progress in nucleic acids application in monitoring environmentally and clinically important metal ions in the electrochemical or optical manner. In addition, several examples of such biosensor applications in portable microfluidic devices are shown.

Colorimetric detection of Aflatoxin B1 by using smartphone-assisted microfluidic paper-based analytical devices

Aflatoxin B 1 (AFB 1 ) is a powerful carcinogen, and the pollution it causes has become a global public health safety issue. Therefore, microfluidic paper-based analytical devices (μPADs) with smartphones to read colorimetric signals were constructed for rapid AFB 1 detection. The colorimetric mechanism of μPADs was based on the common starch–iodine reaction, and the addition of gelatin/chitosan enhanced the colorimetric effect. During detection and analysis, the competitive analysis occurred in the process of AFB 1 monoclonal antibody's recognition of free AFB 1 and functionalized antigen. The glucose oxidase-labeled IgG was used as the secondary antibody. Hydrogen peroxide, an oxidation product of the glucose, caused a chroma change in μPADs. Simultaneously, the change in chroma was consistent with the change in AFB 1 concentration. Under the best optimized conditions, the limits of detection and quantitation of AFB 1 in the buffer were 9.45 and 12.00 ng mL −1 , respectively. A comparison of the detection performance of commercial kits with that of the method introduced herein confirmed the superiority of our method in actual food samples with known AFB 1 concentrations. This colorimetric μPADs analysis met the detection requirements of some countries, such as China, and had great potential for development in the direction of daily extensive food safety control and environmental monitoring. • Portable and low-cost smartphone-based microfluidic paper-based analytical devices (μPADs). • The colorimetric μPAD detection system can detect aflatoxin B1 as low as 9.45 ng mL −1 through Lab data analysis. • The colorimetric mechanism is based on the common starch–iodine reaction. • The addition of gelatin and chitosan mixture enhances the colorimetric effect.

A visual portable microfluidic experimental device with multiple electric field regulation functions.

High portability and miniaturization are two of the most important objectives pursued by microfluidic methods. However, there remain many challenges for the design of portable and visual microfluidic devices (e.g., electrokinetic experiments) due to the use of a microscope and power supply. To this end, we report a visual portable microfluidic experimental device (PMED) with multiple electric field regulation functions, which can realize the electric field regulation functions of various basic microfluidic experiments through modular design. The internal reaction process of the microfluidic chip is displayed by a smartphone, and the experimental results are analyzed using a mobile phone application (APP). Taking the induced-charge electroosmosis (ICEO) particle focusing phenomenon as an example, we carried out detailed experiments on PMED and obtained conclusions consistent with numerical simulations. In addition to ICEO experiments, other functions such as alternating electroosmosis (ACEO), thermal buoyancy convection, and dielectrophoresis (DEP) can be realized by replacing module-specific covers. The device expands the application of microfluidic experiments and provides a certain reference for the further integration and portability of subsequent microfluidic experiment devices.

Modernization of Control of Pathogenic Micro-Organisms in the Food-Chain Requires a Durable Role for Immunoaffinity-Based Detection Methodology-A Review.

Food microbiology is deluged by a vastly growing plethora of analytical methods. This review endeavors to color the context into which methodology has to fit and underlines the importance of sampling and sample treatment. The context is that the highest risk of food contamination is through the animal and human fecal route with a majority of foodborne infections originating from sources in mass and domestic kitchens at the end of the food-chain. Containment requires easy-to-use, failsafe, single-use tests giving an overall risk score in situ. Conversely, progressive food-safety systems are relying increasingly on early assessment of batches and groups involving risk-based sampling, monitoring environment and herd/flock health status, and (historic) food-chain information. Accordingly, responsible field laboratories prefer specificity, multi-analyte, and high-throughput procedures. Under certain etiological and epidemiological circumstances, indirect antigen immunoaffinity assays outperform the diagnostic sensitivity and diagnostic specificity of e.g., nucleic acid sequence-based assays. The current bulk of testing involves therefore ante- and post-mortem probing of humoral response to several pathogens. In this review, the inclusion of immunoglobulins against additional invasive micro-organisms indicating the level of hygiene and ergo public health risks in tests is advocated. Immunomagnetic separation, immunochromatography, immunosensor, microsphere array, lab-on-a-chip/disc platforms increasingly in combination with nanotechnologies, are discussed. The heuristic development of portable and ambulant microfluidic devices is intriguing and promising. Tant pis, many new platforms seem unattainable as the industry standard. Comparability of results with those of reference methods hinders the implementation of new technologies. Whatever the scientific and technological excellence and incentives, the decision-maker determines this implementation after weighing mainly costs and business risks.

Influence of core–shell structured conductive fillers on the electromechanical properties of ferroelectric nanocomposites

Although the ultra-large induced strain of ferroelectric polymers has been achieved under an electric field near breakdown strength, the high driving voltage has been a long-standing obstacle for their safety applications in the actuator, transducer, wearable devices. To resolve this issue, novel core–shell structured CNT–Al2O3 nanoparticles grown via atomic layer deposition are utilized to improve the electromechanical properties of P(VDF-TrFE)-based nanocomposites under low applied fields. The electric coercive field (Ec) of nanocomposites substantially decreased with increase in fillers, giving rise to the boosted polarization and induced strain at low electric field. Accordingly, the nanocomposite with 1.1 wt% CNT–Al2O3 exhibited a 600% increase in transverse induced strain than that of the neat P(VDF-TrFE). For an identical induced strain, the required driving voltage of the nanocomposite could be effectively reduced by up to 200%. By numerically calculating electric field distribution and polarization, it was revealed that the higher ferroelectric property and lower driving voltage resulted from the enhanced interfacial polarization and the reduced field intensity to switch the polarization of the nanocomposites. This study provides a new route to improve the performance of ferroelectric polymers and holds great promise for using ferroelectric nanocomposites with low operating voltage in practical applications, including portable microfluidic and electronic devices. To reduce the driving voltage, a novel core–shell structured CNT–Al2O3 nanoparticle filler was utilized to improve the electromechanical properties of P(VDF-TrFE)-based nanocomposites under the low applied electric field. The introduction of CNT–Al2O3 filler in nanocomposite could significantly reduce the required electric field intensity for switching the polarization. Owning to its lowest Ec and higher polarization, the transverse induced strain of 1.1 wt% nanocomposite increased by 600% than that of the neat P(VDF-TrFE).

Recent advances in nanomaterials integrated immunosensors for food toxin detection.

For the management and prevention of many chronic and acute diseases, the rapid quantification of toxicity in food and feed products have become a significant concern. Technology advancements in the area of biosensors, bioelectronics, miniaturization techniques, and microfluidics have shown a significant impact than conventional methods which have given a boost to improve the sensing performance towards food analyte detection. In this article, recent literature of Aflatoxin B1 (AFB1), worldwide permissible limits, major outbreaks and severe impact on healthy life have been discussed. An improvement achieved in detection range, limit of detection, shelf-life of the biosensor by integrated dimensional nanomaterials such as zero-dimension, one-dimension and two-dimension for AFB1 detection using electrical and optical transduction mechanism has been summarized. A critical overview of the latest trends using paper-based and micro-spotted array integrated with the anisotropic shape of nanomaterials, portable microfluidic devices have also been described together with future perspectives for further advancements.

Portable microfluidic devices for in-field detection of pharmaceutical residues in water: Recent outcomes and current technological situation – A short review

The presence of pharmaceutical residues in urban and non-urban water is a reflection of the continuous growth of an essential industry, whose residues are often released into the environment throughout the manufacturing processes of commercial drugs, and by the human and veterinarian consumption of these products. Pharmaceutical residues are currently classified as emerging contaminants, and their documented occurrence in waters has rocketed since recent discoveries have demonstrated a series of toxicological and ecotoxicological effects associated with the interaction with these molecules. Moreover, several complications have been identified in the instrumental techniques traditionally used for their detection, although their remarkable analytical characteristics, deriving in the absence of response when these emerging contaminants are present at low concentrations in aquatic matrices. Microfluidic analytical platforms are practical, portable, and low-cost solutions that have been successfully used for the screening of pharmaceuticals in environmental matrices in situ. In this short review, various essential aspects of these technologies were presented. Additionally, recent approaches for water analysis using different types of microfluidic technologies for detecting drugs were exposed, finding a promising potential on the exploiting of the features offered by these platforms for this particular application.

Evaluation of Mass Sensitive Micro-Array biosensors for their feasibility in multiplex detection of low molecular weight toxins using mycotoxins as model compounds

Mycotoxins produced by Fusarium species including trichothecenes, zearalenone and fumonisins, can co-contaminate food and feed throughout the supply chain, including cereal grains and animal feeds. There is an increasing demand to enhance global food security by improving the monitoring of mycotoxins throughout our food supply chain. For time and cost-efficient analysis, rapid tests capable of detecting multiple toxins from a single sample are ideal. Considering these current trends in mycotoxin testing, this project examined the feasibility of using both a portable and non-portable mass-based biosensor for multiplex mycotoxin detection. The biosensor was a mass sensitive microarray (MSMA) which consisted of 4 × 16 miniaturized mass sensitive transducer pixels based on solidly mounted resonator (SMR) technology. Functionalisation of individual pixels on the sensor surface using nano-spotting technology for the simultaneous and semi-quantitative detection of three regulated mycotoxins: T2-toxin (T2) zearalenone (ZEN), and fumonisin B1 (FumB1) was examined. With the integration of portable and non-portable microfluidic devices for antibody and standard sample injections, competitive inhibition assays were developed followed by singleplex and multiplex calibration curves. The characteristics and performance of the MSMA were evaluated including sensitivity which was determined as the concentration causing 50% inhibition. Sensitivity of singleplex assays using the portable microfluidic device (PMD) were 1.3 ng/ml, 2.0 ng/ml and 6.8 ng/ml for T2, FumB1 and ZEN, respectively. Sensitivity of the multiplex assay again using the PMD was 6.1 ng/ml, 3.6 ng/ml and 2.4 ng/ml for T2, FumB1 and ZEN, respectively. The PMD was an easy to use and highly sensitive screening tool which has been demonstrated for the multiplex detection of three regulated mycotoxins. Analysis was in real time and results were fully digital. Since detection of analytes was by mass it was both a label-free and cost-efficient method proposed method of analysis for mycotoxins.

Microfluidic Point-of-Care Devices: New Trends and Future Prospects for eHealth Diagnostics.

Point-of-care (PoC) diagnostics is promising for early detection of a number of diseases, including cancer, diabetes, and cardiovascular diseases, in addition to serving for monitoring health conditions. To be efficient and cost-effective, portable PoC devices are made with microfluidic technologies, with which laboratory analysis can be made with small-volume samples. Recent years have witnessed considerable progress in this area with “epidermal electronics”, including miniaturized wearable diagnosis devices. These wearable devices allow for continuous real-time transmission of biological data to the Internet for further processing and transformation into clinical knowledge. Other approaches include bluetooth and WiFi technology for data transmission from portable (non-wearable) diagnosis devices to cellphones or computers, and then to the Internet for communication with centralized healthcare structures. There are, however, considerable challenges to be faced before PoC devices become routine in the clinical practice. For instance, the implementation of this technology requires integration of detection components with other fluid regulatory elements at the microscale, where fluid-flow properties become increasingly controlled by viscous forces rather than inertial forces. Another challenge is to develop new materials for environmentally friendly, cheap, and portable microfluidic devices. In this review paper, we first revisit the progress made in the last few years and discuss trends and strategies for the fabrication of microfluidic devices. Then, we discuss the challenges in lab-on-a-chip biosensing devices, including colorimetric sensors coupled to smartphones, plasmonic sensors, and electronic tongues. The latter ones use statistical and big data analysis for proper classification. The increasing use of big data and artificial intelligence methods is then commented upon in the context of wearable and handled biosensing platforms for the Internet of things and futuristic healthcare systems.

Triplex Inertia-Magneto-Elastic (TIME) sorting of microparticles in non-Newtonian fluids

Detection of harmful biological substances in food at the Point of Use (PoU) is very important to the prevention of foodborne diseases. Sample and reagent preparation at the PoU, as a necessary step before detection, is urgently needed. Portable and field-deployable sample preparation microfluidic devices for manipulating particles and biological substances in Newtonian fluids like water have been developed. However, understanding the mechanism of particle focusing and multiplex sorting of particles with various properties in prominent non-Newtonian fluids such as milk, blood, and DNA solutions have lagged behind. Accordingly, we developed a microfluidic device to investigate the effect of inertial, magnetic and elastic forces on the focusing of magnetic (9 and 15 µm) and non-magnetic (15 µm) particles in synthetic viscoelastic fluids with various viscosities. We also demonstrated the application of our studies in Triplex Inertia-Magneto-Elastic (TIME) sorting of these particles with purity and efficiency of more than 92%. The device included a square microchannel with a side permanent magnet, expanding symmetrically downstream to a wider channel to drop the particles velocity for on-chip imaging. We investigated the effect of multiple parameters on the focusing of each particle experimentally and analytically, in order to obtain understanding of particle dynamics and the best recipe in which triplex particle separation could be achieved with high efficiency. The studied parameters included the microchannel dimension, flow rate, fluid viscoelasticity, and magnetic field strength and exposure time. This study provides the foundation for development of devices for separation of bio-substances in viscoelastic fluids, immunologically attached to microparticles. Our device has the potential to be used for on-site sample preparation along with a variety of biosensors to render biodetection possible at the PoU.

Microfluidic devices powered by integrated elasto-magnetic pumps.

We show how an asymmetric elasto-magnetic system provides a novel integrated pumping solution for lab-on-a-chip and point of care devices. This monolithic pumping solution, inspired by Purcell's 3-link swimmer, is integrated within a simple microfluidic device, bypassing the requirement of external connections. We experimentally prove that this system can provide tuneable fluid flow with a flow rate of up to 600 μL h-1. This fluid flow is achieved by actuating the pump using a weak, uniform, uniaxial, oscillating magnetic field, with field amplitudes in the range of 3-6 mT. Crucially, the fluid flow can be reversed by adjusting the driving frequency. We experimentally prove that this device can successfully operate on fluids with a range of viscosities, where pumping at higher viscosity correlates with a decreasing optimal driving frequency. The fluid flow produced by this device is understood here by examining the non-reciprocal motion of the elasto-magnetic component. This device has the capability to replace external pumping systems with a simple, integrated, lab-on-a-chip component.

Microfluidic Passive Valve with Ultra-Low Threshold Pressure for High-Throughput Liquid Delivery.

The microvalve for accurate flow control under low fluidic pressure is vital in cost-effective and miniaturized microfluidic devices. This paper proposes a novel microfluidic passive valve comprising of a liquid chamber, an elastic membrane, and an ellipsoidal control chamber, which actualizes a high flow rate control under an ultra-low threshold pressure. A prototype of the microvalve was fabricated by 3D printing and UV laser-cutting technologies and was tested under static and time-dependent pressure conditions. The prototype microvalve showed a nearly constant flow rate of 4.03 mL/min, with a variation of ~4.22% under the inlet liquid pressures varied from 6 kPa to 12 kPa. In addition, the microvalve could stabilize the flow rate of liquid under the time-varying sinusoidal pressures or the square wave pressures. To validate the functionality of the microvalve, the prototype microvalve was applied in a gas-driven flow system which employed an air blower or human mouth blowing as the low-cost gas source. The microvalve was demonstrated to successfully regulate the steady flow delivery in the system under the low driving pressures produced by the above gas sources. We believe that this new microfluidic passive valve will be suitable for controlling fluid flow in portable microfluidic devices or systems of wider applications.

A sheathless inertial focusing technique for optofluidic devices

Here, we demonstrate single-position, three-dimensional (3D) focusing of cells or micron-sized particles in the range 0.175 < a/w < 0.9 (a, cell or particle diameter; w, width of the microchannel), on a single-layer, single-channel microfluidic chip, which totally alleviates the need of using any sheath flow or external force, making the microfluidic chip standalone operational. The focusing is a result of inertial microfluidic hydrodynamic forces such as inertial lift forces and Dean drag forces, which are determined by the geometry of microchannel. With the microfluidic channel comprising a series of radially increasing, uniform semi-arcs interleaved by linear sections, sheathless focusing at flow rates up to 700 μL/min is achieved in our study. The result can be well explained by a developed empirical model relating the ratio of inertial lift forces and Dean drag forces, and the geometrical parameters of the microchannel. Following this approach, we illustrate experimental characterization of micron-sized sample focusing using fluorescent microparticles, pancreatic cancer cells, and macrophages under Reynolds number flows ranging between 20 and 153. We foresee the single-position focusing outcome of the proposed sheathless chip design in developing portable microfluidic and optofluidic devices for in vitro theranostics.

Visual Quantitative Detection of Circulating Tumor Cells with Single-Cell Sensitivity Using a Portable Microfluidic Device.

Immunocytological technologies, molecular technologies, and functional assays are widely used for detecting circulating tumor cells (CTCs) after enrichment from patients' blood sample. Unfortunately, accessibility to these technologies is limited due to the need for sophisticated instrumentation and skilled operators. Portable microfluidic devices have become attractive tools for expanding the access and efficiency of detection beyond hospitals to sites near the patient. Herein, a volumetric bar chart chip (V-Chip) is developed as a portable platform for CTC detection. The target CTCs are labeled with aptamer-conjugated nanoparticles (ACNPs) and analyzed by V-Chip through quantifying the byproduct (oxygen) of the catalytic reaction between ACNPs and hydrogen peroxide, which results in the movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Thus, the CTC number is decoded into visually quantifiable information and a linear correlation can be found between the distance moved by the ink and number of cells in the sample. This method is sensitive enough that a single cell can be detected. Furthermore, the clinical capabilities of this system are demonstrated for quantitative CTC detection in the presence of a high leukocyte background. This portable detection method shows great potential for quantification of rare cells with single-cell sensitivity for various applications.

New advances in microfluidic flow cytometry.

In recent years, researchers are paying the increasing attention to the development of portable microfluidic diagnostic devices including microfluidic flow cytometry for the point-of-care testing. Microfluidic flow cytometry, where microfluidics and flow cytometry work together to realize novel functionalities on the microchip, provides a powerful tool for measuring the multiple characteristics of biological samples. The development of a portable, low-cost, and compact flow cytometer can benefit the health care in underserved areas such as Africa or Asia. In this article, we review recent advancements of microfluidics including sample pumping, focusing and sorting, novel detection approaches, and data analysis in the field of flow cytometry. The challenge of microfluidic flow cytometry is also examined briefly.

Microfluidic Paper-based Analytical Devices for Determination of Creatinine in Urine Samples.

Simple, low-cost and portable microfluidic paper-based analytical devices (μPADs) for determination of creatinine in urine samples were developed. The methodology was based on Jaffé reaction between the creatinine and picric acid in alkaline conditions, generating a colorimetric creatinine-alkaline picrate complex. The product exhibits an orange color that is clearly visible on the μPADs. The color intensity of the complex, which is indicative of the concentration of creatinine, is then quantitatively determined using ImageJ software. Various experimental parameters were optimized to achieve the best performance of the μPADs. Under the optimum conditions, a wide linear range was obtained in the range of 0.2 - 1 mM with a limit of detection and limit of quantitation of 0.08 and 0.26 mM, respectively. The accuracy of the proposed method was in good agreement with the standard Jaffé method. Finally, the developed devices were successfully applied for the determination of creatinine in urine samples.

Direct enrichment of pathogens from physiological samples of high conductivity and viscosity using H-filter and positive dielectrophoresis.

The full potential of microfluidic techniques as rapid and accurate methods for the detection of disease-causing agents and foodborne pathogens is critically limited by the complex sample preparation process, which commonly comprises the enrichment of bacterial cells to detectable levels. In this manuscript, we describe a microfluidic device which integrates H-filter desalination with positive dielectrophoresis (pDEP) for direct enrichment of bacterial cells from physiological samples of high conductivity and viscosity, such as cow's milk and whole human blood. The device contained a winding channel in which electrolytes in the samples continuously diffused into deionized (DI) water (desalination), while the bacterial cells remained in the samples. The length of the main channel was optimized by numerical simulation and experimentally evaluated by the diffusion of fluorescein into DI water. The effects of another three factors on H-filter desalination were also investigated, including (a) the flow rate ratio between the sample and DI water, (b) sample viscosity, and (c) non-Newtonian fluids. After H-filter desalination, the samples were withdrawn into the dielectrophoresis chamber in which the bacterial cells were captured by pDEP. The feasibility of the device was demonstrated by the direct capture of the bacterial cells in 1× PBS buffer, cow's milk, and whole human blood after H-filter desalination, with the capture efficiencies of 70.7%, 90.0%, and 80.2%, respectively. We believe that this simple method can be easily integrated into portable microfluidic diagnosis devices for rapid and accurate detection of disease-causing agents and foodborne pathogens.

A portable, hand-powered microfluidic device for sorting of biological particles

Manually hand-powered portable microfluidic devices are cheap alternatives for point-of-care diagnostics. Currently, on-field tests are limited by the use of bulky syringe pumps, pressure controller and equipment. In this work, we present a manually operated microfluidic device incorporated with a groove-based channel. We show that the device is capable to effectively sort particles/cells by manual hand powering. First, the grooved-based channel with differently sized polystyrene particles was characterized using syringe pumps to study their distributions under various flow rate conditions. Afterward, the particle mixtures were sorted manually using hand power to verify the capability of this device. Finally, the manually operated device was used to sort platelets from peripheral blood mononuclear cells (PBMCs). The platelets were collected with a purity of ~ 100%. The purity of PBMCs was enhanced from 0.8 to 10.4% after multiple processes which results in an enrichment ratio of 13.8. During the process of manual hand pumping, the flow fluctuation caused by unstable injection will not influence the sorting performance. Due to its simplicity, this manually operated microfluidic chip is suitable for outfield settings.

Sensitive optical detection of clinically relevant biomarkers in affordable microfluidic devices: Overcoming substrate diffusion limitations

Abstract One of the biggest challenges in miniaturization of optical immunoassays is the short light path distance of microchannels/microcapillaries. Protein biomarkers are often presented in circulating blood in the picomolar-femtomolar range, requiring exceptional levels of sensitivity that cannot be met with traditional chromogenic substrates and without sophisticated, bulky detection systems. This study discloses an effective strategy for increasing the sensitivity and shorten the total test time for sandwich ELISAs in microfluidic devices optically interrogated, based on enhancing enzymatic amplification. We found that activity of Horseradish Peroxidase (HRP) in mesofluidic systems is highly limited by diffusion, therefore increasing the concentration of enzymatic substrate in these systems does not translate into an enhancement in enzymatic conversation. The opposite happens in microfluidic systems due to short diffusion distances, however increased concentration of the second enzymatic substrate, hydrogen peroxide (H2O2), leads to enzyme inhibition as herein reported. Consequently, we found that the molar ratio of o-phenylenediamine (OPD) to hydrogen peroxide from commercially substrate formulations is not suitable for miniaturized systems. Sandwich ELISA quantitation of a cancer biomarker PSA and human cytokine IL-1β in fluoropolymer microfluidic strips revealed over one order of magnitude increase in sensitivity and 10-fold decrease in incubation time by simply changing the molar ratio of OPD:H2O2 from 1:3 to 1:1 and increasing OPD concentration from 1 to 4 mg/ml. This enhancement in enzymatic amplification offers finally the sensitivity required for optical interrogation of novel portable and affordable microfluidic devices with inexpensive and ubiquitous smartphones and flatbed scanners.