ABSTRACT
Current diagnostic tests for high sensitivity detection of protein biomarkers involve long incubation times or require bulky/expensive instrumentation, hindering their use for point-of-care testing. Here, we report a microfluidic electrochemical immunosensor that employs a unique finger-actuated mixer for rapid, ultrasensitive measurements of protein biomarkers. Mixing was implemented during the incubation steps, which accelerated biomolecular transport and promoted immunocomplex formation, leading to enhanced analytical sensitivity and a shortened detection time. Electrochemical measurements were performed using a handheld diagnostic device consisting of a smartphone and miniature potentiostat. Proof of principle was demonstrated by using this platform for quantitative measurements of C-X-C motif chemokine ligand 9 (CXCL9), a serological biomarker for autoimmune and inflammatory diseases, which could be detected in human plasma at concentrations as low as 4.7 pg mL-1 in <25 min. The ability to rapidly detect protein biomarkers with high sensitivity in a point-of-care format makes this device a promising tool for diagnostic testing, particularly in resource-limited settings.
Subject(s)
Biomarkers , Electrochemical Techniques , Point-of-Care Testing , Humans , Biomarkers/blood , Biomarkers/analysis , Electrochemical Techniques/instrumentation , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/instrumentation , Equipment Design , Limit of Detection , Biosensing Techniques/instrumentation , Immunoassay/instrumentation , Immunoassay/methodsABSTRACT
Interstitial fluid (ISF) contains a wealth of biomolecules, yet it is underutilized for diagnostic testing due to a lack of rapid and simple techniques for collecting abundant amounts of fluid. Here, we report a simple and minimally invasive technique for rapidly sampling larger quantities of ISF from human skin. A microneedle array is used to generate micropores in skin from which ISF is extracted using a vacuum-assisted skin patch. Using this technique, an average of 20.8 µL of dermal ISF is collected in 25 min, which is an â¼6-fold improvement over existing sampling methods. Proteomic analysis of collected ISF reveals that it has nearly identical protein composition as blood, and >600 medically relevant biomarkers are identified. Toward this end, we demonstrate the detection of SARS-CoV-2 neutralizing antibodies in ISF collected from COVID-19 vaccinees using two commercial immunoassays, showcasing the utility of this technique for diagnostic testing.
ABSTRACT
Malaria is a major cause of illness and death worldwide. Rapid diagnostic tests are the most widely used tool for detecting malaria infection, however, they only provide binary results and lack the sensitivity needed to detect many asymptomatic infections. Molecular assays for quantifying malaria biomarkers offer higher detection sensitivity, however, they are time-consuming, and require expert training and expensive equipment, making them unsuitable for use in most of Africa. To address the need for simple, accurate and field-deployable malaria diagnostic tests, we have developed a microfluidic point-of-care (mPOC) immunoassay for rapid quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, in whole blood. This device features two diagnostic modes for detecting PfHRP2 at low (100's pg/mL) and high (1,000's ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples from malaria patients demonstrates that this platform offers similar accuracy as an ultra-sensitive PfHRP2 enzyme-linked immunosorbent assay (ELISA) test, while being 12× faster and simpler to use. This mPOC immunoassay can be deployed in rural health centers to assist clinicians in diagnosing and triaging malaria patients, ultimately improving patient outcomes.
Subject(s)
Biosensing Techniques , Malaria, Falciparum , Malaria , Humans , Malaria, Falciparum/diagnosis , Malaria, Falciparum/parasitology , Plasmodium falciparum , Microfluidics , Point-of-Care Systems , Sensitivity and Specificity , Antigens, Protozoan , Protozoan Proteins , Malaria/diagnosis , Prognosis , Enzyme-Linked Immunosorbent Assay/methodsABSTRACT
The detection and/or quantification of biomarkers in blood is important for the early detection, diagnosis, and treatment of a variety of diseases and medical conditions. Among the different types of sensors for detecting molecular biomarkers, such as proteins, nucleic acids, and small-molecule drugs, affinity-based electrochemical sensors offer the advantages of high analytical sensitivity and specificity, fast detection times, simple operation, and portability. However, biomolecular detection in whole blood is challenging due to its highly complex matrix, necessitating sample purification (i.e., centrifugation), which involves the use of bulky, expensive equipment and tedious sample-handling procedures. To address these challenges, various strategies have been employed, such as purifying the blood sample directly on the sensor, employing micro-/nanoparticles to enhance the detection signal, and coating the electrode surface with blocking agents to reduce nonspecific binding, to improve the analytical performance of affinity-based electrochemical sensors without requiring sample pre-processing steps or laboratory equipment. In this article, we present an overview of affinity-based electrochemical sensor technologies that employ these strategies for biomolecular detection in whole blood.
Subject(s)
Biosensing Techniques , Nanoparticles , Nucleic Acids , Electrochemical Techniques/methods , Electrodes , Biomarkers , Biosensing Techniques/methodsABSTRACT
Protein-based diagnostics are the standard of care for screening and diagnosing a broad range of diseases and medical conditions. The current gold standard method for quantifying proteins in clinical specimens is the enzyme-linked immunosorbent assay (ELISA), which offers high analytical sensitivity, can process many samples at once, and is widely available in many diagnostic laboratories worldwide. However, running an ELISA is cumbersome, requiring multiple liquid handling and washing steps, and time-intensive (â¼2 - 4 h per test). Here, we demonstrate a unique magneto-ELISA that utilizes dually labeled magnetic nanoparticles (DMPs) coated with horseradish peroxidase (HRP) and an HRP-conjugated detection antibody, enabling rapid immunomagnetic enrichment and signal amplification. For proof of concept, this assay was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which exhibited a lower limit of detection of 2 pg mL-1 (33 fM) in human serum. Measurements of PfHRP2 in clinical blood samples from individuals with and without P. falciparum infection revealed that this magneto-ELISA offers a superior diagnostic accuracy compared to a commercial PfHRP2 ELISA kit. We also demonstrate the versatility of this platform by adapting it for the detection of SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 8 pg mL-1 (174 fM) in human serum. In addition to its high analytical performance, this assay can be completed in 30 min, requires no specialized equipment, and is compatible with standard microplate readers and ELISA protocols, allowing it to integrate readily into current clinical practice.
Subject(s)
COVID-19 , Malaria, Falciparum , Nanoparticles , Enzyme-Linked Immunosorbent Assay/methods , Humans , Malaria, Falciparum/diagnosis , Malaria, Falciparum/parasitology , Plasmodium falciparum , SARS-CoV-2ABSTRACT
Current diagnostic tests for sensitive protein detection rely on immunological techniques, such as ELISA, which require sample purification, multiple washing steps and lengthy incubation, hindering their use for rapid testing. Here, we report a simple electrothermal flow-enhanced biosensor for ultrafast, high sensitivity measurements of protein biomarkers in whole blood. Magnetic nanobeads dually-labeled with a detection antibody and enzyme reporter are used to form immunocomplexes with the target protein, which are readily transported to the sensor via magnetic concentration. The incorporation of electrothermal flows enhances immunocomplex formation, allowing for rapid and sensitive detection without requiring blood purification or lengthy incubation. Proof of concept was carried out using Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which could be detected at concentrations as low as 5.7â pg mL-1 (95â fM) in whole blood in 7â min. The speed, sensitivity and simplicity of this device make it attractive for rapid diagnostic testing.
Subject(s)
Biosensing Techniques , Malaria, Falciparum , Antibodies , Enzyme-Linked Immunosorbent Assay , Humans , Malaria, Falciparum/diagnosis , Plasmodium falciparum , Protozoan ProteinsABSTRACT
Cytokines are soluble proteins secreted by immune cells that act as molecular messengers relaying instructions and mediating various functions performed by the cellular counterparts of the immune system, by means of a synchronized cascade of signaling pathways. Aberrant expression of cytokines can be indicative of anomalous behavior of the immunoregulatory system, as seen in various illnesses and conditions, such as cancer, autoimmunity, neurodegeneration and other physiological disorders. Cancer and autoimmune diseases are particularly adept at developing mechanisms to escape and modulate the immune system checkpoints, reflected by an altered cytokine profile. Cytokine profiling can provide valuable information for diagnosing such diseases and monitoring their progression, as well as assessing the efficacy of immunotherapeutic regiments. Toward this goal, there has been immense interest in the development of ultrasensitive quantitative detection techniques for cytokines, which involves technologies from various scientific disciplines, such as immunology, electrochemistry, photometry, nanotechnology and electronics. This review focusses on one aspect of this collective effort: electrochemical biosensors. Among the various types of biosensors available, electrochemical biosensors are one of the most reliable, user-friendly, easy to manufacture, cost-effective and versatile technologies that can yield results within a short period of time, making it extremely promising for routine clinical testing.
Subject(s)
Biosensing Techniques , Cytokines/analysis , Autoimmune Diseases/diagnosis , Electrochemical Techniques , Electrochemistry , Electronics , Humans , Nanotechnology , Neoplasms/diagnosis , Neurodegenerative Diseases/diagnosisABSTRACT
The COVID-19 pandemic has highlighted the importance and urgent need for rapid and accurate diagnostic tests for COVID-19 detection and screening. The objective of this work was to develop a simple immunosensor for rapid and high sensitivity measurements of SARS-CoV-2 nucleocapsid protein in serum. This assay is based on a unique sensing scheme utilizing dually-labeled magnetic nanobeads for immunomagnetic enrichment and signal amplification. This immunosensor is integrated onto a microfluidic chip, which offers the advantages of minimal sample and reagent consumption, simplified sample handling, and enhanced detection sensitivity. The functionality of this immunosensor was validated by using it to detect SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 50 pg/mL in whole serum and 10 pg/mL in 5× diluted serum. We also adapted this assay onto a handheld smartphone-based diagnostic device that could detect SARS-CoV-2 nucleocapsid protein at concentrations as low as 230 pg/mL in whole serum and 100 pg/mL in 5× diluted serum. Lastly, we assessed the capability of this immunosensor to diagnose COVID-19 infection by testing clinical serum specimens, which revealed its ability to accurately distinguish PCR-positive COVID-19 patients from healthy, uninfected individuals based on SARS-CoV-2 nucleocapsid protein serum levels. To the best of our knowledge, this work is the first demonstration of rapid (<1 h) SARS-CoV-2 antigen quantification in whole serum samples. The ability to rapidly detect SARS-CoV-2 protein biomarkers with high sensitivity in very small (<50 µL) serum samples makes this platform a promising tool for point-of-care COVID-19 testing.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/blood , SARS-CoV-2 , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Electrochemical Techniques , Humans , Immunoassay , Magnetic Phenomena , MicrofluidicsABSTRACT
Lateral flow immunochromatographic assays (LFIAs) are analytical devices used to detect the presence of one or more target analytes in a liquid sample. While LFIAs are one of the simplest and inexpensive types of immunoassays, they consist of multiple components (sample pad, conjugate pad, membrane, absorbent pad, backing card) and materials, requiring time-consuming device assembly. Here, we report a unique lateral flow immunochromatographic assay constructed from a single piece of cellulose paper, which is fabricated via laser cutting. Compared with conventional lateral flow immunochromatographic devices, this single-layer immunoassay enables simpler and faster fabrication, while minimizing material consumption and overall device costs. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a biomarker for malaria infection, which could be detected at concentrations as low as 4 ng mL-1 by the naked eye with no cross reactivity with other common Plasmodium protein biomarkers. While offering similar speed and ease-of-use as conventional LFIAs with a higher detection sensitivity than existing LFIAs for PfHRP2 detection, this single-layer lateral flow immunoassay has the potential to improve malaria testing, as well as the detection of other important protein biomarkers for point-of-care testing.
Subject(s)
Malaria , Plasmodium , Antigens, Protozoan , Humans , Immunoassay , Malaria/diagnosis , Protozoan ProteinsABSTRACT
Rapid diagnostic tests are one of the most commonly used tests to detect and screen for infectious diseases in the developing world. While these tests are simple, inexpensive, and readily available, they rely on finger-prick blood sampling, which requires trained medical personnel, poses risks of infection, and can complicate cooperation in young children, asymptomatic individuals, and communities with blood taboos. Here, we report a novel microneedle-based skin patch for the rapid detection of protein biomarkers in dermal interstitial fluid. Sample collection is facilitated by a hydrophilic hollow microneedle array that autonomously extracts and transports interstitial fluid to an antibody-based lateral flow test strip via surface tension for colorimetric antigen detection. We employ a simple gold enhancement treatment to enhance the detection sensitivity of this colloidal gold-based lateral flow assay and elucidate the underlying mechanism of this enhancement mechanism through experimental investigation. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2, a biomarker for malaria infection, which could be detected at concentrations as low as 8 ng/mL. Each test can be completed in <20 min and requires no equipment. To the best of our knowledge, this work is the first demonstration of a microneedle-based lateral flow assay for rapid protein detection in dermal interstitial fluid. In addition to its simplicity, minimally invasive nature, and low cost, this diagnostic device can be readily adapted to detect other protein biomarkers in interstitial fluid, making it a promising tool for point-of-care testing.
ABSTRACT
Currently, the diagnosis of many diseases relies on laboratory-based immunoassays (ELISA, Western Blot), which are laborious, time-consuming and expensive. To address these limitations, we report a wash-free and label-free electrochemical immunoassay for rapid measurements of protein biomarkers in blood samples. This immunosensor employs a unique detection scheme based on electrochemical-chemical (EC) redox cycling for signal amplification combined with an affinity-based protein quantification strategy. All of the reagents required for this assay are dried and stored on a stacked membrane assembly, consisting of a Vivid Plasma Separation membrane and two cellulose membranes situated above the sensor, enabling excellent stability at room temperature for up to 2 months. Proof of concept was carried out by performing measurements of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in whole blood samples, which could be detected from 100 ng/mL to 100 µg/mL with excellent specificity and reproducibility. Each measurement requires only two liquid dispensing steps and can completed in 5 min, making this diagnostic platform promising for point-of-care testing in resource-limited settings.
Subject(s)
Antigens, Protozoan/blood , Immunoassay/methods , Protozoan Proteins/blood , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Equipment Design , Humans , Immunoassay/instrumentation , Limit of Detection , Oxidation-Reduction , Reproducibility of Results , Ruthenium/chemistry , Sensitivity and SpecificityABSTRACT
We report a new enzyme-free electrochemical sensor for ultrasensitive measurements of protein biomarkers in plasma and whole blood samples based on a unique electrochemical-chemical-chemical (ECC) redox cycling signal amplification scheme. This scheme uses methylene blue (MB) as a redox indicator which undergoes an endergonic reaction with Ru(NH3)63+ and a highly exergonic reaction with tris(2-carboxyethyl)phosphine (TCEP). This approach offers improved detection sensitivity and sensor stability compared with enzyme-based ECC redox cycling techniques, while involving a simpler sensor modification process and detection protocol. This redox cycling scheme was combined with a robust immunosandwich assay for quantitative measurements of protein biomarkers. For proof of principle, Plasmodium falciparum histidine-rich protein 2 (PfHRP2) was measured in human plasma and whole blood samples, which could be detected down to 10 fg mL-1 and 18 fg mL-1, respectively. Furthermore, this immunosensor exhibits high selectivity, excellent reproducibility and good stability for up to 2 weeks, making it a promising platform for point-of-care testing, especially for detecting extremely low biomarker concentrations in raw biofluids.
Subject(s)
Biosensing Techniques , Electrochemical Techniques , Immunoassay , Methylene Blue/chemistry , Antigens, Protozoan/blood , Biomarkers/blood , Humans , Limit of Detection , Oxidation-Reduction , Protozoan Proteins/blood , Reproducibility of ResultsABSTRACT
Electrochemical sensors are an attractive platform for analytical measurements due to their high sensitivity, portability and fast response time. These attributes also make electrochemical sensors well suited for wearable applications which require excellent flexibility and durability. Towards this end, we have developed a robust electrochemical sensor on gauze via a unique embroidery fabrication process for quantitative measurements of wound biomarkers. For proof of principle, this biosensor was used to detect uric acid, a biomarker for wound severity and healing, in simulated wound fluid which exhibits high specificity, good linearly from 0 to 800µM, and excellent reproducibility. Continuous sensing of uric acid was also performed using this biosensor which reveals that it can generate consistent and accurate measurements for up to 7h. Experiments to evaluate the robustness of the embroidered gauze sensor demonstrate that it offers excellent resilience against mechanical stress and deformation, making it a promising wearable platform for assessing and monitoring wound status in situ.
Subject(s)
Biosensing Techniques , Uric Acid/isolation & purification , Wounds and Injuries/diagnosis , Biomarkers/metabolism , Electrochemical Techniques , Humans , Uric Acid/metabolism , Wound Healing/genetics , Wounds and Injuries/pathologyABSTRACT
Plastics are one of the most commonly used materials for fabricating microfluidic devices. While various methods exist for fabricating plastic microdevices, hot embossing offers several unique advantages including high throughput, excellent compatibility with most thermoplastics and low start-up costs. However, hot embossing requires metal or silicon molds that are fabricated using CNC milling or microfabrication techniques which are time consuming, expensive and required skilled technicians. Here, we demonstrate for the first time the fabrication of plastic microchannels using 3D printed metal molds. Through optimization of the powder composition and processing parameters, we were able to generate stainless steel molds with superior material properties (density and surface finish) than previously reported 3D printed metal parts. Molds were used to fabricate poly(methyl methacrylate) (PMMA) replicas which exhibited good feature integrity and replication quality. Microchannels fabricated using these replicas exhibited leak-free operation and comparable flow performance as those fabricated from CNC milled molds. The speed and simplicity of this approach can greatly facilitate the development (i.e. prototyping) and manufacture of plastic microfluidic devices for research and commercial applications.
ABSTRACT
Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.
Subject(s)
Electrochemical Techniques/methods , Malaria, Falciparum/diagnosis , Metal Nanoparticles/chemistry , Plasmodium falciparum/isolation & purification , Platinum/chemistry , Proteins/analysis , Protozoan Proteins/analysis , Saliva/parasitology , Antibodies, Protozoan/chemistry , Antibodies, Protozoan/immunology , Biosensing Techniques/methods , Humans , Hydrazines/chemistry , Immunoassay/methods , Malaria, Falciparum/parasitology , Metal Nanoparticles/ultrastructure , Methylene Blue/chemistry , Models, Molecular , Plasmodium falciparum/immunology , Proteins/immunology , Protozoan Proteins/immunology , Reproducibility of ResultsABSTRACT
Thermoplastics are becoming a popular material for fabricating microfluidic devices and there is an increasing need for robust surface modification strategies. UV/ozone (UVO) treatment is a simple and effective method for making plastic surfaces more hydrophilic. Prior reports on the stability of UVO-treated plastics are limited to four weeks, which is not sufficient for applications requiring long-term storage. Here, we present new findings on the long-term stability of UVO-treated plastics for up to 16 weeks and show that the storage condition has a significant impact on the surface stability. Static contact angle measurements, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were performed on UVO-treated cyclic olefin copolymer (COC), polycarbonate (PC) and poly(methyl methacrylate) (PMMA) stored in air, dehumidified and vacuum conditions. We found that the hydrophobic recovery of UVO-treated COC and PC can be inhibited by storing them in dehumidified or vacuum conditions, whereas the stability of PMMA is not significantly influenced by the storage condition. Protein adsorption studies were carried out and showed that there is a significant reduction in the amount of protein adsorption on UVO-treated plastics compared with untreated plastics. Lastly, UVO-treated PMMA microchannels were fabricated and used for capillary-driven flow, which revealed that longer treatment durations generate faster flow rates. These collective results offer new insights into the utility of UVO-treated plastics for microfluidic analytical applications.
ABSTRACT
Electrochemical sensors are powerful analytical tools which possess the capacity for rapid detection of biomarkers in clinical specimens. While most electrochemical sensors are fabricated on rigid substrates, there is a growing need for sensors that can be manufactured on inexpensive and flexible materials. Here, we present a unique embroidered electrochemical sensor that is capable of quantitative analytical measurements using raw biofluid samples. Conductive threads immobilized with enzyme probes were generated using a simple and robust fabrication process and used to fabricate flexible, mechanically robust electrodes on textiles. For proof of concept, measurements were performed to detect glucose and lactate in buffer and whole blood samples, which exhibited excellent specificity and accuracy. We also demonstrate that our embroidered biosensor can be readily fabricated in two-dimensional (2D) arrays for multiplexed measurements. Lastly, we show that this biosensor exhibits good resiliency against mechanical stress and superior repeatability, which are important requirements for flexible sensor platforms.
Subject(s)
Electrochemistry/instrumentation , Blood Glucose/analysis , Equipment Design , Humans , Stress, Mechanical , Time FactorsABSTRACT
Biosensors utilizing living tissues and cells have recently gained significant attention as functional devices for chemical sensing and biochemical analysis. These devices integrate biological components (i.e. single cells, cell networks, tissues) with micro-electro-mechanical systems (MEMS)-based sensors and transducers. Various types of cells and tissues derived from natural and bioengineered sources have been used as recognition and sensing elements, which are generally characterized by high sensitivity and specificity. This review summarizes the state of the art in tissue- and cell-based biosensing platforms with an emphasis on those using taste, olfactory, and neural cells and tissues. Many of these devices employ unique integration strategies and sensing schemes based on sensitive transducers including microelectrode arrays (MEAs), field effect transistors (FETs), and light-addressable potentiometric sensors (LAPSs). Several groups have coupled these hybrid biosensors with microfluidics which offers added benefits of small sample volumes and enhanced automation. While this technology is currently limited to lab settings due to the limited stability of living biological components, further research to enhance their robustness will enable these devices to be employed in field and clinical settings.
Subject(s)
Micro-Electrical-Mechanical Systems , Neurons/metabolism , Smell , Taste , Animals , Bioengineering/instrumentation , Bioengineering/methods , Biosensing Techniques/instrumentation , Humans , Light , Microfluidics/instrumentation , Neural Networks, Computer , Potentiometry/instrumentation , Potentiometry/methods , Reproducibility of Results , Semiconductors , Temperature , TransducersABSTRACT
Paper-based electrochemical biosensors are a promising technology that enables rapid, quantitative measurements on an inexpensive platform. However, the control of liquids in paper networks is generally limited to a single sample delivery step. Here, we propose a simple method to automate the loading and delivery of liquid samples to sensing electrodes on paper networks by stacking multiple layers of paper. Using these stacked paper devices (SPDs), we demonstrate a unique strategy to fully immerse planar electrodes by aqueous liquids via capillary flow. Amperometric measurements of xanthine oxidase revealed that electrochemical sensors on four-layer SPDs generated detection signals up to 75% higher compared with those on single-layer paper devices. Furthermore, measurements could be performed with minimal user involvement and completed within 30 min. Due to its simplicity, enhanced automation, and capability for quantitative measurements, stacked paper electrochemical biosensors can be useful tools for point-of-care testing in resource-limited settings.