Paper-based Analytical Devices Research Articles

Trace nickel analysis in water samples via paper-based devices coupled with co-precipitation

A simple, fast, low-cost and sensitive microfluidic paper-based analytical device (u-PADs) integrated with the co-precipitation enrichment procedure was developed to analyse Ni(II) in tap and mineral water samples. The impacting factors, including pH, centrifugation (5 min at 4000 rpm), and amounts of reagents were optimized. The limit of detection of 0.009 mg L-1 and linear range of 0.03-2.00 mg L-1 were achieved with good intra- and inter-day precision (4.7 and 5.6% RSD, respectively). The recovery tests were conducted by spiking tap and mineral water samples and analyzed using the u-PADs after co-precipitation enrichment. The results obtained by the proposed method were validated by inductively coupled plasma-optical emission spectrometry (ICP-OES). The recoveries of the present method and ICP-OES were ranged from 92.4-106.8% and 92.9-97.2%, respectively. The two sets of (u-PADs and ICP-OES) results were in good agreement, as a paired t-test indicated no significant differences. The proposed method could be utilized for analyzing trace levels of Ni(II) in water samples in developing countries where the availability of conventional analytical instruments are significant problems.

Capacitive platform for real-time wireless monitoring of liquid wicking in a paper strip.

Understanding the phenomenon of liquid wicking in porous media is crucial for various applications, including the transportation of fluids in soils, the absorption of liquids in textiles and paper, and the development of new and efficient microfluidic paper-based analytical devices (μPADs). Hence, accurate and real-time monitoring of the liquid wicking process is essential to enable precise flow transport and control in microfluidic devices, thus enhancing their performance and usefulness. However, most existing flow monitoring strategies require external instrumentation, are generally bulky and unsuitable for portable systems. In this work, we present a portable, compact, and cost-effective electronic platform for real-time and wireless flow monitoring of liquid wicking in paper strips. The developed microcontroller-based system enables flow and flow rate monitoring based on the capacitance measurement of a pair of electrodes patterned beneath the paper strip along the liquid path, with an accuracy of 4 fF and a full-scale range of 8 pF. Additionally to the wired transmission of the monitored data to a computer via USB, the liquid wicking process can be followed in real-time via Bluetooth using a custom-developed smartphone application. The performance of the capacitive monitoring platform was evaluated for different aqueous solutions (purified water and 1 M NaCl solution), various paper strip geometries, and several custom-made chemical valves for flow retention (chitosan-, wax-, and sucrose-based barriers). The experimental validation delivered a full-scale relative error of 0.25%, resulting in an absolute capacitance error of ±10 fF. In terms of reproducibility, the maximum uncertainty was below 10 nl s-1 for flow rate determination in this study. Furthermore, the experimental data was compared and validated with numerical analysis through electrical and flow dynamics simulations in porous media, providing crucial information on the wicking process, its physical parameters, and liquid flow dynamics.

The Paper-Based Biosensor for Early Detection of Oral Cancer

The significance of paper-based biosensors for the early stage detection of oral cancer and their compatibility with affordable and low-cost diagnostics have been discussed in this article. Oral cancer is a major public health concern in India, and early-stage detection can be a great boon to the public. Among all the available methods for diagnosis, paper-based biosensors have proven to be very efficient in all aspects. It requires a very small volume of sample and very little time, and the process of detection is very simple. The diagnostic platform described here employs a hydrophobic material (wax) to pattern microfluidic channels on a hydrophilic substrate (paper) to detect the desired biomarker in saliva as biological fluid. Biomarker detection in saliva is advantageous because it is a noninvasive and potentially diagnostic sample. One of the primary benefits of using saliva in diagnosis is that sample collection is simple and noninvasive, significantly reducing the discomfort associated with blood collection and the privacy concerns associated with urine collection. The presence of biomarkers such as salivary cotinine and nitrite can be detected on a single paper-based analytical device, allowing for parallel quantification of the biomarkers. Calorimetric assays are used to analyze the biomarkers qualitatively, and ImageJ software is used to analyze the biomarkers quantitatively

Self-assembly of protein-DNA superstructures for alkaline phosphatase detection in blood.

We designed a paper-based analytical device by integrating horseradish peroxidase (HRP)-encapsulated 3D DNA for visual detection of alkaline phosphatase (ALP). This device allows on-paper sample pre-treatment, target recognition and signal readout, enabling simple (without additional pre-treatment of blood samples) and rapid (within 23 min) determination of ALP in clinical samples.

Real Time Paper Based Detection of Streptococcus bovis using Chromogenic Substrate in Resource Constrained Environments

Healthcare management faces a variety of difficulties in Circumstances with limited resources. Accurate diagnosis is the first step in disease prevention and therapy that works However, the diagnostic tools that are available in the economically advanced world are frequently of little utility in underdeveloped nations including Pakistan. The gram-positive bacterium Streptococcus bovis is an opportunistic pathogen that can cause various infections from superficial skin infections to severe and potentially fatal invasive diseases. Objective: To design a platform in environments with limited resources for the quick detection of Streptococcus bovis. Methods: A paper based analytical device (PAD) has been created with enrichment in sterile PYP broth for 1.5 hours and used for the detection of alkaline phosphatase activity using the chromogenic substrate Para-Nitrophenyl Phosphate (PNPP), the specie could be found in clinical samples. A coagulase test and sample Gram staining was conducted with the test. Qualitative detection was evaluated by visual detection while quantitative analysis is carried out using Image J software. Results: Alkaline Phosphatase ( S. bovis) reacts with the PNPP substrate (5.7 mM) in the presence of PAD. Concentrations below 4.5x104 cfu mL resulted in the detection of a color change. The micro PADs were incubated at 37 °C for 3–4 hours before reaction. Colored product (yellow) indicated the presence of S.bovis. Conclusion: Within 2 hours including enrichment time the test may identify Streptococcus bovis up to 104 CFUmL-1

Microfluidic paper-based analytical devices for cancer diagnosis

Cancer is a leading cause of death worldwide. Early diagnosis of cancer is crucial for successful treatment which, in turn, will decrease mortality. The development of low-cost, accurate, and easy to operate point-of-need (PON) devices to be used for cancer diagnosis and treatment follow-up is a worldwide need, especially in developing countries. Paper-based analytical devices (PADs) are considered a key solution, as they provide a low-cost platform for developing PON biosensors for cancer biomarker detections. There are various types of 2D and 3D PADs according to the type of paper substrate (filter paper, chromatographic paper, nitrocellulose membranes, etc.), fabrication method (wax printing, screen printing, cutting, etc.), detection technique used (colorimetry, fluorescence, chemiluminescence, electrochemiluminescence, electrochemical, etc.), the assay principle and recognition element used (antibodies, aptamers, DNA, nanoparticles, enzymes, etc.). Controlling all these factors determines the performance, accuracy, and sensitivity of the developed devices. This review discusses all these factors in the different PADs used for detection of cancer biomarkers and summarizes the advantages and disadvantages of each one.

A prototype microfluidic paper-based chromatic device for simultaneous determination of copper(II) and zinc(II) in urine

The present work proposes the development of a simple and easy-to-make microfluidic paper-based analytical device (µPAD) as a prototype chromatic device for the simultaneous determination of copper and zinc in urine. The µPAD comprises a main circular sampling zone with two identical arms, each containing a circular pretreatment zone and a circular detection zone. Copper(II) and zinc(II) react with 1-(2-pyridylazo)-2-naphthol (PAN) reagent, at pH 6.0 and room temperature, to form chelates colored red and pink, respectively, that can be quantified by digital imaging analysis. The colorimetric reaction sensitivity significantly improved by coating the paper substrate surface with 5% 3-aminopropyltriethoxysilane (APTES) and applying three successive additions of standard or sample onto the sampling zone, without any prior sample preparation step. These combined analytical strategies increased the color intensity of the metal chelates formed on µPAD surface, allowing to detect the desired elements at trace level concentrations. The reaction interferences associated with the presence of foreign elements were eliminated or reduced through the addition of masking agents at different concentrations for each case. Under optimized conditions, excellent linear relationships were obtained in the range 0.1−10 µg mL−1, with determination coefficients (R2) greater than 0.9957. The limits of detection (LOD) and quantification (LOQ), respectively, were 27.1 µg L−1 and 90.3 µg L−1 for copper and 35.9 µg L−1 and 119.7 µg L−1 for zinc. The developed prototype device was validated by analyzing urine certified material samples, obtaining a good concordance without significant difference between the certified values and the measured values. Its application for clinical analysis offers simple, quick, cheap, noninvasive, and environmentally friendly measurements, which are desirable analytical features for point-of-care testing.

Paper based microfluidic devices: a review of fabrication techniques and applications.

A wide range of applications are possible with paper-based analytical devices, which are low priced, easy to fabricate and operate, and require no specialized equipment. Paper-based microfluidics offers the design of miniaturized POC devices to be applied in the health, environment, food, and energy sector employing the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment free and Deliverable to end users) principle of WHO. Therefore, this field is growing very rapidly and ample research is being done. This review focuses on fabrication and detection techniques reported to date. Additionally, this review emphasises on the application of this technology in the area of medical diagnosis, energy generation, environmental monitoring, and food quality control. This review also presents the theoretical analysis of fluid flow in porous media for the efficient handling and control of fluids. The limitations of PAD have also been discussed with an emphasis to concern on the transformation of such devices from laboratory to the consumer.

Tetrahedral DNA framework assisted rotational paper-based analytical device for differential detection of SARS-CoV-2 and influenza A H1N1 virus

Coronavirus disease 2019 (COVID-19) and influenza A are two respiratory infectious diseases with similar clinical manifestations. Because of the complex global epidemic situation of COVID-19, the distinction and diagnosis of COVID-19 and influenza A infected persons is crucial for epidemic prevention and control. In this study, tetrahedral DNA framework (TDF) was combined with a rotational paper-based analytical device, and the color change generated by the reaction between horseradish peroxidase (HRP) and 3,3′5,5′-tetramethylbenzidine (TMB)–H2O2 was used for grayscale signal analysis by ImageJ software. The quantitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A H1N1 virus were realized simultaneously. Under the optimal conditions, the paper-based analytical device showed a good linear relationship between the two viruses in the range of 10-14-10-8g/mL, and the two viruses were not affected by cross reaction. This sensor provides a convenient and reliable method for clinical rapid differentiation and diagnosis of COVID-19 and influenza A.

Development of Mia Phenotyping Using Paper-Based Device.

The MNS7 (Mia) blood group antigen is found at a different prevalence among different ethnic groups. Anti-Mia can cause hemolytic disease of the fetus and newborn (HDFN) and both acute- and delayed-type hemolytic transfusion reactions (HTR). Mia typing should be performed in donors to prevent life-threatening hemolytic transfusion reactions. The gel card and standard tube methods still need specialized equipment, centrifugation, and expertise for result interpretation. We used a novel paper-based analytical device (PAD) pre-coated with monoclonal IgM anti-Mia for Mia phenotyping. We measured grey pixel intensity in blood typing results for interpretation processing using OpenCV at the sample (SP) and elution parts (EP); furthermore, we used the SP: EP ratio and F-score as analysis criteria. We typed 214 blood EDTA samples with PAD-Mia and then compared with gel card results for setting an analysis criterion. We observed 100% sensitivity, specificity, and accuracy when we applied the SP: EP ratio and F-score with the optimal criterion (1.07 and 0.17 for SP: EP ratio and F-score, respectively). The validation of PAD-Mia typing for blood donor samples (n = 150) via F-score gave 100% sensitivity and specificity when compared with the gel card method; therefore, we argue that PAD-Mia typing can be used for Mia phenotyping without sero-centrifugation. Moreover, to study the correlation between genotype and phenotype, PCR-SSP was performed to identify GYP(B-A-B) hybrids. The results revealed that all Mia+ blood samples gave a positive with GP. Hut, GP. HF, GP. Mur, GP. Hop, and GP. Bun. Results of the gel card method and PCR-SSP were concordant. Hence, using PAD-Mia typing in blood donors would be helpful for creating a phenotype database of blood donors for reducing alloimmunization risks.

Point-of-care assay platform for uric acid, urea, and triglycerides with a microfluidic paper device (μPAD) controlled by stimulus-sensitive valves

A microfluidic biosensor paper-based analytical device, or μPAD, was developed as a point-of-care assay platform for the simultaneous determination of three clinically important markers: uric acid, urea, and triglycerides. To achieve a simultaneous determination, thermosensitive valves based on N-isopropylacrylamide polymers controlled the flow of fluid in the μPAD. The evaluation of the analytical characteristics of the μPAD was done using a photographic camera at room temperature.The detection limits were 4.5·10−5, 2.5·10−3, and 1.5·10−3 mg mL−1, for uric acid, urea, and triglycerides, respectively, and the precision expressed as relative standard deviation (% RSD) 3.2, 1.2, and 1.6%. The obtained μPAD devices were validated with real human plasma samples, demonstrating high accuracy and precision. Multiple analyte point-of-care tests such as those developed here have excellent characteristics, being easy to manufacture, cost-effective, easy to use, and highly sensitive.

Poly-(MMA-IL) filter paper: A new class of paper-based analytical device for thin-film microextraction of multi-class antibiotics in environmental water samples using LC-MS/MS analysis

A paper-based polymeric ionic liquid (p-Poly-(MMA-IL)) was successfully developed by grafting the polymeric ionic liquid on the surface of commercial filter paper (FP) by using the dipping method, presenting a new cost-effective film. The newly developed p-Poly-(MMA-IL) FP was then applied as a paper-based thin-film microextraction (p-TFME) analytical device to extract 14 compounds as representative of five groups of antibiotic drugs, which were sulfonamides, tetracyclines, fluoroquinolones, penicillin and macrolides in environmental water samples. Besides, p-Poly-(MMA-IL) FP, p-Poly-(MMA) FP, and unmodified filter paper were successfully characterised by FTIR, NMR, FESEM, TGA, and XRD techniques. They underwent significant parameters optimisation, which affected the extraction efficiency. Under optimal conditions, the proposed (p-Poly-(MMA-IL) FP-TFME) device method was evaluated and applied to analyse multi-class antibiotic drugs in environmental water samples by using a liquid chromatography-mass spectrometry (LC-MS). The validation method showed that a good linearity (0.1 μg L−1 - 500 μg L−1) was noted (R2 > 0.993, n = 3). Detection and quantification limits were within 0.05 μg L−1 – 4.52 μg L−1 and 0.15 μg L−1 – 13.6 μg L−1, respectively. The relative standard deviation (RSD) values ranged at 1.4%–12.2% (intra-day, n = 15) and 4.4%–11.0% (inter-day, n = 10). The extraction recoveries of environmental water samples ranged from 79.1% to 126.8%, with an RSD of less than 15.4% (n = 3). The newly developed paper-based polymeric ionic liquid (p-Poly-(MMA-IL) FP) for analysis of multi-class antibiotic drugs under the p-TFME analytical device procedure was successfully achieved with limited sample volume and organic solvent, fast extraction, and feasible in daily analysis. The detection concentration and relative RSD of multi-class antibiotics determined in various environmental water samples by the proposed method (n = 5) were within 0.44 μg L−1 – 54.41 μg L−1 and 0.69%–15.56%, respectively. These results signified the potential of the p-Poly-(MMA-IL) FP-TFME device as an efficient, sensitive and environmentally friendly approach for analysing antibiotics.

Energy transfer mediated rapid and visual discrimination of tetracyclines and quercetin in food by using N, Cu Co-doped carbon dots

The appearance of multi-drug resistant Escherichia coli makes the combination of tetracyclines (TCs) and quercetin (QCT) more common to fight stubborn bacterial infections so that the effective detections of TCs and QCT are essential and necessary. Here, a novel fluorescence probe for differentiating TCs and QCT is developed based on the nitrogen and copper co-doped carbon dots (N, Cu-CDs). The N, Cu-CDs are prepared from ethylene diamine tetraacetic acid (EDTA) and anhydrous copper chloride as precursors through hydrothermal process and exhibit bright blue fluorescence with excellent optical stability. With the presence of four tetracyclines (DOX, TC, CTC and OTC), the fluorescence intensity of N, Cu-CDs is quenched directly due to the internal filtration effect (IFE), and the detection limit obtained through single-signal fluorescence sensing is as low as 23.8 nM for DOX, 37.2 nM for TC, 43.8 nM for OTC and 28.8 nM for CTC. More remarkably, three dimensional ratiometric fluorescence probe for detecting QCT is proposed based on the appearance of another emission at (410 nm, 490 nm) due to electron transform (ET) process. This new method shows a good linear relationship in the range of 10–100 μM with a low detection limit of 59.3 nM. Furthermore, a dual-channel fluorescence sensing platform based on microfluidics paper-based analytical devices (μPADs) is developed for simultaneously visual discrimination of TCs (DOX is chosen as the typical detecting model for TCs) and QCT. This investigation provides a new way for the development of CDs as multifunction fluorescence probes.

A 3D microfluidic paper-based analytical device with smartphone-assisted colorimetric detection for iodine speciation in seaweed samples

The present work reports on the development of a 3D origami microfluidic paper-based analytical device (3D µPAD) for the determination of iodide and iodate in edible seaweeds by smartphone-based colorimetric detection. In addition, a methacrylate holder was designed and fabricated to enhance interlayer contact in 3D µPADs, obtaining excellent sensitivity and precision, also allowing real-time monitoring in a straightforward and expeditious way. The reported assay, based on the formation of a blue colored triiodide-starch complex at the detection areas of the 3D µPAD, represents an affordable, fast and greener alternative for the simultaneous determination of inorganic iodine species. Under optimal conditions, the proposed method showed limits of detection and quantification of 9.8 and 32.7 µM for I- and 0.6 and 1.8 µM for IO3-, respectively. The repeatability, expressed as relative standard deviation, was 1.7% and 3.3% for I- and IO3-, respectively. The proposed 3D µPAD was applied to the determination of iodine species in extracts of edible seaweeds and related food additives, showing satisfactory recoveries (90–109%).

Enhancing of detection resolution via designing of a multi-functional 3D connector between sampling and detection zones in distance-based microfluidic paper-based analytical device: multi-channel design for multiplex analysis.

One of the problems in the distance-based microfluidic paper-based analytical device (DB-µPAD) that limits the detection resolution is the mixing of reagents from the detection to the sampling zone or vice versa due to spreading by capillary action. In the present paper, to overcome mixing of the reagents in the zones, a multi-functional connector using a three-dimensional (3D) design has been developed externally to connect the two zones. Using such a novel design, it is acertained that there is no any mixing due to the dispersion of the reagents in the two zones. Interestingly, the simple 3D connector has other functions, such as its potential to be used as a masking zone and/or reaction zone whenever is needed. Based on this proposed 3D connector-based DB-µPAD, three parallel microchannels were built as detection zones with one sampling zone for multiplex analysis for the detection of Fe2+, Ni2+, and hardness of water. In the Ni2+ channel, the connector piece worked as both masking part and connection part. In the Fe2+ line, the connector served as the connector and reducing pad (Fe3+ to Fe2+ ions). While in the third channel, the connector has connection function only. Satisfactory limit of detection (LoD), accuracy, and precision were obtained using this design. The LODs obtained with the proposed design were 1.13, 0.62, and 1.87mg.L-1 for total hardness, Fe2+, and Ni2+, respectively.

A fully integrated 3D printed platform for sulfite determination in beverages via gas diffusion membrane extraction and digital video treatment

In this study, we have described a miniaturized, simple, and low-cost device for sulfite determination in beverages by coupling Gas Diffusion Microextraction to paper-based analytical devices. The color change of an acid-base indicator – promoted by the generated gaseous SO2 – impregnated onto the paper surface was monitored in the function of time by video recording using a smartphone. The analytical information was related to the Hue, Saturation, Value (HSV) color space extracted from the video file. The complete analytical platform was built using a 3D printer, allowing the easy fabrication of a low-cost tailored device. Under optimized conditions, a linear relation from 5 to 90 mg L-1 was obtained using 30 µL of the reagent, 1 mL of sample, and 10 min of analysis. The relative standard deviation and the limit of detection were 2.2 % and 1.6 mg L-1, respectively. The method was successfully employed in several beverages, such as juices, soda, and coconut water.

Simultaneous Cross-type Detection of Water Quality Indexes via a Smartphone-App Integrated Microfluidic Paper-Based Platform.

Water quality guarantee in remote areas necessitates the development of portable, sensitive, fast, cost-effective, and easy-to-use water quality detection methods. The current work reports on a microfluidic paper-based analytical device (μPAD) integrated with a smartphone app for the simultaneous detection of cross-type water quality parameters including pH, Cu(II), Ni(II), Fe(III), and nitrite. The shapes, baking time, amount, and ratios of reaction reagent mixtures of wax μPAD were optimized to improve the color uniformity and intensity effectively. An easy-to-use smartphone app was established for recording, analyzing, and directly reading the colorimetric signals and target concentrations on μPAD. The results showed that under the optimum conditions, the current analytical platform has reached the detection limits of 0.4, 1.9, 2.9, and 1.1 ppm for nitrite, Cu(II), Ni(II), and Fe(III), respectively, and the liner ranges are 2.3-90 ppm (nitrite), 3.8-400 ppm (Cu(II)), 2.9-1000 ppm (Ni(II)), 2.8-500 ppm (Fe(III)), and 5-9 (pH). The proposed portable smartphone-app integrated μPAD detection system was successfully applied to real industrial wastewater and river water quality monitoring. The proposed method has great potential for field water quality detection.

Smartphone-based colorimetric detection of cardiac troponin T via label-free aptasensing

We report an aptasensing platform for the detection of cardiac troponin T (cTnT) in the immediate and early phases of acute myocardial infarction (AMI). High-flow filter paper was used to fabricate a microfluidic paper-based analytical device (μ-PAD), which was further modified with gold-decorated polystyrene microparticles functionalized with a highly specific cTnT aptamer. Herein, cTnT detection is presented in two linear ranges (0.01-0.8 μg/ml and 6.25-50 μg/ml) with an LoD of 3.9X10−4 μg/ml, which is in agreement with reference values determined by the American Heart Association. The proposed platform showed remarkable selectivity against AMI-associated cardiac biomarkers such as TNF-alpha, interleukin-6, cardiac troponin I, and reactive protein-C. This aptasensor is a label-free assay that relies only on smartphone-based image analysis and takes less processing time in comparison with traditional methods like ELISA. Furthermore, it exhibits outstanding stability over 23 days when devices are stored at 4 °C. The reported platform is a stable and cost-effective method for the on-site and user-friendly detection of cTnT in normal saline buffer and diluted human serum.

Evaluation of chromogenic substrates for horseradish peroxidase on paper-based microfluidic devices

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring. A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates, which produce colored products and enable the qualitative and quantitative detection of analytes. Horseradish peroxidase (HRP) is one of the most commonly used enzymes for this application. While a wide variety of chromogenic substrates and substrate solutions are available for HRP, these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs. In this work, 33 chromogenic substrate solutions for HRP containing one of six different substrate molecules were evaluated in a standardized colorimetric assay and compared on the basis of limit of detection (LOD), lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), sensitivity, dynamic range, stability, and cost. While the substrates tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were found to achieve the best overall analytical performance, each substrate had characteristics that may make them appealing for specific applications. The results of this work will facilitate the selection of substrate molecules and the formulation of substrate solutions for future colorimetric paper-based assays involving HRP.

Facile and Compact Electrochemical Paper-Based Analytical Device for Point-of-Care Diagnostic of Dual Carcinogen Oxidative Stress Biomarkers through a Molecularly Imprinted Polymer Coated on Graphene Quantum-Dot Capped Gold.

Nanoscale imprinting significantly increases the specific surface area and recognition capabilities of a molecularly imprinted polymer by improving accessibility to analytes, binding kinetics, and template removal. Herein, we present a novel synthetic route for a dual molecularly imprinted polymer (dual-MIP) of the carcinogen oxidative stress biomarkers 3-nitrotyrosine (3-NT) and 4-nitroquinolin-N-oxide (4-NQO) as coatings on graphene quantum-dot capped gold nanoparticles (GQDs-AuNPs). The dual-MIP was successfully coated on the GQDs-AuNPs core via a (3-mercaptopropyl) trimethoxysilane (MPTMS) linkage and copolymerization with the 3-aminopropyltriethoxysilane (APTMS) functional monomer. In addition, we fabricated a facile and compact three-dimensional electrochemical paper-based analytical device (3D-ePAD) for the simultaneous determination of the dual biomarkers using a GQDs-AuNPs@dual-MIP-modified graphene electrode (GQDs-AuNPs@dual-MIP/SPGE). The developed dual-MIP device provides greatly enhanced electrochemical signal amplification due to the improved electrode-specific surface area, electrocatalytic activity, and the inclusion of large numbers of dual-imprinted sites for 3-NT and 4-NQO detection. Quantitative analysis used square wave voltammetry, with an oxidation current appearing at -0.10 V for 4-NQO and +0.78 V for 3-NT. The dual-MIP sensor revealed excellent linear dynamic ranges of 0.01 to 500 μM for 3-NT and 0.005 to 250 μM for 4-NQO, with detection limits in nanomolar levels for both biomarkers. Furthermore, the dual-MIP sensor for the simultaneous determination of 3-NT and 4-NQO provides high accuracy and precision, with no evidence of interference from urine, serum, or whole blood samples.