Paper-based Analytical Devices Research Articles

A digital bar pH indicator based on a microfluidic paper-based analytical device

A pH value of an aqueous medium is an essential parameter in the environment and biology. Nowadays, it is possible to measure using a colorimetric pH paper or an electrochemical pH meter. However, since the two methods have drawbacks of the limited precision in pH determination and the careful management of a glass membrane electrode, respectively, it still remains as a challenge to discover a user-friendly, reliable pH-measuring device. Herein, a novel digital bar pH indicator was developed using a microfluidic paper-based analytical device (μPAD) to determine with the naked eye the pH value of an aqueous sample. The fabricated digital indicator consists of seven independent pH sensors displaying a color change from yellow to greenish blue above specific pH values. These sensing elements were vertically aligned to form a bar shape, and their color-changing pH points were regulated to increase upward from 4.5 to 7.5 with an interval of 0.5 units. For fast and simultaneous detection of the colorimetric sensors, sample addition to an inlet of the μPAD was quickly transported vertically along a hollow paper channel, horizontally divided, and arrived at the detection zones at similar times via delay channels. Through independent experiments, control of the color-changing pH point and flow rate were confirmed by addition of a surfactant additive into the pH indicator and inserting empty spaces in a flow channel, respectively. Reliable operation of the fabricated μPAD was verified using standard pH buffer samples. Finally, the feasibility of our digital bar pH indicator was successfully demonstrated for determining the pH value of a urine sample under the resolution of 0.5 units.

Single-step batch fabrication of microfluidic paper-based analytical devices with a 3D printer and their applications in nanoenzyme-enhanced visual detection of dopamine.

Wax printing is the most widely used method for fabricating microfluidic paper-based analytical devices (μPADs), but it still suffers from disadvantages like discontinuation of wax printers and need for additional equipment for heating treatment. To address these issues, this work initially describes a new class of wax printing approach for high-precision, batch fabrication of μPADs using a household 3D printer. It only involves a one patterning step of printing polyethylene wax into rice paper body. Under optimized parameters, a fabrication resolution, namely the minimum hydrophilic channel width, down to ~189 ± 30 μm could be achieved. In addition, the analytical applicability of such polyethylene wax-patterned μPADs was demonstrated well with enhanced colorimetric detection of dopamine as a model analyte by combining metal-organic framework (MOF) based nanoenzymes (ZIF-67) with a smartphone (for portable quantitative readout). The developed nanosensor could linearly detect dopamine over a concentration range from 10 to 1000 μM, with a detection limit of ca. 2.75 μM (3σ). The recovery results for analyzing several real samples (i.e., pig feed, chicken feed, pork and human serum) were between 91.82 and 102.79%, further validating its good detection accuracy for potential practical applications in food safety and medical diagnosis.

Molecularly Imprinted Polymers Coupled with Cellulosic Paper-Based Analytical Devices for Biosensing Applications

Molecularly Imprinted Polymers Coupled with Cellulosic Paper-Based Analytical Devices for Biosensing Applications

A Microfluidic Paper-Based Lateral Flow Device for Quantitative ELISA

This study presents an innovative lateral flow microfluidic paper-based analytical device (μPAD) designed for conducting quantitative paper-based enzyme-linked immunosorbent assays (p-ELISA), seamlessly executing conventional ELISA steps in a paper-based format. The p-ELISA device utilizes a passive fluidic circuit with functional elements such as a multi-bi-material cantilever (B-MaC) assembly, delay channels, and a buffer zone, all enclosed within housing for autonomous, sequential loading of critical reagents onto the detection zone. This novel approach not only demonstrates a rapid assay completion time of under 30 min, but also boasts reduced reagent requirements, minimal equipment needs, and broad applicability across clinical diagnostics and environmental surveillance. Through detailed descriptions of the design, materials, and fabrication methods for the multi-directional flow assay (MDFA), this manuscript highlights the device’s potential for complex biochemical analyses in a user-friendly and versatile format. Analytical performance evaluation, including a limit of detection (LOD) of 8.4 pM for Rabbit IgG, benchmarks the device’s efficacy compared to existing p-ELISA methodologies. This pioneering work lays the groundwork for future advancements in autonomous diagnostics, aiming to enhance global health outcomes through accessible and reliable testing solutions.

Bifunctional oxidase-peroxidase mimicking Fe–Ce MOF on paper-based analytical devices to intensify luminol chemiluminescence: Application for measuring different sugars with a smartphone readout

This study presents a potent paper-based analytical device (PAD) for quantifying various sugars using an innovative bi-nanozyme made from a 2-dimensional Fe/Ce metal-organic framework (FeCe-BTC). The MOF showed excellent bifunctional peroxidase-oxidase activities, efficiently catalyzing luminol's chemiluminescence (CL) reaction. As a peroxidase-like nanozyme, FeCe-BTC could facilitate the dissociation of hydrogen peroxide (H2O2) into hydroxyl radicals, which then oxidize luminol. Additionally, it was also discovered that when reacting with H2O2, the MOF turns into a mixed-valence MOF, and acts as an oxidase nanozyme. This activity is caused by the generated Ce4+ ions in the structure of MOF that can directly oxidize luminol. The MOF was directly synthesized on the PAD and cascaded with specific natural enzymes to establish simple, rapid, and selective CL sensors for the measurement of different sugars. A cell phone was also used to record light intensities, which were then correlated to the analyte concentration. The designed PAD showed a wide linear range of 0.1–10 mM for glucose, fructose, and sucrose, with detection limits of 0.03, 0.04, and 0.04 mM, respectively. It showed satisfactory results in food and biological samples with recovery values ranging from 95.8 to 102.4 %, which makes it a promising candidate for point-of-care (POC) testing for food control and medicinal purposes.

3D Origami-Inspired Electrochemical Paper-Based Analytical Devices with Flow-Through Electrodes and Quasi-Steady Flow

3D Origami-Inspired Electrochemical Paper-Based Analytical Devices with Flow-Through Electrodes and Quasi-Steady Flow

Microscale titration of acetic acid using digital colorimetry and paper-based analytical devices

Microscale titration of acetic acid using digital colorimetry and paper-based analytical devices

An exonuclease I-assisted quencher-free 2-aminopurine aptasensor based on a multipath paper-based device for ultrasensitive detection of kanamycin

A quencher-free multipath microfluidic paper-based analytical device (µPAD) was constructed for ultrasensitive detection of kanamycin based on exonuclease I (Exo I)-assisted signal enhancement of 2-aminopurine (2-AP). Here, Exo I, a single-stranded DNA-specific nuclease, was introduced to fully liberate 2-AP mononucleotides to greatly enhance biosensing sensitivity. 2-AP, a fluorescent adenine analogue embedded in single-stranded DNA (ssDNA), was employed as the detection signal source. The fluorescence of 2-AP is strong in the mononucleotide state, while it can have low fluorescence and even no fluorescence in ssDNA and dsDNA, respectively. The 2-AP fluorescence probe included 2-AP DNA and kanamycin aptamer. When kanamycin was present, binding occurred between kanamycin and the aptamer, leading to ssDNA, which was further digested by Exo I. In this case, free 2-AP mononucleotides were liberated, indicating strong fluorescence. In addition, the captured kanamycin was released for binding with the new aptamer, which resulted in the formation of a binding-hydrolysis-release cycle with the aid of Exo I. Under optimal conditions, this µPAD exhibited sensitive and multipath detection of kanamycin at concentrations as low as 1.26 × 10−14 M with a wide range of 10−13∼10−7 M. Furthermore, satisfactory results were achieved for analysing spiked kanamycin in milk and honey samples. This strategy is a very promising tool for monitoring antibiotics and evaluating the safety of animal-derived foods.

Boron-doped g-C3N4 supporting Cu nanozyme for colorimetric-fluorescent-smartphone detection of α-glucosidase

BackgroundDue to that the higher activity of nanozymes would bring outstanding performance for the nanozyme-based biosensing strategies, great efforts have been made by researchers to improve the catalytic activity of nanozymes, and novel nanozymes with high catalytic activity are desired. Considering the crucial role in controlling blood glucose level, strategies like colorimetric and chemiluminescence to monitor α-glucosidase are developed. However, multi-mode detection with higher sensitivity was insufficient. Therefore, developing triple-mode detection method for α-glucosidase based on great performance nanozyme is of great importance. ResultsIn this work, a novel nanozyme Cu-BCN was synthesized by loading Cu on boron doped carbon substrate g-C3N4 and applied to the colorimetric-fluorescent-smartphone triple-mode detection of α-glucosidase. In the presence of H2O2, Cu-BCN catalyzed the generation of 1O2 from H2O2, 1O2 subsequently oxidized TMB to blue colored oxTMB. In the presence of hydroquinone (HQ), the ROS produced from H2O2 was consumed, inhibiting the oxidation of TMB, which endows the possibility of colorimetric and visual on-site detection of HQ. Further, due to that the fluorescence of Mg-CQDs at 444 nm could be quenched by oxTMB, HQ could also be quantified through fluorescent mode. Since α-glucosidase could efficiently hydrolyze α-arbutin into HQ, the sensitive detection of α-glucosidase was realized. Further, colorimetric paper-based device (c-PAD) was fabricated for on-site α-glucosidase detection. The LODs for α-glucosidase via three modes were 2.20, 1.62 and 2.83 U/L respectively, high sensitivities were realized. SignificanceThe nanozyme Cu-BCN possesses higher peroxidase-like activity by doping boron to the substrate than non-doped Cu-CN. The proposed triple-mode detection of α-glucosidase is more sensitive than most previous reports, and is reliable when applied to practical sample. Further, the smartphone-based colorimetric paper-based analytical device (c-PAD) made of simple materials could also detect α-glucosidase sensitively. The smartphone-based on-site detection provided a convenient, instrument-free and sensitive sensing method for α-glucosidase.

Distance-based qualitative determination of pyrophosphate ions with a Haus-made microfluidic paper-based device using the metal–organic framework NH2-MIL-88 (Fe)

This study initially reports a distance-based quantitative assay of pyrophosphate ion (PPi) based on analyte-triggered decomposition of metal–organic framework (MOF) in microfluidic paper-based analytical devices (µPADs). The µPADs were created using a mini-cutter machine and a home-use hot-pressing machine for loading one type of MOF of NH2-MIL-88 (Fe) prepared with Fe3+ and fluorescent ligand of 2-aminoterephthalic acid (NH2-BDC). Upon sample introduction, the PPi could compete with NH2-BDC to bind Fe3+, leading to disintegration of the MOF and releasing of the fluorescent ligand in paper channel. The length (distance) of the NH2-BDC’s flow trace, when irradiated by a money tester pen, is proportional to the PPi level. The results show that the developed method only requires a ruler to linearly quantify PPi in a concentration range of 0–1000 μM, with a limit estimated to be ∼0.25 μM. Furthermore, recovery of PPi in orange juice and human urine was between 95.72 and 108.11 %, further validating its high detection accuracy. The fabrication method for µPADs offers many attractive advantages kike simplicity, batch production capability and low cost. Moreover, to our knowledge, this may be the first report of a portable quantitative MOF-based PPi assay with distance-based readout in µPADs.

Preconcentrating extractive polymeric network in paper-based sensing of copper

The concept of using an extractive polymeric sensing network on a paper-based substrate for the pretreatment of samples in the colorimetric analysis of heavy metals is demonstrated with the determination of Cu2+ in aqueous samples. The newly developed paper-based device combines the advantages of both liquid–liquid extraction-based separation and the analytical capabilities of microfluidic paper-based analytical devices (μPADs). The preconcentrating extractive polymeric network composed of 53.5 wt% poly(vinyl chloride) (PVC), 45 wt% extractant (di-(2-ethylhexyl)phosphoric acid (D2EHPA)) and 1.5 wt% dye (1-(2′-pyridylazo)-2-naphthol (PAN)), coated on filter paper, shows good water-wicking properties, allowing the transmission of high sample volumes. This results in significant analyte preconcentration. The extractant (D2EHPA) selectively binds Fe3+ and Zn2+ which otherwise will compete with Cu2+ in its colour reaction with PAN at the chosen pH of 3.0. The device’s capacity to process higher sample volumes resulting in higher sensitivity is increased by the integration of several filter paper absorption layers. The optimized device is characterized by a limit of detection (LOD) for tap and environmental waters of 3.4 and 9.6 µg/L, respectively; stability of 51 days if stored in a refrigerator; and total analysis time (from sample insertion to colour detection) of less than 5 min.

Sensitive assessment of bilirubin using various color space signals derived from captured images of microfluidic paper-based analytical devices

Many neonates’ manifest hyperbilirubinemia and jaundice during the first week following birth because of bilirubin accumulation in the blood. Newborns with severe hyperbilirubinemia develop neurotoxicity. Therefore, sensitive, accurate, cost-effective, and timely determination of bilirubin levels is highly desired. Herein, we present three simple systems of bilirubin assessment based on spectrophotometric, and digital imaging tools. Image-capturing devices pose a formidable challenge to sophisticated spectrophotometers and field-monitoring devices because of their simplicity and sensitivity. The first and second systems of bilirubin assessment, in solution, relied on spectrophotometric and conventional color responses of digital images collected with conventional or android smartphone camera, with LODs of 0.060–0.082 and LOQs of 0.200–0.273 ppm bilirubin. The third system utilized a microfluidic paper-based analytical device (μ-PAD) that is patterned with a wax printer on Whatman grade-1 chromatographic papers, with linear calibration graphs of up to 2.70,s and LODs of 0.035–0.040 and LOQs of 0.117–0.133 μg bilirubin/μ-PAD.

Wearable, disposable and non-enzymatic fluorescence nanosensor for monitoring sweat glucose through smartphone

The promise of non-invasive health monitoring has fuelled the development of wearable biosensors for in situ detection of biological analytes in sweat. This paper presents a new fluorescent nanosensor probe based on boric acid functionalized and heteroatom doped carbon quantum dots (CQDs) embedded in paper-based analytical devices and integrated with a hydrophilic cotton thread-based microfluidic channel for the development of a flexible, wearable, biocompatible, and biodegradable sweat sensor for real-time measurement of sweat glucose concentration via smartphone readout. The morphological, physiochemical, compositional, crystallinity, and optical properties of nanosensors were extensively investigated. CQD particle sizes were found to be widely dispersed, ranging from 2.6 to 7.1 nm, with an average diameter of 4.38 nm. A dual response via fluorescence and colorimetric change was demonstrated for on-site sweat glucose monitoring using a smartphone camera to capture images in RGB (red, green, and blue) format and analyse them simultaneously by a customized android app, with quantitative results displayed on the smartphone. The limit of detection value for in situ measurement of sweat glucose concentration was 1.40 to 2.00 μM (25.25 to 36.13 μg/dL) and the limit of quantification value was 4.67 to 6.69 μM (84.16–120.44 μg/dL) in the pH range of 4.5 to 7.5. The developed sensor's performance metrics were validated using a standard clinical technique, which indicates strong correlation between two methods with Pearson correlation coefficient values of 0.83 and 0.80 for non-diabetic and type- II diabetes patients, respectively. Besides, interference, spike, and recovery studies were carried out with recovery rates ranging from 96.5 to 105.8 %, demonstrating good repeatability and reproducibility. Furthermore, feasible fluorescence quenching processes have been explored. In addition, the disposable and biodegradable nature of the nanosensor probe was studied by throwing it into the soil in the natural environment, where it dissolves almost spontaneously after three weeks.

Microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum

BackgroundCalcium and magnesium ions are highly abundant and important cations in human body. At the same time, both dyscalcemia and dysmagnesemia are frequently encountered in the clinical practice. As deficiency or excess of Ca(II) or Mg(II) can cause severe symptoms, determining these ions in serum is of great importance. Concentration of these ions in biological samples is typically assayed in clinical laboratories with the use of expensive and specialized equipment. Since those methods cannot be easily adapted for self-diagnosis purposes, there is a great need to develop a convenient tool for reliable determination of calcium and magnesium in serum at the point-of-care. ResultsThe colorimetric methods employed for calcium and magnesium analysis were o-cresophtalein complexone assay and xylidyl blue assay, respectively. Analytical signal acquisition was accomplished using an ordinary flatbed scanner or smartphone and free software. For increased user-friendliness the device was optimized to perform simultaneous determination of calcium and magnesium ions in only 10 min. In the optimized conditions, the limit of detection for calcium ions was 0.09 mmol L−1, while for magnesium it was 0.04 mmol L−1. Determination of both ions requires only 4 μL of serum sample. The developed paper-based sensors were validated with control human serum samples and the obtained relative errors for majority of samples were below 20 %. SignificanceIn this paper, a microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum is reported for the first time. Additionally, this is also the first report on colorimetric determination in serum of any of these ions in paper-based format. Simultaneous detection of both ions allows for fast and user-friendly screening of disturbance in calcium and magnesium homeostasis.

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/μL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/μL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (μPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

Two-dimensional metal-organic framework nanozyme-mediated portable paper-based analytical device for dichlorophen assay

The metal-organic frameworks (MOFs) nanozyme-mediated paper-based analytical devices (PADs) have shown great potential in portable visual determination of phenolic compounds in the environment. However, most MOF nanozymes suffer from poor dispersibility and block-like structure, which often prompts deposition and results in diminished enzymatic activity, severely hindering their environmental applications. Here, we proposed colorimetric PADs for the visual detection of dichlorophen (Dcp) based on its significant inhibitory effect on the two-dimensional (2D) MOF nanozyme activity. Specifically, we synthesized a 2D Cu TCPP (Fe) (defined as 2D-CTF) MOF nanozyme exhibiting excellent dispersibility and remarkable peroxidase-like (POD-like) activity, which could catalyze the oxidation and subsequent color change of 3,3′,5,5′-tetramethylbenzidine even under neutral conditions. Notably, the POD-like activity of 2D-CTF demonstrated a unique response to Dcp because of the occupation of Fe–N4 active sites on the 2D-CTF. This property enables the use of 2D-CTF as a highly efficient catalyst to develop colorimetric PADs for naked-eye and portable detection of Dcp. We believe that the proposed colorimetric PADs offer an efficient method for Dcp assay and open fresh avenues for the advancement of colorimetric sensors for analyzing of phenolic toxic substances in real samples.

Superhydrophobic eggshell for fabrication of hydrophobic barrier of paper-based analytical device for colorimetric determination of ammonium ion in water

This work demonstrates the first application of the superhydrophobic eggshell as an environmental-friendly material for fabricating the hydrophobic barrier of a paper-based analytical device (PAD). The eggshell surface was modified by mixing the pulverized chicken eggshell biowaste with stearic acid and a polystyrene binder to accomplish superhydrophobicity. The PAD (10 × 10 mm2) is comprised of the circular-shaped hydrophilic reservoir (Ø 5 mm). The liquid barrier was fabricated by painting both the topside and the underside of the filter paper with the dispersed superhydrophobic eggshell solution. The SEM images revealed that the microsized superhydrophobic eggshell particles absorbed onto the porous surface of the cellulose fibril. A coated surface with a water contact angle of 155.39° ± 1.02° (n = 10) on the paper substrate was achieved, and this indicated superhydrophobicity. The eggshell barrier enabled excellent water and chemical resistance. The fabricated PAD was applied for the colorimetric determination of ammonium cations (NH4+), based on the modified Berthelot reaction. Samples and chromogenic solutions were aliquoted onto the hydrophilic reservoir, with subsequent capture of the optical image of the stable, green-colored product by a smart phone under a light-controlled studio. The green color intensities were evaluated by ImageJ™ and plotted against the standard NH4+ concentrations. A wide linear calibration range 5.0 to 100 mg N/L was achieved with good linearity (r2 > 0.99). The PAD confirmed satisfied analytical recovery (Mean ± SD: 100.6 % ± 0.97, n = 11 samples) and high precision (RSD = 0.85 %: 10-PAD replicate measurements of 1.0 mg N/L). The limit of detection (3 SD of blank/slope) of 1.05 mg N/L was found sensitive enough to monitor the NH4+ contents in freshwater. The results, determined by the developed PAD and ion chromatography, showed no significant difference under the statistical paired t-test at 95 % confidence (tstat = -1.80, tcri = 2.57, n = 6 samples).

Digitally Controlled Printing of Bioink Barriers for Paper-Based Analytical Devices: An Environmentally Friendly One-Step Approach.

The patterning of hydrophilic paper with hydrophobic materials has emerged as an interesting method for the fabrication of paper-based devices (PADs). Herein, we demonstrate a digitally automated, easy, low-cost, eco-friendly, and readily available method to create highly hydrophobic barriers on paper that can be promptly employed with PADs by simply using a bioink made with rosin, a commercially available natural resin obtained from conifer trees. The bioink can be easily delivered with the use of a ballpoint pen to produce water- and organic solvent-resistant barriers, showing superior properties when compared to other methods such as wax-printing or permanent markers. The approach enables the pen to be attached to a commercially available cutting printer to perform the semiautomated fabrication of hydrophobic barriers for PADs. With the aid of digitally controlled optimization, together with features of machine learning and design of experiments, we show a thorough investigation on the barrier strength that can be further adjusted to the desired application's needs. Then, we explored the barrier sturdiness across various uses, such as wide range aqueous pH sensing and the harsh acidic/organic conditions needed for the colorimetric detection of cholecalciferol.

Fabrication of flexible paper-based conducting molecularly imprinted polymer as analytical devices: Electrochemically assisted solid phase microextraction and selective flexible sensor for determination of naproxen

An electrochemical method is presented for the preparation of paper-based conductive molecular imprinted polymer (CMIP) film as a recognition element of a sensor and a selective absorber in separation methods. The paper became conductive by chemically incorporating polypyrrole (PPy) doped with anion dopant. The effects of the anion dopant and oxidant type on the conductivity of the modified paper were studied. Afterward, a CMIP film based on PPy and an anionic drug as a template was electrochemically synthesized on conductive paper surface, which was then utilized as a selective sorbent for electrochemically assisted solid phase microextraction (EC-SPME) and a recognition element in fabricating flexible drug-selective sensor. Factors affecting the performance of the paper-based EC-SPME device were studied in naproxen (NAP) analysis. All quantitative analytical parameters in the EC-SPME device were obtained by UV–vis absorption spectroscopy. Under the optimal conditions, linearity was achieved within the range of 2.0 × 10−7–2.0 × 10−5 mol/L with determination coefficient (R2 = 0.99) and the limit of detection (LOD) was 0.6 × 10−7mol/L. Moreover, a fabricated paper-based NAP-selective sensor, showed Nernstian response slopes of −61.6 ± 0.5 mV decade−1 over the linear range of 4.0 × 10−7–1.0 × 10−2 mol/L and LOD was 2.0 × 10−7 mol/L. The applicability and efficiency of the paper-based analytical devices were investigated for NAP determination as a model drug in human serum samples. The recoveries were obtained in the range of 90.0–103.0 %. Accordingly, paper-based CMIPs were found to be a novel fabrication method for efficient, selective, flexible and cost-effective material for analytical devices.