Paper-based Test Strips Research Articles

Construction of rhodamine-based fluorescent sensor for fast, on-site quantitative detection of hazardous salicylic acid in practical sample analysis

Salicylic acid (SA) is widely used in food storage, preservatives, additives, healthcare, and the pharmaceutical industry. However, various poisoning symptoms are frequently reported upon ingestion of a large amount of SA. Therefore, discovering new tools for sensing SA with fast, simple, and portable performance is imperative. Herein, five rhodamine-based fluorescent sensors were constructed, and investigated their SA detection profiles. Probe 1 was excellent selective with a rapid response, highly sensitive (LOD = 2.5 μM), good interference resistance, and unaided eye recognition. The spray experiment and paper-based test strips indicating that probe 1 enables to the on-site and quantitatively detect SA on actual food surfaces by using a smartphone identifying the RGB values. The sensing performance was validated in soil samples, water, and various agricultural food samples. Overall, the constructed SA sensor can function as a promising, convenient, and affordable tool for point-of-care detection of SA in diversiform environmental samples.

A Copper-Incorporated meso-(N'-acetyl-hydrizide)-BODIPY as a "Off-On" Fluorescent Probe for Histidine.

We present a highly sensitive and selective fluorescence "turn-on" sensor for l-histidine (His) detection in aqueous solutions utilizing a 1-Cu2+ complex. This sensing platform employs a fluorescence-based ligand displacement approach, featuring a meso-(N'-acetyl-hydrizide)-based BODIPY derivative (1) complexed with Cu2+. Initially highly fluorescent, compound 1 is selectively quenched by Cu2+ ions, forming the 1-Cu2+ complex. The high affinity between His and Cu2+ effectively displaces 1 from the complex, restoring fluorescence. The system exhibits rapid response (within 5 minutes), excellent sensitivity (detection limit of 78 nM), operational simplicity, and a large fluorescence "turn-on" signal. It demonstrates remarkable selectivity for His over other amino acids, with maleimide masking cysteine interference. Notably stable in complex biological matrices, the sensor has successfully quantified His in artificial urine samples. Its practical applicability extends to paper-based test strips, offering portability and potential for real-time His monitoring in clinical diagnostics and biological systems.

A portable optical sensor combining smartphone with phycocyanin-based fluorescent test paper for rapid, visual and on-site detection of CO32–

With the development of global industry, carbon dioxide emissions surged. The conversion of carbon dioxide from the air results in some CO32−, which can exacerbate environmental disasters like ocean acidification. Therefore, the content of CO32− in seawater is an important indicator of the degree of ocean acidification. In this study, natural fluorescent protein phycocyanin (PC) was used as a fluorescent probe, and a fluorescence detection method was established for quantitative monitoring of CO32− with quick response time (within 50 s), high sensitivity, and selectivity. The fluorescence quenching phenomenon between PC and CO32− was mainly attributed to static quenching. The limit of detection (LOD) was 0.42 μM and the method was successfully applied to monitor CO32− in tap water and seawater, acquiring satisfactory recovery between 99.28 % and 106.40 %. More importantly, paper-based test strips were easily fabricated using PC, enabling the rapid, visual, and on-site detection of CO32− with the aid of a smartphone. The visual detection integrated with the smartphone was converted to data information (RGB value) through a Color Picker APP and successfully used for quantitative identification of CO32−. By capturing fluorescent images and analyzing the corresponding RGB value via a smartphone, the linear calibration ranged from 0.5 μM to 500.0 μM with LOD of 0.11 μM was obtained. Satisfactory recoveries were acquired in tap water (98.00 %-107.50 %) and seawater (97.30 %-101.74 %), respectively. Therefore, integrating the PC fluorescent paper with a smartphone realizes the rapid, visual, and on-site detection of CO32− in the water environment, which is expected to broaden application prospects of monitoring ocean acidification degree.

Colorimetric pH-sensing of artificial gastric fluid using naphthalimide-based CH acids

In this study, we introduce novel colorimetric pH-sensing probes based on naphthalimide malonate derivatives. These probes were synthesized by reacting 4-bromo-1,8-naphthalimide with various malonates, including malononitrile, ethyl cyanoacetate, and diethyl malonate. Each derivative exhibited distinct pH-sensing characteristics due to their differing CH acidities. The malononitrile-based probe, NPI-N2, demonstrated pronounced chromogenic pH-signaling behavior, transitioning from colorless to red-violet, accompanied by a decrease in fluorescence intensity. Notably, NPI-N2 retained its pH-sensing capability in the presence of common metal ions, anions, and pepsin, a key component of gastric fluid. The pKa of NPI-N2 was determined to be 3.08 through pH-dependent absorbance curve fitting. To modulate the pH-sensing range, ester-nitrile (NPI-EN) and diethyl ester (NPI-E2) subunits were incorporated into the naphthalimide framework, resulting in increased pKa values of 6.73 and 10.76, respectively. The pH-signaling mechanism of NPI-N2 was elucidated by 1H and 13C NMR spectroscopy, revealing deprotonation of the malononitrile moiety and subsequent resonance extension through the naphthalimide structure. To facilitate practical pH determination, NPI-N2 was integrated into a paper-based test strip, enabling convenient and reliable pH measurement of artificial gastric fluid.

Paper-Based procalcitonin and Interleukin-6 test strip with Spectrum-Based optical reader for enterovirus severity differentiation in children

Infectious diseases significantly impact global health, necessitating prompt diagnosis to mitigate life-threatening sepsis risk. Identifying patients at risk of severe neurological complications from enterovirus infections is challenging due to nonspecific initial presentations. Point-of-care testing (POCT) has emerged as a transformative tool, with low-cost lateral-flow colorimetric assays showing promise in deployable POCT devices. We developed a PCT/IL-6 rapid diagnostic system integrating lateral flow assay (LFA) test strips and a portable optical spectrum reader, allowing simultaneous semi-quantitative measurement of serum PCT and IL-6 within 30 min at the point of care. The system demonstrated a strong correlation with traditional ELISA and effectively differentiated severe pediatric enterovirus cases using serum samples. IL-6 showed superior discriminatory ability over PCT in identifying patients with severe neurological complications. This novel diagnostic platform holds great potential for early sepsis recognition and infectious disease management, especially in resource-limited settings.

Development of a simple and rapid dipstick paper-based test strip for colorimetric determination of nitrate and nitrite in water and foodstuffs

A rapid user-friendly paper-based test strip using zinc microparticles in conjugation with Griess reagent was developed for nitrite and nitrate detection. Test strips were fabricated using a simple and fast method of step-by-step immersion into reagents so that each strip contained a single detection pad for nitrite detection and another separate pad for nitrate detection. To reduce nitrate to nitrite, zinc microparticles suspended in ethanolic solution of polyvinylpyrrolidone (PVP) were uniformly immobilized on the paper strips that were previously impregnated in the Griess reagent and dried. The Griess reagent components were optimized to reach the highest color intensity. The optimized test strip was able to determine both nitrite and nitrate with respective detection limits of 0.43 and 9.43 mg/kg and a detection time of 60 s. The performance of the new test strips was evaluated for the simultaneous colorimetric detection of nitrite and nitrate in water and different food samples.

Selective sensing of paraquat by simple off-the-shelf compounds: Applications using composite hydrogel beads and smartphone

The massive use of paraquat (PQ) in agricultural practices has posed serious threats to food safety and human health. Real-time monitoring of PQ remains an obstacle. Herein, a fluorometric as well as smartphone-based very cheap sensing assay is discussed for the detection of this non-selective contact herbicide PQ based on simple off-the-shelf commercially available dyes PTSA (1), HPTS (2) and Eosin Y (3) in pure aqueous media. Owing to their sensitivity towards PQ through electrostatic interactions, these dyes undergo a fluorescence quenching process. Dyes (2) and (3) show a rapid colorimetric change in the presence of PQ as shown by UV-Vis absorption spectral studies. 1 exhibits high sensitivity for PQ in comparison to 2 and 3 with the detection limit as low as 182 nM. All three probes exhibit high sensitivity, high selectivity for PQ over other analytes and excellent anti-interference properties. The Stern-Volmer plots and fluorescence lifetime studies confirm the static quenching process of the probes in the presence of PQ. Furthermore, sensor-coated paper-based test strips were successfully used for the ultrafast detection and quantification of PQ in spiked real water, fruit and vegetable samples. For the first time, silica-sodium alginate (SA) polymer hybrid hydrogel beads incorporated with probes 1, 2 and 3 were used for the detection and adsorption of PQ from water samples. For assessing the reliability of the proposed sensing system for the on-site detection of PQ, an easy-to-use portable test cassette kit integrated with a smartphone was used. The signal outputs were captured and analysed via RGB color value to use the probes for the practical application by using a smartphone as a portable analytical device for the rapid point-of-care monitoring of PQ.

A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 μm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

Ion-selective paper-based chromogenic strip and electrochemical sensor for the detection of ammonium ions

The fraudulent practice of adding ammonium salts to milk in order to enhance the nitrogen content has serious health implications including renal failure. Ammonium ion detection has been targeted through the conventional analytical methods however, lack of portability and complex operation limits their use for field applications. In this scenario, paper-based test strips and electrochemical sensors can provide strategies for point-of-test applications. Here, an ion-selective paper-based chromogenic strip and an electrochemical sensor has been developed for detection and quantification of ammonium ions. The developed paper-based chromogenic strip showed detection limit of 0.008 % (w/v) for ammonium ions, whereas, electrochemical sensor showed an LOD of 0.0062 ± 0.0023 % (w/v) with a sensitivity of 1.6 × 10−5 A.% −1.mm−2. The developed paper-based chromogenic strip and electrochemical sensor can be employed by regulatory bodies and dairy industries to ensure the safety and quality of milk.

An Encyclopedic Compendium on Chemosensing Supramolecular Metal-Organic Gels.

Chemosensing, an interdisciplinary scientific domain, plays a pivotal role ranging from environmental monitoring to healthcare diagnostics and (inter)national security. Metal-organic gels (MOGs) are recognized for their stability, selectivity, and responsiveness, making them valuable for chemosensing applications. Researchers have explored the development of MOGs based on different metal ions and ligands, allowing for tailored properties and sensitivities, and have even demonstrated their applications as portable sensors such as paper-based test strips for practical use. Herein, several studies related to MOGs development and their applications in the chemosensing field via UV-visible or luminance along with electrochemical approach are presented. These papers explored MOGs as versatile materials with their use in sensing bio or environmental analytes. This review provides a foundational understanding of key concepts, methodologies, and recent advancements in this field, fostering the scientific community.

A paper-based colorimetric sensor using Au/Ag nanocages for sensitive detection of hydrazine in water samples by smartphone

In this work, Au/Ag nanocages have been used as an effective platform for sensitive colorimetric detection of hydrazine. By adding hydrazine together with silver ions to the hollow Au/Ag nanocages, they undergo a morphological transformation to closed nanoboxes. This leads to a noticeable blue shift in the localized surface plasmon resonance (LSPR) peak position and a distinct color change from blue to purple in the solution. The extent of shift in the LSPR peak of the Au/Ag nanocages exhibited a direct correlation with the concentration of hydrazine within the range of 0.1 to 10.0 µM. The limit of detection (LOD) achieved was 0.03 µM, with a relative standard deviation (RSD) of 1.7% at a concentration of 0.5 µM. Furthermore, we developed a paper-based sensor with smartphone readout for hydrazine detection within the range of 0.5 to 10.0 µM, with an LOD of 0.3 µM and an RSD of 3.3% at a concentration of 2.0 µM. The effectiveness of the sensor was successfully demonstrated in the analysis of hydrazine in environmental samples. This is the first time that Au/Ag nanocages are used for simple paper-based assay. With its high sensitivity, low cost, and rapid analysis time, this method offers a promising solution for the quantitative measurement of hydrazine in environmental samples by portable paper-based test strips.

A novel dual-activatable ultrasensitive chemiluminescent probe for mercury (II) monitoring: From rational design to multiple application

Real-time optical sensing of mercury has been developed rapidly in recent years but remains challenging such as bearing background interference. Herein, a Hg2+ and base dual-activatable ultrasensitive chemiluminescent probe CL-Hg based on benzothiazole-phenoxyl-dioxetane with profits of excitation light-free and minimal interference is presented. The photophysical properties study and sensing performance verified CL-Hg is coupled with unique advantages of long-term detection (more than 400 min), ultrahigh sensitivity (LOD = 0.52 nM), and high specificity to Hg2+, and visualization detection by the paper-based test strips. More importantly, CL-Hg showed the qualitative and quantitative detection capability for Hg2+ with great recyclability in real samples of water, seafood, and beverages, holding great potential for on-site monitoring of Hg2+ levels in the actual samples. To our knowledge, this is the first work achieving the detection of Hg2+ by chemiluminescence. Overall, the Hg2+-activated visualization platform offers a practical method for detecting Hg2+ in various application scenarios.

Enhanced Molecularly Imprinted Fluorescent Test Strip for Rapid and Visual Detection of Norfloxacin via a Smartphone.

Paper-based test strips with on-site visual detection have become a hot spot in the field of target detection. Yet, low specific surface area and uneven deposition limit the further application of test strips. Herein, a novel "turn-on" ratio of molecularly imprinted membranes (Eu@CDs-MIMs) was successfully prepared based on a Eu complex-doped polyvinylidene fluoride membrane for the selective, rapid and on-site visual detection of norfloxacin (NOR). The formation of surface-imprinted polymer-containing carbon dots (CDs) improves the roughness and hydrophilicity of Eu@CDs-MIMs. Fluorescence lifetimes and UV absorption spectra verified that the fluorescence enhancement of CDs is based on the synergistic effect of charge transfer and hydrogen bonding between CDs and NOR. The fluorescent test strip showed a linear fluorescent response within the concentration range of 5-50 nM with a limit of detection of 1.35 nM and a short response time of 1 min. In comparison with filter paper-based test strips, Eu@CDs-MIMs exhibit a brighter and more uniform fluorescent color change from red to blue that is visible to the naked eye. Additionally, the applied ratio fluorescent test strip was combined with a smartphone to translate RGB values into concentrations for the visual and quantitative detection of NOR and verified the detection results using high-performance liquid chromatography. The portable fluorescent test strip provides a reliable approach for the rapid, visual, and on-site detection of NOR and quinolones.

A Metal-Free Triazacoronene-Based Bimodal VOC Sensor.

Developing suitable sensors for selective and sensitive detection of volatile organic compounds (VOCs) is crucial for monitoring indoor and outdoor air quality. VOCs are very harmful to our health upon inhalation or contact. Bimodal sensor materials with more than one transduction capability (optical and electrical) offer the ability to extract complementary information from the individual analyte, thus improving detection accuracy and performance. The privilege of manipulating the optoelectronic properties of the polycyclic aromatic hydrocarbon-based semiconducting materials offers rapid signal transduction in multimodal sensing applications. A thiophene-functionalized triazacoronene (TTAC) donor-acceptor-donor (D-A-D) type sensor is reported here for VOC sensing. The single-crystal X-ray structure analysis of the TTAC revealed that a distinctive supramolecular polymer architecture was formed because of cooperative π-π and intermolecular D-A interactions and exhibited rapid signal transduction upon exposure to specific VOCs. The TTAC-embedded green luminescent paper-based test strip exhibited an on-off fluorescence response upon nitrobenzene vapor exposure for 120 s. The selective and rapid response is due to the fast photoinduced electron transfer, as is evident from the time-resolved excited-state dynamics and density functional theory studies. The thick-film-based prototype chemiresistive sensor detects harmful VOCs in a custom-made gas sensing system including benzene, toluene, and nitrobenzene. The TTAC sensor rapidly responds (200 s) at relatively low temperatures (180 οC) compared to other reported metal-oxide-based sensors.

Simple colorimetric paper-based test strip for point-of-use quality testing of ethanol-based hand sanitizers.

A novel, simple, affordable, and reliable colorimetric paper-based analytical device (PAD) was developed for the point-of-use quality testing of ethanol-based hand sanitizers, mainly against adulteration by water. The principle was based on the novel solvatochromism of methylparaben (MPB)-Fe3+ complex, where water is essential for complex formation and ethanol is necessary for MPB solubility. The intensity of the formed violet color, measured at 528 nm, showed a good correlation (R2 = 0.996) with the percentage water in the reaction media over a range from 40% to 100% (0-60% ethanol), with excellent accuracy and precision as indicated by the percent recovery within 100.00% ± 2% and %RSD of <2%. A PAD was prepared by the sequential immobilization of Fe3+ ions and MPB on chitosan-modified filter paper. The developed PAD was successfully applied for the quality testing of ethanol-based hand sanitizers using an established color index, where clearly distinct colors were observed as a function of the percentage ethanol (0-100%). The developed test strips could achieve on-site lab-quality results without expensive or sophisticated instruments using a few milligrams of FeCl3 and MPB in addition to regular filter paper. Accordingly, it can be used as a test strip for the quality checking of ethanol-based hand sanitizers by end users.

GSH-AuNCs/Cu2+ as selective fluorescent sensing probe for sensitive determination of glyphosate in the environment

The sensitive and selective detection of glyphosate (Gly) in the environment is urgently need because it has significant implications for human health and environmental safety. In this work, a GSH-AuNCs/Cu2+ sensing probe was fabricated via the coordination between Cu2+ and GSH-AuNCs, which resulted in the suppression of the fluorescence of GSH-AuNCs. Glyphosate can competitively grasp Cu2+ from GSH-AuNCs/Cu2+, leading to fluorescence recovery. Thereby, a simple and sensitive “turn-on” fluorescence strategy was developed for the quantitative detection of glyphosate. The linear range of glyphosate detection is 0.25 ∼ 1.25 μM, with a detection limit of 92.16 ng/mL. This method was demonstrated selectivity, fast response, and sensitivity, making it suitable for the quantitative determination of glyphosate in real water samples. Furthermore, a paper-based test strip was constructed using the sensing probe, which enabled on-site visual and semiquantitative detection of Gly. Overall, our strategy provides new opportunities for determination of organophosphorus pesticides in the environment.

A ratiometric fluorescent indicator-displacement assay for on-site determination and intracellular imaging of nitroxinil

Nitroxinil (NIT) is a widely using veterinary medicine to protect cattle and sheep yet may threaten human health when ingested through food chain. Developing fluorescent analytical methods in ratiometric manners was essential for the on-site detection and in-situ monitoring of NIT but still challenging. Here, we improved the indicator-displacement assay (IDA)-based method and designed the first ratiometric fluorescent probe for NIT by using an albumin host and an Aggregation-induced emission (AIE) guest. This probe exhibited fast response (10 s), high sensitivity (limit of detection: 4.6 ppb), good selectivity (over twelve medicines) and eye-discriminable fluorescent color change (green–red) upon responding to NIT. Based on these properties, this probe enabled quantitative determination of NIT in real food samples, on-site analysis via a paper-based test strip, and fluorescence imaging of NIT in living cells.

Multifunctional light-controllable nanozyme enabled bimodal fluorometric/colorimetric sensing of mercury ions at ambient pH

Nanomaterials with enzyme-like catalytic features (nanozymes) find wide use in analytical sensing. Apart from catalytic characteristics, some other interesting functions coexist in the materials. How to combine these properties to design multifunctional nanozymes for new sensing strategy development is challenging. Besides, in nanozymes it is still a challenge to conveniently control the catalytic process, which also hinders their further applications in advanced biochemical analysis. To remove the above barriers, here we design a light-controllable multifunctional nanozyme, namely manganese-inserted cadmium telluride (Mn–CdTe) particles, that integrates oxidase-like activity with luminescence together, to achieve the fluorometric/colorimetric dual-mode detection of toxic mercury ions (Hg2+) at ambient pH. The Mn–CdTe exhibits a light-triggered oxidase-mimicking catalytic behavior to induce chromogenic reactions, thus enabling one to start or stop the catalytic progress easily via applying or withdrawing light irradiation. Meanwhile, the quantum dot material can exhibit bright photoluminescence, which provides the fluorometric channel to sense targets. When Hg2+ is introduced, it rapidly leans toward Mn–CdTe through electrostatic interaction and Te–Hg bonding and induces the aggregation of the latter. As a result, the luminescence of Mn–CdTe is dynamically quenched, and the masking of active sites in aggregated Mn–CdTe leads to the decrease of light-initiated oxidase-mimetic activity. According to this principle, a new fluorometric/colorimetric bimodal method was established for Hg2+ determination with excellent performance. A 3D-printed portable platform combining paper-based test strips and an App-equipped smartphone was further fabricated, making it possible to achieve in-field sensing of the analyte in various matrices.

From lab to life: Safe and efficient optical based dual-mode chemosensor for the detection of aluminium (III) and copper (II) ions

In order to create a new Schiff base sensor R4P, rhodamine B hydrazide was directly combined with 4-propoxybenzaldehyde. The structure of this new sensor was completely revealed by spectroscopic methods, and a single crystal that could be used for X-ray diffraction techniques was produced. Under a UV lamp, the colourless and non-fluorescent R4P solution exhibits an orange fluorescence enhancement when Al3+ is present and showed a pink colour change when Cu2+ is present. Spectroscopic tools like UV, fluorescence, MS and NMR spectroscopy were used to study the recognition performance and complexation process of the sensor. The findings demonstrated that sensor R4P, with a limit of detection as low as 10-8 M and an association constant of ×104 M−1, is highly sensitive and selective towards both Al3+ and Cu2+ in two different solvent systems. Additionally, the designed system had a reasonable recovery range of above 89% for Al3+ and Cu2+ in two chosen water samples of natural mineral water and tap water. Moreover, a paper-based test strip that can detect target metal ions both colorimetrically and fluorometrically was created utilising chromatography paper. Aside from that, R4P's minimal cytotoxicity marks it appropriate for bio-imaging studies on metal ion sensing using the fluorescence response in the HCT 116 cell line.

A Low-cost Creatinine Biosensor by Differential Optical Signal Readout for the Whole Blood Analysis.

This study developed a low-cost paper-based biosensor for point-of-care (POC) detection of blood creatinine by using differential optical signal readout. Dual-channel photochemical paper-based test strips were fabricated with stackable multilayer films containing pre-immobilized enzymes and reagents for the identification and conversion of creatinine and creatine. Enzyme-linked reactions generated hydrogen peroxide (H2O2), which formed a blue oxidized condensate with aniline derivatives. The color depth was quantified via the differential optical signal of the two channels and positively correlated with the concentration of the analyte. This method was first proposed to address the issue of endogenous interferences in the enzymatic assay of creatinine, greatly improving the detection accuracy. The proposed biosensor was calibrated with spiked blood samples, and achieved a wide detection range of 31-1483 μmol/L, showing superior detection performance to general enzymatic methods, especially in the low concentration range. Creatine interference testing demonstrated that the biosensor could resist the interference of ≤ 300 μmol/L endogenous creatine. It is believed that the proposed optical differential biosensor for blood creatinine could enable to pave the way for a daily monitoring system for renal diseases.Clinical Relevance- This stackable multilayer paper-based biosensor provides an enzymatic colorimetric assay of creatinine in whole blood, which can be read out by the differential optical signal to exclude interference from endogenous creatine.