論文

Paper-based analytical devices (PADs) are promising platforms to achieve Point-of-Care Testing (POCT) due to their low cost, ease of operation, disposability, and simplicity of assaying. Inspired by the bioanalytical potential of marine luciferins as excellent optical indicators, we evaluated a panel of 30 coelenterazine (CTZ) analogs for the specific imaging of important serum proteins from various species. This panel included 20 novel CTZ analogs, recently developed by the authors and designated as the “G-series”. Remarkably, several G-series CTZ analogs are exclusively bright with specific serum albumins and collectively create unique intensity patterns for these proteins. These G-series CTZ indicators produced highly quantitative luminescence signals with distinct spectral profiles across both trace and physiological albumin concentrations. This luminescence was utilized to determine kinetic constants, identify reaction sites, and establish dose–response curves for the various albumins. Finally, the optimized imaging system was transitioned to paper microplates for a rapid, on-site assay of serum albumins. HSA and BSA deposits dried on paper microplates were successfully imaged with CTZ indicators, achieving high signal-to-background ratios. This platform offers a cost-effective and portable approach to the rapid assay of serum albumins from various animal sources.

Thermo-responsive polymers are widely applied in various fields, including drug delivery and surface coating. Dual thermo-responsive polymers, which exhibit both lower and upper critical solution temperatures (LCST and UCST), enable notable temperature-dependent behaviors within a tunable range. However, the structure–property relationships governing dual temperature-responsiveness are still insufficiently understood, particularly for polymers including pH-sensitive zwitterionic groups. In this study, LCST–UCST polymers, P(MEO2MAxMEOMAyGAASt20), composed of mono/di(ethylene glycol) methyl ether methacrylate (MEOMA/MEO2MA) and a zwitterionic monomer derived from glutamic acid (GAASt), were designed. By varying the ethylene glycol (EG) side chain length, pH, and salts, it was investigated how the balance between hydrophilic and electrostatic interactions governs the thermo-responsive behavior of the copolymers. In 10 mM phosphate buffer (PB, pH 7), UCST behavior was observed with only P(MEOMA79GAASt21), likely because shorter EG chains increased the relative contribution of zwitterionic units. The UCST of this polymer increased with the addition of salt, unlike typical zwitterionic UCST-type polymers. Polymers containing MEO2MA exhibited LCST behavior in 10 mM PB (pH 7) containing 100 mM NaCl, and the LCST decreased with shorter EG chains and kosmotropic ions. Under acidic and basic conditions, the thermo-responsive transition was not observed. Furthermore, P(MEO2MA19MEOMA61GAASt20) exhibited LCST–UCST behavior induced by NaCl. The diameter of the polymer in solution varied with temperature: terpolymer chains below the LCST, the largest aggregates between the LCST and UCST, and small globules above the UCST. Our findings provide insight into the molecular design strategies for thermo-responsive polymers in bioapplications.

A portable colorimetric platform was developed for rapid, selective, and sensitive detection of cholesterol-enriched low-density lipoprotein (LDL-C) in plasma by exploiting the aptamer-enhanced catalytic activity of NiMnFe layered double hydroxides (NiMnFe-LDHs). Unlike conventional enzymatic colorimetric assays and ELISA, which are costly, time-consuming, and reliant on sophisticated instrumentation, this system enables direct image acquisition and automated analysis using a single-board computer, thereby reducing assay time, operational complexity, and user dependence. Functionalization of NiMnFe-LDHs with an LDL-specific aptamer improves nanomaterial dispersion through electrostatic repulsion, increasing exposure of catalytic active sites and enhancing hydroxyl radical (•OH) generation. In addition, π–π interactions and hydrogen bonding between the aptamer and 3,3′,5,5′-tetramethylbenzidine (TMB) improve substrate affinity, further strengthening the peroxidase-like activity of the NiMnFe-LDHs@aptamer system. Upon LDL-C binding, formation of an LDL-C/aptamer corona partially blocks the LDH surface, limiting access of TMB and H2O2 to catalytic sites. This suppresses •OH generation and TMB oxidation, resulting in a concentration-dependent decrease in colorimetric signal. The portable device achieved a linear detection range of 0.05–1.25 mg dL⁻¹ with a detection limit of 0.04 mg dL⁻¹. Although compatible with 96-well plates, the system’s key advantage lies in its integrated imaging and processing workflow, enabling scalable, high-throughput, and decentralized LDL-C diagnostics with potential extension to other biomarkers.

Cancer antigen 125 (CA-125) is a glycoprotein associated with cholangiocarcinoma, making it a valuable biomarker for diagnosis. However, conventional detection methods have limitations due to their reliance on antibodies for specificity and natural enzymes as labels for signal amplification. Both are costly and unstable under extreme pH conditions, and enzymes exhibit a low catalytic efficiency. Addressing these limitations could enhance the diagnostic accuracy of CA-125 detection in cholangiocarcinoma diagnosis. In this work, Ni-MnFe-layered double hydroxides (Ni-MnFe-LDHs) conjugated with an aptamer (Ni-MnFe-LDHs@aptamer) are demonstrated as a superior peroxidase mimic to enhance the sensitivity for the colorimetric detection of CA-125 on a paper-based analytical device (PAD) platform. Ni-MnFe-LDHs exhibited efficient peroxidase-like activity, catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into an intense blue product in the presence of hydrogen peroxide (H2O2). Upon modification with a CA 125-specific aptamer, Ni-MnFe-LDHs became more dispersed due to electrostatic repulsion, exposing more active sites and generating more hydroxyl radicals (·OH). Moreover, π–π stacking and hydrogen bond interactions between the aptamer and TMB increased the substrate affinity of Ni-MnFe-LDHs, thereby enhancing their catalytic performance. This resulted in a darker blue signal in the absence of CA-125. When CA-125 was present in the sample, it was captured by the aptamer on Ni-MnFe-LDHs, resulting in a decreased blue color signal. Using CA-125 as the target analyte, a linear relationship between the scanner-recorded signal intensity and analyte concentration was observed in the range of 10–25 U/mL. The results obtained from real human sample application using the developed method were consistent with those from a clinical laboratory. This method can be easily implemented and holds great potential as a prototype for various diagnostic applications, especially for detecting biomarkers in serum or plasma samples. It offers significant benefits for point-of-care testing.

Accurate monitoring of sodium (Na⁺) and potassium (K⁺) ions is vital for diagnosing electrolyte imbalances associated with physiological disorders. We report a dual-ion potentiometric sensing platform that integrates laser-induced graphene (LIG), in situ laser-synthesized gold nanoparticles (AuNPs) on LIG substrate, and inkjet-printed all-solid-state ion-selective electrodes (ISEs) for simultaneous Na⁺ and K⁺ detection. Fabricated on a miniaturized microfluidic substrate with a dual-channel design, this device enables simultaneous measurement of both ions from a single sample. A CO2 laser irradiation first converts polyimide (PI) into LIG, while a second laser pass simultaneously synthesizes AuNPs in situ and re-engraves the LIG surface. This interface produces a porous and hydrophobic morphology that enhances the electroactive area, charge-transfer kinetics, and potential stability. Potentiometric signals are acquired within 100 s using a portable bipotentiostat and transmitted wirelessly via Bluetooth to a smartphone. The sensor displayed near-Nernstian sensitivities of 59.5 mV decade−1 (Na⁺) and 59.8 mV decade−1 (K⁺), wide linear ranges, excellent repeatability, and low detection limits of 10 µм (Na⁺) and 7.9 µм (K⁺). Validation with human urine samples confirmed its analytical reliability. This work demonstrates a simple and cost-effective strategy for electrolyte monitoring, offering strong potential for point-of-care diagnostics and field-based health monitoring.

The beginning of this century has witnessed the emergence of many disruptive technologies changing the ways of sensing target analytes. Among these, smartphone-enabled analytical devices (SEADs) have gained attention as a promising technology, particularly in resource-limited settings. SEADs are recognized for being portable, cost-effective, user-friendly, and reliable platforms for detecting a wide range of analytes. In parallel, paper-based analytical devices (PADs) primarily relying on colorimetric or fluorescence signal transduction, have also attracted significant research interest across various applications. This review highlights the potential of integrating these two emerging analytical platforms, a combination that could revolutionize future onsite sensing. An overview of SEADs and PADs is provided, with focus on recent developments in optical detection. Advantages and limitations of integrating smartphones with PADs in different scenarios are presented. Additionally, recent applications of smartphone-coupled PADs are presented. Finally, key challenges associated with merging these two technologies are discussed.

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The rapid and accurate detection of foodborne pathogens, particularly Salmonella Typhimurium (S. Typhimurium), is essential for ensuring food quality and safety. This work presents a portable, label-free potentiometric aptasensor using an ion flux polymer membrane for the sensitive and rapid detection of S. Typhimurium in meat and water, operated via a smartphone-based potentiostat. The sensor employs a specific aptamer immobilized on a solid-state contact ion-selective electrode, featuring a cationic marker and a carboxylated poly(vinyl chloride) membrane modified on a laser-induced graphene electrode. Binding of outer membrane protein D from S. Typhimurium to its aptamer hinders cation flux across the membrane, generating measurable potential changes that enable quantitative detection within 8 min. The assay offered a linear range of 1–10,000 CFU/mL, a detection limit of 1 CFU/mL, and a sensitivity of 16.572 mV/decade. Sensor performance was validated in various meat and water matrices, including groundwater, tap water, drinking water, pork, and chicken, yielding results consistent with ISO 19250:2010 and a commercial lateral flow immunoassay. Recovery rates from spiked meat samples ranged from 96 to 108%, with relative standard deviations below 5.7%, demonstrating good accuracy in complex matrices. The aptasensor also exhibited excellent specificity toward S. Typhimurium and retained over 90% of its sensitivity after three weeks of storage, confirming its good storage stability and practical applicability. These findings indicate that the proposed portable aptasensor is well suited for routine on-site detection of S. Typhimurium in food products and represents a valuable tool for rapid food safety monitoring.

Since their first report in 2007, microfluidic paper-based analytical devices (μPADs) have continued to gain attention as promising tools for point-of-care diagnostics due to their low cost, portability, ease of operation, and design flexibility. This review summarizes and discusses recent advances in the field, mostly based on works published between 2017 and 2025, with a focus on progress and remaining challenges in bridging the gap between proof-of-concept demonstrations in academic laboratories and real-world implementation. Special emphasis is placed on devices validated with clinical samples and capable of true sample-in–answer-out operation. To comprehensively assess recent developments, nearly one hundred reported examples were analysed not only in terms of analytical figures of merit but also with respect to practical criteria such as real-sample testing, long-term storage stability, the need for off-device sample pretreatment, reagent handling complexity, time-control requirements, and the number of operation steps. In parallel, topics of ongoing academic interest are highlighted, including automated sequential reagent delivery, strategies for accelerating liquid flow, and robust signal readout methods going beyond purely qualitative approaches to enhance assay sensitivity, precision, rapidity, and instrument-free usability. Finally, the review introduces emerging analytical technologies newly integrated into μPAD platforms, such as surface-enhanced Raman scattering (SERS), bioluminescence, CRISPR-based assays, and machine learning-driven data interpretation, which further expand the analytical capabilities and scope of μPADs.

Two selective methods were developed for detecting mercury ions (Hg2⁺) in food and water samples using catalytic fluorometry based on Au-Hg amalgamation with enhanced peroxidase-like activity. These approaches include (i) a laser-printed microfluidic paper-based analytical device (LP-µPAD) and (ii) a paper-based microcentrifuge tube test kit. The LP-µPAD was fabricated via commercial laser printing and integrated gold nanoparticles (AuNPs) with o-phenylenediamine (OPD) and hydrogen peroxide (H₂O₂). The test kit enabled Hg2⁺ detection in food by reducing Hg2⁺ to Hg⁰ with stannous chloride, facilitating Au-Hg amalgam formation in the detection zone. Both approaches utilized the Au-Hg amalgam to catalyze H₂O₂-mediated oxidation of OPD, generating fluorescent 2,3-diaminophenazine (DAP), further enhancing fluorescence intensity in proportion to Hg2⁺ concentrations. The LP-µPAD exhibited a detection range of 3.0–20.0 µg L⁻1 with a limit of detection (LOD) of 1.65 µg L⁻1, whereas the test kit provided a detection range of 0.1–1.0 mg L⁻1 with an LOD of 0.08 mg L⁻1. Both sensors showed high selectivity for Hg2⁺ over other ions and performed well in real sample analyses, aligning closely with results from conventional methods.

In clinical diagnosis, the determination of target proteins at low concentration levels is generally performed by immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), which is a time-consuming process. To date, paper-based ELISA platforms enabling faster and less expensive analysis have been developed, but their important issue for clinical applications is the limited sensitivity compared to conventional ELISA. To address this challenge, this paper introduces a simple, rapid, and highly sensitive detection method for non-nucleic acid targets achieved by integrating the CRISPR/Cas12a system into paper-based ELISA. An origami-type paper-based device enabling simple assay operation has been designed, and the detection of targets on the paper substrates is based on observing the fluorescence signal induced by the CRISPR/Cas12a enzyme cleaving a probe single-stranded DNA (ssDNA) labeled with fluorophore and quencher (FQ reporter). To enhance sensitivity, antibodies labeled with a network of multiple DNA activating the CRISPR/Cas12a enzyme have been utilized as detection antibodies. As a result, the developed device successfully boosted the detection sensitivity for both human IgG and the hepatitis B virus surface antigen (HBsAg). In particular, the limit of detection (LOD) for HBsAg was estimated to be 12 pg/mL, representing over 10-fold higher sensitivity compared with commercially available HBsAg ELISA kits (LOD: 200 pg/mL). In addition, the fluorescence response toward porcine whole blood samples containing different HBsAg concentrations was also confirmed by capturing images with a smartphone, followed by quantitative data analysis. These results demonstrate the potential applicability of the proposed platform for clinical tests at the point of care.

Although CRISPR-based nucleic acid detection has great potential in point-of-care testing due to its simplicity, it has been rarely integrated into paper-based analytical devices (PADs), which are attractive platforms to simplify assays. This work introduces a CRISPR-assisted nucleic acid quantification approach integrated into a PAD with signal readout by a personal glucose meter (PGM). Retention of magnetic beads by filter paper and pre-deposition of all required reagents by freeze-drying stabilized with trehalose enabled the indirect quantification of human papilloma virus (HPV) DNA through a PGM readout without complicated user intervention and complex reagent handling. The calculated limit of detection was 57 pM, which is comparable with other amplification-free CRISPR-based assays detecting nucleic acids. The fully integrated device exhibited good storage stability for up to 4 weeks, suggesting its applicability toward practical point-of-care nucleic acid quantification.

A paper-based potentiometric sensor integrated with a polymeric hydrogel has been developed for sodium ion (Na+) determination in human urine. The construction of an all-solid-state ion selective electrode (s-ISE) and an all-solid-state reference electrode (s-RE) on a photo paper substrate was achieved using an inkjet printing method. For s-ISE fabrication, carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) were printed on the substrate as a nanocomposite solid contact. A polymeric hydrogel containing lithium acetate (CH3COOLi) was then prepared and used as an intermediate layer to improve the adhesion between the ion selective membrane (ISM) and the AuNP/CNT solid contact, leading to enhanced detection sensitivity. The printed s-RE consisted of a pseudo silver/silver chloride electrode (p-Ag/AgCl) coated with a polymeric hydrogel containing KCl to improve the potential stability of the sensor. Under the optimal conditions, the hydrogel-integrated paper-based potentiometric sensor provided a response toward Na+ over a linear range of 10−7 M to 1 M with a near Nernstian slope of 56.42 ± 0.68 mV per decade. This sensor exhibited fast response, good sensitivity, and reasonable selectivity for Na+ measurement. Furthermore, the developed sensor was effectively applied for the detection of Na+ in urine samples with high accuracy. The presented work can be considered as a good addition to the growing field of potentiometric analytical platforms suitable for large-scale production using inkjet printing technology.

The convergence of smartphones with chemiluminescence and electrochemiluminescence (CL/ECL) assays marks a transformative leap in the realm of sensing technologies. The traditional CL/ECL assays, known for their high sensitivity and versatility, find extensive applications in medical diagnostics, environmental monitoring, food safety, and forensic sciences. However, these techniques have long been constrained due to the requirement of expensive instrumentation and complex reagent handling and hence their accessibility within certain environments is limited. In an era where rapid, accurate, and routine analysis is critical, smartphone-enabled CL/ECL systems offer substantial advantages over conventional analytical methods. By leveraging the universal accessibility and technological sophistication of smartphones and combining them with CL/ECL-based sensing, the smartphone has evolved into a cost-efficient and accessible analytical platform. The ability of the combined platform to conduct on-site analysis in real-time with minimal effort has emerged as a game-changer, particularly in low-resource settings. This mini-review explores the rapid evolution of smartphone-coupled CL/ECL systems over the last five years. The article covers the areas where the combined platform has been implemented in recent years for various sensing applications. The review further identifies key challenges that are associated with such combined platforms and finally highlights the future perspectives of the present topic.

A highly sensitive ratiometric fluorescence sensing system was developed for simultaneous glucose and total cholesterol (TC) detection in whole blood using MnFe-layered double hydroxides (MnFe-LDHs) as a peroxidase mimic, combined with an o-phenylenediamine (OPD) substrate and nitrogen-doped graphene quantum dots (N-GQDs). The detection platform, an X-shaped laminated microfluidic paper-based analytical device (XL-μPAD), was fabricated via laser printing and cutting. The MnFe-LDHs' large surface area and layered structure provide a high affinity for OPD, with a Michaelis–Menten constant (KM) of 0.0127 mmol L−1. Upon placing a drop of blood on the XL-μPAD sample pad, the enzymatic reactions of glucose and TC produce H2O2, which MnFe-LDHs convert to hydroxyl radicals (•OH). These radicals oxidize OPD into fluorescent 2,3-diamino phenazine (DAP) with emission at 560 nm. Meanwhile, the N-GQDs emit fluorescence at 415 nm, which is quenched by DAP through the inner filter effect (IFE) and dynamic quenching, enabling ratiometric sensing via the intensity ratio (I560/I415). As H2O2 levels increase, a visible green emission appears, correlating with glucose and TC levels. This XL-μPAD system demonstrates promising potential as a portable device for multiplex biomarker detection and diagnostic applications.

The availability of various calcium ion (Ca2+) fluorescent probes has contributed to revealing physiological events related to intracellular Ca2+. However, conventional probes face challenges for quantitatively and selectively visualizing high Ca2+ concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca2+ homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca2+ probe, KLCA-Fura, utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a ligand, for which we recently demonstrated the suitability as a new low-affinity ligand for Ca2+. KLCA-Fura showed a blue shift in excitation wavelength with increasing Ca2+ concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca2+ is lower than commercially available conventional Ca2+ probes. Furthermore, the selectivity for Ca2+ and the fluorescence intensity were considered sufficient to accurately detect Ca2+. The corresponding acetoxymethyl ester, KLCA-FuraAM, was synthesized for intracellular imaging and applied to Ca2+ quantification in neurons. KLCA-FuraAM enabled quantitative ratiometric monitoring of the two-step Ca2+ concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca2+ probe, Fura-FF, KLCA-FuraAM has demonstrated the potential to quantitatively visualize the behavior of high Ca2+ concentrations. The ratiometric low-affinity Ca2+ probe, KLCA-Fura, is expected to be a powerful tool for discovering new functions of Ca2+ in neurons.

Ecological surveys of living things based on DNAs from environmental samples are attractive. However, despite simplicity of water sampling from the target environment, it is still necessary to transport the samples to the laboratory for DNA analysis based on skillful next-generation sequencers. To perform DNA-oriented surveys based on a simple protocol without any special training, we demonstrated, in this study, the detection of genes from cell-containing environmental waters using gene sensor arrays that require no DNA labeling and no external indicators. Cell-suspended PBS or river water were used as models of environmental waters containing living things, and DNA samples were prepared by PCR amplification. Ferrocene-terminated probes were synthesized and immobilized on an electrode array to develop a sensor array. The sensor array showed a large response to a target DNA complementary to the probe and no response to a mismatched DNA, indicating sequence-specific detection. For DNA samples prepared from the cells in PBS, they showed good responses similar to those for the target DNA. They also significantly detected DNA samples from the cells in river water at a general environmental concentration (38 cells mL−1) with 28-fold larger responses than those for 0 cells mL−1.

Lateral flow immunoassays (LFIAs) are widely used for the simple and rapid detection of various targets at the point of need. However, LFIAs enabling the simultaneous detection of multiple analytes and the possibility for naked-eye semi-quantitative analysis are facing various challenges, including the requirement of large sample volumes, low efficiency, and accuracy. This is particularly the case for the competitive immunoassay format targeting the detection of low molecular weight compounds, such as, for example, drugs. Due to limited space for multiple reaction zones on a single planar nitrocellulose membrane, conducting multiplexed tests requires the addition of more test strips, which consequently increases the size of the whole device. To overcome these spatial constraints, two 3D-printed devices fitting eight assay lanes of both backed and unbacked nitrocellulose membranes have been designed. For proof of concept, 8-OHdG, caffeine, and acetaminophen were used as model analytes. Inkjet printing was applied to deposit capture reagents in the form of text symbols while controlling the concentration thresholds for text readability to achieve an intuitive result expression. A comparably small sample volume of 350 mL was sufficient to simultaneously visually distinguish 4 concentration levels of caffeine (0, 4, 10, 175 ng mL-1) and acetaminophen (0, 4, 8, 12 ng mL-1) in mixed solutions without crosstalk. This study demonstrates the potential of 3D-printed LFIA devices for multiplex and semiquantitative analyte detection.

Background: Carcinoembryonic Antigen (CEA) and 4-nitroquinoline-N-oxide (4-NQO) are cancer markers that play a crucial role in tumor risk assessment and early cancer diagnosis. Therefore, it is in demand to develop a fast, accurate, simple, and cost-effective method to detect these cancer markers for quick and early stage-cancer diagnosis and treatment. Results: Herein, we report a dual signaling approach for direct and indirect signal transduction of cancer biomarker binding on molecularly imprinted-electrodes integrated on an ePAD, enabling sensitive and selective quantitative analysis of 4-NQO and CEA. Multiple test zones on a single device based on artificial recognition sites using core-shell Cu-MOF@MIP were developed to create a selective “Dual-signal-ePAD”. An effective and facile imprinting method on a Cu-MOF surface based on the synthesized of 4-VPBA/MAA/TRIM polymer. It resulted in a novel MIP useable as a high-performance sensing tool for the ultrasensitive and specific quantification of the target cancer biomarkers. Multiple tests consisted of voltammetric analysis of 4-NQO via oxidation of the analyte on a WE1, and impedance analysis of CEA via the molecular interaction with the imprinted WE2. Significance: The proposed device provides enhanced electrochemical signal amplification due to its high conductivity, electrocatalytic activity, and the target-specific recognition sites for accurate determination of 4-NQO and CEA targets. The Dual-signal-ePAD exhibits good voltammetric and EIS response, oxidation currents of 4-NQO at −0.06 V and RCT of CEA, resulting in high selectivity and sensitivity with a linear response range covering the concentration range of clinical interest for both analytes. Dual-signal ePAD is a good candidate for clinical screening and POCT.

Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM – among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.