Abstract
Olfactory dysfunction has emerged as an important clinical issue, particularly following the global rise in post-viral anosmia. Recent research suggests an association between altered calcium homeostasis and poor olfactory signaling, but there is no validated method for assessing calcium levels in nasal secretions. Herein, a novel paper-based electrochemical sensor for the direct determination of free calcium ions in nasal secretions, providing potential diagnostic biomarker in olfactory dysfunction. The sensor integrates multi-walled carbon nanotubes, carbon dots synthesized from guava fruit, and the calcium-selective ionophore ETH 1001 to improve conductivity and selectivity. It exhibits a Nernstian response with a slope of 29.14 ± 0.3 mV/decade over a linear range from 10−7 to 10−1 M, and a detection limit of 7.5 × 10−8 M. The sensor has good consistency, with less than 1 mV fluctuation in potential over 180 days, and strong selectivity against interfering nasal electrolytes. Applied to nasal samples from 166 participants, the sensor demonstrated significantly elevated calcium levels in patients with anosmia compared to healthy controls (7.30 ± 0.004 × 10⁻2 M vs. 1.84 ± 0.01 × 10⁻2 M, p < 0.05). Compared to existing technologies, the proposed sensor achieves superior sensitivity, broader linearity, and greater pH tolerance.
Introduction
Olfactory function plays a vital role in human well-being, deeply influencing quality of life, nutrition, and social interaction 1,2,3. In clinical settings, the precise measurement of ions is fundamental for timely diagnosis and understanding of various physiological and pathological processes. Among these ions, calcium plays a role in numerous biological functions, including signal transduction, neurotransmission, and cellular homeostasis.
Calcium plays a specific role in the olfactory transduction. Generally, odorant compounds activate G-protein–coupled receptors on the olfactory receptor neurons (ORNs), providing an influx of calcium through cyclic nucleotide-gated (CNG) channels. Signal termination and adaptation are improved by this calcium entry, which not only intensifies the odorant signal but also initiates negative feedback through calcium-calmodulin interactions that desensitize the CNG channels and activate calcium dependent chloride channels. This finely tuned regulation confirms that olfactory neurons remain sensitive to new stimuli without becoming oversaturated4,5. In pathological conditions, post-viral or traumatic anosmia, calcium homeostasis becomes dysregulated. Rise of extracellular calcium in the nasal secretions may dampen olfactory signaling through altering the electrochemical gradient across ORNs, thereby decreasing calcium influx during signal detection. This causes impaired depolarization and weakened odor signal transmission to the olfactory bulb. Moreover, in Sino-nasal disease, epithelial damage and nasal secretion composition changes can elevate local calcium levels, which may worsen inhibitory feedback and lead to olfactory dysfunction5,6,7,8. Therefore, quantifying free calcium in the nasal secretions provides a functional readout of the ionic environment at the olfactory epithelium.
Despite the growing recognition of calcium’s role in olfaction, no validated analytical method currently exists for its direct determination in nasal secretions. This gap is particularly critical in light of the global rise in olfactory disorders, especially post COVID-19, which have highlighted the urgent need for reliable biomarkers and diagnostic tools. Developing a simple, accurate method for determining nasal calcium could not only contribute in the pathophysiology study of olfactory dysfunction but also provide insights into prognosis and guide therapeutic interventions aimed at restoring the sense of smell through calcium modulation.
The World Health Organization (WHO) identifies seven essential characteristics that analytical and diagnostic tools should possess: affordability, sensitivity, specificity, ease of use, rapid and robust performance, equipment-free operation, and efficient deliverability9. In alignment with these criteria, portable paper-based analytical devices have gained significant attention across various fields, including clinical diagnostics, environmental monitoring, and food and pharmaceutical analysis, owing to their low cost, simplicity, and suitability for point-of-care testing10,11. To further improve the performance of these devices, particularly in electrochemical sensing, nanomaterials have been increasingly integrated into their design. Among them, carbon nanoparticles and carbon dots have shown promise in improving the transduction mechanisms of ion-selective electrodes by enhancing electrochemical performance, conductivity, mechanical strength, and overall adaptability. Additionally, carbon nanotubes have demonstrated a unique function in ink formulation, acting as efficient electrical conductors in solid-contact ion-selective electrodes, thereby contributing to the development of more sensitive and robust paper-based sensors12,13.
Recent advances in nanomaterials and nanocomposites have significantly improved the development of electrochemical biosensors. Two-dimensional (2D) nanomaterials, including graphene derivatives and metal–organic frameworks, have been highlighted as promising building blocks due to their large surface area, high conductivity, and mechanical flexibility enabling ultra-sensitive point-of-care biomarker detection and lab-on-chip applications14. Beyond 2D systems, novel nanocomposite strategies have been reported to address specific biosensing challenges. For example, albumin-fouling presents a major limitation in electrochemical analysis of complex biofluids. To circumvent this, single-walled carbon nanotubes dispersed in poly (aniline-N-propane sulfonic acid) have been successfully utilized, demonstrating antifouling behavior and high selectivity for purine metabolites, with potential for renal disease diagnostics15. Similarly, polymer–nanocomposite hybrid approaches have been tailored for cancer biomarker detection. A portable amperometric biosensor integrating chitosan–polyaniline nanocomposites with mesoporous carbon achieved highly sensitive sarcosine detection, providing rapid screening of prostate cancer biomarkers while maintaining excellent bioactivity retention and stability16. Another emerging direction integrates smartphone-based sensing platforms. A tunable tyrosinase-mimicking copper–mesoporous carbon nanocomposite enabled real-time catechol monitoring, exemplifying how electrochemical sensors can be miniaturized into portable, user-friendly devices for field diagnostics17.
This study aims to develop a low-cost, portable, and reliable analytical sensor for the determination of calcium ions in nasal secretions, targeting its potential role as a biomarker in olfactory dysfunction. In line with the WHO guidelines for ideal diagnostic tools, a nanoparticle-based, paper-based solid-state sensor was carefully designed and fabricated. Although the synthesis of fruit-derived carbon dots has been validated in previous studies, their application in paper-based electrochemical devices remains largely unexplored. To the best of our knowledge, this is the first work to employ guava-derived carbon dots in combination with highly conductive multi-walled carbon nanotubes and the calcium-selective ionophore ETH 1001 to fabricate a sensor specifically designed for calcium measurement in nasal secretions. This unique combination improves signal conductivity, stability, and selectivity toward free calcium ions. The sensor exhibits a Nernstian response with a slope of 29.14 ± 0.3 mV/decade over a wide linear range of 10−7 to 10−1 M, with a detection limit of 7.5 × 10−8 M. This sensitive and robust platform enables accurate comparison of calcium levels in nasal secretions from both healthy individuals and patients with olfactory impairment, thereby introducing a novel diagnostic approach to elucidate the biochemical link between calcium homeostasis and olfactory function.
Experimental
Materials and reagents
Analytical-grade reagents, including tetrahydrofuran, dioctylphthalate (DOP), poly (vinyl chloride) (PVC), and multi-walled carbon nanotubes (MWCNT), were purchased from Sigma-Aldrich, Germany. Acetonitrile, calcium chloride, gum Arabic, magnesium chloride, methanol, potassium chloride, propylene glycol, polyvinyl butyral, sodium chloride, sodium carbonate, sodium phosphate, silver nitrate and zinc chloride were sourced from El-Nasr Company, Egypt. The calcium-selective ionophore ETH 1001 (ethylene glycol bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid) was obtained from Fluka, Switzerland. Carbon conductive paint was acquired from Garfield Ave, West Chester, PA, USA. Guava fruits were purchased from the Egyptian market.
Apparatus
The Jenway pH meter model 3510, USA. JEOL JEM-M2100 transmission electron microscope operating at 200 kV, USA. PerkinElmer FTIR spectrophotometer, USA. Field-emission scanning electron microscope QUANTA FEG 250, Netherlands.
Standard solution
A standard calcium solution (10−l M) was made by dissolving calcium chloride in 50 mL of distilled water and adding water to make it 100 mL. Different working solutions (10–7 to 10–2 M) were prepared by diluting the stock standard solution with distilled water.
Procedures for designing a paper-based calcium sensor
Two filter paper strips were modified with MWCNT and carbon dots to develop paper based analytical sensor. Figure 1 illustrates the schematic diagram of the proposed sensor, which includes a working and reference electrode on the same disposable paper platform.
Preparing of carbon dots from guava fruit
Guava fruit slices were carbonized at 250 °C for 50 min. The final substance was coarsely ground and 200 mg was diluted in 60 mL of distilled water. The solution was heated for 10 min and centrifuged for 20 min. After adding water, the yellow-colored solution was filtered into a 100-mL volumetric flask18,19.
Preparation of ink solution containing MWCNT and carbon dots
500 mg of MWCNT and 500 mg of gum Arabic were dissolved in 50 mL of deionized water in a 100 mL volumetric flask. After adding 5 mL of propylene glycol, the liquid was agitated for 30 min, then 10 mL of carbon dots solution was added and mixed for a further 30 min20.
Design of the paper-substrate
Two rectangular filter paper strips (20 × 5 mm) served as a platform. A soft-bristled brush (a fine-tip applicator brush) was used to apply the prepared ink solution containing MWCNT and carbon dots to both sides of the paper strips. The coating thickness was carefully controlled to approximately 50 µm to ensure consistent and uniform application. Sensitivity was evaluated for different coating thicknesses )25 µm, 50 µm, and 100 µm (with the 50 µm coating showing the best performance, which was chosen for the final sensor design due to its optimal balance of performance and ease of fabrication. For the control, the same procedure was applied to graphite-coated filter paper strips, which were then modified with the ink solution. The paper pieces were sandwiched between two plastic insulator sheets, each with two 4 mm orifices on top. One aperture was reserved for the reference electrode, while the other was used for the calcium potentiometric sensor.
Preparation of the reference electrode
350 mg of polyvinyl butyral was combined with methanol solution and ultrasonicated for 30 min. The cocktail was then treated with 50 mg of sodium chloride and 30 mg of silver nitrate, followed by another 30 min of sonication. The resulting cocktail became white, highlighting the presence of silver chloride. Apply 40 μL of the cocktail to the orifice on a paper strip and let it dry in the dark for 60 min. The dried polyvinyl butyral film was treated with one drop of 3 M potassium chloride and allowed to stand for one hour21.
Preparation of the selective calcium sensor
A glass petri dish containing 168 mg of DOP, 168 mg of PVC, and 14 mg of calcium ionophore I (ETH 1001) was thoroughly homogenized after being dissolved in 2 mL of tetrahydrofuran. A 30 μL aliquot of the homogenous mixture was then dropped onto the orifice of calcium filter paper and allowed to dry, forming a dried membrane.
Analytical method calibration and validation
Two calibration curves generated: solvent calibration and biological sample calibration. For the solvent calibration curve, the sensor was immersed in standard calcium solutions (10–7, 10–6, 10–5, 10–4, 10–3, 10–2, 10–1 M). After 20 s of equilibration with stirring, the electromotive force values were recorded and plotted against the logarithm of calcium ion activity.
For the biological sample calibration curve, specific quantities of standard calcium solutions containing quality control samples were added to tubes containing 1 mL of healthy human nasal secretion. The concentrations used were as follows: lower limit of quantification (LLOQ) at 7.5 × 10–8 M, lower quantifiable concentration (LQC) at 10–7 M, middle quantifiable concentration (MQC) at 10–3 M and high quantifiable concentration (HQC) at 1 × 10–1 M. The samples were treated with acetonitrile, centrifuged to remove cellular debris and secretions protein content, and the resulting supernatants were dried and dissolved in borate buffer solution at pH 8. The electromotive force values of these samples were recorded and plotted against the logarithm of calcium ion activity to construct the calibration curve.
In addition, nine replicates of the prepared LQC, MQC and HQC samples were analyzed on the same day for intra-day accuracy and precision, and on three different days for inter-day accuracy and precision.
Clinical sample assessment
Participants selection
All participants were contacted in advance and provided with comprehensive information about the study before giving written informed consent. A total of 166 adult subjects (102 females and 64 males), aged between 18 and 69 years, were recruited from the Department of Otolaryngology et al.-Azhar University in Damietta, Egypt. Olfactory function was assessed using the validated Sniffin’ Sticks test22,23. Based on the test results, participants were categorized into well-defined groups. Normal subjects (n = 66) had intact olfactory function with no history of smell disorders. Patients with anosmia (n = 100) were classified into four subgroups based on etiology: Post COVID-19 (n = 55), reflecting viral-induced damage to olfactory pathways; Post traumatic (n = 10), due to head trauma impairing olfactory structures; Sino-nasal (n = 25), related to chronic inflammation or obstruction within the nasal cavity; and Idiopathic (n = 10), where the cause of anosmia was unknown. The study protocol was reviewed and approved by the Ethical Committee of the Faculty of Medicine, Al-Azhar University, Damietta, Egypt, and was conducted in accordance with the Declaration of Helsinki. The institutional review board (IRB) approval number is DFM-IRB 00,012,367-23-07-009.
Analytical of nasal secretion samples
Nasal secretion samples were collected by gently instilling 5 mL of sterile borate buffer solution (pH 8) into each participant’s nostril, followed by collecting the expelled secretions in a sterile plastic petri dish. The collected samples were immediately treated with acetonitrile, centrifuged to remove cellular debris and mucus proteins, and the clear supernatant was used for calcium analysis. The pretreatment protocol was standardized and repeated across all samples, and its reproducibility was confirmed during method validation to ensure accuracy and reliability of the measurements. The calcium concentrations in nasal samples were determined using the designed sensor.
Results and discussion
Transformation of filter paper into conductive platform
To convert insulating filter paper into a conductive platform, it was coated with a conductive ink solution containing MWCNT and carbon dots. FESEM was used to examine the surface morphology of the coated paper and compare it to standard graphite-coated paper. Figure 2a depicts the FESEM image of the graphite-coated paper, which revealed a smooth and uniform surface with a continuous coating of graphite. Figure 2b depicts the modified paper coated with MWCNT and carbon dots, which had a rough and heterogeneous surface. The coating was made up of an extensive network of interconnected MWCNT and distributed carbon dot particles that could be seen on a large fraction of the cellulose fibers.
Synthesis and characterization of carbon dots from guava fruit slices: mechanism, structure, and optical properties
Carbon dots were synthesized through the thermal carbonization of guava slices. Sucrose, present in guava slices, was hydrolyzed to glucose and fructose. Glucose can undergo isomerization into fructose, and both sugars are subsequently dehydrated to form intermediate soluble compounds such as furfural derivatives, organic acids, aldehydes, and phenolic compounds. These intermediates then undergo polymerization and condensation to form soluble polymeric structures. Through aldol condensation, cycloaddition, and hydroxymethyl-mediated furan resin condensation, aromatic clusters are generated. Once the concentration of these aromatic structures surpasses a critical supersaturation threshold, a burst nucleation event occurs, leading to the formation of carbon dots24.
The XRD pattern of the synthesized carbon dots, Fig. 3a, exhibited a sharp diffraction peak (002) at 2θ = 19.11, confirming an interlayer spacing of 0.460 nm, which is attributed to carbon dots presence. TEM imaging, Fig. 3b, indicated a non-uniform spherical morphology with diameters ranging from 2.4 to 11.3 nm. Statistical analysis revealed that the majority of particles measured between 6 and 8 nm.
FTIR spectroscopy was used to identify the functional groups on the surface of the produced particles, as shown in Fig. 3c. The measured peaks strongly indicated the production of carbon dots from guava slice elements. The spectrum revealed a broad, sharp peak at 2435 cm-1, indicating the presence of hydroxyl groups and absorbed water molecules on the carbon dot surface. Peaks at 2894 cm-1 and 2932 cm-1 corresponded to C-H stretching vibrations, indicating the presence of organic compounds. The distinct peaks at 1714 cm-1 revealed the presence of carbonyl groups, which might be aldehydes, ketones, or carboxylic acids. Furthermore, the signal at 1650 cm-1 showed the presence of aromatic carbon–carbon double bonds. The peaks at 1043 cm-1 and 1081 cm-1 were carbon–oxygen stretching vibrations, indicating the presence of alcohol, ether, or ester groups18,25.
Sensor cocktail composition
The sensing matrix composition should have adequate selectivity to allow for successful complexation at the electrode surface. The calcium-selective ionophore ETH 1001 was chosen due to its strong affinity for calcium ions, which is essential for providing high sensitivity and stability in the sensor. This ionophore has been widely used in similar electrochemical sensing applications and has provided superior performance for calcium ion detection. In addition, it was selected based on its proven track record in calcium sensing and its ability to produce a Nernstian response with a stable response, enhancing the performance of the sensor. When ETH 1001 is present, the carboxylic acid and amino ethyl ether groups bond to calcium ions, forming a compound that can cause electrical potential shifts across a membrane. The percentage of ETH 1001 was varied in the sensor composition, while the percentages of PVC and DOP were kept equal across all formulations, as shown in Table 1, where each formulation contains identical percentages of PVC and DOP. The sensor with 4% (w/w) ETH 1001 exhibited the best performance. However, when the concentration of ETH 1001 was increased beyond 4%, the sensor exhibited slightly reduced slopes and sensitivity. This reduction is likely due to inhomogeneities in the ionophore distribution, which can lead to non-uniform interaction with calcium ions. As a result, the sensor’s ability to provide a consistent Nernstian response is compromised, reducing the overall sensitivity and altering the calibration slope.
pH conditions and selectivity study of the proposed sensor
The influence of pH on a range of 2–12 was investigated using 10–3 and 10–4 M calcium solutions. The potential was determined at various pH levels, Fig. 4a. The potential remained steady in the pH range of 2 to 9.
One of the sensor key characters is its selectivity behavior, which determines whether the suggested sensor can be used for the analysis of real samples. The separate solution method was used to calculated the potentiometric selectivity coefficient values, \({\text{k}}_{{\text{a},\text{ b }}^{\text{pot}}}.\) It reflects the sensor response to a specific ion (a) in comparison to a solution containing interfering chemicals (b). Two different ion solutions with equal activity were used: one for ion (a) alone at activity (αa) and another for ion (b) at activity (αb). The potentiometric selectivity coefficients were then calculated using an equation26:
where \({E}_{a}\) and \({E}_{b}\) are the measured potential of 10–3 M of calcium solution and interfering compounds including sodium chloride, potassium chloride, magnesium chloride, sodium phosphate, zinc chloride and sodium carbonate. S is the slope,\({Z}_{a}\) and \({Z}_{b}\) are the charges of ions and \(\alpha a\) is the activity of 10–3 M of calcium solution. The estimated potentiometric selectivity coefficients suggested that the proposed sensor exhibited strong selectivity for calcium ions, as shown in Table 2.
Analytical method calibration and validation
Solvent calibration and biological sample calibration plots were shown in Fig. 4b. The matrix effect was determined by comparing the biological sample calibration curve to the solvent calibration curve and computing the slope ratio. The ratio of 101.92% suggested that the matrix had no significant impact on evaluating biological samples. Table 2 summarizes the results of linearity, accuracy, and precision tests. The sensor exhibited a linear relationship between potential and calcium ion activity over the concentration range of 10−7 to 10−1 M, with a super-Nernstian slope of 29.14 ± 0.3 mV/decade and a dynamic response time of 20 s. The sensor’s potential and Nernstian slope varied by less than 1 mV within a single day and across 180 days.
To calculate the limit of detection (LOD), the standard formula LOD = blank signal + 3 × STD was used, where STD is the standard deviation of the blank signal. The LOD was determined to be 7.5 × 10−8 M, indicating the increased sensitivity of the manufactured sensor. To evaluate the method’s accuracy and precision, LQC, MQC, and HQC samples were tested in nine replicates on the same day and three different days. The results presented in Table 2, indicated the method’s repeatability, reproducibility, and consistency over time, ensuring reliable performance of the sensor across different conditions.
Determination of calcium concentration in the nasal secretions
The developed sensor was used to measure calcium levels in human nasal secretions from healthy participants and olfactory dysfunction patients. Table 3 summarizes participant characteristics. The calcium concentrations in the individual samples were determined as described in the experimental section. The results demonstrated the presence of calcium in the nasal secretions of both groups, with considerably higher levels observed in patients with olfactory impairment than in healthy subjects (7.30 ± 0.004 × 10⁻2 M vs. 1.84 ± 0.01 × 10⁻2 M, p = 0.031). Subgroup analysis revealed elevated calcium levels across different etiologies of olfactory dysfunction compared to controls: post-COVID-19 patients (7.24 ± 0.007 × 10⁻2 M for men; 7.38 ± 0.003 × 10⁻2 M for women, p = 0.344), post-traumatic patients (7.80 ± 0.003 × 10⁻2 M for men; 7.58 ± 0.003 × 10⁻2 M for women, p = 0.070), Sino-nasal patients (7.61 ± 0.004 × 10⁻2 M for men; 7.35 ± 0.003 × 10⁻2 M for women, p = 0.069), and idiopathic cases (7.42 ± 0.004 × 10⁻2 M for both men and women, p = 0.216). The data are presented in Fig. 5 as a bar graph to enhance visual comparison.
Comparative assessment of the proposed sensor and previously reported sensor
A clear advancement in calcium sensing performance is demonstrated by the proposed paper-based sensor modified with MWCNTs and carbon dots, as summarized in Table 4. Compared with previously reported methods27,28,29, the presented method exhibits the lowest detection limit (7.5 × 10−8 M) and the broadest linearity range (1.0 × 10−7–1.0 × 10−1 M), enabling accurate quantification from trace to relatively high calcium levels. The obtained slope (29.14 mV decade−1) is close to the ideal Nernstian response and markedly superior to that of the coated carbon-brush electrode (15.09 mV decade−1), confirming efficient ion-to-electron transduction. In addition, the sensor operates reliably over a wide pH range (2–9), highlighting its robustness in diverse sample matrices. Although its response time (20 s) is slightly longer than that of the carbon-brush sensor (8 s), this is compensated by the remarkable sensitivity and broader applicability. In contrast, paper sensor modified with nano-optodes, while attractive for cost and naked-eye readout, remain restricted by higher detection limits and narrower working ranges. Overall, the proposed sensor offers the most balanced profile of sensitivity, selectivity, and operational flexibility, representing a significant step for calcium monitoring in both field and laboratory settings.
Study limitations and future prospectives
Although the developed paper-based calcium sensor demonstrated high sensitivity, selectivity, and stability, certain limitations should be acknowledged. The response time of approximately 20 s, while acceptable for practical applications, is relatively longer than some nanomaterial-based sensors. This slower response may be linked to the diffusion dynamics of calcium ions through the paper substrate and the sensing membrane. Moreover, the fabrication process, although low-cost and simple, may introduce variability due to inhomogeneities in the coating. To address these challenges, future studies could focus on optimizing the coating thickness, improving membrane homogeneity, or incorporating alternative ionophores with faster binding kinetics. Additionally, the integration of advanced nanomaterials or microfluidic platforms could further enhance both response time and reproducibility, thereby increasing the competitiveness of the proposed sensor compared to existing electrochemical platforms.
Conclusion
A paper-based sensor has been developed incorporating multi-walled carbon nanotubes, carbon dots, and the calcium-selective ionophore ETH 1001 for calcium determination in nasal secretions. The sensor showed high sensitivity (slope of 29.14 ± 0.3 mV/decade), a wide linear range (10−7–10−1 M), and a low detection limit (7.5 × 10−8 M). Applied to clinical samples, it revealed significantly higher calcium levels in patients with olfactory impairment (7.30 ± 0.004 × 10⁻2 M) compared to healthy controls 184 ± 0.01 × 10⁻2 M, p < 0.05). A limitation of this study is that ETH 1001 was tested as an ionophore, and future work may explore alternative ionophores or other nanomaterials to further improve performance. Moreover, larger multicenter studies are recommended to validate the clinical applicability of this sensor.
Data availability
The datasets generated and/or analysed during the current study are not publicly available due to privacy and ethical restrictions, but are available from the corresponding author on reasonable request.
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MSI and AAMI and NMA conceived and designed the study. RMAA, AMAA, FKSN, and NAH conducted the statistical analysis and writing original manuscript. RBSB, SSA, and NASA, BMNA contributed to data interpretation and manuscript editing. AIZ conducted the experimental work and data collection. All authors reviewed and approved the final version of the manuscript.
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This work was approved by the Committee of Research Ethics in the Faculty of Medicine, Al-Azhar University, Damietta, Egypt. All participants signed informed consent statements before participation in the study. All described procedures were performed in accordance with relevant guidelines and regulations, and in compliance with the Declaration of Helsinki.
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Imam, M.S., Aldekhail, N.M., Alhashemi, R.M.A. et al. Nanoparticle modified paper-based analytical sensor for calcium determination in human nasal secretions and its association with olfactory dysfunction. Sci Rep 15, 35574 (2025). https://doi.org/10.1038/s41598-025-23104-w
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DOI: https://doi.org/10.1038/s41598-025-23104-w