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This research was supported by the Regional Innovation System &#38; Education (RISE) program through the Gangwon RISE Center, funded by the Ministry of Education (MOE) and the Gangwon State (G.S.), Republic of Korea (2026-RISE-10-001). The present research has been conducted by the Excellent Researcher Support Project of Kwangwoon University in 2026

Commercially obtained de-identified pooled human saliva was used in this study. No human participants were recruited, and institutional ethics approval was not required.
1. Saliva sample preparation
<ol>
	<li>Instruct participants to refrain from eating or drinking for at least 30 min prior to sample collection.</li>
	<li>Prepare the saliva sample according to the supplier&#8217;s instructions. 
	NOTE: This study used commercially available pooled human saliva for protocol-level validation.</li>
	<li>Gently mix the saliva sample before use to ensure homogeneity.</li>
	<li>Store the saliva samples at 4 &#176;C and use them within the supplier&#39;s recommended storage period.</li>
</ol>
2. Preparation of the RBCM-coated membrane (Figure 1A)
<ol>
	<li>Isolate RBCM from whole blood according to a previously reported protocol16.
	<ol>
		<li>Use commercially obtained human whole blood anticoagulated with K2EDTA as the RBCM source.</li>
		<li>Centrifuge the whole blood at 800 &#215; g for 5 min and collect the red blood cell fraction after removing the plasma and buffy coat.</li>
		<li>Wash the isolated red blood cell pellet three times with ice-cold 1&#215; phosphate-buffered saline (PBS) using gentle manual mixing.</li>
		<li>Resuspend the washed red blood cell pellet in ice-cold 0.25&#215; PBS for 30 min to induce hemolysis.</li>
		<li>Separate free hemoglobin by centrifuging the lysed suspension at 20,000 &#215; g for 30 min and collecting the RBCM pellet.</li>
		<li>Wash the collected RBCM pellet three additional times with 0.25&#215; PBS and store the pale pink RBCM pellet at &#8722;80 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Prepare a polyethersulfone (PES) membrane with a pore size of 30 nm and a diameter of 13 mm.</li>
	<li>Deposit 100 &#181;L of 2% (w/v) RBCM solution onto one side of the PES membrane. Gently spread or dispense the solution dropwise until the entire membrane surface is fully wetted.</li>
	<li>Incubate the membrane at 50 &#176;C for 30 min to ensure uniform coating and drying.</li>
	<li>Store the coated membrane at room temperature and use it within 15 days after preparation17. 
	NOTE: Detailed characterization of the RBCM-coated PES membrane has been described in our previous study16.</li>
</ol>
3. Fabrication of the SaliFilter device (Figure 1B)
<ol>
	<li>Place the RBCM-coated membrane into the designated filter holder.</li>
	<li>Assemble the device by securely connecting the inlet and outlet components.</li>
	<li>Attach a 1 mL syringe to the inlet of the device. 
	CAUTION: Ensure that all components are tightly sealed to prevent leakage during operation.</li>
</ol>
4. Saliva pretreatment 
<ol>
	<li>Load 500 &#181;L of saliva into the syringe connected to the SaliFilter device.</li>
	<li>Apply gentle manual pressure to pass the saliva sample through the membrane (~ 2 min).</li>
	<li>Collect the pretreated saliva from the outlet.</li>
	<li>Use the pretreated sample immediately or store it at 4 &#176;C until further analysis.</li>
</ol>
5. SDS-PAGE analysis of salivary proteins
<ol>
	<li>Mix 15 &#181;L of the sample with 5 &#181;L of Laemmli sample buffer.</li>
	<li>Load the mixture into separate wells of an 8% SDS-PAGE gel.</li>
	<li>Perform electrophoresis at 150 V for 40 min.</li>
	<li>Stain the gel with Coomassie blue for 2 h.</li>
	<li>Destain the gel in distilled water overnight.</li>
	<li>Capture images of the gel using a smartphone or imaging system.</li>
</ol>
6. Cortisol detection using Lateral Flow Assay (LFA)
<ol>
	<li>Apply 150 &#181;L of pretreated saliva to the sample pad of the LFA strip.</li>
	<li>Allow the sample to migrate along the strip for 10 min.</li>
	<li>Capture images of the test results using a smartphone or imaging system.</li>
	<li>Open the LFA image in ImageJ and use Image &#62; Color &#62; Split Channels to separate the RGB channels.</li>
	<li>Use the green channel image for test-line intensity analysis.</li>
	<li>Select a rectangular Region of Interest (ROI) of 100 &#215; 10 pixels covering the test-line and apply the same ROI size to all strip images.</li>
	<li>Measure the mean intensity value of the test-line ROI. Select an adjacent blank membrane region with the same ROI size and measure the background intensity.</li>
	<li>Calculate the normalized test-line intensity as follows: normalized intensity = test-line intensity &#8722; background intensity.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Present quantitative data as mean &#177; standard deviation (SD), unless otherwise stated. Define error bars as SD. 
	NOTE: In this study, all reported N values represent technical replicates performed using aliquots of commercially obtained pooled human saliva.</li>
	<li>Perform linear regression analysis using the normalized test-line intensity values obtained from cortisol concentrations of 0&#8211;8 ng mL&#8722;1 and calculate the coefficient of determination (R2) from the fitted curve.</li>
	<li>Calculate the limit of detection (LOD) using the following equation: LOD = 3.3 &#215; SE / |slope|, where SE is the standard error of the y-intercept and |slope| is the absolute value of the slope obtained from the linear regression equation. Use the absolute value of the slope because the competitive LFA produces a negative slope. 
	NOTE: In this study, no inferential statistical tests were performed, and no significance criteria were applied.</li>
</ol>

The authors have no conflicts of interest to declare.

Saliva-based diagnostics offer a noninvasive alternative to blood analysis. However, their broader adoption has been constrained by the intrinsic viscosity of saliva and the presence of interfering components such as mucins and proteins. These factors disrupt capillary-driven flow and compromise the performance of simple analytical platforms, including LFAs, thereby necessitating effective sample pretreatment.
In this study, we demonstrate that the proposed handheld filtration device, SaliFilt...

Cortisol, a glucocorticoid hormone secreted by the adrenal cortex, is a key biomarker for assessing physiological stress1,2. It can be measured in multiple biological fluids, including serum, plasma, urine, interstitial fluid, and saliva, with blood and saliva being the most commonly used in clinical practice3. Although blood-based measurements provide reliable quantitative information, they require invasive venipuncture and trained personnel, which limits their suitability for frequent or longitudinal monitoring4,5,6. In contrast, salivary cortisol measurement offers a noninvasive and convenient alternative, enabling point-of-care and self-monitoring applications7,8. Notably, salivary cortisol levels correlate strongly with blood concentrations, supporting saliva as a reliable surrogate sample9. However, the intrinsic viscosity of saliva and the presence of interfering components significantly hinder analytical performance, necessitating effective pretreatment prior to analysis10,11,12.
Various pretreatment strategies have been explored to address these challenges. Centrifugation is widely used to remove large components such as mucins, proteins, and cellular debris. However, it requires bulky laboratory equipment and multiple processing steps, which limits its applicability in decentralized settings13,14. Dilution with buffer solutions can reduce viscosity and improve sample flow15. Nevertheless, it simultaneously lowers analyte concentration, thereby compromising analytical sensitivity. As a result, existing approaches remain insufficient for point-of-care applications, where rapid, equipment-free processing and preservation of target analytes are essential.
To overcome these limitations, we developed SaliFilter, a handheld device for saliva pretreatment tailored to point-of-care cortisol detection. The device employs a filtration mechanism that selectively removes high-molecular-weight interfering substances while permitting the passage of small molecules, including cortisol16. Operated manually, the device enables rapid, user-friendly processing without the need for external instrumentation. By eliminating interfering components, this approach improves capillary flow in lateral flow assays, thereby enhancing analytical reliability. This protocol describes the fabrication of the device and its application for rapid saliva pretreatment prior to cortisol detection using lateral flow assays.

<table><tbody><tr><td>Artificial saliva</td><td>Solarbio, China</td><td>A7990</td><td></td></tr><tr><td>Bio-Rad Protein Assay Dye Reagent</td><td>Bio-Rad, USA</td><td>5000006</td><td>For bradford assay</td></tr><tr><td>Coomassie blue</td><td>Abcam, UK</td><td>ab119211</td><td>For protein band visualization</td></tr><tr><td>Cortisol ELISA Kit</td><td>Abcam, UK</td><td>ab154996</td><td></td></tr><tr><td>Cortisol LFA</td><td>In-house</td><td></td><td></td></tr><tr><td>Hot plate</td><td>Daihan Scientific, Republic of Korea)</td><td></td><td></td></tr><tr><td>Hydrocortisone (Cortisol)</td><td>Sigma-Aldrich, USA</td><td>PHR1014</td><td></td></tr><tr><td>Laemmli sample buffer</td><td>GenDEPOT, USA</td><td>L1200-001</td><td>For protein denaturation prior to SDS-PAGE</td></tr><tr><td>Membrane holder</td><td>GVS Filter Technology</td><td>1220950</td><td>13mm diameter holder</td></tr><tr><td>Mini-PROTEAN&amp;reg; Tetra Cell for 0.75 mm Gels</td><td>Bio-Rad, USA</td><td>1658000</td><td>For gel electrophoresis</td></tr><tr><td>Mucin</td><td>Sigma-Aldrich, USA</td><td>M2378</td><td></td></tr><tr><td>NanoDrop 2000 spectrophotometer</td><td>Thermo Fisher Scientific, USA</td><td></td><td>For protein assay</td></tr><tr><td>PES membranes</td><td>STERLITECH, USA</td><td>PES00313100</td><td>0.03 Micron, 13 mm PES membrane</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>DUKSAN, Republic of Korea</td><td>LB004</td><td></td></tr><tr><td>Pooled Human Saliva</td><td>Innovative Research Inc., USA</td><td>IRHUSL5ML</td><td></td></tr><tr><td>RBCM</td><td>In-house</td><td></td><td></td></tr><tr><td>Resolving Gel Buffer for PAGE</td><td>Bio-Rad, USA</td><td>1610798</td><td>For resolving gel preparation</td></tr><tr><td>Salivette Cortisol</td><td>SARSTEDT, Germany</td><td>51.1534.500</td><td>Saliva collection kit</td></tr><tr><td>Stacking Gel Buffer for PAGE</td><td>Bio-Rad, USA</td><td>1610799</td><td>For stacking gel preparation</td></tr><tr><td>Super&amp;bull;SAL&amp;trade;</td><td>Oasis Diagnostics, USA</td><td>SSAL-601</td><td>Saliva collection kit</td></tr><tr><td>Syringe</td><td>koreavaccine, Republic of Korea)</td><td></td><td>1 mL volume</td></tr><tr><td>TEMED</td><td>Bio-Rad, USA</td><td>1610800</td><td>For acrylamide polymerization</td></tr></tbody></table>

handheld device for saliva pretreatment to improve cortisol detection

Handheld Device for Saliva Pretreatment to Improve Cortisol Detection

Salivary cortisol detection offers a noninvasive alternative to blood-based analysis. However, its practical implementation remains limited by the intrinsic viscosity of saliva and the presence of interfering components. In particular, mucins and salivary proteins impede capillary-driven flow and compromise the reliability of subsequent lateral flow assays (LFAs), thereby necessitating effective and accessible pretreatment strategies. Here, we present a handheld saliva pretreatment device, termed SaliFilter, for point-of-care cortisol detection. The device employs a filtration mechanism that selectively removes high-molecular-weight interfering components while allowing the passage of small molecules, including cortisol. Operated by simple manual pressure, it enables rapid sample processing (~2 min) without the need for external equipment, making it suitable for decentralized settings. The performance of the device was evaluated using turbidity measurements, protein quantification, and SDS-PAGE, which confirmed efficient removal of mucin and salivary proteins. Treated samples exhibited enhanced capillary flow, resulting in stable and reliable LFA operation. Quantitative analysis demonstrated improved signal reproducibility, and cortisol detection achieved a limit of detection of 1.47 ng mL&#8315;1 over clinically relevant concentration ranges. This protocol establishes an effective saliva pretreatment strategy that improves analytical sensitivity and reliability without compromising analyte integrity. The proposed approach provides a practical, portable solution for saliva-based diagnostics, with potential applicability for point-of-care testing and self-monitoring.

Effective saliva pretreatment requires removing interfering components, including mucins and proteins, that compromise downstream analytical performance. The filtration capability of SaliFilter was first evaluated by comparing turbidity changes with those obtained using commercial saliva collection kits (Figure 2A). Untreated saliva exhibited high turbidity, whereas SaliFilter-treated samples appeared visibly clear, indicating efficient removal of mucins and suspended components. In contrast...

This protocol describes a rapid saliva pretreatment method for improving cortisol detection. A handheld filtration device is used to remove mucins and other interfering substances while preserving the target analyte. The pretreated saliva exhibits improved capillary flow, thereby enabling more consistent and reproducible assay performance.

Salivary cortisol detection offers a noninvasive alternative to blood-based analysis. However, its practical implementation remains limited by the ...

Our research focuses on saliva pretreatment to improve the reliability of cortisol detection. This protocol can be applied to saliva-based biomarker analysis using the lateral flow assay. To begin, gently mix the commercially obtained de-identified pooled human saliva sample before use to ensure homogeneity.Store the saliva samples at four degrees Celsius and use them within the supplier's recommended storage period. Thaw the pale pink red blood cell membrane or RBCM pellet prepared and stored at 80 degrees Celsius as per a standard protocol. Prepare a polyethersulfone or PES membrane with a pore size of 30 nanometers and a diameter of 13 millimeters.Deposit 100 microliters of 2%weight per volume RBCM solution onto one side of the PES membrane. Gently spread or dispense the solution drop-wise until the entire membrane surface is fully wetted. Incubate the membrane at 50 degrees Celsius for 30 minutes to ensure uniform coating and drying.Store the coated membrane at room temperature and use it within 15 days after preparation. To fabricate the SaliFilter device, place the RBCM-coated PES membrane into the designated filter holder. Assemble the device by securely connecting the inlet and outlet components.Attach a one milliliter syringe to the device inlet. Load 500 microliters of saliva into the syringe connected to the SaliFilter device. Apply gentle manual pressure to pass the saliva sample through the membrane for approximately two minutes.Collect the pretreated saliva from the outlet and use it immediately or store it at four degrees Celsius until further analysis. After protein detection by SDS-PAGE, apply 150 microliters of pretreated saliva to the sample pad of the LFA strip for cortisol detection. Allow the sample to migrate along the strip for 10 minutes.Capture images of the test results using a smartphone or imaging system. Drag and drop the captured LFA file into the ImageJ software window. Click on image, choose color, and then select split channels to separate the red, green, and blue channels.Close the red and blue channels and use the green channel image for test line intensity analysis. Use the rectangle tool to select the region of interest or ROI of 100 by 10 pixels covering the test line. Open the other LFA strip images and apply the same ROI size across all samples.Click on analyze and choose measure to obtain the mean intensity value of the test line ROI. Next, select an adjacent blank membrane region with the same ROI size and measure the background intensity. Export the values to an Excel spreadsheet and calculate the normalized test line intensity by subtracting the background intensity from the test line intensity.Evaluation of filtration capability of SaliFilter by comparing turbidity changes against commercial saliva collection kits showed high turbidity in untreated saliva, whereas SaliFilter treated samples appeared visibly clear. Samples processed using commercial kits showed only limited improvement in clarity. Salivary protein quantification by absorbance at 280 nanometers showed an approximate 69%reduction following filtration.A Bradford assay showed a marked decrease in color intensity in SaliFilter treated samples corresponding to an approximately 80%reduction in total protein concentration. Capillary flow analysis demonstrated that removing mucins restored flow performance in pretreated saliva. SDS-PAGE analysis confirmed that mucins were retained upstream of the membrane and were absent from the processed samples.This protocol allows researchers to evaluate soluble cortisol after removing proteins and mucins. After filtration, samples can be analyzed by SDS-PAGE, protein assays, ELISA, or Future studies can validate this platform using clinical saliva samples with different cortisol concentrations.

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112 Views.  Kwangwoon University. Our research focuses on saliva pretreatment to improve the reliability of cortisol detection. This protocol can be applied to saliva-based biomarker analysis using the lateral flow assay. To begin, gently mix the commercially obtained de-identified pooled human saliva sample before use to ensure homogeneity.Store the saliva samples at four degrees Celsius and use them within the supplier's recommended storage period. Thaw the pale pink red blood cell membrane or RBCM pellet prepared an...

Handheld Device for Saliva Pretreatment to Improve Cortisol Detection

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