Handheld Device for Saliva Pretreatment to Improve Cortisol Detection
June 26th, 2026
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.
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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Overview
This article introduces SaliFilter, a handheld saliva pretreatment device designed to enhance the reliability and sensitivity of salivary cortisol detection at the point of care. By efficiently removing high-molecular-weight interfering components such as mucins and salivary proteins, the device enables improved performance of lateral flow assays (LFAs) for cortisol measurement in decentralized and self-monitoring settings.
Key Study Components
Area of Science
- Analytical chemistry
- Point-of-care diagnostics
- Bioengineering
Background
- Salivary cortisol is a valuable biomarker for stress and endocrine disorders.
- Saliva offers a noninvasive alternative to blood sampling.
- Viscosity and interfering proteins in saliva hinder capillary-driven flow in LFAs.
- Effective pretreatment is needed to improve assay reliability and sensitivity.
Purpose of Study
- To develop a simple, portable device for saliva pretreatment prior to cortisol detection.
- To selectively remove mucins and proteins that interfere with LFA performance.
- To enable rapid, equipment-free sample processing suitable for point-of-care use.
Methods Used
- Design and fabrication of the SaliFilter handheld device.
- Manual pressure-driven filtration to process saliva samples (~2 min per sample).
- Evaluation of filtration efficiency using turbidity measurements, protein quantification, and SDS-PAGE.
- Assessment of LFA performance with treated versus untreated saliva samples.
Main Results
- SaliFilter effectively removed mucins and high-molecular-weight salivary proteins.
- Treated samples showed improved capillary flow and stable LFA operation.
- Quantitative analysis demonstrated enhanced signal reproducibility.
- The device enabled cortisol detection with a limit of detection of 1.47 ng mL⁻1 across clinically relevant ranges.
Conclusions
- SaliFilter provides a rapid, accessible pretreatment solution for saliva-based diagnostics.
- The device improves analytical sensitivity and reliability of salivary cortisol LFAs.
- This approach is practical for decentralized point-of-care testing and self-monitoring applications.
Frequently Asked Questions
Executive Industry Relevance
Reliable salivary cortisol detection is critical for noninvasive biomarker strategies in decentralized and point-of-care settings. The SaliFilter device addresses a key bottleneck by enabling rapid, standardized pretreatment of saliva, improving assay reproducibility and analytical sensitivity. This capability supports robust biomarker workflows and expands the utility of saliva-based diagnostics across early discovery and translational research pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables accurate quantification of salivary cortisol for stress-related biomarker validation.
- Reduces biological noise by removing mucins and interfering proteins, supporting mechanistic de-risking.
- Improves predictive confidence in biomarker-driven target selection and triage.
Screening & Assay Development
- Facilitates preparation of low-viscosity, interference-free saliva samples for LFA development.
- Supports assay standardization and reproducibility by minimizing sample variability.
- Enables reliable, quantitative readouts for compound or biomarker screening workflows.
Translational & Preclinical Research
- Improves continuity from discovery to preclinical validation by supporting robust, noninvasive biomarker measurement.
- Aligns with translational biomarker strategies for stress and endocrine research.
- Reduces risk of assay failure in decentralized or self-monitoring studies.
Pipeline & Workflow Integration
The SaliFilter device integrates at the sample preparation stage, upstream of lateral flow assay-based cortisol quantification, supporting workflows from early biomarker discovery through translational validation.
- Discovery Biology: Enhances hypothesis testing by enabling reliable measurement of salivary cortisol in diverse settings.
- Screening: Provides standardized, interference-free samples for assay development and screening campaigns.
- Analytics: Delivers quantitative, reproducible outputs that facilitate condition comparison and statistical analysis.
- Translational Research: Supports noninvasive biomarker continuity from research to preclinical and decentralized applications.
- Enterprise Reuse: Offers a portable, scalable pretreatment solution adaptable across biomarker and diagnostic programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in salivary biomarker studies.
- Operational Value: Delivers rapid, equipment-free sample processing with high reproducibility.
- Strategic Value: Enables robust go/no-go decisions and reduces late-stage assay risk in decentralized workflows.
- Portfolio Impact: Supports risk-adjusted prioritization of noninvasive biomarker programs.
Implementation Considerations
- Requires basic laboratory or point-of-care training for manual device operation.
- Minimal instrumentation needs, as filtration is manual and rapid (~2 min per sample).
- Standardization of sample handling protocols is essential for cross-team reproducibility.
- Adaptable to various saliva-based biomarker assays beyond cortisol, pending validation.
- Potential limitations include analyte-specific compatibility and throughput for high-volume studies.
