Saliva stamp – a disposable, paper-based device for measuring oxidative stress biomarkers

Abstract

The Saliva Stamp is a novel, non -invasive, and user -friendly device for measuring oxidative stress biomarkers in saliva. This point -of-care diagnostic tool utilizes a proprietary reagent and a unique filter paper design to detect 8 -OHdG biomarkers. The device provides rapid, accurate, and sensitive results, enabling early detection and monitoring of oxidative stress -related diseases. The Saliva Stamp overcomes the drawbacks of existing methods, offering a convenient, accessible, and environmentally friendly solution for oxidative stress assessment.

Description

[0001] Saliva Stamp - Description

[0002] of the invention including prior and highlights of tie invention Background and Prior Art B ackground O xidative stress is a state of in balance betv een tie production of reactive oxygen species RO S and tie body s ability to detoxify these harm fol com pounds C hronic oxidative stress is linked to various diseases including cancer diabetes and neurodegene rative disorders E arty detection and m onitoring of oxidative stress are crucialfor preventing and m anaging these conditions Prior A rtC urrentm ethods form easuring oxidative stress brm arkers are invasive expensive and ti e consuming Sone of tie existing methods include 1 Blood tests Require venipuncture causing discern tort and risk of infection 2 U rine tests M ay not accurately reflect oxidative stress levels due to variability in urine concentration 3 Sali / a tests Existing saliva based diagnostic devices require separate reagents con plicated analysis procedures and are not user friendly 4 Laboratory based assays Enzyne linked imm unosorbent assay ELEA high perfoimance liquid chromatography HPLC and mass spectrometry M S are complex tine consuming and expensive Li itations of PriorArtl Jhvasiveness Blood testsand other invasi / em etnods cause discern tort and risk of infection 2 Cost Laboratory based assays and existing diagnostic devices are expensive 3 Complexity C urrentm etnods require specialized training and equipm ent 4 Tin e consum ing E xisting m etnods involve lengthy analysis procedures N eed for tie Invention There is a need for a sin pie cost effective and non invasive m ethod -form easuring oxidative stress bion arkers The Saliva Siam p invention addresses these lin iiations by providing a user friendly paper based device for easy and accuratemeasuren ent of oxidative stress bion arkers in saliva H ghlights of tie Invention 1 N ovel disposable paper based device M easures oxidative stress bion arkers in saliva 2 Proprietary reagent Specifically reactswiti 8 OHdG bimarkers 3 U nique stam p design Enableseas / mess free saliva collection and analysis 4 Non invasive cost effective and user friendly Addresses limitations of prior artmetiods 5 Quick and easy analysis Provides accuratem easuram ent of oxidative stress bim arkers 9 Novelty Non Obviousness Invention Step and U tilityOftiis invention w iti brief justification N ovelty 1 U nique com bination The Saliva Siam p com bines a paper based platfotm proprietary reagent and stam p design which is new and original 2 Proprietary reagent The reagents com position and specific reaction with 8 OH dG bim arkers are novel 3 Stam p design The design enables easy sali / a collection and analysis which is a new and innovative feature Non Obviousness 1 U npredictable com bination The com bination of a paper based platfotm proprietary reagent and stam p design is unexpected and non obvious 2 Reagents specific reaction The proprietary reagents specific reaction with 8 OHdG bimarkers is surprising and non obvious 3 Sim pie yeteffective design The stam p design s sin plicity and effectiveness are non obvious and innovative Invention Step 1 N ew and innovative solution The Saliva Stam p provides a new and innovatk / e solution form easuring oxidative stress bimarkers 2 Overcomes prior art limitations The invention overcomes tie limitations of prior artmetiods such as invasiveness complexity and cost 3 Enables easy and accurate

[0003] m easuram ent The Saliva Stam p enables easy and accuratem easuram entof oxidative stress bim arkers U tility 1 M easures oxidative stress bim arkers The Saliva Stam p m easures oxidative stress bim arkers which is usefolfor diagnosing and m onitoring various diseases 2 Non invasive and user friendly The invention is non invasive and user friendly making it suitable forwidespread use 3 Cost effective The Saliva Stam p is cost effective reducing tiefinancial burden on individuals and healthcare systems Claims 1 A disposable paper based device form easuring oxidative stress bion arkers in saliva con prising a proprietary reagent specifically reacting with 8 OH dG bim arkers and a unique stam p design enabling easy saliva collection and analysis 1 1 The device of claim 1 wherein the proprietary reagent com prises 05 mgmL 33 55 T etram ethylbenzidine TM B 5U mL Horseradish peroxidase HRP and 5 μg / mL monoclonal antibody specific to 8 OHdG 1 2 The device of claim 1 wherein 1he unique stam p design ism ade of a paper based m ateriallhatis biodegradable and envirorm entally friendly 1 3 The device of claim 1 further con prising a reference chart for interpreting resulting color changes 2 A m ethod form easuring oxidatve stress bim arkers in saliva using the device of claim one 3 A kitform easuring oxidative stress bim arkers in saliva con prising 1he devie of claim 1 a reference chart and instructions for use 10 O bjectves of 1he Invention O bjective of 1he Invention The prin ary objective of 1he Saliva Stamp invention is to provide a sin pie cost effective and non invasivem ethod form easuring oxidative stress bim arkers in saliva Specific Objectives 1 To develop a disposable paper based device for easy saliva collection and analysis 2 To create a proprietary reagent that specifically reacts with 8 OHdG bimarkers 3 To design a unique stam p design that enables mess free saliva collection and analysis 4 To provide a user friendly and accuratem ethod form easuring oxidative stress bim arkers 5 To enable early detection and monitoring of oxidative stress related diseases 11 Detailed description of tie invention Drawing Fig 1 Saliva stam p frontview with tie saliva collection area and a filter paper The Saliva Stamp holder for tie saliva collection area ism ade from an eco friendly product specifically Bio-PMMA (Biodegradable Polymethylmethacrylate) A sustainable biodegradable and non toxic material PLA Poly lactic Acid A biodegradable and renewable thermoplastic derived from natural resources O tier bioplastics Such as PB AT Polybutylene Adipate co Butylene Terephthalate orPHA Polyhydroxyalkanoates Thiseco friendly m aterial ensures tie Saliva Stamp holder is Biodegradable Non toxic Sustainable

[0004] E nvirorm entally friendly F ig 2 Proprietary reagent Proprietary Reagent Composition

[0005] 1 33 55 Tetramethylbenzidine TM B 05mgmL A chromogenic substrate that reacts with the enzyme Horseradish Peroxidase HRP 2 Horseradish Peroxidase HRP 5U mL An enzym e that catalyzes the oxidation of TM B resulting in a color change 3 Monoclonal Antibody specific to 8 OHdG 5 μg / mL A highly specific antibody that binds to 8 OHdG a biomarker for oxidative stress 4 Glycerol 10% (v / v) A stabilizer tnathelpsm aintain tie reagents activity and shelf life 5 Trehalose 1 w v A sugar that helps stabilize proteins and m aintain tie reagents activity 6 Phosphate Buffered Saline PBS solution Asa base A buffer solution tiat m aintains tie reagents pH and stability Proprietary R eagentC haracteristics 1 High specificity: The monoclonal antibody ensures high specificity for 8 OHdG m inin izing false positives 2 High sensitivity: The optimized reagent com position enables detection of low levels of 8 O H dG 3 Stability The reagent is stable fora prolonged period ensuring consistent performance 4 Ease of use The reagentis sin pie to use with a straightforward protocolfor saliva sam pie analysis Advantages of tie Proprietary R eagent 1 Accurate detection Enables accurate detection of oxidative stress bim arkers in saliva 2 C onvenient E asy to use and integrate into tie Saliva Stamp devie 3 Cost effective Reduces tie need for expensive equipment and trained personnel 4 E nvirorm entally friendly The reagents com position and packaging are designed to m inin ize envirorm entalin pactFig3 Dipping Process Preparation 1 Proprietary Reagent Preparation The proprietary reagent is prepared according to the optimized formulation 2 Filter Paper Preparation The filter paper is cut to the desired size and shape for the Saliva Stamp device D ipping Process 1 D ipping The filter paper is carefully dipped into the proprietary reagent solution ensuring complete saturation 2 Coating The reagent solution evenly coats tie filter paper allowing the reagents to bind to the paper's surface 3 E xcess Ren oval Excess reagent solution is gently removed from the filter paper using a pipette or a gentle shaking m otion Drying and Storage 1 Drying The dipped filter paper is allowed to air dry at room ten perature ensuring tie reagents are stable and effective 2 Storage The dried filter paper is stored in a sealed container or pouch tomaintain its stability and shelflife

[0006] Fig 4 Saliva Collection and A natysis Adding Saliva to tie Filter Paper 1 Saliva Collection A small saliva sample approximately 50 100 L is collected from the user 2 Placing Saliva on F liter Paper The saliva sam pie is carefully placed onto tie dipped filter paperw ithin tie Saliva Siam p holder W ailing Period 1 Incubation The saliva sam pie is allowed to incubate on tie filter paper for a specified period approximately 5-10 minutes 2 R eaction D uring this tin e tie proprietary reagent on tie filter paper reacts with the 8-OHdG biomarkers presentin tie saliva sample Color Change 1. Color Development: As the reaction occurs a visible color change is observed on tie filter paper indicating tie presence of oxidative stress bim arkers 2 Result Interpretation The resulting color change is com pared to a reference chart to determine tie level of oxidative stress bim arkers present in tie saliva sam pie

[0007] Fig 5 interpretation of tie C hart Color Chart The chart displays a range of colors each corresponding to a specific levelof oxidative stress bim arkers Color Gradations 1 Negative (Colorless) N o oxidative stress bim arkers detected 2 Light Yellow L cw levels of oxidative stress bim arkers detected 3 Medium Yellow M oderate levels of oxidative stress bim arkers detected 4 Dark Yellow / Brown H igh levels of oxidative stress bim arkers detected Result Interpretation 1 Match the Color: Compare tie resulting cobr change on tie filter paper to tie cobr chart 2 Determine the Level: Determine tie levelof oxidative stress bim arkers present in the saliva sample based on the matching color E xam pie If tie resulting cobr change on tie filter paperm atches tie Medium Yellow cobr on tie chart it indicates moderate levels of oxidative stress bim arkers are present in the saliva sample. This interpretation provides a quick and easy way to determine oxidative stress levels, enabling users to take proactive steps towards maintaining their health and wellbeing

[0008] 12 D raw backs b tie Existing State of Art Invasive and UncomfortabteM etiodsl Blood draws C urrentm ethods form easurbg oxidative stress bim arkers often require invasive blood draws which can be painful and uncom fortable for patients 2 Saliva collection W hite some existing methods use saliva collection they often require large sample volumes or specialized equipm ent Time Consumbg and Expensive 1 Lab based testing M any existing methods require lab based testing which can be time consuming and expensive 2 Specialized equipm ent C urrent m etnods often require specialized equipm ent such as spectrophotom eters or E L ISA readers Limited Accessibility 1 Limited availability Existingm etiodsm ay not be widely available particularly b resource lim ited settings 2 T rabed personnel M any existingm etnods require trabed personnel to adm bister and interpret the tests O tierD raw backs 1 V ariable accuracyE xisting m etiodsm ay have variable accuracy depending on "factors such as sam pie handling and storage 2 L in ited sensitivity Son e existing m etiodsm ay not be sensitive enough to detect low levels of oxidative stress bimarkers These drawbacks highlight tie need -for a more convenient accessible and accuratem etiod "form easuring oxidative stress bim arkers such as tie Sali / a Stamp 13 How these D raw backs have been overcom e by your invention how tie Saliva Sta p invention overcom es tie draw backs of existing m etnods O vercom ing D raw backs N on Invasive and Comfortable 1 Painless saliva collection: The Saliva Stamp uses a simple, non-invasive saliva collection method 2 N o blood draw s E lin inates tie need for painful blood draws Quick Eas / and Affordable 1 Rapid results Provides results within minutes 2 No specialized equipment Uses a sin pie low cost design 3 Affordable Reduces costs associated with lab based testing Increased A ccessbility 1 Portable The Saliva Stamp is a portable, point-of-care device 2 User friendly Easy to use with minimal training required 3 W ide availability C an be widely distributed even in resource limited settings In proved A ccuracy and Sensitivity 1 Highly sensitive: Detects low levels of oxidative stress bim arkers 2 A ccurate results Provides accurate reliable results Additional Benefits 1 Environmentally friendly Uses biodegradable m aterials 2 Easy disposal Sin pie safe disposal of used devices The Saliva Stamp invention addresses tie drawbacks of existing m etiods providing a convenient accessible and accurate solution form easuring oxidative stress bim arkers Summary Abstract The Saliva Stamp is a novel, non-invasive and user friendly device for measuring oxidative stress biomarkers in saliva. This point-of-care diagnostic tool utilizes a proprietary reagent and a unique filter paperdesign to detect 8-OHdG biomarkers The devie provides rapid accurate and sensitive results enabling early detection and m onitoring of oxidative stress related diseases The Saliva Stamp overcomes tiedrawbacks of existing methods offering a convenient accessible and envirorm entalty friendly solution for oxidative stress assessn ent

Claims

AMENDED CLAIMSreceived by the International Bureau on 17 February 2026 (17.02.2026)Claims

1. A disposable, paper-based diagnostic device for rapid, point-of- care detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in an untreated saliva sample, comprising:(a) a cellulose-based substrate formed as a stamp-type contact element configured to receive saliva by direct static compression, the substrate being free of lateral-flow channels and capillary- driven transport paths; and(b) a single-step reaction zone integrated within the substrate and comprising a dry-state stabilized reagent matrix including(i) an anti- 8-OHdG monoclonal antibody,(ii) horseradish peroxidase (HRP), and(iii) 3,3',5,5'-tetramethylbenzidine (TMB);wherein mechanical compression of the substrate against a salivabearing surface causes localized rehydration of the reagent matrix and initiates an enzyme-mediated colorimetric reaction, and wherein the device generates a visually interpretable color signal within 5 minutes without sample pretreatment, external power, or analytical instrumentation.

2. The device of claim 1, wherein the dry-state stabilized reagent matrix further comprises glycerol at about 10% (v / v) and trehalose at about 1% (w / v), the combination being configured to preserve enzymatic activity of HRP and antigen-binding functionality of the antibody during storage at ambient temperature.

3. The device of claim 2, wherein the reagent matrix further comprises a phosphate-buffered saline (PBS) component that establishes a controlled pH environment upon saliva-induced rehydration to ensure reproducible colorimetric development.

4. The device of claim 1, wherein the cellulose-based substrate has a predetermined thickness and porosity selected to absorb a defined saliva volume under static compression, enabling semi- quantitative interpretation of the colorimetric response relative to a reference standard.

5. A method for non-invasive detection of 8-hydroxy-2'- deoxy guano sine (8-OHdG) in saliva, comprising:(a) contacting a saliva sample with the diagnostic device of claim 1 by direct static compression;(b) allowing saliva-mediated rehydration of the dry-state reagent matrix to initiate a colorimetric reaction; and(c) visually assessing the resulting color intensity within 1to 5 minutes, wherein the color intensity correlates with the concentration of 8-OHdG in the saliva sample.

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