Paper-based Colorimetric Assay: Rapid Detection of HVA in Urine Samples

Paper-based Colorimetric Assay: Rapid Detection of HVA in Urine Samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Paper-based Colorimetric Assay: Rapid Detection of HVA in Urine Samples Vrushali Bhalchim, Vaishali Undale, Sunil Shewale This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4455995/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Diagnosing and monitoring of Parkinson's disease, schizophrenia, and certain mood disorders involves detecting homovanillic acid an essential aspect excreted through urine. In this study, we explored novel approaches for detecting homovanillic acid (HVA) in urine samples, aiming for cost-effective and on-site analysis methods. Traditional techniques like spectrophotometric determination and HPLC, while sensitive and selective, are often impractical due to their expense and lack of portability. Instead, we investigated the utility of paper-based analytical devices (PADs), which have gained popularity for various analytical applications including chemical element identification and environmental contamination assessment. We utilized different types of Whatmann filter papers (No. 1, No. 2, No. 4) and chromatography paper as substrates for the PADs, and employed combination of potassium ferrocyanide and ferric chloride reagent for HVA detection. Our experiments revealed positive results, indicating the efficacy of this approach. Optimization experiments identified 30 minutes to 3 hours as the optimal immersion times, with a subsequent 30-minute drying period at room temperature. Notably, variations in performance were observed among the different filter papers, with Whatmann CF6 paper exhibiting significant differences compared to the others in terms of HVA detection efficiency. HVA paper-based analytical devices urine diagnosis and monitoring Parkinson’s disease etc Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Homovanillic acid (HVA) monitoring is often done in clinical settings to assess dopamine metabolism. It's particularly useful in conditions where dopamine levels are implicated, such as Parkinson's disease, schizophrenia, and certain mood disorders [ 1 ]. In medical practice, measuring HVA levels can provide insights into the activity of dopamine-producing neurons in the brain. Abnormal levels of HVA may indicate dysfunction in dopamine regulation, which can help clinicians in diagnosing and managing various neurological and psychiatric conditions. Many HPLC methods, utilizing diverse sample extraction methods alongside analytical columns and either electrochemical or fluorimetric detection, have been developed. HPLC coupled with electrochemical detection is the preferred approach for detecting HVA due to its high sensitivity. However, this method is relatively nonspecific and can produce complex chromatograms if isolation steps are omitted. Therefore, most HPLC techniques using electrochemical detection to identify biogenic amines first separate interfering compounds using extraction columns. Though time-consuming, these procedures could provide significant advantages in routine clinical investigations. Various analytical methods, such as spectrophotometric determination [ 2 ] and high-performance liquid chromatography (HPLC) [ 3 ], are commonly employed to detect homovanillic acid (HVA). However, despite their precision and sensitivity, these techniques necessitate expensive equipment and are impractical for on-site analysis. To address this limitation, there's a growing interest in developing indicator strips or optical sensor membranes for detecting chemical substances in herbal medicine. Some researchers opt for polymers as substrate materials for this purpose [ 4 , 5 ]. Paper-based analytical devices (PADs) have recently emerged as a straightforward, cost-efficient, and user-friendly tool for environmental and biological analyses [ 6 – 8 ]. They have been successfully applied for on-site analysis of organic molecules [ 9 ], metals [ 6 ], and pesticides[ 10 ]. A prevalent detection method in PADs involves a colorimetric approach, utilizing specific reagents to identify and detect analytes in samples [ 11 – 13 ]. In this study, we opted combination of Potassium Ferric cyanide + Ferric chloride as the colorimetric reagent for detecting HVA. Potassium Ferric cyanide + Ferric chloride was chosen as metal ions forms complex with HVA compound, making it a suitable colorimetric reagent. We screened the reagent and applied it to various types of Whatman filter papers, including Whatman No. 1, No. 4, and No. 6 [ 14 ]. The selectivity, sensitivity, and practicality of this paper-based analytical device (PAD) for HVA detection were also evaluated. The results indicate that this PAD is effective for detecting HVA in urine samples. 2. MATERIAL AND METHODS All chemicals utilized were of analytical grade and were employed without additional purification. HVA was obtained from Sigma Aldrich Pvt. Ltd. Potassium ferro cyanide was purchased from Himedia whereas Ferric chloride was purchased from Lobachemie. Different grades of whatmann papers were purchased from whatmann grade 1 (whatmann), Whatmann CF4 (Cytiva), Whatmann CF6 (Cytiva) respectively. The absorbance was measured using a UV-Visible spectrophotometer model 1900 Shimadzu, utilizing a 1.0 cm quartz cell. 2.1 Reagent preparation Weighed quantity of Potassium Ferric cyanide 24 mg + Ferric chloride 257 mg was dissolved in 10 ml of distilled water and stirred completely at room temperature. 2.2 Design and Optimization of Specific Reagent Tests on Paper 2.3 Optimization and selection of dip strip base for qualitative detection of HVA At different time interval (i.e 30, 60, 120, 180 mins) the different grade of Whatmann filter paper grade A: Whatmann No 1, B: Whatmann CF4, C: Whatmann CF6 strips were soaked in reagent reaction mixture and dried at R.T. The developed strip for HVA were dipped in 100 µg/ml of standard HVA sol. and time required for color development were recorded. 2.4 Qualitative detection by dip strip at different HVA concentrations The optimized strip for HVA were treated with different concentration of HVA (25, 10, 1 mg/ml) and (1 µg/ml) for determining the minimum concentration to be detected by developed dip strip for HVA. 2.5 Real time analysis by dip strip in urine sample Before analysis the urine samples were extracted 0.1 M perchloric acid and then dip strip was dipped into the sample then any color change on strip was observed for qualitative detection of HVA. 2.6 Prototype design of dip strip Based on the earlier studies reported the material to be used was selected [ 15 ]. From point of care diagnosis approach the design of the strip was done as dip strip from point of ease of use, qualitative detection and economical use. 2.7 Concentration dependent color codes From stock solution of standard HVA (10 mg in 10 ml) different dilutions of HVA were prepared from 10 µg/ml to 1 mg/ml and treated with reaction mixture to identify the detection limit of HVA concentration with developed strip. 2.8 Validation of dip strip Stability testing of strip was performed at 1, 2, 3 months to assess the stability of dip strip to produce the color change when dipped in HVA solution. Human ethical permission For human pilot study the proposal was submitted to Institutional Ethics committee at Dr. D. Y. Patil Homoeopathic Medical College and Research Center, Pimpri, Pune 411018. The proposal was sanctioned by IEC protocol no DYPHMCRC/E-04/2023, dated 30/4/2023. The enrolment of participants was done by taking their oral informed consent from patient by the neurologist and guiding them about the risks, benefits, awareness, and purpose of study experiment procedure. The study was performed in accordance with relevant guidelines and regulations as suggested by the ethics committee. 4. RESULT AND DISCUSSION 3.1 Design and Optimization of dip strip for qualitative detection of HVA in urine sample paper In this study, Whatman filter papers No. 4 and No. 6, along with Whatman chromatography paper No. 1, were employed. Whatman No. 4 filter paper features pore sizes ranging from 20 to 25 µm and exhibits rapid capillary action. Whatman chromatography paper No. 1 is widely used for chromatography analysis, while Whatman chromatography paper No. 6, with its pore size of 3 µm, is specifically utilized for water analysis. Figure 1 illustrates the color change of Whatman filter paper in response to varying immersion times in the presence of HVA. Whatmann filter paper grade A: Whatmann No 1, B: Whatmann CF4, C: Whatmann CF6 The outcome demonstrates that 30 mins is the ideal immersion time for strips reaction mixture pot. ferrocyanide + ferric chloride since the color intensity is higher during this period. Particµlarly the pore size, every Whatman filter paper is unique. All 3 Whatmann paper no 1, CF4, CF6 were able to absorb HVA on paper, absorb chemicals in essentially the same ways. The ideal drying period was completed at room temperature. Approximately one hour is required for the paper to dry completely at room temperature thus used as drying time for the paper utilized. Figure 2 depicts the paper-based analytical device's design. This design's material is simple and cost-effective. This pattern was created for every filter paper. 3.2 Performance of Paper-Based Analytical Device To perform the sensitivity test for each paper strip prepared, we determined the minimum detectable concentration of HVA that the PAD can identify. The varying concentrations of HVA was weighed. In the PAD, (25, 10, 1 mg/ml, 1 µg/ml) of HVA was added. It was noted that the presence of HVA caused a color shift in the PAD. Whatmann CF6 paper changes color quickly when it is submerged in an HVA solution. Significant change in color was absorbed as per concentration. According to the sensitivity data, the lowest concentration that may be measured is 1mg/ml because at this concentration reagent still exhibit the proper color changes; at concentrations below this, the reaction mixture pot. ferrocyanide + ferric chloride does not produce any discoloration. A selectivity test was carried out to ascertain the ability of the paper-based analytical device (PAD) to detect HVA. The test was conducted by dipping PAD into a HVA solution added in artificial urine. Figure 3 demonstrates that the reaction mixture pot. ferrocyanide + ferric chloride showed change in color when placed on a PAD that had been dipped in artificial urine. The reaction mixture pot. ferrocyanide + ferric chloride produces a green to blue color change on PAD dipped in HVA. This data shows that PAD is selective for detecting HVA because it reacts differently with other interfering chemicals, resµlting in a different color change. 3.3 Real time application of developed dipstrip for HVA in urine sample The real time application of this established qualitative & quantitative method was performed by analysis in human urine. The fasting urine sample of human was collected for qualitative analysis by developed strip and quantitative analysis by UV spectroscopy was performed. The results obtained by UV method was found to be of significant difference in levels for biogenic amines & its metabolites concentration when normal group was compared with PD, AD, Depression patients, whereas non significant difference in results obtained by UV method & developed strip for HVA (Fig. 4,5). 3.4 Concentration based color codes Qualitative detection of HVA was possible based on color intensity the concentration can be depicted in Table 1 . 3.5 Stability of paper-based analytical device's (PAD) The stability of paper strip was checked at 1, 2, 3 months for reproducibility of detection of HVA in urine sample. The detection of HVA by paper strip was possible upto 3 months (Fig. 6 ) as the pot. ferrocyanide + ferric chloride present on paper base was possible to change color from green to blue. After 3 months the pot. ferrocyanide + ferric chloride did not show any color change. 5. CONCLUSION The combination of potassium ferrocyanide and ferric chloride was chosen for HVA detection and subsequently applied to the PAD. The PAD exhibited favorable sensitivity and selectivity towards 5-HIAA. Moreover, the developed PAD successfully detected HVA in urine samples, corroborating the UV data. This method offers simplicity, rapidity, instrument-free operation, and selectivity for on-site HVA analysis in urine samples. Declarations CONFLICT OF INTEREST NIL. FUNDING SOURCE Self-funded. Author Contribution Vrushali Bhalchim: literature review, conceptualization, experimental work, manuscript draftingVaishali Undale: Conceptualization, Critical reviewsSunil Shewale: Manuscript drafting and analysis ACKNOWLEDGMENT The authors are thankful to the Management and Principal Dr. Sohan Chitlange for providing excellent research facilities. Data Availability All data generated or analysed during this study are included in this published article. Yes, the data is available for supporting the research findings in the previously reported studies for determination of catecholamines and their metabolites in urine samples by HPLC (ref no 3). The concept of PAD for qualitative determination has been developed earlier for "Design and optimization of colorimetric paper-based analytical device for rapid detection of allopurinol in herbal medicine" (ref no 14). The present research work is novel as there are no reported studies available on development of PAD for determination of catecholamine and metabolites. References Bhalchim V, Undale V. Assessment Of Metabolites: A New Era In Neurology. Turkish Journal of Physiotherapy and Rehabilitation. 2021 oct;32:3: 19580-90. Salihović M, Sofić E. HVAand HVA in the coma cerebri, hydrocephalus and tumor cerebri. InCMBEBIH 2017: Proceedings of the International Conference on Medical and Biological Engineering 2017 2017 (pp. 396-401). Springer Singapore. Doi: https://doi.org/10.1007/978-981-10-4166-2_61. Saracino MA, Mandrioli R, Mercolini L, Ferranti A, Zaimovic A, Leonardi C, Raggi MA. Determination of homovanillic acid (HVA) in human plasma by HPLC with coulometric detection and a new SPE procedure. Journal of pharmaceutical and biomedical analysis. 2006 Sep 11;42(1):107-12. Doi: https://doi.org/10.1016/j.jpba.2005.11.030. Dalli I, Ramdhani D, Hasanah AN. Design of indicator strip using polystyrene (PS) and polymethylmethacrylate (PMMA) for detection of diclofenac sodium in traditional pain relief herbal medicines. Indonesian Journal of Chemistry. 2017 Mar 1;17(1):71-8. Doi: https://doi.org/10.22146/ijc.23612. Pratiwi R, Suherman SE, Poongan RA, Mutakin M, Hasanah AN. Design of optical sensor membrane based on polymer poly (methyl methacrylate) for paracetamol detection in traditional herbal medicine. International Journal of Analytical Chemistry. 2018 Jun 6;2018. Doi: https://doi.org/10.1155/2018/8918329. Lin Y, Gritsenko D, Feng S, Teh YC, Lu X, Xu J. Detection of heavy metal by paper-based microfluidics. Biosensors and Bioelectronics. 2016 Sep 15;83:256-66. Doi: https://doi.org/10.1016/j.bios.2016.04.061. Cate DM, Adkins JA, Mettakoonpitak J, Henry CS. Recent developments in paper-based microfluidic devices. Analytical chemistry. 2015 Jan 6;87(1):19-41. Doi: https://doi.org/10.1021/ac503968p. Martinez AW, Phillips ST, Nie Z, Cheng CM, Carrilho E, Wiley BJ, Whitesides GM. Programmable diagnostic devices made from paper and tape. Lab on a Chip. 2010;10(19):2499-504. Doi: https://doi.org/10.1039/C0LC00021C. Meredith NA, Quinn C, Cate DM, Reilly TH, Volckens J, Henry CS. based analytical devices for environmental analysis. Analyst. 2016;141(6):1874-87. Doi: https://doi.org/10.1039/C5AN02572A. Yang Y, Noviana E, Nguyen MP, Geiss BJ, Dandy DS, Henry CS. based microfluidic devices: Emerging themes and applications. Analytical chemistry. 2017 Jan 3;89(1):71-91. Doi: https://doi.org/10.1021/acs.analchem.6b04581. Martinez AW, Phillips ST, Carrilho E, Thomas III SW, Sindi H, Whitesides GM. Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis. Analytical chemistry. 2008 May 15;80(10):3699-707. Doi: https://doi.org/10.1021/ac800112r. Nery EW, Kubota LT. Sensing approaches on paper-based devices: a review. Analytical and bioanalytical chemistry. 2013 Sep;405:7573-95. Doi: https://doi.org/10.1007/s00216-013-6911-4 Chaiyo S, Siangproh W, Apilux A, Chailapakul O. Highly selective and sensitive paper-based colorimetric sensor using thiosulfate catalytic etching of silver nanoplates for trace determination of copper ions. Analytica chimica acta. 2015 Mar 25;866:75-83. Doi: https://doi.org/10.1016/j.aca.2015.01.042. Pratiwi R, Septyani RN, Febriany R, Saputri FA, Nuwarda RF. Design and optimization of colorimetric paper-based analytical device for rapid detection of allopurinol in herbal medicine. International Journal of Analytical Chemistry. 2019 Mar 19;2019. Doi: https://doi.org/10.1155/2019/4682839. Wang W, Wu WY, Zhu JJ. Tree-shaped paper strip for semiquantitative colorimetric detection of protein with self-calibration. Journal of Chromatography A. 2010 Jun 11;1217(24):3896-9. Doi: https://doi.org/10.1016/j.chroma.2010.04.017. Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviewers agreed at journal 18 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 01 Jul, 2024 Editor assigned by journal 31 May, 2024 Submission checks completed at journal 31 May, 2024 First submitted to journal 21 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4455995","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312150134,"identity":"5da6bd17-6c71-4b73-8bce-c7dfbabce2f5","order_by":0,"name":"Vrushali Bhalchim","email":"data:image/png;base64,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","orcid":"","institution":"Dr. D. 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INTRODUCTION","content":"\u003cp\u003eHomovanillic acid (HVA) monitoring is often done in clinical settings to assess dopamine metabolism. It's particularly useful in conditions where dopamine levels are implicated, such as Parkinson's disease, schizophrenia, and certain mood disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In medical practice, measuring HVA levels can provide insights into the activity of dopamine-producing neurons in the brain. Abnormal levels of HVA may indicate dysfunction in dopamine regulation, which can help clinicians in diagnosing and managing various neurological and psychiatric conditions. Many HPLC methods, utilizing diverse sample extraction methods alongside analytical columns and either electrochemical or fluorimetric detection, have been developed. HPLC coupled with electrochemical detection is the preferred approach for detecting HVA due to its high sensitivity. However, this method is relatively nonspecific and can produce complex chromatograms if isolation steps are omitted. Therefore, most HPLC techniques using electrochemical detection to identify biogenic amines first separate interfering compounds using extraction columns. Though time-consuming, these procedures could provide significant advantages in routine clinical investigations.\u003c/p\u003e \u003cp\u003eVarious analytical methods, such as spectrophotometric determination [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and high-performance liquid chromatography (HPLC) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], are commonly employed to detect homovanillic acid (HVA). However, despite their precision and sensitivity, these techniques necessitate expensive equipment and are impractical for on-site analysis. To address this limitation, there's a growing interest in developing indicator strips or optical sensor membranes for detecting chemical substances in herbal medicine. Some researchers opt for polymers as substrate materials for this purpose [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Paper-based analytical devices (PADs) have recently emerged as a straightforward, cost-efficient, and user-friendly tool for environmental and biological analyses [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They have been successfully applied for on-site analysis of organic molecules [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], metals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and pesticides[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A prevalent detection method in PADs involves a colorimetric approach, utilizing specific reagents to identify and detect analytes in samples [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we opted combination of Potassium Ferric cyanide\u0026thinsp;+\u0026thinsp;Ferric chloride as the colorimetric reagent for detecting HVA. Potassium Ferric cyanide\u0026thinsp;+\u0026thinsp;Ferric chloride was chosen as metal ions forms complex with HVA compound, making it a suitable colorimetric reagent. We screened the reagent and applied it to various types of Whatman filter papers, including Whatman No. 1, No. 4, and No. 6 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The selectivity, sensitivity, and practicality of this paper-based analytical device (PAD) for HVA detection were also evaluated. The results indicate that this PAD is effective for detecting HVA in urine samples.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cp\u003eAll chemicals utilized were of analytical grade and were employed without additional purification. HVA was obtained from Sigma Aldrich Pvt. Ltd. Potassium ferro cyanide was purchased from Himedia whereas Ferric chloride was purchased from Lobachemie. Different grades of whatmann papers were purchased from whatmann grade 1 (whatmann), Whatmann CF4 (Cytiva), Whatmann CF6 (Cytiva) respectively. The absorbance was measured using a UV-Visible spectrophotometer model 1900 Shimadzu, utilizing a 1.0 cm quartz cell.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 \u003cstrong\u003eReagent preparation\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eWeighed quantity of Potassium Ferric cyanide 24 mg\u0026thinsp;+\u0026thinsp;Ferric chloride 257 mg was dissolved in 10 ml of distilled water and stirred completely at room temperature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 \u003cstrong\u003eDesign and Optimization of Specific Reagent Tests on Paper\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 832px; height: 244.077px;\" width=\"832\" height=\"244.077\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 \u003cstrong\u003eOptimization and selection of dip strip base for qualitative detection of HVA\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eAt different time interval (i.e 30, 60, 120, 180 mins) the different grade of Whatmann filter paper grade A: Whatmann No 1, B: Whatmann CF4, C: Whatmann CF6 strips were soaked in reagent reaction mixture and dried at R.T. The developed strip for HVA were dipped in 100 \u0026micro;g/ml of standard HVA sol. and time required for color development were recorded.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 \u003cstrong\u003eQualitative detection by dip strip at different HVA concentrations\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe optimized strip for HVA were treated with different concentration of HVA (25, 10, 1 mg/ml) and (1 \u0026micro;g/ml) for determining the minimum concentration to be detected by developed dip strip for HVA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 \u003cstrong\u003eReal time analysis by dip strip in urine sample\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eBefore analysis the urine samples were extracted 0.1 M perchloric acid and then dip strip was dipped into the sample then any color change on strip was observed for qualitative detection of HVA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 \u003cstrong\u003ePrototype design of dip strip\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eBased on the earlier studies reported the material to be used was selected [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. From point of care diagnosis approach the design of the strip was done as dip strip from point of ease of use, qualitative detection and economical use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Concentration dependent color codes\u003c/h2\u003e\n \u003cp\u003eFrom stock solution of standard HVA (10 mg in 10 ml) different dilutions of HVA were prepared from 10 \u0026micro;g/ml to 1 mg/ml and treated with reaction mixture to identify the detection limit of HVA concentration with developed strip.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e2.8 Validation of dip strip\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eStability testing of strip was performed at 1, 2, 3 months to assess the stability of dip strip to produce the color change when dipped in HVA solution.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003eHuman ethical permission\u003c/strong\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eFor human pilot study the proposal was submitted to Institutional Ethics committee at Dr. D. Y. Patil Homoeopathic Medical College and Research Center, Pimpri, Pune 411018.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eThe proposal was sanctioned by IEC protocol no DYPHMCRC/E-04/2023, dated 30/4/2023.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eThe enrolment of participants was done by taking their oral informed consent from patient by the neurologist and guiding them about the risks, benefits, awareness, and purpose of study experiment procedure.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eThe study was performed in accordance with relevant guidelines and regulations as suggested by the ethics committee.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. RESULT AND DISCUSSION","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Design and Optimization of dip strip for qualitative detection of HVA in urine sample\u0026nbsp;\u003cstrong\u003epaper\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eIn this study, Whatman filter papers No. 4 and No. 6, along with Whatman chromatography paper No. 1, were employed. Whatman No. 4 filter paper features pore sizes ranging from 20 to 25 \u0026micro;m and exhibits rapid capillary action. Whatman chromatography paper No. 1 is widely used for chromatography analysis, while Whatman chromatography paper No. 6, with its pore size of 3 \u0026micro;m, is specifically utilized for water analysis. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the color change of Whatman filter paper in response to varying immersion times in the presence of HVA.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eWhatmann filter paper grade A: Whatmann No 1, B: Whatmann CF4, C: Whatmann CF6\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe outcome demonstrates that 30 mins is the ideal immersion time for strips reaction mixture pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride since the color intensity is higher during this period. Partic\u0026micro;larly the pore size, every Whatman filter paper is unique. All 3 Whatmann paper no 1, CF4, CF6 were able to absorb HVA on paper, absorb chemicals in essentially the same ways. The ideal drying period was completed at room temperature. Approximately one hour is required for the paper to dry completely at room temperature thus used as drying time for the paper utilized. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the paper-based analytical device\u0026apos;s design. This design\u0026apos;s material is simple and cost-effective. This pattern was created for every filter paper.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Performance of Paper-Based Analytical Device\u003c/h2\u003e\n \u003cp\u003eTo perform the sensitivity test for each paper strip prepared, we determined the minimum detectable concentration of HVA that the PAD can identify. The varying concentrations of HVA was weighed. In the PAD, (25, 10, 1 mg/ml, 1 \u0026micro;g/ml) of HVA was added. It was noted that the presence of HVA caused a color shift in the PAD. Whatmann CF6 paper changes color quickly when it is submerged in an HVA solution. Significant change in color was absorbed as per concentration. According to the sensitivity data, the lowest concentration that may be measured is 1mg/ml because at this concentration reagent still exhibit the proper color changes; at concentrations below this, the reaction mixture pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride does not produce any discoloration.\u003c/p\u003e\n \u003cp\u003eA selectivity test was carried out to ascertain the ability of the paper-based analytical device (PAD) to detect HVA. The test was conducted by dipping PAD into a HVA solution added in artificial urine. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e demonstrates that the reaction mixture pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride showed change in color when placed on a PAD that had been dipped in artificial urine. The reaction mixture pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride produces a green to blue color change on PAD dipped in HVA. This data shows that PAD is selective for detecting HVA because it reacts differently with other interfering chemicals, res\u0026micro;lting in a different color change.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Real time application of developed dipstrip for HVA in urine sample\u003c/h2\u003e\n \u003cp\u003eThe real time application of this established qualitative \u0026amp; quantitative method was performed by analysis in human urine. The fasting urine sample of human was collected for qualitative analysis by developed strip and quantitative analysis by UV spectroscopy was performed. The results obtained by UV method was found to be of significant difference in levels for biogenic amines \u0026amp; its metabolites concentration when normal group was compared with PD, AD, Depression patients, whereas non significant difference in results obtained by UV method \u0026amp; developed strip for HVA (Fig. 4,5).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Concentration based color codes\u003c/h2\u003e\n \u003cp\u003eQualitative detection of HVA was possible based on color intensity the concentration can be depicted in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Stability of paper-based analytical device\u0026apos;s (PAD)\u003c/h2\u003e\n \u003cp\u003eThe stability of paper strip was checked at 1, 2, 3 months for reproducibility of detection of HVA in urine sample. The detection of HVA by paper strip was possible upto 3 months (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) as the pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride present on paper base was possible to change color from green to blue. After 3 months the pot. ferrocyanide\u0026thinsp;+\u0026thinsp;ferric chloride did not show any color change.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe combination of potassium ferrocyanide and ferric chloride was chosen for HVA detection and subsequently applied to the PAD. The PAD exhibited favorable sensitivity and selectivity towards 5-HIAA. Moreover, the developed PAD successfully detected HVA in urine samples, corroborating the UV data. This method offers simplicity, rapidity, instrument-free operation, and selectivity for on-site HVA analysis in urine samples.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\n\u003cp\u003eNIL.\u003c/p\u003e\n\u003ch2\u003eFUNDING SOURCE\u003c/h2\u003e\n\u003cp\u003eSelf-funded.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eVrushali Bhalchim: literature review, conceptualization, experimental work, manuscript draftingVaishali Undale: Conceptualization, Critical reviewsSunil Shewale: Manuscript drafting and analysis\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGMENT\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful to the Management and Principal Dr. Sohan Chitlange for providing excellent research facilities.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article. Yes, the data is available for supporting the research findings in the previously reported studies for determination of catecholamines and their metabolites in urine samples by HPLC (ref no 3). The concept of PAD for qualitative determination has been developed earlier for \u0026quot;Design and optimization of colorimetric paper-based analytical device for rapid detection of allopurinol in herbal medicine\u0026quot; (ref no 14). The present research work is novel as there are no reported studies available on development of PAD for determination of catecholamine and metabolites.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBhalchim V, Undale V. Assessment Of Metabolites: A New Era In Neurology. Turkish Journal of Physiotherapy and Rehabilitation. 2021 oct;32:3: 19580-90.\u003c/li\u003e\n\u003cli\u003eSalihović M, Sofić E. HVAand HVA in the coma cerebri, hydrocephalus and tumor cerebri. InCMBEBIH 2017: Proceedings of the International Conference on Medical and Biological Engineering 2017 2017 (pp. 396-401). Springer Singapore. Doi: https://doi.org/10.1007/978-981-10-4166-2_61.\u003c/li\u003e\n\u003cli\u003eSaracino MA, Mandrioli R, Mercolini L, Ferranti A, Zaimovic A, Leonardi C, Raggi MA. Determination of homovanillic acid (HVA) in human plasma by HPLC with coulometric detection and a new SPE procedure. Journal of pharmaceutical and biomedical analysis. 2006 Sep 11;42(1):107-12. Doi: https://doi.org/10.1016/j.jpba.2005.11.030.\u003c/li\u003e\n\u003cli\u003eDalli I, Ramdhani D, Hasanah AN. Design of indicator strip using polystyrene (PS) and polymethylmethacrylate (PMMA) for detection of diclofenac sodium in traditional pain relief herbal medicines. Indonesian Journal of Chemistry. 2017 Mar 1;17(1):71-8. Doi: https://doi.org/10.22146/ijc.23612.\u003c/li\u003e\n\u003cli\u003ePratiwi R, Suherman SE, Poongan RA, Mutakin M, Hasanah AN. Design of optical sensor membrane based on polymer poly (methyl methacrylate) for paracetamol detection in traditional herbal medicine. International Journal of Analytical Chemistry. 2018 Jun 6;2018. Doi: https://doi.org/10.1155/2018/8918329.\u003c/li\u003e\n\u003cli\u003eLin Y, Gritsenko D, Feng S, Teh YC, Lu X, Xu J. Detection of heavy metal by paper-based microfluidics. Biosensors and Bioelectronics. 2016 Sep 15;83:256-66. Doi: https://doi.org/10.1016/j.bios.2016.04.061.\u003c/li\u003e\n\u003cli\u003eCate DM, Adkins JA, Mettakoonpitak J, Henry CS. Recent developments in paper-based microfluidic devices. Analytical chemistry. 2015 Jan 6;87(1):19-41. Doi: https://doi.org/10.1021/ac503968p.\u003c/li\u003e\n\u003cli\u003eMartinez AW, Phillips ST, Nie Z, Cheng CM, Carrilho E, Wiley BJ, Whitesides GM. Programmable diagnostic devices made from paper and tape. Lab on a Chip. 2010;10(19):2499-504. Doi: https://doi.org/10.1039/C0LC00021C.\u003c/li\u003e\n\u003cli\u003eMeredith NA, Quinn C, Cate DM, Reilly TH, Volckens J, Henry CS. based analytical devices for environmental analysis. Analyst. 2016;141(6):1874-87. Doi: https://doi.org/10.1039/C5AN02572A.\u003c/li\u003e\n\u003cli\u003eYang Y, Noviana E, Nguyen MP, Geiss BJ, Dandy DS, Henry CS. based microfluidic devices: Emerging themes and applications. Analytical chemistry. 2017 Jan 3;89(1):71-91. Doi: https://doi.org/10.1021/acs.analchem.6b04581.\u003c/li\u003e\n\u003cli\u003eMartinez AW, Phillips ST, Carrilho E, Thomas III SW, Sindi H, Whitesides GM. Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis. Analytical chemistry. 2008 May 15;80(10):3699-707. Doi: https://doi.org/10.1021/ac800112r.\u003c/li\u003e\n\u003cli\u003eNery EW, Kubota LT. Sensing approaches on paper-based devices: a review. Analytical and bioanalytical chemistry. 2013 Sep;405:7573-95. Doi: https://doi.org/10.1007/s00216-013-6911-4\u003c/li\u003e\n\u003cli\u003eChaiyo S, Siangproh W, Apilux A, Chailapakul O. Highly selective and sensitive paper-based colorimetric sensor using thiosulfate catalytic etching of silver nanoplates for trace determination of copper ions. Analytica chimica acta. 2015 Mar 25;866:75-83. Doi: https://doi.org/10.1016/j.aca.2015.01.042.\u003c/li\u003e\n\u003cli\u003ePratiwi R, Septyani RN, Febriany R, Saputri FA, Nuwarda RF. Design and optimization of colorimetric paper-based analytical device for rapid detection of allopurinol in herbal medicine. International Journal of Analytical Chemistry. 2019 Mar 19;2019. Doi: https://doi.org/10.1155/2019/4682839.\u003c/li\u003e\n\u003cli\u003eWang W, Wu WY, Zhu JJ. Tree-shaped paper strip for semiquantitative colorimetric detection of protein with self-calibration. Journal of Chromatography A. 2010 Jun 11;1217(24):3896-9. Doi: https://doi.org/10.1016/j.chroma.2010.04.017.\u003c/li\u003e\n\u003c/ol\u003e\n"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HVA, paper-based analytical devices, urine, diagnosis and monitoring, Parkinson’s disease etc","lastPublishedDoi":"10.21203/rs.3.rs-4455995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4455995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiagnosing and monitoring of Parkinson's disease, schizophrenia, and certain mood disorders involves detecting homovanillic acid an essential aspect excreted through urine. In this study, we explored novel approaches for detecting homovanillic acid (HVA) in urine samples, aiming for cost-effective and on-site analysis methods. Traditional techniques like spectrophotometric determination and HPLC, while sensitive and selective, are often impractical due to their expense and lack of portability. Instead, we investigated the utility of paper-based analytical devices (PADs), which have gained popularity for various analytical applications including chemical element identification and environmental contamination assessment. We utilized different types of Whatmann filter papers (No. 1, No. 2, No. 4) and chromatography paper as substrates for the PADs, and employed combination of potassium ferrocyanide and ferric chloride reagent for HVA detection. Our experiments revealed positive results, indicating the efficacy of this approach. Optimization experiments identified 30 minutes to 3 hours as the optimal immersion times, with a subsequent 30-minute drying period at room temperature. Notably, variations in performance were observed among the different filter papers, with Whatmann CF6 paper exhibiting significant differences compared to the others in terms of HVA detection efficiency.\u003c/p\u003e","manuscriptTitle":"Paper-based Colorimetric Assay: Rapid Detection of HVA in Urine Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 07:08:59","doi":"10.21203/rs.3.rs-4455995/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-07-23T05:04:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199602497564176622938596618694648724783","date":"2024-07-19T08:33:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T16:32:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312918550224223251729903256436676009236","date":"2024-07-18T08:51:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54487609771708030849768302610673642456","date":"2024-07-17T13:21:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-05T09:07:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250795835418898949068056339624952902141","date":"2024-07-02T14:18:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-01T18:49:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-31T19:28:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-31T19:27:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2024-05-21T15:52:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b894cce8-6459-4203-baa2-8958adb4b958","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T07:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-13 07:08:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4455995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4455995","identity":"rs-4455995","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}