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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Addressing urgent dengue diagnostic needs, here we introduce a smartphone app-integrated Dengue NS1 Paper-based Analytical Device (DEN-NS1-PAD) for quantifying Dengue NS1 antigen concentration in clinical serum/blood samples. This innovation enhances dengue management by aiding clinical decision-making in various healthcare settings, even resource-limited ones.

Abstract

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an annual incidence of approximately 10 million cases and 20,000-25,000 deaths, particularly among children, there is an urgent need for practical diagnostic tools. The presence of dengue non-structural protein 1 (NS1) during early infection has been linked to cytokine release, vascular leakage, and endothelial dysfunction, making it a potential marker for severe dengue.

Paper-based immunoassays such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices (PADs) have gained popularity as diagnostic tests due to their simplicity, rapidity, inexpensiveness, specificity, and ease of interpretation. However, conventional paper-based immunoassays for dengue NS1 detection typically rely on visual inspection, yielding only qualitative results. To address this limitation and enhance sensitivity, we proposed a highly portable NS1 dengue detection assay on a Paper-based Analytical Device (PAD), namely, DEN-NS1-PAD, that integrates a smartphone application as a colorimetric and quantitative reader. The development system enables direct quantification of NS1 concentrations in clinical samples.

Serum and blood samples obtained from patients were utilized to demonstrate the system prototype performance. The results were obtained immediately and can be employed for clinical assessment, both in well-equipped healthcare facilities and resource-limited settings. This innovative combination of a paper-based immunoassay with a smartphone application offers a promising approach for enhanced detection and quantification of dengue NS1 antigen. By augmenting sensitivity beyond the capabilities of the naked eye, this system holds great potential for improving clinical decision-making in dengue management, particularly in remote or underserved areas.

Introduction

Dengue virus (DENV) infection is the fastest-spreading mosquito-borne disease1, and more than 390 million people are infected with 96 million symptomatic infections, 2 million cases of severe disease, and more than 25,000 deaths per year occur in the world1,2. According to the World Health Organization (WHO), an estimated 3.9 billion people are at risk for dengue; ~70% live in Asia Pacific countries and mainly in Southeast Asia3. In 2019, the number of dengue cases reported to WHO was 4.2 million, and Thailand contributed at least 136,000 dengue cases and 144 dea....

Protocol

The Ethics Committee of the Institutional Review Board, Royal Thai Army Medical Department, Phramongkutklao Hospital, Bangkok, Thailand (IRBRTA 1218/2562) granted approval. In carrying out this study, we complied with all necessary ethical regulations.

1. Device fabrication of the paper-based Immunoassay

NOTE: The paper-based immunoassay device was fabricated following previously established methods36,

Representative Results

Selecting a fabrication method is pivotal to ensure reproducible assay performances in paper-based immunoassay devices. In our study, we explored various manufacturing processes and materials in the context of demonstrating a paper-based immunoassay. Our chosen method utilizes a wax printing system to create hydrophobic barriers within paper-based microfluidic devices. This approach stands out due to its simplicity, speed, and consistent results. Of note, it offers the advantage of avoiding the use of photoresist chemica.......

Discussion

One of the important design parameters for a smartphone-based reader system is the ability to provide reproducible imaging processing of samples. In this study, for simplicity and convenience, the images were captured from three different smartphone brands with 12-13 MP cameras without using an imaging box or accessories. Variable conditions of image capturing, such as the resolution of the camera, image capturing time, lighting conditions, and environment, can influence the color intensity of the test and control spots .......

Acknowledgements

M.H.P. gratefully acknowledges the scholarship research fund from Universitas Islam Indonesia (UII). The authors extend their gratitude to Mr. Nutchanon Ninyawee for his valuable expertise and assistance throughout the development of the mobile application and his contributions to the manuscript. Furthermore, the authors appreciate the financial support provided by Thailand Science Research and Innovation (TSRI), Basic Research Fund: Fiscal year 2023 (project no. FRB660073/0164) under Program Smart Healthcare of King Mongkut's University of Technology Thonburi.

....

Materials

NameCompanyCatalog NumberComments
Materials
0.1 M phosphate-buffered saline (PBS, pH 7.2) 
BBS containing 0.1% Tween 20, 10% sucrose, and 1% casein  the conjugate area treatment and blocking buffer
Borate buffered saline (BBS) (25 mM sodium borate and 150 mM sodium chloride at pH 8.2) supplemented with 1% BSA the washing buffer during the conjugation process AuNPs with the antibody
Boric acidMerck10043-35-3
Bovine serum albumin fraction V (BSA)  PAA Lab GmbH (Germany)K41-001 
CaseinMerck9005-46-3
Chromatography paper Grade 2 GE Healthcare3002-911 
Clear laminate film3M (Stationery shops)
Disodium hydrogen phosphateMerck7558-79-4
Double tape sideStationery shops
Goat anti-mouse IgG antibody MyBiosource (USA)MBS435013
Gold nanoparticles (40 nm)  Serve Science Co., Ltd. (Thailand)
Human IgG polyclonal antibody  MerckAG711-M
Mouse dengue NS1 monoclonal antibody MyBiosource (USA)MBS834415
Mouse dengue NS1 monoclonal antibody MyBiosource (USA)MBS834236
NS1 serotype 2 antigensMyBiosource (USA)MBS 568697
PBS 1X containing 0.1% Tween 20 was used as telution buffer
Plastic backing card 10x30 cmPacific Biotech Co., Ltd. (Thailand)
Poly-L-lysine (PLL)Sigma AldrichP4832
Potassium ChlorideMerck104936
Potassium monophosphateMerck104877
Sodium ChlorideMerck7647-14-5
Sodium tetraborate Sigma Aldrich1303-96-4
SucroseMerck57-50-1
Tween 20Sigma Aldrich9005-64-5
Instruments
CytationTM 5 multimode readerBioTek
Mobile phonesHuawei Y7, iPhone 11, Samsung a20
Photo scannerEpson Perfection V30
OvenMemmert
Wax printer Xerox ColorQube 8880-PS
Software
Could AutoML Vision Object Detection documentationGoogle Cloud
ImageJNational Institute of Health, Bethesda, MD, USA
Inkscape 0.91 Software

References

  1. Cattarino, L., Rodriguez-Barraquer, I., Imai, N., Cummings, D. A. T., Ferguson, N. M. Mapping global variation in dengue transmission intensity. Science Translational Medicine. 12 (528), 1-11 (2020).
  2. World Health Organization (WHO). . Treatment, prevention an....

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