detection of dengue ns1 antibody using paper based immunoassay

Smartphone-geïntegreerd Dengue NS1 diagnostisch apparaat op papier

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 death cases from dengue infection4. The dengue outbreak in Thailand occurs during the rainy season, from April to December, in both urban and rural areas, especially in the northeastern area.
DENV infections have different clinical manifestations ranging from subclinical symptoms, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). The main characteristic of severe DHF condition is increased vascular permeability followed by shock and organ dysfunction1. Understanding the molecular pathway that can cause the vascular leak is very important in developing effective dengue treatments. Dengue non-structural protein 1 (NS1) is a secreted glycoprotein during early virus infection5,6, and it functions as a cofactor for viral RNA replication7. NS1 can trigger cytokine release and contribute to vascular leak by binding to toll-like receptor 4 (TLR4) and endothelial glycocalyx8,9. In vitro research has shown that NS1 interacts with endothelial cells and induces apoptosis. This condition can contribute to endothelial dysfunction and vascular leak10. NS1 antigen levels, correlated with serum Interleukin (IL)-10 levels, were increased significantly in patients with severe clinical disease11. Dengue NS1 also contributes to disease pathogenesis by inducing IL-10 and suppressing DENV-specific T-cell responses12,13. In addition, dengue NS1 protein was related to severe clinical disease, and the concentration of NS1 &#62; 600 ng mL-1 in the first 3 days of illness was associated with the development of DHF14.
The persistence of the dengue NS1 antigen in patients with DHF could be used as a marker of severe dengue6. There are several methods to detect NS1 in clinical samples such as enzyme-linked immunosorbent assay (ELISA)and the rapid test15. The gold standard for measuring the concentration of NS1 proteins in a clinical setting is the ELISA method. However, the ELISA method is expensive and requires skilled personnel, and laboratory facilities16. Therefore, the development of technology for detecting and quantifying NS1 proteins in the point-of-care test (POCT) is still ongoing. In the last decade, paper-based immunoassays such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices (&#181;PADs) have become popular as diagnostic tests because of their simplicity, rapidity, inexpensiveness, and specificity17,18,19. In a paper-based immunoassay, several labels have been used to generate signals, such as gold nanoparticles (AuNPs)20, magnetic nanoparticles21,22, quantum dots23, and fluorescence materials24,25. AuNPs are the most common labels used in paper-based immunoassays due to their inexpensive cost of production, ease of manufacture, stability, and simple readout. Currently, lateral flow assays (LFAs) for dengue NS1 are famously used in the clinical setting26,27. However, conventional LFA label detection commonly uses the naked eye and only provides qualitative results.
In the last decade, more than 5 billion smartphones have been widely used globally, and there is potential for developing portable detection28,29. Smartphones have multi-functional capacities such as built-in physical sensors, multi-core processors, digital cameras, USB ports, audio ports, wireless, and application software, making them suitable for use in various biosensor platforms30. In addition, wireless technologies allow data to be sent quickly and can be used for real-time and on-site monitoring31. Mudanyali et al. combined the paper-based immunoassay and smartphones to develop a portable, equipment-free, rapid, low-cost, and user-friendly POCT platform for malaria, tuberculosis, and HIV32. Ling et al. reported a lateral flow assay combined with a smartphone camera to detect alkaline phosphatase activity in milk quantitatively33. Hou et al. also developed a smartphone-based, dual-modality imaging system for quantitative signals from color or fluorescence in the lateral flow assay34. In addition, using the smartphone as a colorimetric and quantitative reader can improve the sensitivity while the naked eye cannot confidently report the presence of the target35.
Presenting a breakthrough in dengue diagnostics, the DEN-NS1-PAD36,37,38 (referred to as the device henceforth) offers a portable and efficient solution. Using wax-printed microfluidic paper-based technology, this device quantifies NS1 with high sensitivity and specificity through image processing. To further enhance its utility, we have developed a user-friendly smartphone app for colorimetric and quantitative reading. Clinical validation using patient samples from Thai hospitals underscores its immediate impact on real-time patient assessment. Our innovation marks a pivotal advancement in streamlined, point-of-care-dengue management, promising to revolutionize diagnostics in resource-limited healthcare landscapes.

Auteur in de schijnwerpers: Ontwikkeling van een smartphone-verbeterd apparaat op papier voor snelle detectie van dengue NS1

Draagbare op papier gebaseerde immunoassay gecombineerd met smartphone-applicatie voor colorimetrische en kwantitatieve detectie van dengue NS1-antigeen

The scope of the research is detecting dengue using a specific biomarker NS1, with a paper-based analytical device. Our research focus on developing a simple, cheap, sensitive, and specific device to detect dengue. We also develop smartphone application to improve sensitivity and measurement of NS1 in real sample.Our sensitivity is developing a method to fabricate microfluidic method based on our coning analysis called PEC. Using formula is in our team, were some high techniques, aimed to embody commercialization of various PEC designs, offering scalable solutions for more supply applications. Our research combines PEC with smartphone application, which is very promising for dengue NS1 measurement.The mobile application significantly enhanced sensitivity and efficiency in qualifying NS1 in serum samples, compared to observation with the naked eye. This protocol is simple, cheap and reproducible to produce PEC to detect dengue NS1. The PEC is sensitive and specific for detecting dengue NS1, and will increase the sensitivities after combining with their smartphone.Our goal is to improve dengue NS1 past performance with the blood samples, evaluating the cross-infections cell types, and incubating it into the paper-based devices for multiple diseases. Addressing technology culture trend is crucial for successful commercialization in bring all the disease screening and medical care.

Detectie van Dengue NS1-antilichaam met behulp van op papier gebaseerde immunoassay

Breng om te beginnen 300 microliter perifeer bloed van 30 patiënten op de eerste dag van ziekenhuisopname over in paarse EDTA-buizen. Centrifugeer het bloed bij 2.884 G bij vier graden Celsius gedurende 20 minuten. Breng het plasma over in een schone polypropyleen buis.Breng vervolgens 20 microliter plasma aan op het monstergebied van het immunoassay-apparaat. Pipetteer nu 30 microliter van de wasbuffer naar het monstergebied. Nadat u het monster volledig hebt laten ontwaken voor het apparaat, legt u de beelden van het apparaat vast met een smartphone.Analyseer de test- en controlegebieden met behulp van ImageJ en de aangepaste mobiele applicatie. Om de intensiteit van de test in de mobiele applicatie te kwantificeren, start u de ontwikkelde mobiele applicatie op de smartphone. Selecteer camera gebruiken of uploaden vanuit galerij om de gegevensbron te kiezen of te uploaden.Navigeer nu naar het analysegedeelte en druk op analyseren. De applicatie zal nu de gegevens verwerken en de resultaten weergeven. Het op papier gebaseerde immunoassay-apparaat was in staat om snel dengue te diagnosticeren, vergelijkbaar met een in de handel verkrijgbare diagnostische test.

Research

JoVE Journal

Immunology and Infection

Detection of Dengue NS1 Antibody Using Paper Based Immunoassay

Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an ...

Designing a Paper-Based Immunoassay Device for the Detection of Dengue Antibody NS1

Ontwerp van een op papier gebaseerd immunoassay-apparaat voor de detectie van dengue-antilichaam NS1