**3. Conclusion**

disk was employed for transporting the fluid from one chamber to another. The main reason behind choosing this LOCD platform was the low cost, rapid detection, fully automation, and multiplex detection of target analytes. In addition, this platform provides high surface area to volume ratio and micromixing facility which enhance the biosensing of the assay in terms of sensitivity and specificity. After the successful development of the assay, it was evaluated by detecting dengue IgG antibody from the several hospitalized patients. The study claimed that LOCD successfully detected dengue IgG antibodies with 95% sensitivity and 100% specificity [63]. Using the microfluidic dielectrophoresis platform, Iswardy et al. developed a bead-based immunofluorescence assay for the detection of dengue virus. During the assay development, mouse anti-*Flavivirus*-capture antibodies were modified with beads, and DENV was modified with fluorescence label. The principle of this assay was based on employing the DEP to capture modified beads in the microfluidic chip which will later interact with modified DENV to form immune complex on these beads. Fluorescence microscopy was used to detect fluorescent signals, and later these signals were quantified by Image J freeware. It was found that incorporation of microfluidic platform speeds up the immuno-reactions and target analyte was detected in a short period of 5 minutes. Interestingly, this assay used ~15 μL of dengue sample to test the dengue virus presence. The study showed that the developed assay was

PFU/mL [64].

Paper offers several unique advantages than conventional device materials such as powerfree liquid transport through capillary force and evaporation, high surface area to volume ratio, and storing reagent in active form within the fiber network [65, 66]. Lo et al. combined RT-LAMP with paper-based diagnostic devices for the detection of dengue virus. First, cDNA was amplified using RT-LAMP at 63°C. Later, the amplified products were mixed with detection probes and then moved in paper-based test zone constructed on paper-based diagnostic device. Afterwards, the fluorescent signals were examined and analyzed by image recoding system and Image J. The developed assay demonstrated high sensitivity in paper-based diagnostic device with a LOD of 31.75 μg/mL of amplified products [67]. Zhang et al. worked on to improve the flow of salivary fluid in paper-based immunoassay. This study believes that paper-based immunoassay is more often compromised due to the formation of aggregates between conjugates and specimen and, thus, inhibits the labeled target molecule to reach at test line. To resolve this issue, this study developed a stacking flow immunoassay to detect dengue-specific IgG antibody in salivary fluid. The stacking flow architecture was aimed to bypass the sample pretreatment step which is often required for testing the salivary fluid. To achieve this goal, two different paths were designed for guiding the sample and reagents separately in the test strip. According to study, application of this tactic prevented the interference of salivary substances with particle-based sensing system, and these substances were omitted before making any contact with the detection reagents, therefore, resulting in low background. Moreover, study showed equipping the strip with flow regulator enables the uniform flow in the strip which produces even test line. It was found the developed immunoassay successfully detected the dengue IgG antibodies which are important biomarker for the secondary dengue infection [68]. As we know that low sensitivity is a great disadvantage of

able to detect DENV with a LOD of 10<sup>4</sup>

138 Dengue Fever - a Resilient Threat in the Face of Innovation

*2.1.4. Novel paper-based diagnostic devices*

In this chapter, we discussed the advanced diagnostic methods for the diagnosis of dengue. In terms of analytical sensitivity and rapidity, these novel methods showed remarkable achievements. However, most of these studies ignored the specificity criteria of diagnostic test. Specificity is a very important aspect of diagnostic test which discriminates true negative from false positive and true-positive from false-negative detection of an infectious disease. So, this aspect should be also investigated along with the sensitivity of the diagnostic test. In addition, diagnostic sensitivity and specificity should be also investigated using well-referenced samples. Moreover, all the four serotypes should be tested to see whether this method is equally effective for all serotypes. Testing of these parameters of diagnostic test will give much broad picture to analyze its potential for evolving as a point-of-care test.
