**Optical Density Optimization of Malaria Pan Rapid Diagnostic Test Strips for Improved Test Zone Band Intensity**

**Prince Manta 1, Rupak Nagraik 2, Avinash Sharma 2, Akshay Kumar 3, Pritt Verma 4, Shravan Kumar Paswan 4, Dmitry O. Bokov 5, Juber Dastagir Shaikh 6, Roopvir Kaur 7, Ana Francesca Vommaro Leite 8, Silas Jose Braz Filho 8, Nimisha Shiwalkar 9, Purnadeo Persaud 10 and Deepak N. Kapoor 1,\***


Received: 30 July 2020; Accepted: 14 September 2020; Published: 29 October 2020

**Abstract:** For the last few decades, the immunochromatographic assay has been used for the rapid detection of biological markers in infectious diseases in humans and animals The assay, also known as lateral flow assay, is utilized for the detection of antigen or antibody in human infectious diseases. There are a series of steps involved in the development of these immuno-chromatographic test kits, from gold nano colloids preparation to nitrocellulose membrane coating (NCM). These tests are mostly used for qualitative assays by a visual interpretation of results. For the interpretation of the results, the color intensity of the test zone is therefore very significant. Herein, the study was performed on a malaria antigen test kit. Several studies have reported the use of gold nanoparticles (AuNPs) with varying diameters and its binding with various concentrations of protein in order to optimize tests. However, none of these studies have reported how to fix (improve) test zone band intensity (color), if di fferent sized AuNPs were synthesized during a reaction and when conjugated equally with same amount of protein. Herein, di fferent AuNPs with average diameter ranging from 10 nm to 50 nm were prepared and conjugated equally with protein concentration of 150 μg/mL with KD = 1.0 × 10−3. Afterwards, the developed kits' test zone band intensity for all di fferent sizes AuNPs was fixed to the same band level (high) by utilization of an ultraviolet-visible spectrophotometer. The study found that the same optical density (OD) has the same test zone band intensity irrespective of AuNP size. This study also illustrates the use of absorption maxima (λ max) techniques to characterize AuNPs and to prevent wastage of protein while developing immunochromatographic test kits.

**Keywords:** lateral flow assay; immuno-chromatographic; gold nanoparticles sensor; UV/Vis spectrophotometer; malaria pan rapid diagnostic strip; point-of-care
