**1. Introduction**

Malaria is caused by parasites that are transmitted to humans via the bites of the infected female Anopheles mosquito. While preventable and curable, it still remains a paramount cause of morbidity and mortality in developing countries. Malaria is estimated to kill between 1.5 to 2.7 million people annually [1]. Malaria morbidity is estimated at about 300–500 million annually, and malaria clinical diagnosis is most e ffective at 50%. Malaria immunoassays use the inherent sensitivity, specificity and binding a ffinity of antibodies to respective antigens for the detection of antigens in a sample. In immunoassays, the sample tested includes whole blood, urine, saliva, serum, etc. [2]. In the Malaria Pan Antigen rapid test kit, the sample used is Red Blood cells containing specific antigens of *P. vivex and P. malariae*/*P. ovale* [3]. The red blood cells ge<sup>t</sup> lysed by a bu ffer solution to allow antigen–antibody binding at the test site. Immunoassay signals emanate from the gold-labeled antibody set for the antigen on a substratum at the binding site (Test line). Typical antibody labels include fluorescent molecules, nano- or microparticles, or enzymes. Gold nanoparticles (NPs) are the most widely used label [4]. Such immunoassays can be used in industry, clinical or laboratory settings, doctor's o ffices, or as over-the-counter tests [2]. At the test line, the naked eye will see a gold-labelled marker as a pink/red line [5]. In most countries, the diagnosis of malaria challenges multiple laboratories [3]. The laboratories require longer than one hour to analyze the findings, leading to less consistency in the analysis of the results.

#### *1.1. Components of Immuno-Chromatographic Test Kits*

The Immuno-Chromatographic kit is composed of components shown in Figure 1. The parts of the kits are attached on an inert polyvinyl chloride (PVC) backing material and further packed in a plastic cassette with a specimen port and reaction window displaying the capture and control zones [2]. The Immunochromatographic Test Kit has a sample pad, conventionally composed of glass fibres. The sample pad is selected to have zero cross-reactivity with the specimen. The sample pad is pretreated with a bu ffer for specimen pH adjustment and extraction of unspecific antigen form specimens [6]. One of the vital parts of the strip is nitrocellulose membrane (NCM). In this, the interaction between antigen and antibody takes place. Typically, a hydrophobic nitrocellulose membrane is used on which anti-target analyte antibodies are immobilized in a line that crosses the membrane to act as a capture zone on the test line [2]. The NCM membrane should be chosen based upon pore size [7]. Other parts of test strips are glass fibres or non-woven fibres based conjugate pads which can be pre-treated to avoid any cross-reactivity [8]. Conclusively, the conjugate pad is prepared by dipping the glass fibers into a colloidal solution of gold protein and then used after drying. In addition, an absorbent pad is present in the kit, which is designed to collect extra specimen samples passing the reaction membrane [9].

## *1.2. The Protein*

In the Malaria Pan immunoassay, antibody protein is used for AuNP conjugation. Plasmodium lactate dehydrogenase (pLDH) and goa<sup>t</sup> anti-mouse (GAM) protein are used for binding at test and control lines, respectively. An ultraviolet-visible spectrophotometer optimization technique was demonstrated in this work by formulating an immuno-chromatographic detection kit for Malaria Pan using AuNPs as an indicator. Various research works attempted to optimize the AuNP size [10–13], and the AuNPs of about 30–40 nm were reported to be optimal [11,12]. Khlebtsov and Byzova et al. also tried to determine the optimum concentration of protein required for AuNP conjugation [14,15].

In present research, gold nanoparticles (AuNPs) were utilized as labels, and the concentration of AuNPs with conjugate antibodies was tailored to a fine-tuned optical density (OD). The gold nanoparticles of various sizes (10 nm to 50 nm) were prepared, by quantifying λ max (absorption maxima) and dynamic light scattering (DLS). The relationship of AuNP diameters with a concentration of target protein was monitored to develop a better test kit. Finally, the developed immuno-chromatographic test kit test zone band intensity was tested using RGB and HSV color models. The reason to select a malaria test kit for the study is to create a more cost-effective rapid diagnostic test kits because malaria cases are found in countries where cost-effectiveness is significant. The study aim to improve test band intensity irrespective of AuNP size using a fixed quantity of protein while optimizing the optical density.

**Figure 1.** Presentation of lateral flow strip that works on sandwich assay. Blood sample lysed with buffer solution is added to the sample pad. *P. vivex*/*P. malariae*/*P. ovale* malaria antigens attach to antibodies in the red colored gold conjugate pad and the complex formed attaches to test line monoclonal anti-PAN specific pLDH antibodies. The excess labeled antibodies bind with Goat anti-mouse IgG antibodies in the control line. The extra lysed red blood cells ge<sup>t</sup> absorbed in the absorbent pad.

#### **2. Materials and Methods**

#### *2.1. Reagents, Instruments and Other Support Materials*

For the fabrication of immunochromatographic strip assay, sodium hydrogen phosphate, sucrose, disodium hydrogen phosphate, sodium chloride and bovine serum albumin (BSA) were purchased from Merck, Darmstadt, Germany. The gold chloride used for the synthesis of gold nanoparticles was purchased from Sigma-Aldrich, Tokyo, Japan. The Plasmodium lactate dehydrogenase (pLDH) antibodies' molecules and control line Goat anti-mouse protein were purchased from Fapon Biotech, Shenzhen, China. All the other chemicals and reagents used in the present study were of analytical grade reagents. The Delsa™ Nano Submicron Particle Size Zeta Potential instrument of Beckman Coulter, Brea, CA, USA was used for analyzing AuNP diameters. An ultraviolet-visible spectrophotometer 1900i of Shimadzu, Kyoto, Japan was used to measure optical density and absorbance maxima. The nitro cellulose membrane was coated with XYXYZ3210™ dispense platform of Bio-dot, Irvine, CA, USA. The centrifuge of Remi RM-12C, Mumbai, India was utilized for AuNP–protein conjugate centrifugation, and the magnetic stirrer of Remi, Mumbai, India was also used in the study. The nitrocellulose membrane was purchased form Nupore System Pvt. Ltd., Ghaziabad, India and Glass fibre sample pad and conjugate pad were purchased from Advanced Micro Devices, Ambala, India.
