*2.2. Experimental*

#### 2.2.1. Preparation of Gold Nanoparticles (AuNPs)

The gold nanoparticles were prepared by classical classical Turkevich and Fern methods by citrate reduction. In general, the Turkevich and Fern process reaction leads to formation of AuNPs of size range 10 nm to 100 nm [16,17]. Utilizing raw gold chloride (AuHCl4) [18], the 1% light yellowish color solution was prepared by dissolving 1 gm of gold chloride in 100 mL of ultra-mili-Q water. The aforementioned 1% gold solution was furthermore dissolved into ultra-milli-Q water in order to obtain the optical density between 0.7 and 0.9 at λ max (absorption maxima) by taking a solution spectrum scan at wavelength between 700 nm and 400 nm. Now, the final gold solution had been refluxed for 30 min at 100 ◦C. The above-diluted gold solution was reduced by adding 1% (1 g of sodium citrate in 100 mL of water) sodium citrate solution of pH 7.80 ± 0.5 with refluxing until bright red color develops. Initially, the addition of a 1% solution of sodium citrate turns the color of the solution black. The color change from mildly yellowish to brick red confirms the synthesis of nanoparticles. The change in colour solution is due to the surface plasmon resonance e ffect (SPR) in which electrons excited to its higher state and produces a colour change. During the reduction mechanism, metal salts ge<sup>t</sup> converted into their ionic form when it combines with water. Di fferent chemical functional groups of reducing agents combine with metal ions whether they are bivalent or monovalent and reduce it into a zerovalent state of small size [19].

The color transforms from red to pink to blue as the reaction proceeds. As the solution color turns pink, the reaction was stopped by decreasing the temperature to room temperature in the ice bath. Particle size distribution of synthesized nanoparticles was analysed by dynamic light scattering.

The five gold nanoparticles of the sizes 10 nm, 20 nm, 30 nm, 40 nm and 50 nm were chosen for protein conjugation after AuNP characterisation. The prepared pink-colored gold solution was characterized by spectrophotometric absorbance maxima (λ max) by scanning in a visible wavelength range of 700 nm to 400 nm.

#### 2.2.2. Protein Conjugation with Gold NPs

For all the above-prepared AuNPs of size 10 nm to 50 nm, pH was adjusted separately to 7.00 ± 0.1 with 0.2 M Potassium Carbonate solution of pH 12.00 ± 0.5. The pH adjustment is predicated on the protein's isoelectric point, which varies from protein to protein. The pH was measured with the help of pH paper. The antibody pLDH (Plasmodium lactate dehydrogenase) reagents have been diluted to 150 μg/mL from the stock solution with 10 mM of Sodium Dihydrogen Phosphate bu ffer of pH 8.50 ± 0.1. Afterwards, the protein pLDH of 150 μg/mL concentration was conjugated to all five AuNPs (10 nm to 50 nm). Conjugation of the AuNPs and protein was achieved by stirring the solution to 10 ± 2 min. Following this, 1% BSA (Bovine Serum Albumin) was added into the gold conjugate solution and stirred for 30 ± 2 min for stabilisation and abstraction of unbound protein. For all five sizes of AuNPs, the single tuned protein concentration was used to detect the e ffect of gold nanoparticle size on the band intensity of developed kits.
