2.2.3. Centrifugation

The above five separately prepared AuNP–protein conjugate solutions were centrifuged. The centrifugation was performed with a Relative Centrifugal Force (RCF) of 7000× *g* for 45 min at 4 ◦C to 8 ◦C temperature. The centrifugation of an AuNPs–protein conjugate solution at a force higher than 7000× *g* RCF can sometimes shows aggregation while centrifugation at a force slower than 7000× *g* RCF may give less residue with a dark supernatant. Centrifugation at 7000× *g* RCF gives a stable and good yield of the residue or pellet. The supernatant's aspiration was performed in a di fferent beaker, and gold pellets were resuspended in the phosphate-bu ffered saline (PBS) bu ffer. The absorbance of the supernatant was measured at a 520 nm wavelength. If the OD was greater than 0.05, then the supernatant's re-centrifugation is performed one more time. The supernatant was discarded if the OD was less than 0.05. This will enable conjugate recovery and prevent wastage. The supernatant aspiration is performed in a separate beaker, accumulating the AnNP–protein pellets. Carefully, the supernatant aspiration and the residue re-suspension was accomplished in a re-suspension bu ffer. Figure 2 represents the protein conjugation and centrifugation procedure.

**Figure 2.** The diagrammatical representation of the protein conjugation and centrifugation methodology.

#### 2.2.4. Conjugate Pad Prepration

All five separately re-suspended AuNPs—protein conjugate above solutions were diluted (ultra-pure mili-Q water) to a constant OD of 3.00 at 520 nm wavelength.

Following the dilution, five conjugate pads were prepared by dipping the glass fibre pad into the conjugate solution. In comparison, the other changes through the entire development of the kit was held constant, e.g., test line concentration and control line protein.

## 2.2.5. Membrane Coating

The five nitrocellulose membranes were coated at the test (Pan) and control line (C). The test and control line coating on the nitrocellulose membrane (NCM) was achieved with the use of a Bio-dot dispensing machine. First of all, the bio-dot machine stripping system (tubing and jets) was flushed with de-ionized water over ten cycles. The control and test solutions were then coated on NCMs. For drying, membrane sheets were kept in the oven at 30 ◦C for 30 min after coating. The concentrations of test and control line reagents were as follows:

#### 2.2.6. Test Line Reagents Concentration

To obtain the final test solution, the pLDH (Plasmodium lactate dehydrogenase) antibody was diluted from the stock solution to 50 μg/mL with 1% sucrose solution in the PBS bu ffer. The antibody protein mixing in PBS bu ffer was performed with a magnetic stirrer, and a 0.22-micron filter was used to eliminate the suspended particles.

#### 2.2.7. Control Line Reagents Concentration

To obtain the final control line solution, Goat Anti Mouse IgG was diluted from stock solution to 400 μg/mL with a 0.5 percent sucrose in the PBS bu ffer. To extract the suspended particles, mixing and filtration were achieved using a 0.22-micron filter.

#### **3. Results and Discussion**

#### *3.1. Gold NP Characterization*

Firstly, we prepared the most stable AuNPs of an average of of 10 nm to 50 nm in diameter. The AuNPs with absorbance maxima (λ max) ranging between 520 to 570 wavelengths were considered for the development of the Malaria Pan Antigen detection test kit. In this range of λ max, the AuNP size ranges from 10 nm to 50 nm as determined by the particle sizer. Figure 3A–E represent the AuNP size measured in dynamic light scattering (DLS). AuNPs of this range were selected due to their smaller particle size and lower polydispersity index (PDI). It has been observed that smaller sized nanoparticles have better conjugation with the protein [10–12,20]. The size of gold NPs depends on the sodium citrate content used [18]. The concentration of sodium citrate in gold solution a ffects the size of AuNPs, which can be controlled by measuring absorbance maxima (λ max). The sodium citrate of viz 0.2, 0.4, 0.6, 0.75 and 0.90 mg/mL in gold solutions produces nanoparticles (NPs) with average diameters of 10 nm (size distribution of 8 to 12 nm), 20 nm (size distribution of 17 to 23 nm), 30 nm (size distribution of 26 to 35 nm), 40 nm (size distribution of 32 to 50 nm) and 50 nm (size distribution of 36 to 80 nm), respectively (Figure 3). AuNPs shows λ max at varying wavelengths of 520 (10 nm), 530 (20 nm), 540 (30 nm), 560 (40 nm) and 570 (60 nm). Figure 4A–E represent the λ max of AuNPs. The optical density of prepared AuNPs at λ max ranges between 8.0 to 9.0. When sodium citrate concentration increases in AuNP solution, AuNP size does too. When the size of the gold NPs increases, the absorbance maxima (λ max) shift to higher wavelengths (Figure 5), and the color of the solution turns from pink to blue, reflecting nanoparticles' instability.

**Figure 3.** Synthesized gold nanoparticles' size distribution measured by the Zeta Seizer. The sodium citrate solutions of 0.2, 0.4, 0.6, 0.75 and 0.90 mg/mL in gold solutions' produced nanoparticles (NPs) with average diameters of (**A**) 10 nm (size distribution of 8 to 12 nm); (**B**) 20 nm (size distribution of 17 to 23 nm); (**C**) 30 nm (size distribution of 26 to 35 nm); (**D**) 40 nm (size distribution of 32 to 50 nm); and (**E**) 50 nm (size distribution of 36 to 80 nm).

**Figure 4.** Optical density spectrum of synthesized gold nanoparticles. The gold nanoparticles (AUNPs) show different λ max (absorbance maxima) at different sizes; i.e., (**A**) λ max 520 nm at diameter of 10 nm; (**B**) λ max 530 nm at diameter of 20 nm; (**C**) λ max 540 nm at diameter of 30 nm; (**D**) λ max 560 nm at diameter of 40 nm; (**E**) λ max 570 nm at diameter of 50 nm.

**Figure 5.** The graph shows a correlation of synthesized gold nanoparticles (10 nm, 20 nm, 30 nm, 40 nm and 50 nm) λ max (absorbance maxima) and Optical Density. With an increase in the size of the gold NPs, the absorbance maxima (λ max) shifts to higher wavelengths (520 nm to 570 nm), reflecting nanoparticle instability.

As the average diameters increase, the nanoparticles' size distribution increases, which causes the instability of AuNPs (Figure 3). This technique is widely used for the determination of particle size in colloidal solution, which, in turn, used to measure the thickness of capping or stabilizing agen<sup>t</sup> along with its actual size of metallic core. These studies also determined the hydrodynamic diameter of the synthesized nanoparticles. These results also suggested that there is an absence of large aggregates when these nanoparticles were dispersed in aqueous medium [21].

#### *3.2. Monitoring the Protein Loss*

After centrifigation of AuNP–protein conjugate, a small portion of re-suspended solution was diluted (1 in 100) into ultra-pure mili-Q water to facilitate OD measurement at 520 nm. The OD values obtained are shown in Table 1.

**Table 1.** Optical density (OD) measurement results of synthesized gold nanoparticles–protein centrifuged re-suspension conjugate solution when diluted in 1–100.


Figure 6 and Table 1 indicate that 40 nm AuNPs have high OD, and thus AuNPs have maximum protein binding with AuNPs of 40 nm size. In contrast, the other AuNPs had less binding. This indicates that the supernatant lost extra unbound protein. This means that the additional unbound protein was lost in the supernatant. The protein depletion can also be checked with the use of UV/Vis spectrophotometer OD analysis.

**Figure 6.** The spectrum overlay graph shows optical density of gold nanoparticles (10 nm, 20 nm, 30 nm, 40 nm and 50 nm) and protein conjugate solution (1–100 mL) measured at 520 nm of wavelength. The gold nanoparticles of average diameter of 40 nm have maximum optical density (0.426), which reflects the maximum protein binding.

#### *3.3. Test Line Intensity Analysis*

The developed kits were tested to find out the kit test zone band intensity (results) when equivalent protein ratios are conjugated with different AuNP sizes. The five immunochromatographic rapid test kits were formulated using a conjugate pad (10 nm to 50 nm) prepared above. Then, all five of the immunechromatographic test kits were assembled. Now, the five developed test kits were tested for band intensity using 5 μL of malaria (*P. vivax*) positive blood specimens of concentration 150 parasites/μL. The three specifications were given to test line viz. high test line intensity was ranked as +3, medium test line intensity was ranked as +2, and weak test line intensity was ranked as +1. Upon testing all kits with the same specimen samples, all kits showed an equal band intensity of +3 (high) as shown in Table 2. In this analysis, it was noticed that, if the final OD is tuned to one point, there will be no effects of AuNPs sizes on kit results (test zone intensity). OD-adjustment will refine the final test zone band intensity. Figure 7 is the systematic representation of the results. All five test kits developed after OD tuning to 3.0 were additionally verified for their specificity with 5 μL of Malaria Pf (Falciparum) antigen blood specimens of concentration 40 parasites/μ<sup>L</sup> to find out test kit susceptibility for *P. Falciparum*. The specificity results of developed kits (Table 3) were found without any false positive indication (no Pan line appears), and the the control band intensity was high (+3).

**Table 2.** Test line intensity results of five Malaria Pan Ag immunochromatographic rapid test kits formulated using different diameter AuNPs. The kit final OD is tuned to one point (3.00) after conjugation with protein. Kit's test line intensity was tested using *P. vivax* positive blood specimen of a concentration of 150 parasites/μL. The band intensity for test (Pan) and control (C) line is ranked as: high test (+3), medium (+2) and weak (+1). Upon testing, all developed kits showed an equal sensitivity of +3 (high).


**Figure 7.** The diagram systematically represented the results.

**Table 3.** Specificity results of five Malaria Pan Ag immunochromatographic rapid test kits formulated using different sizes AuNPs. Kit's specificity was tested using *P. Falciparum* positive blood specimens of concentration 40 parasites/μL. The color band intensity for control (C) line was +3 (high) and no line appears on the test zone (Pan) indicating absence of any cross reactivity.
