Results

1.1. Covalent conjugation of IgG anti-rat in goat 1mg/mL to standard gold nanoparticles

Our standard gold nanoparticles 40nm at 1OD was activated into EDC/NHS buffer to create the thioester bond as a covalent bond on the gold nanoparticles surface to conjugate with antibody IgG anti-rat in goat.
However, there was a transparently colorless solution, which could be understood that the buffer did wash away the gold nanoparticles, which left the clear solution.

The UV-vis result of unfunctionalized standard gold nanoparticles showed that the peak of it appeared at wavelength 525 nm in figure 2. However, for the functionalized standard gold nanoparticles with IgG did not show any peak from the range of 520 nm to 540 nm in figure 1, which can be understood that there was no appearance of gold nanoparticles in the solution to conjugate with IgG.
Besides that, we also found out that the procedure we used was not suitable for standard gold nanoparticles; it was supposed to be used for carboxyl gold nanoparticles instead with the concentration of IgG had to be 0.5mg/mL for standard gold nanoparticles 40nm.

1.2. Passive adsorption to make ionic conjugation of IgG anti-rat in goat 0.5mg/mL to standard gold nanoparticles

From our first experiment to functionalize gold nanoparticles with IgG, we found out that passive adsorption to create the ionic bond with IgG was the correct one. According to the procedure, the best optimal buffer pH at 5.5 in MES buffer as it still remained the red color of gold nanoparticles solution with the IgG concentration at 20μg/mL. While the gold nanoparticles in other buffers at higher pH are (figure 3).

To evaluate the functionalized gold nanoparticles with IgG in MES buffer pH 5.5, the test was performed on a lateral flow test strip as a reverse test with anti-CD117 anti-human in mouse at the control line and IgG anti-rat in goat at the test line. The evaluation was successful as two red dots which presented the functionalized gold nanoparticles bound to the IgG anti-rat in goat and anti-CD117 anti-human in mice (figure 4).

1.3 Passive adsorption to make ionic conjugation of IgG anti rat in goat at 0.1mg/mL, 0.2mg/mL and 0.5mg/mL to standard gold nanoparticles

From the success with functionalized gold nanoparticles with IgG anti-rat in goat in MES buffer pH 5.5, We repeat the same procedure with IgG anti-rat in goat in acetate buffer at pH 5.3 and pH 5.1 evaluate which one is more specific for our passive adsorption method and different concentrations of IgG anti-rat in goat at 0.1mg/mL, 0.2mg/mL and 0.5mg/mL. After the functionalization, the most stable among the rest with more pinkish color are IgG 0.2mg/mL in acetate buffer pH 5.3, IgG 0.5mg/mL in both acetate buffer pH 5.3 and MES buffer pH5.5 (figure 5).

1.4 Passive adsorption to make ionic conjugation of anti-CD117 anti mouse in rat to standard gold nanoparticles

According to the previous result, we repeat the procedure to functionalize gold nanoparticles with anti-CD117 anti-mouse in rat 0.2mg/mL in acetate buffer pH 5.3, anti-CD117 0.5mg/mL in both acetate buffer pH 5.3 and MES buffer pH5.5 (in figure 6). However, the color shades of all the solutions are less pink as they almost turn colorless. This is assumed that the passive adsorption method for our standard gold nanoparticles with anti-CD117 anti-mouse in rat at different concentrations and different adjusted pH buffer is not successful.

2.1. Covalent conjugation of IgG anti-rat in goat 0.5mg/mL to carboxyl gold nanoparticles

The activation of carboxyl gold nanoparticles with EDC/NHS to create the covalent bond did not show color washed away as the color still remained in pink after first washing with PBST to eliminate the EDC/NHS excess on gold nanoparticles.
After the first wash and centrifugation to eliminate the washing buffer, the gold nanoparticles are conjugated in two and three hours of incubation time to enhance the effectiveness of conjugating with antibodies on the surface. Compared to the two-hour incubation and three-hour incubation to conjugate the antibody, the three-hour conjugation solution has a darker red color via observation.
To validate the functionalized gold nanoparticle, we did the microplate reading. 50μL of each solution has been added into Costar 96 well-plate for the reading at wavelength 530 nm with 1% BSA in PBS buffer as the blank sample.
As can be seen in Table 1, the absorbance of functionalized gold nanoparticles with IgG in 3h incubation is much higher than 2h incubation after it got subtracted with the buffer 1% BSA in PBS as the blocking buffer in the final addition. This is assumed that the color of gold nanoparticles in 3h incubation is darker as more gold nanoparticles in the solution.

Nevertheless, the functionalized gold nanoparticles have to be tested for the quantification to make sure that they were actually crosslinked with IgG. Therefore, we tested the functionalized gold nanoparticles solutions with an immunodot blot test as a recommendation from Cytodiagnostics company to confirm that our gold nanoparticle solution was functionalized successfully. The anti-CD117 was dotted on the nitrocellulose membrane with 0.5μL, and the membrane with the anti-CD117 dot was saturated in a solution of functionalized gold nanoparticles with IgG, and then incubated in 3h. The saturated nitrocellulose membrane was washed with 0,05% Tween solution for washing; nevertheless, there was no sign of dot to prove that the functionalization succeeded (figure 8).

2.2. Functionalized carboxyl AuNPs 40nm with anti-CD117 by using EDC/NHS for activation at 3h of incubation

The same procedure for covalent conjugation of gold nanoparticles with anti-CD117 (anti-mouse in rat) is applied with the optimal incubation time as 3h for the conjugation according to the previous result with covalent conjugation with IgG. Moreover, we also did the same immunodot blot test to confirm that our functionalized gold nanoparticles with anti-CD117 anti-mouse in rat was also successful with the same procedure as the previous test with functionalized gold nanoparticles with IgG.

From which can be seen in figure 9, the immuno dot blot assay did not show any result of success as it was supposed to show the color of red dot on nitrocellulose membrane with IgG and anti-C117. Thus, the first functionalization of gold nanoparticles with both IgG and anti-CD117 anti-mouse in rat was not successful.

2.3. Refunctionalized carboxyl AuNPs 40nm with anti-CD117 by using EDC/NHS for activation at 3h/4h of incubation

For the previous functionalization, an immunodot blot test has shown that there was no reaction to approve the unctionalization was successful. For that reason, we decided to refunctionalized the carboxyl gold nanoparticles again with anti-CD117 anti-mouse in rat in higher incubation time for conjugation with 3h and 4h by following the same procedure without any modification.

However, instead of using the immunodot blot assay the same as the previous time, the UV-Vis microplate reader is applied to validate our functionalized gold nanoparticle with anti-CD 117 anti- mouse in rat at 3h and 4h of incubation (Figure 10).

The result from the plate reading analyzed that there are two differentiated peaks of functionalized gold nanoparticles in 3h and 4h of incubation in the spectrum range from 525 nm to 540 nm. Additionally, the peaks of two solutions both lie at 535 nm as can be assumed that we successfully functionalize the carboxyl gold nanoparticles with anti-CD117. However, the control spectrum for diluted gold nanoparticles in 1%BSA in the PBS buffer did not show the peak at 530 nm as it was supposed to be, and this can be considered as an error while diluting the control.