Biotin-Streptavidin Conjugate with GNPs Assisted Modification

We have successfully prepared different sizes of gold nanoparticles by controlling the ratio of applying reagents. We use several instruments and software to confirm the size of gold nanoparticles. The scheme 4.4 shows the detailed processing flow of surface modification.

(a) (b)

position fixing, and may cause serious personal inaccuracy. IPP (Image-Pro Plus) is the ultimate image analysis software package for fluorescence imaging, quality assurance, materials imaging. The software is widely accepted in various scientific researches such as medical and industrial applications. Count/size is one of the most useful function; it can measure the average or dispersion of diameter, area, and circumference, etc. This software greatly eliminates the inaccuracy, enabling us to measure the most accurate experimental data.

As shown in figure 4.6(a), gold nanoparticles from SEM characterization was well dispersed by appropriate and careful sampling. No aggregation existed between gold nanoparticles.

figure 4.6 (b) depicts the number-diameter diagram of gold nanoparticles, we can evaluate the distribution and mean size of gold nanoparticles.

Figure 4.6. (a) The SEM image of gold nanoparticle; (b) number-diameter bar chart of gold nanopartice.

Another method for diameter determination employed UV-visible spectrophotometer.

UV-visible absorption spectra are useful for studying the properties of metal nanoparticles due to the sensitive peak positions and shapes in relation to the particle size and composition.

When white light illustrated through or reflected by a color substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelength(s) absorbed. Thus, absorption of 420-430 nm light

(a (b

renders a substance yellow, and absorption of 500-520 nm light makes it red. Green is unique in that it can be created by absorption close to 400 nm as well as absorption near 800 nm.

Here, we use the above theory. Figure 4.7 shows the adsorption curve of different size of gold nanoparticles. The surface GNP density are 7.84×10¹³ / m² (20nm), 4.76×10¹³ / m² (45nm), 2.01×10¹³ / m² (60nm) and 1.14×10¹³ / m² (75nm), respectively.

Figure 4.7. Absorbance spectra of different sizes of gold nanoparticles.

4.3.1 Characterization with Fluorescence Microscopy and IPP software

We studied the binding efficiency of streptavidin by gold nanoparticles assisted modification. In this thesis, we employed the fluorescence microscopy, UV-visible spectrophotometer and IV characterization to investigate the degree of enhancement.

Firstly, we verified several sizes of gold nanoparticle and chose the suitable size for the

Another important function for the IPP software is “histogram”, which can convert any information of image into a statistical chart. In this study, we have proposed standard procedure for the comparison of the illumination between several fluorescence images. The procedurea were described as below:

Step 1 Open the image you want to deal with.

Step 2 Convert the colored image into grayscale mode image.

Step 3 Choose several area by the function “AOI” by avoiding the doubtful spots and areas.

Step 4 Definition these blocks by the function of “count/size”, determining which blocks will be calculated.

Step 5 Count the brightness of every pixel in blocks by the function of “histogram”.

Step 6 Export the data of step 6, than we can get the average brightness of a pixel.

Figure 4.8. The diagram of brightness after quantification procedure.

As we know, the thio group will successfully immobilize onto the gold nanoparticels attached by APTES through the assistance of DDT. DDT will cleavage and restrain the formation of double sulfur bonding (S-S). Furthermore, there is no other active product after

reaction happened, indicating that there is no other disturbance in this reaction.

Figure 4.9 shows the bar chart for different sizes of gold nanoparticles that react with cysteamine but without DDT assistance. We found that there is no obvious difference for the groups without gold nanoparticles or with gold nanopartices, except for 5 nm gold nanoparticle. The intensity was increased up when the DDT was participated with the reaction between cysteamine and gold nanoparticles (the result is at Figure 4.9). We found that the intensity of fluorescence was increased 58% between system without any modification and system with 20nm GNPs. The observation suggests gold nanoparticles and cysteamine can improve the binding efficiency. Besides, the area which did not cover with gold nanoparticles still possesses free amino groups that can react with NHS-biotin. Enhanced fluorescence of an organic fluorophore using nanoscale gold particles film was already investigated [7], Particulate gold films were deposited on glass substrates by vapor deposition. In this study, fluorescence was enhanced with increasing the Au thickness and reached saturation at 30 nm.

The observation of literature supports our finding.

Another interesting phenomenon is also observed, the intensity of fluorescence at Figure 4.9 and Figure 4.10 both shows the system modification by 5nm gold nanoparticles having the lowest intensity of fluorescence. The Fluorescein has a maximum absorption at 494 nm and maximum emission at 521 nm. However, surface plasmon (SP) extinction band of hydrosol of 5 nm gold nanoparticles is at about 512 nm [8]. Hence, 5nm gold nanoparticle has higher extinction effect. Furthermore, 5nm gold nanoparticle has high aggregation effect. At the same reaction environment, 5nm gold nanoparticles have higher aggregation effect, and the reaction area was reduced when gold nanoparticles was aggregated.

Figure 4.9. The bar chart of brightness after quantification.

none 5nm 20nm 45nm 60nm 75nm

0 25 50 75 100 125 150 175 200 225

Intensity of Fluorescece (a.u.)

Size of GNPs

5 20 45 60 70

none

Figure 4.10. The bar chart of brightness after quantification (with DTT assisted modification).

We have also verified this system on bare Au film, and the results are shown in Figure 4.11. The intensity of fluorescence is 40 a.u. without any modification, but the value will elevate to 68 a.u. with GNPs assisted modification. The enhancement percentage is calculated to be (68-40)/40= 70%.

none 5nm 20nm 45nm 65nm 75nm

0

Figure 4.11. The bar chart of brightness after quantification. The graft of streptavidin-Fict;

without GNPs, with GNPs but without DTT and with GNPs plus DTT.

4.3.2 UV-Visible Spectrophotometer Evaluation

In this work, we study the effect of enhancement of GNPs and cysteamine by using the photonic property of luminol molecular. Luminol has notable absorption at wavelength of 350 nm [9]. When luminol reacts with H2O2 under the catalyst of HPR, the formed were produced and luminal concentration decreaded gradually. The OD value of luminol is a rather suitable index for further application.

The preliminary test for luminol is shown in figure 4.12. The solid line is the absorption curve of luminol before reaction, and a significant absorption peak at 350 nm[10]. which is in accordance with literature. The dotted line is the absorption curve for luminol molecule that has reacted with HRP solution. The OD value at 350 nm was obviously decreased. This observation suggests the luminol molecules were have consumed.

Figure 4.12. The OD value versus wavelength of absorption.

Furthermore, we prepared slides of silicon dioxide by semiconductor processing. We tried to stick the slides on two inside walls and ensure that the light does not pass through it.

The Figure 4.13 depicts the OD value with respect to wavelength of luminol at various times of 0, 30 minutes, 60 minutes, 90 minutes and 14 hours. Figure 4.13 (a) is the luminol reaction with HRP which has previously immobilized onto surface but without any further modification. The slides of which have surface modification have significantly higher rate of consumption. Figure 4.14 denotes the graph of OD value versus time. The solid line stands for the luminol reaction with immobilized HRP but without any modification. The dotted line

of curve will be taken as reaction rate. After linear curve fitting, the slope of solid and dotted line are -0.0022 and -0.005 respectively, and the percentage of enhancement is [(-0.005)－

(-0.0022)]÷(-0.0022) × 100% = 127%.

Figure 4.13. The OD value of luminal decay with reaction time. Conjugation of streptavidin-HRP without (a) and with (b) GNPs and cysteamine assistant.

Figure 4.14. The OD value of luminal decay with reaction time. Conjugation of streptavidin-HRP without (a) and with (b) GNPs and cysteamine assistant.

0 20 40 60 80 100

4.3.3 Investigation by Photo Diode

A photodiode is a semiconductor diode that functions as a photodetector which nothing more than a bipolar transistor that is encased in a transparent case so that light can reach the base-collector junction. A photodiode is a p-n junction or p-i-n structure. When a photon of sufficient energy strikes the diode, it excites an electron thereby creating a mobile electron and a positively charged electron hole. If the absorption occurs in the junction's depletion region, or one diffusion length away from it, these carriers are swept from the junction by the built-in field of the depletion region, producing a photocurrent.

Photodiodes can be used under either zero bias (photovoltaic mode) or reverse bias (photoconductive mode). In zero bias, light falling on the diode causes a current across the device, leading to forward bias which in turn induces "dark current" in the opposite direction to the photocurrent. This is called the photovoltaic effect, and is the basis for solar cells — in fact; a solar cell is just a large number of big photodiodes.

Figure 4.15. shows the dark and photo current detected by photodiode. Prior to pouring luminol into the reaction tank, we detected dark current. The value of dark current is about 1.02 × 10 ^-11A. We detect the photo current once we pouring the luminol. The photo current was 2.63 × 10^-10 ampere in the Si/APTES/NHS-biotin/Streptavidin-HRP system (Figure 4.15

(a)); the photo current was 2.63 × 10^-9 ampere in the

Si/APTES/GNPs/CysteamineNHS-biotin/Streptavidin-HRP system (Figure 4.15 (b)). The difference between the two system is one order of magnitude, this value is much bigger than the results in previous session. We suggest the reason of this phenomenon is that the photodiode is a kind of high sensitive electronic element which can clearly recognize the tiny difference between illumination. This result is much greater than the fluronescence

Figure 4.15. Real time measurement of chemiluminescence substrate. Without GNPs assistant immobilization; (b) with GNPs assistant immobilization.

(a

(b