Surface Modification of a Stable CdSeZnS/ZnS Alloy Quantum Dot for Immunoassay

Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea Company of GLOBAL ZEUS, Osan, Gyeonggi-do 18364, Republic of Korea Department of Urology, Seoul National University Bundang Hospital, Seoul 13620, Republic of Korea Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul 13620, Republic of Korea Department of Chemistry Education, Seoul National University, Seoul 151-019, Republic of Korea


Introduction
Semiconductor quantum dots (QDs) are increasingly being used in both fundamental and applied research because of their optical and electronic properties [1][2][3]. QDs can absorb a broad range of light and emit the specific wavelength of light which depends on their size. With this property, one of the useful applications of QDs is fluorescent probes in biological imaging [4]. While traditional fluorescent probes have disadvantages such as easy photodegradation, low fluorescence, and photobleaching [5], QDs have excellent optical characteristics and chemical stability including higher photostability, wide band-edge absorption, narrow emission, and high quantum yield (QY) [6]. However, QDs also have limitations for bioapplication. Typically, QDs are dispersed only in nonpolar solvents and limited with severe intermittency in emission. Furthermore, their nonspecific binding can lead to misinterpretation of experimental results. The critical issue of QDs is toxicity which can cause acute or chronic diseases. Another issue of QDs is their instability in biological media because of their high hydrophobic property. Thus, surface modification is required to make QDs which have hydrophilic characteristic and more stable in a biological environment [7][8][9][10][11][12][13][14].
Many studies have been sought to overcome these limitations of QDs. The hydrophobic surface of QDs was changed as a hydrophilic surface via encapsulation with a silica shell, and these silica encapsulated QDs were more suitable for biomedical applications compared to bare QDs [7,[15][16][17][18][19]. Amphiphilic polymer-covered QDs displayed improved stability over a wide pH range and were less prone to nonspecific binding [20,21]. Studies which aimed at enhancing the safety of QDs described that a thick shell of ZnS could reduce QDs' toxicity to cells and increase photoluminescence.
Enzyme-linked immunosorbent assay (ELISA) is a biochemical method for detecting or measuring a molecule, mostly a protein. It is based on the antigen-antibody reaction which has been applied in various different fields such as diagnosis, toxicology, food industry, pharmaceutical industry, immunology, and vaccine development [27]. ELISA possesses advantages that include high sensitivity, specificity, and fast reaction, but it also has many disadvantages such as size limitations, disposal of sensitivity to temperature, and easy decay. To overcome these weaknesses, enormous innovations such as ultrasensitive enzymes and better antibodies have been developed. As good fluorescence probes, QDs were utilized for signal enhancement [28][29][30][31][32].
In many studies, CdSe/ZnS core/shell QDs were fabricated and applied in biological detection [33][34][35][36][37][38][39][40][41][42]. Li's group synthesized CdSe/ZnS QDs and proceeded an assay for the detection of C-reactive protein (CRP). The test for CRP detection in the above study had good detection results, but the QY of QDs was 63% in hydrophobic modification and the QY showed an 8% reduction after hydrophilic modification [43]. For another study, the protein was introduced on CdSe/ZnS QDs. The particle that conjugated with antibody was identified with luminescence images on various proteins and compared the efficiency of each particle. It was a good result in biological application. However, in the case of QY of CdSe/ZnS QDs used in the above paper, it was 35-50% before the ligand exchange, and the QY after ligand exchange was not checked correctly [44]. In general, QDs showed a sharp decrease in QY after ligand exchange, so maintaining QY which is not dropped significantly after surface modification is needed [45][46][47].
In this study, we fabricated a cadmium selenide zinc sulfide/zinc sulfide (CdSeZnS/ZnS) alloy QDs that displayed a high QY which was 84.5%; after surface modification, QY was 83.0% (98.2% of QY was maintained). The surface of alloy QDs was modified via conjugation with antibody molecules, and commercial QDs were used as a control. The conjugation of the commercial QDs markedly reduced QY. By contrast, alloy QDs retained a high QY after surface modification and then were successful in sensitive detecting rabbit IgG. The results indicated that alloy QDs have better stability in QY and better capability in bioconjugation than commercial QDs.

Preparation of Alloy QDs.
Hydrophobic QDs were prepared by mixing 75 mL of ODE and 15 mL of OA and adding 1.1 g Zn(Ac) 2 and 0.384 g of CdO in a three-necked flask, and the mixture was incubated under a vacuum at 150°C for 1 h. After 1 h, moisture in the flask was removed and the flask was heated to 300°C in the presence of nitrogen gas, with the addition of 0.6 mL of TOP and 0.048 g of Se for 3 min. This was followed by the addition of 0.5 mL of 1-dodecanethiol and reaction for 20 min. Three milliliters of TOP and 0.192 g of sulfur were added to the flask and reacted for 10 min, followed by 1 mL of 1-dodecanethiol and reacted for another 10 min. The flask was then cooled to room temperature. The QDs were washed in acetone and dispersed in CHCl 3 . The hydrophobic alloy QDs were prepared by mixing 60 mL of ODE and 10 mL of OA, with the addition of 1.1 g Zn(Ac) 2 and 0.384 g of CdO in a three-necked flask under a vacuum at 150°C for 1 h. Moisture was removed, the flask was heated to 300°C in the presence of nitrogen gas, the prepared hydrophobic QDs were added, and the reaction was allowed to proceed for 10 min. Three milliliters of TOP and 0.192 g of S were added and reacted for 10 min, and the flask was cooled to room temperature. The alloy QDs were washed in acetone and dispersed in CHCl 3 .

Characterization of QDs.
The photoluminescence quantum efficiency of heat-treated specimens excited at 390 nm was measured using a quantum efficiency measurement system (QE-2000, Otsuka Electronics Co., Ltd.). While measuring the quantum efficiency, the emission and excitation sources were also collected by the integrated spheres.

Ligand Exchange of QDs.
Ligands of alloy QD and commercial QD were exchanged using the carboxyl groupcontained ligands as per a previous description for modification. First, the reaction solution containing 1.0 mL of MPA, 1 mL of NH 4 OH, and 30 mL of CHCl 3 was prepared in a Falcon tube, which was mixed by rotation for 2 h. Then, 10 mg of each QD, 10 mL of distilled water, and 10 mL of reaction buffer were mixed in a Falcon tube and allowed to react by rotation for 2 h. The supernatant was collected and was washed several times with CHCl 3 . After alloy QD ligands were exchanged with the carboxyl group, they were washed with acetone and then passed through an Amicon filtersized 100,000 MWCO and dispersed in distilled water.   Journal of Nanomaterials ng mL -1 ) were added to each microwell and incubated for 2 h at 37°C. Then, we washed the microwell three times using PBS containing 0.1 wt.% TWEEN® 20 (300 μL well -1 ) and 300 μL of PBS was added into each microwell. After the immunoassay experiment, fluorescence intensity was measured (excitation wavelength filter was used to 385 nm; emission wavelength filter was used to 630 nm).

Synthesis and Characterization of Anti-Rabbit IgG
Antibody-Conjugated QDs. Our new alloy QDs was established via three strategies to improve the final stability of particles. First, the method of depositing a secondary shell after the primary refining core was used in the core synthesis method of the alloy QDs. Second, the ligand of the primary shell in the synthetic method was applied with a mixture of various thiol-contained ligands (1-octanethiol, 1-dodecanethiol, 1-decanrthiol...) which reduced the lattice mismatch in the next response to increase the stability of the core in the alloy QDs. Third, in the synthetic method, the precursor of the shell was added sequentially to enhance particle stability. Figure 1(a) shows a representative transmission electron microscopy image of bare QDs, and Figure 1(b) shows QD size distribution, respectively. The average size of commercial QDs was 5:6 ± 1:0 nm, and its relative standard deviation was 0.16. The average size of alloy QDs was 10:1 ± 2:1 nm, and its relative standard deviation was 0.21. They were also sized through DLS with water as a solvent ( Figures S1 and S2).
The surfaces of QDs were functionalized with MPA to produce a hydrophilic surface with carboxyl groups. To conjugate an antibody, the carboxyl groups were activated using EDC and sulfo-NHS. To evaluate the quality of two kinds of antibody-conjugated QDs, ultraviolet-visible (UV-Vis) absorbance, fluorescence intensity, zeta potential, and QY examination were performed at the same concentrations. The UV-Vis absorbance at 300-400 nm was higher in alloy QDs than in commercial QDs (Figure 2(a)). Fluorescence intensity was measured at an emission and excitation wavelength of 385 nm and 600-650 nm in 0.05 mg mL -1 of QD concentration, respectively (Figure 2(b)). As a result of comparing fluorescence intensity at the same concentration, the fluorescence intensity of alloy QDs was relatively stronger than that of commercial QDs. To confirm the anti-rabbit IgG antibody conjugation, we compared the QY and zeta potential of carboxyl group-functionalized QDs and antibodyconjugated QDs. After conjugating antibodies to QDs, QY was measured to determine how different the efficiency of  Journal of Nanomaterials commercial QDs and alloy QDs were. Carboxyl groupfunctionalized commercial QDs displayed a relatively low level of QY at 11.3%, with QY decreasing to 2.5% after antibody conjugation. On the other hand, carboxyl groupfunctionalized alloy QDs displayed a high QY of 84.5%, and this high QY was maintained even after antibody conjugation (83.0%) (Figures 3(a) and S3). Before antibody conjugation, both QDs showed a higher negative charge due to the carboxyl group on the surface, whereas antibody-conjugated QDs showed less negative charge due to the antibody on the surface (Figure 3(b)). These findings indicated that the antibodies were successfully conjugated onto the surface of carboxyl groupfunctionalized QDs.
Additionally, cell viability experiments were conducted to confirm the suitability of biological experiments and particle stability of alloy QDs and common QDs ( Figure S4). The results indicated the superiority of alloy QDs, which were used in further experiments.
3.2. QD-Based Sandwich Immunoassay Strategy. The antirabbit IgG antibody was used as a capture antibody, and it was used to the detection of rabbit IgG as target. The capture antibody was attached to the wells of high-binding immune plates through physical absorption. BSA was used to block the uncoated active sites to prevent nonspecific adsorption of the target in microplates. Different concentrations of the target were added for testing. Subsequently, detection antibody-conjugated QDs were bound with the targetcapture antibody complex and formed the sandwich immunocomplex. As the decreased target concentration, the amount of capture antibody-conjugated QDs which bound as sandwich immunocomplex was also decreased, and as a result, the fluorescence intensity declined. Thus, it was feasible to determine the concentration of the target by monitoring the fluorescence intensity.

Optimization of Antibody-Conjugated QD Concentration.
To get the optimized concentration of QDs (for sandwich immunoassay), we set the concentration of detection antibody-(anti-rabbit IgG) conjugated alloy QD variously between 0 and 1:0 × 10 7 ng mL -1 . The experiment group proceeded with the capture antibody, target antibody, and detection antibody-conjugated QDs. Control group 1 used alloy QD-conjugated antibody and capture antibody, without target. Control group 2 used alloy QD-conjugated antibody and target, without capture antibody. The concentration of both capture antibody and target antibody was not changed during optimizing the concentration of detection antibodyconjugated QDs. As shown in Figure 4, we performed sandwich immunoassay and achieved strong fluorescence intensity at the experiment group, meanwhile weak fluorescence intensity at the control group. Weak fluorescence intensity of the control group is indicated for proving nonspecific binding. Consequentially, we confirmed that there was distinct fluorescence intensity at the experiment group and no 5 Journal of Nanomaterials significant nonspecific binding at control group 1 below the 2:5 × 10 5 ng mL -1 of detection antibody-conjugated QDs. So, the concentration of detection antibody-conjugated QDs was optimized as 2:5 × 10 5 ng mL -1 , and QDs with optimized concentration were used by the same concentration (2:5 × 10 5 ng mL -1 ) for the rabbit IgG detection experiment.

Performance of the Sandwich Immunoassay Using Alloy
QDs for Rabbit IgG Detection. Under the optimized condition of detecting anti-rabbit IgG antibody-conjugated QDs (2:5 × 10 5 ng mL -1 of detection antibody-conjugated QDs), we performed the sandwich immunoassay experiment for detecting of the rabbit IgG. To determine the limit of detection (LOD) of the rabbit IgG, the concentration of rabbit IgG was increased gradually from 0 (PBS buffer only) to 1:0 × 10 4 ng mL -1 . The fluorescence intensity was gradually increased according to the increase of rabbit IgG concentration. Meanwhile, in the control group in which the capture antibody was absent, fluorescence intensity was almost the same at all concentrations of rabbit IgG. The maximum fluorescence intensity of both the experimental and control groups was compared to achieve the limit of detection; the   Exp.
Con. Figure 5: Fluorescence intensity of anti-rabbit IgG antibody-conjugated alloy QDs for rabbit IgG detection. Compared with the experiment group, the target was absent in the control group. 6 Journal of Nanomaterials LOD for the rabbit IgG was 1:1 × 10 2 ng mL -1 (3 S/N criteria) ( Figures 5 and S5). Although these sensitivities were not exceptional [49], the results are important as they were the demonstration of the potential biological applications of new alloy QDs that QY was rarely dropped after surface modification. Alloy QDs could have diverse uses due to their remarkably bright fluorescence.

Conclusions
We fabricated the alloy QDs which have a high QY of 84.5%, and their ligand has a carboxyl group that can easily be used and versatile in surface modification. After surface modification, the alloy QDs have a QY of 83%. We compared the optical characteristics between commercial QDs and alloy QDs. The QY of commercial QDs decreased sharply after surface modification from 11.3% to 2.5%, whereas the QY of alloy QDs was stable even after surface modification.
These results indicate that alloy QDs are more applicable than commercial QDs. Further experiments established the bioapplication of alloy QDs. In a sandwich immunoassay to detect rabbit IgG, the LOD was 1:1 × 10 2 ng mL -1 . Combining these results, alloy QDs are more suitable for bioapplication than commercial QDs and have the potential for development.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request. Figure S1 shows the DLS analysis result of alloy QDs. For the alloy QDs, the size of the particles in the water solvent was measured at 25.8 nm. Figure S2 shows the DLS analysis result of commercial QDs. For the commercial QDs, the size of the particles in the water solvent was measured at 762.3 nm. Figure S3 checked the digital image of each QDs. Prior to the conjugation of antibodies, the original alloy QDs had the strongest fluorescence intensity in the image. Figure S4 shows cell viability assay to assess cytotoxicity of alloy QDs. Human retinal pigment epithelial ARPE-19 cells were treated with various concentrations of alloy QDs or conventional QDs. Figure S5 shows LOD calculation. We used 3 S/N criteria to calculate LOD. (Supplementary Materials)