Jurkat T Cell Detectability and Toxicity Evaluation of Low-Temperature Synthesized Cadmium Quantum Dots

Early and highly accurate detection of diverse diseases is in urgent demand than ever, especially for cancers and infectious ones. Among possibilities, biosensing by utilizing conjugated nanoparticles is still a method of choice. However, the toxicity of quantum dots remains a big matter of concern in those biooriented applications. In this study, mercaptosuccinic acid-coated cadmium selenide quantum dots of approximately 2.3 nm were synthesized with a simple green method at low temperature and cost-saving chemicals. The influence of synthesis factors was investigated with different spectroscopic methods. The toxicity issue was evaluated on the NIH-3T3 cell line (ATCC® CRL-1658TM) and an MTT assay, revealing a secure threshold of 20 μg/ml. Consequently, successful conjugation to the CD3 antibody including an A/G protein bridge was implemented and verified with fluorescent methods. Finally, Jurkat T cell detectability of conjugated CdSe was successfully validated with fluorescent microscopy. The CdSe-based products are accessible for future biosensing applications.


Introduction
For years, understanding of semiconducting quantum dots along with their unique properties of zero-dimensional structure has been extensively accumulated not only through diverse synthesis methods but also through novel applications [1][2][3][4][5]. Thanks to the primary advantages such as high stability, broad photoluminescence and absorption spectra, large Stoke shift, long lifetime, and an order of luminescent intensity higher than organic dyes and fluorescent protein counterparts [6,7], quantum dots have been employed widely in biology especially in biomarking and biosensing [8][9][10].
A huge effort has been paid out on the fabrication and characterization of highly luminescent quantum dots derived from trioctylphosphine oxide-(TOPO-) or trioctylphosphine-(TOP-) mediated routes of high temperatures and costly chemicals. However, such quantum dots exhibited low dispersion in water and an inability to be directly applied in biology without surface functionalization [11][12][13]. Recent reports have focused on fabricating highly stable CdSe quantum dots in water with surfactants comprising thiol groups [14][15][16][17]. These nanoparticles could be applied straightly into bioapplications on the one hand, but low luminescence intensity due to dislocation or surface defects was a big drawback. Till now, novel synthesis methods have continuously been introduced to achieve nanoparticles of high quality and high luminescence as well as time and cost saving [18]. Following the trend, this work was aimed at synthesizing mercaptosuccinic acid-(MSA-) coated quantum dots of high quality, high luminescence, and high biocompatibility from inexpensive chemicals without metal-organic precursors. Effects of temperature, reaction time, and pH values on optical properties and structure were under investigation with UV-visible absorption, photoluminescence (PL), Fourier transform infrared spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Experimentally, quantum dots cause a greater effect of toxicity compared to bulky counterparts at the same mass dose [19][20][21]. Potential toxicity and hazardous adverse effects of bare CdSe quantum dots on biological samples have always posed a concern much greater than the core/shell CdSe/ZnS counterparts. In practice, toxicity evaluation of CdSe without a protective shell layer is a crucial step toward basic knowledge and further bioapplications. We evaluated the quantum dot toxicity by using the NIH-3T3 cell line (ATCC® CRL-1658™) and an MTT assay to determine a nontoxic threshold of high cell viability. A cadmium selenide quantum dot concentration of 20 μg/ml was found to be the safe level from which cells presented a great chance for survival (97.4% on average). Finally, for the detection of Jurkat T cells, conjugation of the CD3 antibody to MSA-capped CdSe quantum dots was implemented. In this process, the A/G protein played a role as a vital connector to enhance CD3 antibody-CdSe quantum dot association. Successful conjugation of the A/G protein and the CD3 antibody was verified with photoluminescence and time-resolved fluorescence characterizations. The final products are eminently promising and relevant to further development.
Initially, 1.53 mmol of Cd(CH 3 CO 2 ) 2 ·2H 2 O, 1.85 mmol of MSA, and 50 ml of distilled water were added into a three-neck flask to produce a Cd 2+ precursor. The mixture was stirred in a nitrogen atmosphere until MSA was completely dissolved. Nitrogen gas was introduced to eliminate oxygen and the resulting acetic acid. To adjust pH, NaOH was gradually dropped to reach the expected value (6.0-11.5). For the Se 2precursor, 2.64 mmol of NaBH 4 , 1.34 mmol of Na 2 SeO 3 ·5H 2 O, and 5 ml of water were mixed up for 15 minutes. We rapidly injected the Se 2precursor into the Cd 2+ solution at room temperature. The mixture was then stirred for 30 minutes at 600 rpm. The solution was then heated up to three different reaction temperatures (90°, 95°, or 100°C) without stirring. After some time, toluene was injected to rapidly cool down the solution and to intentionally stop the development of particles. The as-synthesized products were finally dispersed in PBS 7.0 buffer solution for long-time preservation. A simple method of preparation, common chemicals, and reasonably low synthesis temperature are the three big advantages of this method.

Toxicity
Evaluation of Quantum Dots. The toxicity of quantum dots was analyzed using the NIH-3T3 cell line (ATCC® CRL-1658™) and the MTT assay. NIH-3T3 cells were grown in DMEM-10 medium (HiMedia) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Sigma).

CD3 Antibody Conjugation to Quantum Dots.
For conjugation of the CD3 antibody to MSA-capped CdSe quantum dots, the A/G protein was employed as an essential bridge to firmly enhance antibody-quantum dot connection efficiency. For this reason, a higher antibody-antigen interaction performance could be achieved later. Initially, acid-coated quantum dots were shaken in 5% BSA at 10°C for two hours to ensure that there was no direct interaction between quantum dots and cells through nonspecific bonds. Consequently, we supplied antibodies to Jurkat T cells [10,22] at a weight ratio of 3 : 1 over the formerly delivered A/G protein. Reaction took place in an antibody incubation solution (Tris-base 50 mM, NaCl 150 mM, pH 8.2) which was shaken at 10°C for 2.5 hours. Finally, the solution went through centrifugation to collect the conjugated quantum dots.

Instrumental Characterization.
Optical properties of nanoparticles were investigated with different spectroscopic measurements such as UV-visible absorption (PB-10 spectrophotometer, Taiwan), photoluminescence (PL, Jobin Yvon Spex Fluorolog 3, Horiba, Japan) of 325 nm excitation wavelength, and Fourier transform infrared spectrometer (FTIR, Tensor 27, Bruker, Germany). The crystal structure and morphology of quantum dots were studied with X-ray diffraction (D8 ADVANCE XRD, Bruker, Germany) taken with an accelerated voltage of 40 kV, a current intensity of 30 mA, a Cu-Kα wavelength of 1.5406 Angstroms, and transmission electron microscopy (TEM, JEM-1400, JEOL, Japan).

Results and Discussion
Synthesis temperature, reaction time, and pH values revealed a significant impact on the optical property of CdSe dots. A brief discussion about these influences will be given, and characterization of the best synthesis condition (temperature 100°C, reaction time 4 hours, and pH 11.5) will be provided.
The reaction time effect on particle size distribution was systematically studied (from 1, 2, and 3 to 4 hours). While synthesis temperature was maintained at 100°C, four-hour synthesis exhibited the best absorption and photoluminescent spectra of high, sharp peaks and zero-approaching baselines. 2 Journal of Nanomaterials In the CdSe synthesis procedure, Se 2ions were easily oxidized during precursor preparation. To hinder that process, NaBH 4 was used to ensure that all SeO 3 2would be transformed into Se 2as described in Dong et al. ([14]): On the other hand, an excessive amount of NaBH 4 was easily hydrolyzed as well as oxidized; it then helped create an inert environment and prevent Se 2ions from oxidation.
From (1), (2), and (3), it could be expected that a considerable amount of B(OH) 3 and OHwas generated. This vigorously affected the pH value of the solution. To preserve sufficient growth conditions for CdSe quantum dots, pH values had to be adjusted subsequently from 7 to 11.5. Absorption peaks shifted to a longer wavelength when pH values increased. This could be explained by the increase in the dot growth rate when pH values increased in the same synthesis condition including time, temperature, and chemical ratio.
Luminescence intensity was likewise affected as pH values changed. CdSe quantum dot quality presented better improvement in higher pH environments. It could be justified as in higher pH values, thiol groups in MSA could efficiently form more complexes with Cd 2ions. This in turn helped to enhance luminescence intensity by suppressing the surface traps of quantum dots [14,15,18].
Existence of MSA as a capping layer of CdSe quantum dots was successfully validated by two noteworthy signals in FTIR spectra (Figure 2): an absorption band around 1639 cm -1 corresponding to vibration bonding C=O in both MSA and CdSe-MSA quantum dots and a peak at around 2571 cm -1 relating to S-H bonding, which disappeared in the CdSe-MSA case. These helped confirm MSA-CdSe bonding through the thiol (-SH) groups located on the one side of the acidic molecular structure. With two carboxyl groups facing out, the CdSe-MSA quantum dots were highly ready for further conjugation to functional biogroups. Ultimately, a broad absorption band at around 3303 cm -1 of O-H bonding ensured great hydrophilicity of coated CdSe dots.
A transmission electron image of MSA-coated CdSe quantum dots (synthesis temperature 100°C, reaction time 4 hours, and pH 11.5) was taken as shown in Figure 3. Fabricated particles presented a spherical shape with good size homogeneity of approximately 2.3 nm.
We plotted in Figure 4 both the absorption and photoluminescence spectra of a sample optimally synthesized at 100°C, pH 11.5, and a reaction time of 4 hours and finally dispersed in PBS 7.0 buffer solution.
The photoluminescence spectrum (red line) presented a high, sharp peak of narrow full width at half maximum. Both sides of the peak approached near zero values. It certified that a 4-hour reaction was optimal for the growth of quantum dots and MSA not only enhanced the luminescence intensity but also helped to passivate surface traps.
A small difference of approximately 0.096 eV existed between absorption and luminescence peaks. Such a tiny deviation verified the high quality of the synthesized quantum dots in which surface traps were inhibited and energy transfer to lattice vibration was limited.    Journal of Nanomaterials viability by more than 30% is considered a cytotoxic effect [19]. Figure 5 shows the viability of NIH-3T3 cells exposed to various concentrations of quantum dots. In accordance with a cytotoxic assay, morphological observation showed the same tendency, as shown in Figure 6. With cells exposed to and incubated in 2 × 10 2 μg/ ml CdSe-MSA or higher, the vitality ratio dropped down to 25.9%, lower than the safety threshold. Cells became smaller and rounder and turned apoptotic. Previous studies show that Cd 2+ ions are released from the surface of cadmiumbased quantum dots. Cadmium is known as a toxic element that induces several adverse effects such as the induction of oxidative stress, mitochondrial dysfunction, apoptosis, and the disruption of intracellular calcium signaling, which leads to their cytotoxic effects [20,21]. However, there was no obvious morphological change in the presence of quantum dots covered by MSA at the concentrations from 2 × 10 1 μg/ml to 2 × 10 −4 μg/ml; cell vitality ratios remained at a remarkably high level of 97.4% on average. Consequently, at a concentration of 20 μg/ml or less, cells had a great chance for survival. Taken together, we conclude that the highest concentrations of quantum dots that are nontoxic to the NIH-3T3 cell line was at 20 μg/ml.

Bioconjugation of the CD3 Antibody to MSA-Capped CdSe.
Successfully conjugated samples were characterized by photoluminescent and time-resolved fluorescent spectroscopy.
The photoluminescent spectra of MSA-coated CdSe (sample A), A/G protein-MSA-coated CdSe (sample B), and CD3 antibody-A/G protein-MSA-coated CdSe (sample C) are depicted in Figure 7. Interestingly, after connecting with a bridging protein and an antibody, sample 5 Journal of Nanomaterials C exhibited a much higher peak than samples A and B. Such big improvement could probably be explained by lower dangling bonds or traps at the quantum dot surfaces, which might lead to the enhancement of radiative recombination rate and reduction of carrier lifetime. To verify such postulation, the radiative lifetime of the three samples was characterized through time-resolved fluorescence measurements.
From time-resolved fluorescence decay curves (Figure 8), the fluorescence lifetime of these samples has been determined by fitting the decay curves with a triexponential function: where τ 1 , τ 2 , and τ 3 are the first, second, and third components of lifetimes and A 1 , A 2 , and A 3 are the corresponding relative weights of these components. The average fluorescence lifetimes for the decay curves were calculated from the decay times and the relative contribution of the components using the following equation: The fluorescence showed a triexponential decay with average lifetimes of 11.9, 11.9, and 9.1 nanoseconds for MSA-coated CdSe, A/G protein-MSA-coated CdSe, and CD3 antibody-A/G protein-MSA-coated CdSe, respectively.
A shorter radiative lifetime and low nonradiative recombination of carriers in sample C also helped to explain the strong luminescence compared with samples A and B (Figure 7). This is quite convenient and suitable for timesaving detection under fluorescent microscopy.

Jurkat T Cell Labeling
Assessment. In addition to other spectroscopic or microscopic methods, confocal laser scanning microscopy has been considered an effective tool to verify the conjugation of bioagents on quantum dots [8,10] and     Journal of Nanomaterials the successful applications of nanoparticles in the biomedical field [9,[23][24][25][26].
To evaluate cell detection capability, CD3-A/G-CdSe was mixed at a volume ratio of 1 : 3 with Jurkat T cells preserved in RPMI 1640 solution. The mixture was delivered into several microcentrifuge tubes and moderately shaken for 30 minutes. Thereafter, those tubes went through centrifugation of 1500 rounds per minute for 5 minutes to eliminate unnecessary supernatants. The final product was characterized by fluorescent microscopy as shown in Figure 9. High fluorescent intensity could be observed on the membrane of Jurkat T cells where the quantum dots were located. This led to two solid conclusions: (1) good coupling between CD3 antibody-A/G protein-CdSe and cell antigen and (2) substantial bonding between quantum dots and cells.

Conclusion
In summary, we successfully synthesized MSA-coated CdSe by a simple green method at low temperature with costsaving chemicals. The influence of reaction time, temperature, and pH values on optical property was spectroscopically characterized. Uniformly distributed 2.3 nm acid-coated quantum dots of high quality, high luminescence, and high biocompatibility were produced at 100°C, a pH value of 11.5, and a four-hour reaction. The toxicity of MSA-capped CdSe quantum dots was evaluated using the NIH-3T3 cell line (ATCC® CRL-1658™) and the MTT assay resulting in a secure threshold of 20 μg/ml. The CD3 antibody was successfully conjugated to the MSA-capped CdSe with the assistance of the A/G protein. Photoluminescence and time-resolved fluorescence were employed for verification. Lifetimes were found to decrease from 11.9 nanoseconds for MSA-coated CdSe and A/G protein-MSA-coated CdSe down to 9.1 ns for the CD3 antibody-A/G protein-MSA-coated CdSe, respectively. The final conjugated CdSe quantum dots were mixed with Jurkat T cells to evaluate detection ability. Fluorescent microscopic results evidently exhibited great coupling of the CD3 antibody and antigens and validated the high possibility of CdSe-based biosensors. The achieved products are ready for the coming steps of development.

Data Availability
The data used to support the findings of this study are included within the article and available from the corresponding authors upon request.