Time-Resolved Visual Chiral Discrimination of Cysteine Using Unmodified CdTe Quantum Dots

Herein, we demonstrate a simple yet novel luminescence assay for visual chiral discrimination of cysteine. Thioglycolic acid (TGA)-capped cadmium-telluride (CdTe) quantum dots (QDs) exposing green emission were directly synthesized in aqueous solution. The interaction between cysteine molecules and CdTe QDs induced the aggregation of QDs via hydrogen bonding. As a result of electronic coupling within these aggregates, a redshift both in the absorption and emission spectra of QDs occured. The difference in the kinetics of the interactions between L- and D-cysteine with CdTe QDs led to chiral recognition of these enantiomers. Addition of D-cysteine to CdTe QDs in a basic media caused a green-to-yellow color change, while no color alteration in QDs emission was observed in the presence of L-cysteine after 2 hours. Notably, the QDs used in the proposed assay are free from any labling/modification, which makes the present strategy highly attractive for sensing applications. Furthermore, the presented chiral assay is able to determine the enantiomeric excess (ee) of D-cysteine in the whole range of ee values (from −100% to 100%).


Results and Discussion
Time-resolved chiral recognition. TGA-capped CdTe QDs exposing green emission were directly synthesized in aqueous solution. The absorption and fluorescence spectra of the as-prepared thioglycolic acid (TGA)capped CdTe QDs are demonstrated in Fig. S1 in Supporting Information (SI). L-and D-cysteine were added to the QDs' solution and after 15 min NaOH was inserted to the mixture. Time-course emission behavior of QDs toward D-and L-cysteine is shown in Fig. 2. The emission of QDs was quenched thoroughly upon the addition of cysteine in the basic media. Recovery in fluorescence-intensity and redshift of the spectral peak were thereafter observed. In the case of L-cysteine, the fluorescence intensity reached a maximum value after 138 min with an intensity very close to the emission of the blank (solution with no cysteine, shown in Fig. S2). However, for D-cysteine, it took 78 min to obtain the maximal fluorescence intensity which was twice as high as the blank. In both cases, after reaching a maximum, fluorescence quenching and further redshift in the emission peak were observed.
As shown in Fig. 2, the presence of cysteine in solution triggered fluorescence quenching which can be attributed to the photoinduced electron transfer (PET) 31 . Unprotonated amino groups are particularly suitable as electron donors in PET mechanism 32 . Therefore, in the presence of cysteine, containing an amino unit, PET can take place between the nitrogen lone pair and QDs, causing quenching (see Fig. 3).
Due to the high binding affinity of thiol groups toward Cd atoms at the surface of CdTe QDs, cysteine can attach to CdTe through the formation of Cd-S bonds 33,34 . Therefore, after the addition of cysteine to QDs solution, cysteine was gradually bonded to the surface of QDs. It is assumed that the affinity of thiol to QDs can actually push the amino group away from the surface of QDs. Thus, considering the strong dependency of PET on the electron donor-fluorophore distance 32 , PET was suppressed and allowed the fluorescence to be switched on (see Fig. 3).
The rate of fluorescence-intensity recovery by D-cysteine was faster than by L-cysteine. In addition, a higher fluorescence-intensity was observed for D-cysteine (see Fig. 2). Compared to L-cysteine, D-cysteine exhibits a stronger interaction affinity to CdTe QDs 34 . Consequently, it is expected that a higher number of D-cysteine compounds can attach to the surface of QDs, which caused enhancement in both the rate of recovery and the emission (due to better passivation of the QDs' surface) 35,36 .
As demonstrated in Fig. 2, the redshift in the emission spectra was initiated upon the entrance of cysteine in the basic media. The presence of cysteine molecules on the surface of QDs induced the aggregation process due to the hydrogen bonding between the adsorbed thiols. Aggregation of QDs then caused a redshift in the emission spectra that can be explained by two possible reasons: (1) electronic coupling between neighbouring QDs in the aggregates which can lead to a redshift in both absorption and emission spectra; (2) exciton energy transfer between QDs in small distance which will not exhibit any redshift in the absorption spectra 37 . The observed redshift in the absorption spectra, cuased by the aggregation of QDs in the presence of cysteine (see Fig. S4), confirms that the redshift appeared in the emission spectra can be originated from electronic coupling between QDs.
Another point to be considered from Fig. 2 is that CdTe QDs displayed a gradual quenching after reaching a maximum emission intensity. In agreement with our findings, Koole et al. 37 had reported that exciton energy transfer is absent in small green QDs, while it can occur in aggregated QDs. Furthermore, a reduction in fluorescence intensity can be induced by exciton energy transfer 37 . Over time, the size and the number of aggregates increases which leads to exciton energy transfer between QDs in one aggregate, and consequently causes emission quenching. Figure 4 illustrates the peak wavelengths related to the spectra given in Fig. 2, against time. In a certain time, the amount of redshift caused by D-cysteine was more than by L-cysteine. As mentioned earlier, the aggregation of QDs caused redshift in the emission spectra. Therefore, we hypothesized that the rate of aggregation is higher in the presence of D-cysteine, rather than L-cysteine. This can be used for visual discrimination between L-and D-forms of cysteine (see inset in Fig. 4). Over time, the difference in redshifts of QDs declined for both D-and   L-cysteine. Dynamic light scattering (DLS) measurements confirm that D-cysteine mediates more aggregated QDs compared to L-cysteine (See Fig. S5). TEM images further confirmed the aggregation of QDs in the presence of cysteine (see Fig. 5). Figure 2c shows representative emitted colors from QDs over time after the addition of D-cysteine and NaOH under UV irradiation.

Fluorometric determination of enantiomeric excess.
Since it is essential to determine enantiomeric excess (i.e., the difference between the mole fractions of enantiomers) in chiral drug and asymmetric catalyst development 38, 39 , our chiral assay was applied to quantify the enantiomeric excess of cysteine. NaOH concentration, as an effective parameter on emission, was optimized. The difference between peak wavelengths of QDs' emission in the presence of D-and L-cysteine (∆λ D, L ) was monitored at different concentrations of NaOH (see Fig. S6). ∆λ D, L rised up to 60 mmol L −1 , and then no further change was observed. Therefore, we selected 60 mmol L −1 of NaOH as the optimal concentration for further experiments. The emission spectra of CdTe QDs in different enantiomeric excess of cysteine (total concentration 1 mmol L −1 ) are demonstrated in Fig. 6. The highest and lowest signals were observed for absolute D-and L-cysteine, respectively. A good linear relationship was obtained between emission signal at 530 nm and enantiomeric excess of D-cysteine.
The response of unmodified CdTe QDs to other α-amino acids. The response of the unmodified CdTe QDs in the presence of other α-amino acids including arginine, tryptophan, tyrosine, proline, and histidine was negligible regrdless to their D-and L-forms (see Fig. 7). However, the response of D-cysteine was the  highest and distinct from L-cysteine. All α-amino acids consist of a carboxylic acid and an amino functional group. However, cysteine is distinctive from the rest due to the presence of its thiol group. Considering that a chiral CdTe QD was employed in the proposed assay, the key role of the thiol group in the differentiation of Land D-cysteine is evident. In fact, both D-and L-cysteine are bonded to the surface of QDs through S-Cd bond,  but with a different strength that makes them distinguishable. The CD measurement (Fig. S7) confirmed that the applied CdTe QDs were achiral. Similarly, the CD spectra of L-and D-cysteine exhibited mirror-image profiles (see Fig. S8). Aditional CD features emerged for the mixture of QDs and cysteine, compared to pure cysteine (see Fig. S9). These features can be attributed to the formation of Cd x (cystine) y complex and to the chirality induced by the absorbed cysteine molecules on QDs surface 34 . This assumption has been experimentally and theoretically proven by Zhou et al. 34 studying CdTe QDs: since an atom with four different substituents produces a chiral center, if all four atomic positions in a tetrahedron formed by Te and Cd atoms are different, a chiral center will be formed. As they have presented, the adsorption of cysteine molecules on the surface of QDs induces four different substituents, resulting in a chiral center.

Conclusions
In summary, a fluorometric chiral recognition assay was developed for detection of cysteine using unmodified CdTe QDs. We found that the rate of QDs' aggregation was quite different in the presence of D-and L-cysteine. The chiral assay took advantage of the aggregation rate of unmodified QDs for enantioselective recognition of cysteines. The emission color variation of QDs revealed its promissing chiral detection of cysteine even by the naked eye. Moreover, this novel chiral sensor can be applied for the measurement of enantiomeric excess. Compared with other developed fluorimetric methods, the present strategy is fascinating mainly because the CdTe QDs employed do not require any labeling or modification with chiral molecules.
Instrumentation. Absorbance spectra were recorded on a Lambda spectrophotometer from Perkin Elmer using 1.0 cm glass cell. The fluorescence spectra were measured on a Cary Eclipse fluorescence spectrometer (Varian) with the use of 1 × l cm quartz cell. All the spectra were recorded at room temperature. Size distributions were obtained using Zetasizer Viscotec 802 at ambient temperature. Circular dichroism (CD) measurements were made using Jasco J-810 CD recorder.
Synthesis of TGA functionalized CdTe QDs. TGA-capped CdTe QDs were prepared according to the procedure described previously (Ghasemi et al. 2016 40 ). Typically, 2.87 mmol of TGA was added to 10 mL of CdCl 2 solution (0.29 mol L −1 ) and the pH was adjusted to 9.0 with NaOH solution. Then, the solution was stirred under argon bubbling for 5 min. 0.76 mmol of Te powder was separately mixed with 75 mL of NaBH 4 solution (64 mmol L −1 ) under argon flow at 50 °C. After 20 min, 10 mL of pH-adjusted CdCl 2 solution was added, and the temperature was increased to 150 °C. The resulting TGA-capped CdTe QDs with green emission (reaction time 45 min) were taken out and allowed to cool in ice bath.