Characterization of CDs
The morphology of the CDs can be observed in HR-TEM images. Typical spherical CDs are presented in Fig. 1, with an average diameter of about 10 nm. DLS tests reveal that the as-prepared CDs possess a good size distribution, and the size results keep pace with those from the TEM tests. The PL of the as-prepared CDs are typically excitation-dependent due to the quantum confinement [17, 18]. With the gradual increase of excitation wavelength from 310 to 390 nm, the fluorescence intensity increases first and then decreases after reaching a maximum value, while the emission wavelength is gradually red-shifted (Fig. 2). The optimal excitation wavelength (λex) of these three CDs, as well as its corresponding maximum fluorescence emission wavelength (λem), is slightly different (G-CDs: λex = 330 nm, λem = 380 nm; H-CDs, λex = 350 nm, λem = 425 nm; L-CDs: λex = 350 nm, λem = 420 nm). All of the CDs show a relatively high fluorescence quantum yield (G-CDs: 15.69%; L-CDs: 17.19%; H-CDs: 10.32%).
PL Assay of Metal Ions
In order to evaluate the metal ion selectivity of CDs, the PL spectra of CDs were monitored in the presence of various metal ions with the same concentration, including Al(III), Ba(II), Ca(II), Cr(III), Mn(II), Pb(II), Hg(II), Ni(I), Co(II), Fe(III), Cu(II), and Ag(I). Results revealed that all of the CDs showed fluorescence fluctuation in varying degrees in the presence of various metal ions (Fig. 3). Most of the metal ions lead to the fluorescence quenching of the CDs. The response was caused by the complicated interaction between the CDs and metal ions, including electrostatic interaction and coordination effect. There exist carboxyl, amino, and hydroxyl groups on the surface of CDs [19–21]. The negative carboxyl groups of CDs can electrostatically bind with metal cations [22]. In this respect, the fact that all of the metal ions could have an effect on the CDs is easy to understand. However, the significant influence in the variation of fluorescence intensity should originate from the specific coordination effect [23–25]. Among these given metal ions, only Fe(III), Cu(II), and Ag(I) had significant effects on the CDs. Many CDs have been reported to be sensitive to Fe(III), due to their oxygen-contained functional groups coordinated with Fe(III) [26, 27]. Cu(II) or Ag(I) responsive CDs have also been reported in some research, respectively [28–31]. However, there are hardly any literatures about the CDs sensitive to both Cu(II) and Ag(I), simultaneously. Therefore, the particular response should depend on the carbon source we chose to prepare CDs. Gly, His and Leu have been confirmed to form stable coordination complexes with Cu(II) and Ag(I) [32–37]. The CDs prepared from these three amino acids could also inherit the coordination capability.
In order to investigate the inherent relationship between the CDs and their corresponding carbon source in the determination of metal ions, we also monitored the fluorescence intensity of CDs with the variation of Cu(II) ang Ag(I) concentration (Fig. 4). With the increase of Cu(II) and Ag(I) concentration, the fluorescence intensity of CDs decreased regularly (Fig. 5). There existed a good linear relationship between the fluorescence intensity of CDs and the concentration of metal ions in the range of low concentrations. Afterwards, the fluorescence intensity of CDs tended to be constant, indicating that the binding of metal ions reached a saturated state. Comparatively speaking, the fluorescence intensity of H-CDs quenched more compared with the other two CDs, and L-CDs took the second place. The fluorescence quenching degree of CDs can reflect their sensitivity to metal ions. Therefore, among these three CDs, H-CDs are the most sensitive CDs to Cu(II) and Ag(I); L-CDs comes second. Moreover, the sensitivity of the CDs to Ag(I) and Cu(II) keeps pace with the binding ability of their corresponding amino-acid carbon source with Ag(I) and Cu(II). The binding ability between amino acids and metal ions can be evaluated through calculating their binding energy, which have been reported in some literatures [38–40]. Table 1 shows the binding energies of different amino acids to silver and copper [13], and the binding energy is Gly < Leu < His.
Table 1
Relative binding energies (kcal/mol) of amino acids to silver(I), copper(II) and the proton[13]
Amino acid
|
△GoAg[a]
|
△HoCu
|
△GB
|
Gly(G)
|
0.0
|
0.0
|
0.0
|
Ala(V)
|
1.4 ± 0.0
|
1.7
|
3.7
|
Val(V)
|
2.4 ± 0.2
|
3.7
|
5.8
|
Leu(L)
|
2.5 ± 0.1
|
4.1
|
6.8
|
Ile(I)
|
2.8 ± 0.0
|
4.3
|
7.6
|
Asp(D)
|
3.0 ± 0.2
|
5.0
|
5.4
|
Ser(S)
|
3.2 ± 0.4
|
3.1
|
6.8
|
Thr(T)
|
4.6 ± 0.1
|
4.6
|
8.8
|
Glu(E)
|
4.6 ± 0.1
|
7.2
|
6.4
|
Pro(P)
|
5.0 ± 0.1
|
4.8
|
8.1
|
Asn(N)
|
8.3 ± 0.3
|
6.7
|
9.4
|
Phe(F)
|
9.5 ± 0.3
|
8.0
|
8.8
|
Tyr(Y)
|
9.6 ± 0.1
|
8.3
|
9.6
|
Gln(Q)
|
10.7 ± 0.2
|
9.8
|
11.4
|
Met(M)
|
13.1 ± 0.1
|
10.4
|
11.8
|
Trp(W)
|
14.5 ± 0.2
|
11.5
|
15.0
|
His(H)
|
18.0 ± 1.0
|
13.3
|
23.1
|
Lys(K)
|
19.8 ± 1.2
|
>13.3
|
23.6
|
Arg(R)
|
>26.8
|
>13.3
|
36.9
|
[a] △G°Ag ≈ △H°Ag except for His, Lys and Arg; uncertainties show standard deviations of 5 measurements; Teff = 902 ± 26 K.
|
Fluorescence Quenching Mechanism
In order to investigate the mechanism of the metal-ion induced fluorescence quenching of CDs, the fluorescence lifetime (FL) of the CDs was monitored before and after adding Cu(II) (Fig. 6). The fluorescence decay curves of three CDs can be fitted by a single-exponential formula respectively with a similar lifetime (FLG−CD = 3.2567 ns; FLL−CD = 3.4580; FLH−CD = 2.8621). The FL of the CDs almost stayed unchanged in the presence of Cu(II), indicating a time-independant mechanism. Therefore, a static quenching mechanism could account for the response behavior of the CDs to Cu(II) [41, 42]. A nonfluorescent ground-state complex should be formed between the CDs and metal ions, which immediately returns to the ground-tate without emission of a photon after absorbing light.