Fabrication of Yellow-Emitting Chiral Silicon Nanoparticles and Fluorescence/Colorimetric Dual-Mode Recognition of Lysine Enantiomers together with Nanobioimaging

Abstract

Long-wavelength emission fluorescent chiral silicon nanoparticles (c-SiNPs) hold significant potential for biological imaging and complex sample analysis due to their superior optical properties. However, the synthesis of these materials remains a considerable challenge. The activity of lysine is intrinsically linked to its configuration, making it crucial to develop a rapid, sensitive, and selective method for differentiating lysine enantiomers in biochemical and biomedical fields. In this study, N-[3-(trimethoxysilyl)propyl]ethylenediamine and chlorogenic acid were innovatively employed as precursors, and the yellow-emitting c-SiNPs with an emission wavelength of 572 nm were synthesized at room temperature for the first time by adjusting experimental parameters. The obtained c-SiNPs exhibited excellent optical properties, stability, and cell compatibility. Furthermore, the c-SiNPs demonstrated outstanding fluorescence and colorimetric recognition capabilities for lysine enantiomers. Consequently, fluorescence/colorimetric dual-mode sensing methods with high selectivity and sensitivity for the recognition of lysine enantiomers were established, and the linear ranges of these methods for d-lysine were 0.050–20 and 0.10–30 mM, with detection limits of 7.5 and 17 μM, respectively. Additionally, the c-SiNPs demonstrated an ability to bioimaging d-lysine within HeLa cells. Using density functional theory to calculate the recognition mechanism and correlating this with fluorescence and ultraviolet–visible (UV–vis) absorption spectra data, it was confirmed that the recognition mechanism was associated with the Gibbs free energy, binding energy, and hydrogen bond number difference between the c-SiNPs and lysine enantiomers. The method developed in this study for preparing c-SiNPs provided a reference for synthesizing fluorescent c-SiNPs with longer emission wavelengths. Moreover, the established method for identifying lysine enantiomers holds significant guiding implications for the use of high-purity lysine.

Introduction

Silicon nanoparticles (SiNPs) have emerged as promising fluorescent nanomaterials due to their unique optical properties, low toxicity, and high water solubility. They have been extensively utilized in fields such as sensing, anticounterfeiting, bioimaging, and disease therapy in recent years.¹⁻³ With the escalating standards of research, the requirements for the luminous properties of SiNPs are becoming increasingly stringent, primarily focusing on the luminous efficiency and emission wavelength. Consequently, researchers have employed various methods such as microwave, hydrothermal, ultraviolet radiation, and room-temperature synthesis to prepare fluorescent SiNPs with varying luminous efficiencies and emission bands. Notably, SiNPs emitting long-wavelength could effectively avoid the shortcomings of sample matrix interference, biological tissue self-fluorescence, potential photodamage, and low tissue penetration.⁴ Chiral nanomaterials possess unique intrinsic properties. If SiNPs are endowed with chiral characteristics and long-wavelength emission properties, they will be of great significance in the fields of cell labeling, optical imaging, and chiral analysis. Currently, the preparation of chiral SiNPs (c-SiNPs) that emit long-wavelength radiation presents a considerable challenge due to the complex luminescence mechanism of SiNPs. In addition to our previous reports on the preparation and application of blue- and green-emitting chiral SiNPs,^2,5 Wang et al. have mixed blue-emitting silicon quantum dots with cellulose nanocrystals to prepare circularly polarized luminescent silicon nanomaterials using the evaporation-induced self-assembly method.⁶ Therefore, the preparation of long-wavelength-emitted c-SiNPs remains extremely challenging.

Amino acids, integral components of proteins, are crucial to the normal functioning of biological systems. Among these, l-lysine is one of eight amino acids that humans cannot synthesize endogenously but require for optimal health. It plays a pivotal role in human development, immune enhancement, and central nervous tissue function. Low levels of l-lysine have been linked to nutritional anemia, stunted growth, and compromised immune response.⁷ It is well known that the body cannot produce l-lysine on its own; it must rely on dietary sources or medicinal supplements containing this amino acid. Given its critical role and medicinal value, accurate quantification of the l-lysine content is paramount. d-Lysine, while not directly associated with human diseases, has applications in drug development and influences bacterial and fungal metabolites. Following the adverse “Thalidomide Incident”, the use of enantiopure lysine in food, pharmaceuticals, and precursor compounds has gained heightened importance. Consequently, there is a pressing need to develop enantioselective methods for identifying both l-lysine and d-lysine. So far, the identification of lysine enantiomers has primarily relied on electrochemical, gas chromatography, synchrotron X-ray powder diffraction, and thermogravimetric analysis.⁸⁻¹⁰ However, as detection demands have advanced, these methods have encountered challenges, such as high costs, intricate operations, expensive equipment, and lengthy procedures. Consequently, there is an urgent need to develop an efficient, economical, and rapid method for the enantioselective identification of lysine enantiomers. The fluorescence method, due to its simplicity, speed, and high sensitivity, has emerged as a more convenient technology for this purpose. In recent years, several fluorescent chiral nanomaterials, including copper nanoclusters (CuNCs), chiral carbon dots (CDs), carbon quantum dots (CQDs), and MIL-53(Al)-NH₂, have been employed for lysine enantiomers detection.¹¹⁻¹⁴ However, most of these materials suffer from complex preparation processes, poor water solubility, and significant background interference caused by short emission wavelengths. Therefore, it is crucial to synthesize fluorescent chiral nanomaterials with superior properties using a simple and gentle method and to utilize them in the efficient recognition of lysine enantiomers.

In this study, N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) and chlorogenic acid (CA) were selected as the silicon source and chiral reagent to synthesize the yellow-emitting c-SiNPs at room temperature for the first time by adjusting experimental parameters. The choice of CA as raw was influenced by several factors. First, the presence of phenolic −OH in its structure allowed CA to serve as an excellent reducing agent for nanomaterial preparation. Second, the p-π conjugation effect, which occurred between the n electrons on the phenolic −OH and the conjugated π bond in the aromatic ring, enhanced the conjugation degree of c-SiNPs. This increase in the conjugation degree promoted electron delocalization, thereby amplifying the fluorescence emission intensity of c-SiNPs. Finally, the −OH and chiral attributed of CA could be transferred to SiNPs, thereby enhancing the ability of c-SiNPs to recognize lysine enantiomers in aqueous environments.¹⁵ The obtained c-SiNPs have demonstrated superior salt tolerance, pH stability, photobleaching resistance, and cell compatibility, which offered unique benefits in chiral recognition and bioimaging. Consequently, the c-SiNPs were employed for the highly selective and sensitive dual-mode recognition of lysine enantiomers via fluorometry/colorimetry and for d-lysine imaging of HeLa cells. The established method for the identification of lysine enantiomers holds significant importance for the evaluation of lysine purity and research on chiral lysine-related drugs.

Experimental Section

Preparation of c-SiNPs

First, 20 mg of CA was added to 5.0 mL of ultrapure water and stirred at room temperature until fully dissolved. Subsequently, 0.50 mL of DAMO was added dropwise, with stirring continuing for 7.0 h. The resultant bright yellow solution was then filtered through a 0.22 μm aqueous filter membrane and placed in a 3000 Da dialysis bag for 4.0 h to obtain c-SiNPs, which were then stored in a refrigerator at 4 °C for further use. For characterization purposes, the c-SiNPs were obtained via freeze-drying.

Identification of Lysine Enantiomers by Fluorescence Method

50 μL of c-SiNPs solution was first added to 1.0 mL of Tris–HCl (50 mM, pH = 7.4) and left to stand for 1.0 h. Subsequently, 50 μL of either l-lysine or d-lysine at varying concentrations was introduced, and the mixture was shaken before being left undisturbed for 10 min at room temperature. The fluorescence emission spectra of the above system were measured under an excitation wavelength of 476 nm, an emission wavelength of 572 nm, and a slit width of 5/5 nm. The standard curve was constructed based on the relationship between F/F₀ and the concentration of the lysine enantiomer, where F and F₀ represent the fluorescence intensities with and without the presence of the lysine enantiomer, respectively. The procedure for the selective experiment was identical to the aforementioned steps. The selected potential interfering substances included l/d-methionine, l/d-threonine, R/S-mandelic acid, l/d-tartaric acid, l/d-tyrosine, l/d-tryptophan, l/d-phenylalanine, l/d-alanine, l/d-valine, l/d-isoleucine, l/d-proline, l/d-serine, l/d-cysteine, l/d-asparaginate, l/d-aspartic acid, l/d-glutamic acid, l/d-arginine, and l/d-histidine.

Colorimetric Identification of Lysine Enantiomers

A 30 μL solution of c-SiNPs, diluted by a factor of 1.75, was introduced into a 3.0 mL Tris–HCl solution (50 mM, pH = 7.4). Following a 1.0 h standing period, an additional 50 μL of either l-lysine or d-lysine at varying concentrations was added. The UV–vis absorption spectrum was subsequently determined after the mixture was shaken and left to stand at room temperature for 10 min. A standard curve was constructed using the relationship between the absorbance (A) value at 292 nm and the concentration of d-lysine.

Results and Discussion

Synthesis and Optical Properties of c-SiNPs

Temperature plays a significant role in the transformation of the configuration of chiral substances. Therefore, DAMO and CA were subjected to varying temperatures, with the fluorescence spectra of the resultant products subsequently determined. The findings indicated that the fluorescence intensity of SiNPs reached the maximum value at 45 °C, and the fluorescence intensity at 25 °C was marginally lower than at 45 °C (Figure SlA). To preserve the configuration of CA, simplify the experimental operation, enhance safety, and ensure the feasibility of synthesizing c-SiNPs in any laboratory setting, c-SiNPs were prepared at room temperature (Figure 1A). The other preparation conditions were optimized to achieve SiNPs with an optimal fluorescence intensity. Figure S1B reveals that when the volume of DAMO was 0.50 mL, the fluorescence intensity progressively increased as the mass of CA escalated from 5.0 to 25 mg but decreased when the mass of CA continued to increase. Considering cost-effectiveness, 0.50 mL DAMO and 20 mg CA were selected as the optimal quantity for SiNPs preparation in this study. The reaction time for SiNPs preparation was then investigated, and Figure S1C demonstrates that as the reaction time extended, the fluorescence of SiNPs gradually intensified, reaching equilibrium at 7.0 h. Consequently, 7.0 h was determined to be the most suitable time for SiNPs preparation. The fluorescence emission wavelength of SiNPs at varying excitation wavelengths was subsequently measured. Figure S2A indicates that the emission wavelength of SiNPs was dependent on the excitation wavelength. The excitation/emission wavelength of 476/572 nm was chosen, considering the baseline height, emission peak location, and intensity. The color coordinate (0.48, 0.51) in Figure 1B stated that SiNPs emitted yellow fluorescence, and the absolute quantum yield was 0.97%. Furthermore, a comparative experiment was conducted. Specifically, 3-aminopropyltriethoxysilane (APTES) was utilized to react with CA in order to synthesize fluorescent nanomaterials, utilizing the same reaction parameters and purification procedures as those for obtaining c-SiNPs. Next, the fluorescence spectra of the reaction products of APTES and CA were determined. Figure S2B shows that the fluorescence emission wavelengths of the nanomaterials obtained from APTES as silicon sources at varying excitation wavelengths fall within the green region. Notably, these emission wavelengths were consistently shorter than those of c-SiNPs synthesized in this work, which was a disadvantage for subsequent biological imaging applications. Consequently, DAMO was chosen as the silicon source for the preparation of c-SiNPs in this work. Both CA and SiNPs were characterized by circular dichroism spectrometer (CD), Figure 1B illustrates that CA exhibited two absorption bands at approximately 232 and 365 nm, corresponding to n–σ* and n–π* transitions within the CA structure, respectively.¹⁶ In contrast, SiNPs displayed only one absorption band at around 232 nm, indicating that DAMO interacted with CA to subsequently form c-SiNPs.

Figure 1.

(A) Diagram illustrating the preparation of c-SiNPs. (B) Circular dichroism (CD) spectra of c-SiNPs and CA (inset is the CIE pattern of the c-SiNPs).

Stability of c-SiNPs

pH, light radiation time, and ionic strength were selected as the key factors for evaluating the stability of yellow-emitting c-SiNPs. Figure S3A demonstrates that the c-SiNPs maintained excellent stability within a pH range of 4.0–10. However, under condition of strong alkalinity (pH = 11), there was a noted decrease in fluorescence intensity. These phenomena may be caused by the protonation–deprotonation of the functional groups on the surface of c-SiNPs.¹⁷Figure S3B illustrates that the fluorescence intensity of the c-SiNPs remained largely unchanged after being exposed to 476 nm irradiation light for 1.0 h. Figure S3C indicates that the fluorescence intensity of the c-SiNPs continued to be relatively stable when the concentration of NaCl reached as high as 2.0 M. To sum up, the c-SiNPs exhibited excellent stability, suggesting significant potential for practical applications.

Morphology and Structure of c-SiNPs

To confirm the successful synthesis of c-SiNPs, a transmission electron microscope (TEM) image was initially used to characterize the morphology and size. Figure 2A,2B reveals that the c-SiNPs possessed a spherical structure, incorporating elements such as C, N, O, and Si. The size of the c-SiNPs ranged from 1.5 to 2.3 nm, with an average size of 1.9 nm (inset in Figure 2A). Fourier transform infrared spectrometer (FTIR) was employed to characterize the functional group information on the c-SiNPs. As illustrated in Figure 2C, the absorption band at 3272–3481 cm^–1 corresponded to the stretching vibration of N–H and O–H bonds, while the absorption band at 2797–2994 cm^–1 was associated with the stretching vibration of the saturated C–H bond. The absorption peak at 1598 cm^–1 represented the stretching vibration of the benzene ring skeleton, 1357–1524 cm^–1 according to the stretching vibration of C–N and C–O bonds, and 984–1177 cm^–1 signified the stretching vibration of Si–O–Si bond.^2,5,17 Combined with the stretching vibration absorption band of the benzene ring skeleton at 1500–1655 cm^–1 of CA, it can be inferred that the interaction between DAMO and CA played a crucial role in the formation of c-SiNPs. Thermogravimetric analysis (TGA) was utilized to assess the stability of the c-SiNPs. As shown in Figure 2D, a temperature increase to 100.0 °C resulted in a loss of 3.4% due to water evaporation. However, when the temperature escalated from 383.8 to 521.7 °C, there was a significant loss of 28.0% due to structural mass, indicating good thermal stability of the c-SiNPs. X-ray photoelectron spectroscopy (XPS) was engaged to determine the elemental composition and chemical bond information on the c-SiNPs. Figure 2E demonstrates that these c-SiNPs primarily contain Si, C, N, and O elements, corresponding to peaks at 101.0, 284.6, 398.1, and 531.2 eV, respectively. High-resolution XPS peaks for different elements were as follows (Figure 2F–I): C 1s, 284.2 eV (C–Si), 284.6 eV (C–C/C=C), 285.0 eV (C–N), and 285.7 eV (C–OH/C–O–C); N 1s, 398.0 eV (N–C) and 398.3 eV (N–H); O 1s, 530.9 eV (O–Si) and 531.4 eV (O–C); Si 2p, 101.1 eV (Si–C) and 101.9 eV (Si–O).¹⁸⁻²⁰ The findings from both XPS and FTIR were found to be consistent, suggesting that the c-SiNPs had been successfully synthesized with a surface rich in −OH and –NH₂.

Figure 2.

(A) TEM image (inset showing the particle size distribution histogram), (B) EDX spectrum, (C) FTIR spectrum, (D) TGA curve, (E) XPS full spectrum, (F) C 1s fine spectrum, (G) N 1s fine spectrum, (H) O 1s fine spectrum, (I) Si 2p fine spectrum of the c-SiNPs.

Sensitivity and Selectivity of the Fluorescence Method for Recognizing Lysine Enantiomers

The response time of c-SiNPs to l- and d-lysine was measured before the sensitivity of c-SiNPs to identify lysine enantiomers was investigated. Figure 3A illustrates that upon the addition of either l- or d-lysine to a solution of c-SiNPs, there was an increase in system fluorescence, with the increase being more pronounced following the addition of d-lysine, reaching equilibrium within 10 min. Subsequently, varying concentrations of d-lysine (0, 0.050, 0.10, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 12, 15, 20, 25, 30, and 40 mM) were added to the solution of c-SiNPs. Figure 3B demonstrates a positive correlation between the fluorescence intensity of system and the concentration of d-lysine, with an excellent linear relationship observed in the range of 0.050–20 mM (R² = 0.997) and the detection limit of this method was calculated to be 7.5 μM based on the formula 3s/k (Figure 3D).⁵ Under identical conditions, the fluorescence response of c-SiNPs to l-lysine (0, 4.0, 8.0, 15, 20, 30, and 40 mM) was examined. Figure 3C reveals that when the concentration of l-lysine reached 40 mM, the influence on the fluorescence intensity of c-SiNPs was still minimal. Additionally, the recognition ability of the reaction products of raw materials DAMO or CA for lysine enantiomers was determined. Figure S4 indicates that neither product possessed chiral recognition ability for lysine enantiomers. Compared with the established methods, the preparation conditions of the fluorescent probe in this work were notably milder, and the detection limit was comparable to existing reports (Table 1).^7,11−14,21 Moreover, the recognition ability of c-SiNPs for 18 amino acids and other enantiomers was investigated. Figure 3E reveals that c-SiNPs exhibited preeminent recognition ability for lysine enantiomers, and the fluorescence recognition difference factor (F_D – F₀)/(F_L – F₀) was 2.0%. These results showed that the fluorescence method based on c-SiNPs had good sensitivity to d-lysine and good selectivity to lysine enantiomers.

Figure 3.

(A) Response time of c-SiNPs to lysine enantiomers. Spectral response of c-SiNPs to (B) d-lysine and (C) l-lysine. (D) Linear relationship of c-SiNPs for L- and d-lysine. (E) Recognition performance of c-SiNPs to different enantiomers at a concentration of 20 mM.

Table 1. Comparison of c-SiNPs with Other Materials for the Identification of Lysine Enantiomers.

materials	synthesis time (h)	synthesis temperature (°C)	detection wavelength (nm)	linear range (l-lysine)	LOD (l-lysine)	refs
CDs	∼4	180	440/542	0.1–700 μM	19 nM	(7)
OVA@CuNCs	∼4	85	440	10.0 μM–1.0 mM	5.5 μM	(11)
chiral CDs	4	200	<450	0–1.0 mM	3.34 μM	(12)
cCQDs	∼12	160	424	0.0–20.0 mM	0.3 mM	(13)
F-PDs	∼16	30	429/513	4–14 mM	0.28 mM	(21)
MIL-53(Al)-NH2	∼29	150	440	0.33–1.37 mM	7.52 μM	(14)
				0.33–2.12 mM (D)	12.2 μM
c-SiNPs	7.0	25	572a	0.050–20 mM (D)	7.5 μM	this work
			292b	0.10–30 mM (D)	17 μM

^aFor fluorescence method.

^bFor UV–vis method.

Sensitivity of the Colorimetry for Recognizing Lysine Enantiomers

The response of c-SiNPs to lysine enantiomers at different concentrations was assessed using UV–vis absorption spectrometry. Figure 4A illustrates that the c-SiNPs exhibited distinct absorption peaks at approximately 292 and 325 nm, which were attributed to π–π* and n–π* transitions, respectively.²²Figure 4A displays that with the gradual increase of d-lysine concentration, the UV–vis absorption peaks of c-SiNPs at ∼292 and ∼325 nm were significantly enhanced, and a good linear relationship (R² = 0.999) was obtained when the concentration of d-lysine was 0.10–30 mM, and the detection limit for this method was established at 17 μM (Figure 4C). Conversely, varying concentrations of l-lysine had a minimal impact on the UV–vis absorption peak intensity of the c-SiNPs (Figure 4B–D). Furthermore, the color of c-SiNPs remained consistent despite varying concentrations of d-lysine being added in daylight. However, an incremental color shift was observed through fluorescence imaging as the concentration of d-lysine escalated, with 488 nm serving as the excitation wavelength for the image (inset in Figure 4C). These results indicated that c-SiNPs can effectively differentiate lysine enantiomers.

Figure 4.

UV–vis absorption spectra of different concentrations of (A) d-lysine and (B) l-lysine added in the c-SiNPs solution. The linear relationship between the absorbance value and concentrations of (C) d-lysine and (D) l-lysine. The inset in (C) depicts the corresponding photographs captured under (up) daylight and (down) fluorescence imaging conditions.

Mechanism of Recognition of Lysine Enantiomers by c-SiNPs

To verify the mechanism of recognition of lysine enantiomers by c-SiNPs, density functional theory (DFT) was employed to calculate the interaction forces between CA and both d-lysine and l-lysine. CA, the primary chiral building block of c-SiNPs, was selected as a model molecule due to the structural similarities between SiNPs and carbon dots. To date, no definitive explanation for their structure has been provided. Consequently, using c-SiNPs as the basis for computational modeling may yield inaccurate outcomes. To determine the lowest energy conformations of molecules and dimers, configurational searches were conducted using Molclus published by Lu Tian.²³ The DFT calculations were performed using molecular mechanics, semiempirical methods, and DFT with the Gauss 09 program. The B3LYP-D3(BJ) functional method combined with the def2-svp basis set was utilized for geometric optimization.²⁴ Vibrational frequencies at the same energy level were calculated to ensure that there were no virtual frequencies at the energy minimum. Single point energies were computed using B3LYP-D3(BJ)/def2-TZVP, molecular structures were visualized with VMD software,²⁵ and data processing was carried out using Lu Tian’s Multiwfn software package.²⁶ Following optimization, the optimal structures of d-lysine, l-lysine, and CA were selected (Figure 5A–5C), with their optimized molecular coordinates presented in Tables S1–S3. Similarly, the configurations of dimers formed by CA and both d-lysine and l-lysine were optimized by using the aforementioned methods. Relevant molecular diagrams and coordinates are depicted in Figure 5D,5E and Tables S4–S5. A comparison of Figure 5D,5E revealed more intermolecular hydrogen bonds between CA and d-lysine than between CA and l-lysine, indicating that the force between them was stronger than that between CA and l-lysine. To illustrate the weaker interaction between CA and either l-lysine or d-lysine, the Hirshfeld divided independent gradient model (IGMH) was employed.²⁷ Different colors in Figure 5F represented varying forces and strengths. By observation of the difference in color gradient between CA and d-lysine versus CA and l-lysine, it could be inferred that the interaction between CA and d-lysine was significantly stronger (Figure 5G,H). Subsequently, the Gibbs free energy for each molecule was determined using eq 1. G_correction was derived from optimizing the molecular structure on B3LYP-D3(BJ)/def2-SVP, while the E_{singlepointenergy} was calculated on B3LYP-D3(BJ)/def2-TZVP. The computed Gibbs free energies for the different molecules are presented in Table S6. These results demonstrated that the Gibbs free energies between CA and d-lysine were more negative, suggesting a stronger interaction between them. Furthermore, the binding energy of the dimer was calculated using eqs 2 and 3, and Table S7 reveals that the binding energy of CA and d-lysine (−7.20 kJ/mol) was more negative than that of CA and l-lysine (−5.58 kJ/mol), indicating a more robust force between CA and d-lysine.

1

2

3

Figure 5.

Minimum energy configuration of (A) d-lysine, (B) l-lysine, (C) CA, (D) CA/d-lysine, and (E) CA/l-lysine. (F) sign(λ₂)ρ colored IGMH model. (G) IGMH diagrams of (G) CA/d-lysine and (H) CA/l-lysine.

In addition, Figure 3 illustrates that the introduction of the lysine enantiomer to the c-SiNPs solution resulted in a notable enhancement of the fluorescence intensity of the system when d-lysine was present, whereas l-lysine exerted minimal influence on the fluorescence intensity, indicating that the interaction force between c-SiNPs and d-lysine may be stronger. This phenomenon may be mainly attributed to the interaction between d-lysine and c-SiNPs, which caused the surface of c-SiNPs to be covered with more d-lysine, thus obstructing the free rotation of covered c-SiNPs and further improving the fluorescence efficiency of c-SiNPs. As shown in Figure 4A,4B, after adding varying concentrations of lysine enantiomers to the c-SiNPs solution, d-lysine progressively enhanced the UV–vis absorption peak at approximately 292 and 325 nm. This enhancement may be attributed to the interaction between the functional groups (C=O, −OH, –NH₂) in d-lysine and the benzene ring, −OH and NH₂ in the c-SiNPs, leading to the enlargement of the absorption peaks in the c-SiNPs due to the π–π* and n–π* transitions. In contrast, l-lysine displayed minimal impact on the absorption peak of the system, suggesting a weaker interaction between l-lysine and c-SiNPs.¹⁵ Furthermore, the UV–vis absorption spectra of lysine enantiomers and the fluorescence excitation and emission spectra of c-SiNPs were determined. As can be seen from Figure S5, the UV–vis absorption spectra of lysine enantiomers have little overlap with the fluorescence spectra of c-SiNPs, indicating that the internal filtration effect and the fluorescence resonance energy transfer mechanism can be excluded. Therefore, it was speculated that the mechanism by which c-SiNPs distinguish between d-lysine and l-lysine was related to the strong interaction between c-SiNPs and d-lysine.

Cell Imaging

As mentioned above, the superior properties of c-SiNPs made them highly suitable for use in the biological field. Consequently, the cytotoxicity of c-SiNPs to HeLa cells was initially evaluated by using the MTT assay. Figure S6 reveals that at a concentration of 1400 μg/mL, the cell activity remained above 95%, suggesting that the c-SiNPs possessed excellent cytocompatibility. In this work, HeLa cells were imaged using 800 μg/mL c-SiNPs. Figure 6A–F demonstrates a concentration-dependent increase in fluorescence intensity within HeLa cells, corresponding to an increased presence of d-lysine. This phenomenon was consistent with the d-lysine can enhance the fluorescence intensity of the c-SiNPs solution system. Therefore, it could be inferred that c-SiNPs have the potential to image extrinsic d-lysine within HeLa cells.

Figure 6.

(A–F) Confocal cell images of HeLa cells incubated with c-SiNPs and different concentrations of exogenous d-lysine (0, 1.0, 4.0, 8.0, 15, and 25 mM).

Conclusions

In this work, yellow-emitting c-SiNPs were innovatively prepared by using a facile room-temperature method. The obtained c-SiNPs demonstrated excellent water solubility, salt resistance, pH stability, photobleaching resistance, and cell compatibility. Particularly, the c-SiNPs exhibited superior fluorescence/colorimetric recognition capabilities for lysine enantiomers; that is, d-lysine could significantly increase the fluorescence intensity and UV–vis absorption peak intensity of c-SiNPs, while l-lysine had minimal impact. Consequently, fluorescence method and colorimetry were employed to identify lysine enantiomers, and the linear detection ranges for d-lysine were 0.050–20 mM and 0.10–30 mM, with detection limits of 7.5 and 17 μM, respectively. Notably, the c-SiNPs could bioimage exogenous d-lysine (concentrations ranged from 0, 1.0, 4.0, 8.0, 15, and 25 mM) in HeLa cells. The recognition mechanism of the established method was associated with the differences in Gibbs free energy, binding energy, and hydrogen bond number between the c-SiNPs and lysine enantiomers. The prepared c-SiNPs not only enriched the luminescence types of chiral SiNPs but also provided a reference for the preparation of fluorescent chiral SiNPs with a longer emission wavelength. Importantly, the constructed method for identifying lysine enantiomers provided valuable guidance for use involving high-purity lysine.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c04172.

• Materials and reagents; apparatus and characterization; MTT assay and cell imaging; normalized fluorescence intensity of SiNPs formed at different temperatures; effects of different concentrations of c-SiNPs on cell viability; molecular coordinates of l-lysine after optimization; and binding energy of dimers (PDF)

The authors declare no competing financial interest.