Restoring Luminescence of Aged CdSe/ZnS‑Cys Quantum Dots via TPPS4‑Induced Disaggregation

Background

Colloidal semiconductor nanocrystals, or quantum dots, combine intense broadband absorption with narrow, size‑dependent photoluminescence and exceptional thermal and photostability, making them attractive for a range of applications from medical imaging to solar fuels (1, 2). Functionalizing QD surfaces with organic ligands enhances water solubility, reduces cytotoxicity, and confers biocompatibility, enabling targeted labeling of cellular structures (8). The high‑intensity, narrow emission bands of QDs also serve as efficient light antennas for photosensitizers (PS) in photodynamic therapy (PDT), improving light energy capture and PS activation efficiency (7, 11). Consequently, QD–PS hybrids are a focus of research in fluorescence diagnostics (FD) and photochemotherapy (PCT) (9, 10).

Cysteine‑coated CdSe/ZnS QDs (QD‑Cys) are particularly appealing because of their small hydrodynamic size (~3 nm core diameter), high photoluminescence QY (~0.75), and low nonspecific adsorption (12, 13). The synthetic porphyrin meso‑tetrakis (p‑sulfonato‑phenyl) porphyrin (TPPS₄) is a water‑soluble, non‑toxic photosensitizer already tested in clinical photodynamic therapy trials (14, 15). Prior work has shown that energy or charge transfer between TPPS₄ and QDs typically quenches QD emission (16), while aggregation of QDs through electrostatic or hydrogen‑bond interactions can also self‑quench their fluorescence (17). Here we report, for the first time, that TPPS₄ can actually enhance the luminescence of aged QDs by promoting disaggregation.

Experimental

Preparation of (CdSe/ZnS)-Cys Quantum Dots

The CdSe/ZnS core–shell QDs (~3.0 mg) were synthesized following an adapted procedure (18) that involves hot‑injection growth of a CdSe core followed by ZnS shell epitaxy. To introduce cysteine ligands, the hydrophobic QDs were purified by threefold chloroform/methanol washing, then re‑dispersed in chloroform (1.0 mL). Dropwise addition of DL‑cysteine (30 mg mL⁻¹, 200 mL) in methanol, vigorous mixing, and centrifugation (10,000 rpm, 5 min) removed excess solvent. Subsequent methanol washes (16,000 rpm, 10 min, 3×) eliminated unbound cysteine, after which the QD precipitate was dried under vacuum and re‑dispersed in Milli‑Q water with 1 M NaOH, filtered through a 0.02 µm syringe filter (Anotop 25 Plus, Whatman).

Preparation of Porphyrin + (CdSe/ZnS)-Cys QD Samples

TPPS₄ (Midcentury Chemicals, USA) was used as received. Experimental solutions were prepared in phosphate buffer (pH 7.3, 7.5 mM) using Milli‑Q water. For aged QDs (stored at 276 K for 3 months), aliquots of a 140 µM TPPS₄ stock were added to the QD solution without dilution. Control experiments involved replacing equivalent volumes with buffer. All measurements were performed at 297 K.

TPPS₄ concentration was monitored by absorbance at 515 nm (ε_515 nm = 1.3 × 10⁴ M⁻¹ cm⁻¹ [19]). The aged QD concentration was calculated from the first excitonic peak at 520 nm using Yu’s empirical relation (ε = 5857 D^2.65) with D derived from the polynomial fit (Eq. 1). For λ = 520 nm, D = 2.6 nm and ε = 7.4 × 10⁴ M⁻¹ cm⁻¹.

Instruments

Absorption spectra were recorded on a Beckman Coulter DU640 spectrophotometer. Steady‑state photoluminescence was measured on a Hitachi F‑7000 spectrofluorimeter (λ_ex = 480 nm, λ_em = 558 nm). The aged QD QY was determined by the relative method using C12‑NBD‑PC (QY = 0.34 in ethanol) as standard (21–23) via Equation (2).

Time‑resolved decay curves were obtained with a time‑correlated single‑photon counting setup: a Tsunami 3950 titanium‑sapphire laser (8 MHz, 480 nm second harmonic) coupled to an Edinburgh FL900 spectrometer and a refrigerated Hamamatsu R3809U microchannel‑plate photomultiplier. The instrument response function (≈100 ps FWHM) provided a 12 ps time resolution. Decay data were fitted using OriginPro9, with goodness assessed by reduced χ² and residual analysis.

Dynamic light scattering (NanoBrook 90Plus) was performed at 640 nm excitation, 40 mW HeNe laser, to determine hydrodynamic diameters.

Results and Discussion

Freshly synthesized (CdSe/ZnS)-Cys QDs display a sharp emission peak at 558 nm (Fig. 1, black) with a QY of 0.75 (13, 24, 25). Upon 3‑month storage at 276 K, the emission blue‑shifts to 556 nm, broadens, and becomes asymmetric, while QY drops to 0.23 ± 0.03 (Fig. 1, red).

Normalized luminescence spectra of (CdSe/ZnS)-Cys 558 QDs in phosphate buffer (7.5 mM, pH 7.3): fresh (black, λ_max = 558 nm), aged (red, λ_max = 556 nm), and aged plus 5.0 µM TPPS₄ (blue, λ_max = 559 nm). Excitation at 480 nm.

Adding TPPS₄ to aged QD solutions produces a pronounced luminescence recovery, with QY rising to 0.75 ± 0.08 (Fig. 2a, inset). The emission band also becomes symmetric, with a red‑shift up to 559 nm, matching the fresh QD spectrum. Time‑resolved measurements fit a tri‑exponential decay (Eq. 3). The fastest component (≈100 ps) corresponds to scattered excitation light. The intermediate (τ₂) and long (τ₃) components, associated with core and shell recombination, are significantly shorter in aged QDs but recover upon TPPS₄ addition (Table 1). The relative contribution of the shell component (I₃, Eq. 4) increases with TPPS₄ concentration and parallels the QY trend, indicating that shell‑related emission is primarily restored.

a Luminescence spectra and QY of aged (CdSe/ZnS)-Cys 558 QDs (570 nM) as a function of TPPS₄ concentration. b Decay kinetics and I₃ ratio versus TPPS₄ concentration.

The observed enhancement cannot be attributed to reverse energy transfer from TPPS₄ (whose fluorescence lies beyond 600 nm) or Förster resonance energy transfer (FRET) because TPPS₄ absorbs negligibly at the excitation wavelength and its absorption spectrum remains unchanged in mixtures. Instead, the data support a disaggregation mechanism: aged QDs aggregate during storage, reducing QY; TPPS₄ binding increases the net negative surface charge, amplifying electrostatic repulsion and driving particles apart (Scheme 1). The interaction is mediated by the π‑conjugated porphyrin core, which binds metal surfaces with high affinity, overriding the electrostatic repulsion between the sulfonate groups and the negatively charged QD surface.

Scheme 1: TPPS₄ adsorption on aged (CdSe/ZnS)-Cys 558 QDs increases negative surface charge, promoting disaggregation via enhanced electrostatic repulsion.

Estimating the surface area of a 2.6 nm QD (≈145 nm²) shows that ~80 TPPS₄ molecules would be needed to cover the entire surface. However, saturation of QY and I₃ occurs at only ~2 µM TPPS₄ (≈4 molecules per QD), implying that a few bound porphyrins suffice to trigger significant repulsion. Calculated charge densities (σ) confirm this: the QD alone has σ_QD = −0.25 mV/nm², whereas a QD bound to four TPPS₄ molecules has σ_QD+TPPS4 = −1.29 mV/nm², more than five‑fold higher, resulting in >25‑fold stronger repulsion.

Consistent with the aggregation hypothesis, diluting aged QD solutions also increases QY and I₃ (Fig. 4). Dynamic light scattering shows hydrodynamic diameters of 330 ± 170 nm for aged QDs, which shrink to 25 ± 6 nm upon dilution, directly evidencing disaggregation.

a Luminescence spectra and QY of aged (CdSe/ZnS)-Cys 558 QDs at varying concentrations. b Decay kinetics and I₃ versus concentration.

Future work will explore whether TPPS₄ can pre‑emptively stabilize freshly prepared QDs during low‑temperature storage, potentially extending shelf life for biomedical applications.

Conclusions

Long‑term storage of CdSe/ZnS‑Cys QDs in aqueous media induces aggregation that diminishes both quantum yield and fluorescence lifetimes. Introducing TPPS₄ disaggregates aged QDs by augmenting surface negative charge, thereby restoring QY and shell‑related emission to fresh‑sample levels. This disaggregation effect is also observed upon simple dilution, underscoring the role of inter‑particle electrostatics. Our results provide a practical strategy to rejuvenate aged QDs, enhancing their suitability for sustained bioimaging and fluorescence diagnostics, while also offering a means to reduce particle size for improved cellular uptake.

Abbreviations

C12-NBD-PC:

1‑Palmitoyl,2‑(12‑[N‑(7‑nitrobenz‑2‑oxa‑1,3‑diazol‑4‑yl)amino]dodecanoyl)‑sn‑glycero‑3‑phosphocholine

FD:

Fluorescence diagnostics

FP:

Fluorescent probes

FWHM:

Full width at half maximum

PCT:

Photochemotherapy

PDT:

Photodynamic therapy

PS:

Photosensitizers

QD:

Quantum dots

QD-Cys:

Cysteine‑coated QD

QY:

Quantum yield

TOPO:

Trioctylphosphine oxide

TPPS₄:

meso‑tetrakis (p‑sulfonato‑phenyl) porphyrin