Phosphine‑Free Synthesis of Reabsorption‑Suppressed ZnSe/CdS/ZnS Core–Shell Quantum Dots and Their Application in Sensitive CRP Detection

Abstract

We describe a phosphine‑free, single‑pot protocol for creating ZnSe/CdS/ZnS core‑shell quantum dots (QDs) that combine type‑II and type‑I band alignments to suppress reabsorption. The resulting red‑emitting QDs achieve an impressive 82 % photoluminescence quantum yield (QY) and exceptional optical stability. Compared with conventional type‑I QDs, the composite QDs exhibit a markedly larger Stokes shift and reduced reabsorption, which translates into higher fluorescence output. Leveraging these properties, we employed the QDs as fluorescent labels in a fluorescence‑linked immunosorbent assay (FLISA) for C‑reactive protein (CRP). The assay achieved a limit of detection (LOD) of 0.85 ng mL⁻¹—15 % more sensitive than a CdSe/ZnS‑based FLISA (1.00 ng mL⁻¹). These findings demonstrate that ZnSe/CdS/ZnS type‑II/type‑I QDs are strong candidates for biomedical diagnostics and photonic applications.

Background

Semiconductor core/shell QDs possess superior optical traits—broad emission ranges, high quantum yields (QYs), and robust chemical stability—making them ideal fluorescent labels for diagnostics, imaging, and optoelectronics [1–7]. Their electronic structure falls into three categories based on band alignment: type‑I, reverse type‑I, and type‑II. Type‑I QDs confine both electrons and holes within the core, boosting radiative recombination and isolating the active core from surface defects [6–9]. However, the small Stokes shift (≈10 nm) common to type‑I QDs causes significant reabsorption, diminishing emission intensity and limiting quantitative assays [10,11]. Type‑II QDs separate electrons and holes across core and shell, producing a red‑shifted emission and a flattened first‑exciton absorption peak. This reduces spectral overlap, suppresses reabsorption, and improves quantitative detection [12–13]. ZnSe/CdS type‑II QDs span from bluish‑violet to red emission with suppressed reabsorption [13], but their Cd‑rich shells can suffer from surface‑trap‑induced quenching and biotoxicity. Coating with a ZnS outer shell both passivates the surface, enhances QY, and limits Cd leaching [14–15]. Prior syntheses of ZnSe/CdS/ZnS QDs have required toxic phosphines and multi‑step purification, with no reported biological applications.

Here we introduce a phosphine‑free, one‑pot route to produce high‑quality red‑emitting ZnSe/CdS/ZnS QDs that suppress reabsorption. The method employs a low‑toxicity Se precursor (ODE‑Se) and zinc oleate to form ZnSe cores, followed by in‑situ growth of CdS and ZnS shells without intermediate purification—facilitating scalable production. The QDs reach an 82 % QY with reduced Cd content, and their large Stokes shift plus flattened absorption profile yield low reabsorption. Finally, we demonstrate their first use as fluorescent probes in a FLISA for CRP, achieving an LOD of 0.85 ng mL⁻¹—outperforming a CdSe/ZnS reference assay.

Methods

Chemicals

All reagents were used as received: CdO (99.99 %), ZnO (99.9 %), Se (99.9 %), 1‑octadecene (ODE, 90 %), 1‑octanethiol (OT, 98 %), oleic acid (OA, 90 %), PMAO, MES, and standard laboratory solvents (paraffin oil, acetone, hexanes, methanol). Buffer constituents (NaOH, HCl, Na₂CO₃, NaHCO₃, KH₂PO₄, Na₂HPO₄, H₃BO₃, Na₂B₄O₇·10H₂O, Tween‑20) were sourced from Shanghai Sangon Co., Ltd. BSA and calf serum were from Sigma. EDC, sulfo‑NHS, and microplates were from Thermo Fisher Scientific. Mouse anti‑CRP monoclonal antibody and CRP antigen were obtained from Abcam. All chemicals were used without further purification.

Se Precursor (0.1 M)

Se (6 mmol) was dissolved in 60 mL ODE and heated to 220 °C for 180 min under nitrogen, yielding a clear yellow solution.

Zn and Cd Precursors

ZnO (30 mmol) was combined with 30 mL OA and 45 mL ODE, then heated to 310 °C under nitrogen to form a clear solution; the mixture cooled to 140 °C for injection. The Cd precursor was prepared identically, but at 240 °C and a 0.2 M concentration.

Synthesis of ZnSe/CdS/ZnS Type‑II/Type‑I QDs

A typical run began by heating 4 mL Se precursor and 15 mL ODE to 310 °C. At this temperature, 2 mL Zn precursor was rapidly injected. Aliquots were taken at intervals to monitor PL shifts linked to particle growth. Once the core reached the desired size, the temperature was lowered to 230 °C for CdS shell growth. Cd precursor and OT (OT : cation = 1 : 1.2) were then added dropwise at 3 mL h⁻¹ while raising the temperature to 310 °C. The ZnS shell was grown by the same method. QDs were purified by acetone precipitation and redispersed in chloroform.

CdSe/ZnS Type‑I QDs

These QDs were synthesized following established protocols [7] and subsequently used for phase transfer and FLISA preparation identical to the ZnSe/CdS/ZnS procedure.

Phase Transfer to Water

Polymaleic anhydride‑octadecene (PMAO) amphiphilic oligomer was used to render the hydrophobic QDs water‑soluble. QDs and PMAO were dissolved in chloroform (QD/PMAO = 1 : 7) with sonication, then chloroform was evaporated at 45 °C. An equal volume of 0.1 M NaHCO₃ (pH 8.5) was added to dissolve the complex. The resulting QDs‑PMAO retained full fluorescence and exhibited excellent aqueous stability across a wide pH range.

QDs–Antibody Conjugation

QDs‑PMAO were activated with EDC/sulfo‑NHS to link carboxyl groups to monoclonal CRP antibodies. After incubation in PBS and BSA blocking, the conjugates were washed by centrifugation and stored in 50 µL BS buffer (5 mM, pH 8.0).

Antibody‑Coated Microplate Preparation

Microplate wells were coated with 1.8 mg mL⁻¹ CRP monoclonal antibody in 50 mM carbonate‑bicarbonate buffer (pH 9.6), incubated at 4 °C for 24 h, washed three times with 0.05 % Tween‑20 in 10 mM PBS, and blocked with 0.5 % BSA in PBS (pH 7.4) overnight. Plates were dried and stored at 4 °C.

CRP Quantification by FLISA

In each well, 100 µL of standard CRP or sample (diluted in buffer) was added to the antibody‑coated plate, incubated at 37 °C for 30 min, and washed five times. Next, 100 µL of QDs‑mAb probe (diluted in 10 % calf serum/PBS) was added, incubated, and washed as before.

Characterization

UV–vis absorption and PL spectra were recorded with an Ocean Optics PC2000‑ISA spectrophotometer. QYs were measured relative to Rhodamine 101 (QY = 100 %) in ethanol. TEM imaging employed a JEOL JEM‑2010 at 200 kV. Phase analysis used a D8‑ADVANCE XRD with Cu‑Kα radiation (λ = 1.54 Å). Hydrodynamic diameters were determined by dynamic light scattering (Nano‑ZS 90, Malvern).

Results and Discussion

The evolution of UV–vis and PL spectra during shell growth is illustrated in Figure 1. The bare ZnSe core shows a 420 nm absorption and 428 nm emission (FWHM = 17 nm). Adding a single CdS monolayer expands the Stokes shift to 54 nm, with absorption at 497 nm and emission at 551 nm (FWHM = 38 nm). Subsequent CdS shell growth further red‑shifts the PL to 629 nm and broadens the FWHM to 52 nm, reflecting enhanced exciton‑phonon coupling. The flattened first‑exciton absorption peak and strong short‑wavelength absorption (<500 nm) together reduce spectral overlap, effectively suppressing reabsorption. The ZnS shell subsequently shifts the emission leftward and narrows the FWHM to 43 nm, while the QY rises from 20 % to 82 % across the CdS/ZnS growth sequence.

Figure 2 confirms the structural evolution: XRD peaks sharpen and shift to wurtzite CdS and ZnS positions, indicating successful multi‑shell growth and a zinc‑blende to wurtzite transition. TEM images (Fig. 2a‑f) reveal monodisperse spherical QDs expanding from 3.90 nm (core) to 11.92 nm (full core/shell). HRTEM lattice spacings (0.32 nm for ZnSe, 0.35 nm for CdS, 0.31 nm for ZnS) match XRD data, confirming compositional fidelity.

EDS analysis (Table S1) shows Cd/(Zn+Cd) ratios increase during CdS shell growth due to cation exchange at >200 °C, yet overall Cd content remains low (~13 %) compared to typical type‑I CdSe/CdS/ZnS QDs (~40 %). This reduced Cd load diminishes biotoxicity.

Phase transfer to aqueous media via PMAO (Fig. S1) preserves PL intensity and morphology, as confirmed by UV–vis, PL, TEM, and FTIR spectra. The FTIR spectra demonstrate successful anhydride ring opening and COOH formation on the QD surface.

Stability tests (Fig. 3) demonstrate that the hydrophobic QDs retain 85 % of PL after repeated purification, and the PMAO‑encapsulated QDs maintain >85 % PL after 400 h in PBS, across pH 1–13, and up to 90 °C (remaining 76 % PL). These results confirm suitability for biological assays.

Conjugation with CRP antibodies (Fig. 4a) reduces PL intensity by ~40 % due to separation losses but preserves spectral shape. DLS shows a hydrodynamic diameter increase from 46 nm (QDs‑PMAO) to 120 nm (QDs‑mAb), confirming successful conjugation without aggregation.

Using the QDs‑mAb in FLISA (Fig. 5a) yields a clear, quadratic dose–response (R² = 0.9991) over 0–400 ng mL⁻¹. The LOD of 0.85 ng mL⁻¹ surpasses the CdSe/ZnS reference by 15 %. Recovery experiments (Table 1) show 83.6–105.9 % accuracy across low, medium, and high CRP concentrations, underscoring assay reliability.

Conclusions

We have developed a phosphine‑free, scalable synthesis of reabsorption‑suppressed ZnSe/CdS/ZnS core/shell QDs that combine type‑II and type‑I band structures. The QDs deliver high QY (82 %), large Stokes shift, and excellent stability, enabling a highly sensitive FLISA for CRP detection (LOD = 0.85 ng mL⁻¹). These properties position ZnSe/CdS/ZnS QDs as promising tools for biomedical diagnostics and optoelectronic devices.