Fluorescent SnO₂/SiO₂ Nanoadsorbents for High‑Efficiency GST‑Tagged Protein Capture

Background

Efficient protein purification underpins advances in drug metabolism, structural biology, and vaccine development. Traditional chromatographic and precipitation methods are laborious and often compromise protein integrity. Affinity purification, exploiting the natural interaction between GST and its ligand, remains the gold standard for recombinant proteins bearing a GST tag. However, conventional GST‑affinity resins (e.g., Glutathione Sepharose 4B) suffer from limited binding capacity, high cost, and lack of intrinsic visual reporting.

Nanomaterials can overcome these limitations by offering high surface area, tunable chemistry, and, when engineered appropriately, optical signatures. SnO₂ QDs are attractive for bioapplications: they are chemically stable, biocompatible, and exhibit defect‑induced fluorescence that can be harnessed for imaging. By anchoring SnO₂ QDs onto silica nanospheres, we created a dual‑function platform that couples affinity capture with fluorescence monitoring.

Experimental

Materials and General Procedures

All reagents were analytical grade and used without further purification. Key reagents include CTAB, SnCl₄·5H₂O, TEOS, MPS, and GSH. The SnO₂ QDs were prepared via a hydrothermal method, followed by surface attachment to SiO₂‑SH NSs and subsequent GSH conjugation.

Synthesis of SnO₂ Quantum Dots

SnCl₄·5H₂O (3.5 g) was dissolved in 50 mL H₂O; ammonia (5 mL) was added to precipitate Sn(OH)₄, which was washed and re‑dispersed in 30 mL H₂O. The pH was adjusted to 12 with 2 M NH₄OH, then transferred to a Teflon‑lined autoclave and heated at 150 °C for 24 h. The resulting product was collected, washed with ethanol‑isopropanol (1:1), and dried to yield SnO₂ QDs (~5 nm diameter).

Fabrication of SnO₂/SiO₂‑SH Nanospheres

SnO₂ QDs (0.2 g) and CTAB (0.09 g) were dispersed in a water–ethanol mixture (42.5 mL + 7 mL). TEA (2.7 mL) was added, followed by TEOS (3.5 mL) and MPS (0.35 mL) under stirring at 60 °C for 5 h. The mixture was centrifuged, washed with HCl‑ethanol and water, and redispersed to produce SnO₂/SiO₂‑SH NSs (~460 nm diameter).

GSH Functionalization

SnO₂/SiO₂‑SH NSs (4 mL, 0.12 g mL⁻¹) were incubated with GSH (16.7 mg mL⁻¹) in PBS (pH 7.4) at 37 °C for 24 h. After centrifugation and washing, the SnO₂/SiO₂‑GSH NSs were stored in 25 % ethanol at 4 °C.

Affinity Capture of GST‑Tagged Proteins

GST‑tagged GPX3, OST1, and ABI2 were expressed in E. coli BL21(DE3) from pGEX‑6p1 constructs. Cell lysates (pH 7.4, 0.01 M) were mixed with SnO₂/SiO₂‑GSH NSs (1 mL) and rotated at 4 °C for 2 h. After centrifugation and washing, bound proteins were eluted with 0.5 M GSH, quantified by BCA assay, and analyzed by SDS‑PAGE.

GPX3 Activity Assay

Purified GPX3 was de‑tagged with PreScission protease and assayed for peroxidase activity by monitoring NADPH consumption at 340 nm in the presence of thioredoxin, thioredoxin reductase, and H₂O₂.

Redox State Analysis

GPX3 samples were treated with H₂O₂ and DTT, then resolved on non‑reducing 15 % SDS‑PAGE to differentiate oxidized and reduced forms.

Characterization

Morphology and composition were examined by TEM (JEM‑2010) and SEM (JSM 5600LV). XRD patterns were recorded on an X’Pert Philips diffractometer. Fluorescence spectra were measured with a FluoroSENS spectrometer (excitation 260 nm). Protein purity was assessed by SDS‑PAGE (Power PAC 300). BCA assays were performed with the Beijing CoWin kit.

Results and Discussion

Morphology, Crystallinity and Fluorescence of the Nanoadsorbents

The SnO₂ QDs displayed a monodisperse, ~5 nm spherical morphology with a lattice spacing of 0.29 nm (110) plane, confirming their high crystallinity (Fig. 1). XRD matched the cassiterite phase (JCPDS 41‑1445). The composite SnO₂/SiO₂‑GSH NSs retained a spherical shape (~430 nm) with a moderately rough surface, as seen in SEM and TEM images (Fig. 2). The presence of SnO₂ QDs on SiO₂ is confirmed by TEM (Fig. 2c,d).

Fluorescence analysis revealed a strong emission peak at 368 nm, attributable to oxygen‑vacancy defects in SnO₂ (Fig. 3b). Fluorescence imaging of the NSs after protein capture (Fig. 3c) demonstrated intact luminescence, enabling visual tracking of bound complexes.

Protein Capture Efficiency

SDS‑PAGE of GST‑GPX3 captured by SnO₂/SiO₂‑GSH NSs showed clear enrichment of the target protein (Fig. 4a). Increasing elution GSH concentrations (10–100 mM) progressively released the bound protein, confirming the specificity of the GSH–GST interaction. The nanoadsorbents exhibited negligible nonspecific binding to the bulk E. coli lysate.

Reusability tests revealed that the NSs maintained their binding capacity over three consecutive capture cycles (Fig. 4b), underscoring their robustness.

Extending the approach to other GST‑tagged proteins (OST1, ABI2) yielded comparable results (Fig. 5). Binding capacities were 7.4 mg g⁻¹ (GPX3), 7.1 mg g⁻¹ (OST1), and 6.8 mg g⁻¹ (ABI2), surpassing commercial Glutathione Sepharose 4B (7.1, 6.9, 5.6 mg mL⁻¹, respectively).

Preservation of GPX3 Redox State and Activity

After GST removal, GPX3 retained both oxidized and reduced forms, as evidenced by distinct migration on non‑reducing SDS‑PAGE (Fig. 6a). The peroxidase activity assay (Fig. 6b) demonstrated that GPX3 isolated via SnO₂/SiO₂‑GSH NSs exhibited activity comparable to that obtained with commercial resin, confirming that the nanoadsorbent preserves enzymatic function.

Conclusions

We have developed a fluorescent, silica‑protected SnO₂ quantum‑dot nanoadsorbent that captures GST‑tagged proteins with high specificity and capacity while preserving their native redox state and enzymatic activity. The platform’s robust reusability, superior binding performance, and intrinsic fluorescence make it an attractive alternative to conventional affinity resins for rapid protein purification.

Abbreviations

ABI2:

ABA insensitive 2

CTAB:

Hexadecyltrimethyl ammonium bromide

DTT:

Dithiothreitol

GPX3:

Glutathione peroxidase 3

GSH:

Reduced glutathione

GST‑tagged:

Glutathione S‑transferase‑tagged

MPS:

3‑Mercaptopropyl‑trimethoxysilane

NADPH:

Dihydronicotinamide adenine dinucleotide phosphate

OST1:

Open stomata 1

QDs:

Quantum dots

SDS‑PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

SEM:

Scanning electron microscopy

SiO₂‑SH NSs:

Thiol‑functionalized silica nanospheres

SnCl₄:

Tin (IV) chloride

TEA:

Triethylamine

TEM:

Transmission electron microscopy

TEOS:

Tetraethyl orthosilicate

XRD:

X‑ray diffraction