Molecular interaction and Temperature-induced Structural alteration of hydrophilic CdSe:CdS:ZnS Quantum Dots-Apoferritin composite

Molecular interaction and Temperature-induced Structural alteration of hydrophilic CdSe:CdS:ZnS Quantum Dots-Apoferritin composite | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular interaction and Temperature-induced Structural alteration of hydrophilic CdSe:CdS:ZnS Quantum Dots-Apoferritin composite Shikha Chaudhary, Anjali Maurya, Uddipan Das, Ravi Mani Tripathi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5318412/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2025 Read the published version in Journal of Nanoparticle Research → Version 1 posted 5 You are reading this latest preprint version Abstract The encapsulation of core-shell quantum dots (QDs) on apoferritin protein and the thermal stability of these composites have been sparingly reported. In this study, we created a quantum dot-apoferritin composite and investigated its interaction and temperature-induced structural changes. The encapsulation of mercaptopropionic acid functionalized CdSe:CdS:ZnS core-shell QDs in apoferritin was validated using a high-resolution transmission electron microscope. The increasing concentrations (0-250 ng/mL) of QDs in composite (using 0.1 mg/mL apoferritin) showed an increase in absorbance, a decrease in tryptophan fluorescence intensity, and a change in circular dichroism characteristic peaks with increasing temperatures (25 °C, 37 °C and 55 °C). HR-TEM image supports these findings, showing an increase in size (12.0±1.0 nm at 25 °C, 12.5±1.0 nm at 37 °C, and 15±1.3 nm at 55 °C) and gradual release of QDs from the core showing 6±1% (37 °C) and 68±5% (55 °C) hollow composite particles. The single particle analysis for molecular structural elucidation using the negative stain sample confirmed the encapsulation of four QD particles at 25 °C. However, it showed multiple 2D class averages at 37 °C and 55 °C. This heterogeneity in 2D class averages confirms the destabilization of this composite at 37 °C and 55 °C. The single particle analysis revealed the molten globule-like structure of the QD-apoferritin composite at 55 °C. This study revealed that QDs induced significant structural alteration in the apoferritin at a much lower temperature than its melting temperature (80 °C). Apoferritin CdSe:CdS:ZnS quantum dots circular dichroism negative stain single particle analysis HR-TEM QDs-apoferritin composite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Studying quantum dots (QDs) and protein interfaces is vital for understanding molecular-level interactions that significantly affect the conformations of secondary and tertiary protein structures and their structure-function relationships [ 1 , 2 ]. QDs, nanoscale semiconductor particles, have garnered immense attention due to their exceptional optical properties, making them valuable for clinical diagnostics, biosensing, and imaging applications [ 3 , 4 ]. However, the specific interactions between QDs and proteins, especially their impact on protein structure and function, remain less explored than other nanoparticles [ 5 ]. This knowledge gap is critical because the efficacy of QDs in biological applications depends on their favourable interactions with biomolecules [ 6 ]. Reports suggest that QDs must be hydrophilic, appropriately sized for encapsulation, and compatible with physiological conditions to function effectively in biological systems [ 7 ]. Protein nanocages, such as those derived from ferritin and viruses, naturally self-assemble into stable structures, making them ideal for studying protein-nanoparticle interactions [ 8 , 9 ]. Apoferritin, a 24-mer protein complex, has been widely investigated for its ability to encapsulate a variety of semiconductor-based QDs instead of its natural iron core [ 10 ]. Understanding the immobilization of QDs within apoferritin cages is essential for elucidating the molecular nature of these interactions [ 11 ]. Prior research has shown that PbS quantum dots can be successfully encapsulated within apoferritin cages, creating stable fluorescent composites that also provide a protective protein coating for the QDs [ 12 ]. Various analytical techniques, including transmission electron microscopy (TEM), fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS), have been used to investigate the structural and conformational changes of apoferritin upon interaction with QDs [ 13 ]. These methodologies are crucial for understanding the biophysical and structural properties of QDs-treated apoferritin at specific temperatures. Apoferritin is known for its remarkable ability to withstand high temperatures (up to 80 °C) without significantly disrupting its quaternary structure [ 8 ]. This study presents a detailed biophysical analysis of the interactions between hydrophilic core-shell CdSe:CdS:ZnS quantum dots and horse spleen apoferritin. Using a combination of spectroscopic and microscopic techniques—including CD, fluorescence spectroscopy, DLS, and high-resolution-TEM, we investigate the structural and conformational changes in apoferritin upon QD encapsulation at various temperatures (25°C, 37°C, and 55°C). Additionally, single-particle analysis via negative staining was used to elucidate the encapsulation process and the structural heterogeneity of the resulting QD-apoferritin composites at elevated temperatures. Our findings reveal that temperature plays a significant role in modulating apoferritin's structural integrity and the encapsulated QDs' stability, with potential implications for designing QD-based nanocomposites for biological applications. Material and methods Material All the chemicals for QDs synthesis and apoferritin (sigma cat no. (A-3660) were procured from Sigma USA. TEM grids were from Agar Scientific UK. Preparation of apoferritin The native horse spleen apoferritin stock solution (25 mg/mL in 50% glycerol) was diluted by 1X phosphate buffer saline (pH 7.4) to make a working solution (1.0 mg/mL), and glycerol concentration was reduced by dialysis under stirring at 400 rpm in 1X phosphate buffer saline (pH 7.4) overnight. Synthesis of CdSe:CdS:ZnS hydrophilic quantum dots The CdSe core and core-shell (CdSe:CdS:ZnS) QD were synthesized with little modification by Zhu et al. [ 14 ] and Chen et al. [ 15 , 16 ] using an indigenously designed low-cost degassing apparatus. Purified CdSe:CdS:ZnS QDs (400 µL) were suspended in 600 µL chloroform. A 50% solution of 2-MPA was added and allowed to stir for 2h at RT. The sample was centrifuged at high speed, and the supernatant was discarded. The pellet was dried in air and resuspended in 200 µL 0.1 M sodium bicarbonate solution. The bicarbonate solution containing shelled QDs was mixed with acetone (three times the volume) and pelleted by centrifugation at room temperature. The supernatant was carefully discarded, and the pellet was allowed to dry. The pellet was finally dissolved in 1.0 mL of deionized water. The absorption scan (200–600 nm), visual fluorescence under UV light, quantum dot fluorescence (Ex:350 nm; Em:400–600 nm), hydrodynamic diameter/zeta potential (dynamic light scattering DLS), TEM imaging along with EDS (energy dispersive X-ray spectroscopy) and crystallinity using selected area electron diffraction (SAED) were used to characterize the hydrophilic CdSe:CdS:ZnS core-shell quantum dots. Synthesis of QDs-apoferritin composite The purified apoferritin (0.1 mg/mL) was incubated with various concentrations of hydrophilic CdSe:CdS:ZnS QDs (12.5 to 250 ng/mL) overnight to determine the formation of composite to QDs and apoferritin. These samples were characterized by TEM and HR-TEM to confirm the incubation-induced encapsulation of QDs on apoferritin to form the QDs-apoferritin composite. Biophysical characterization of QDs-apoferritin composite at selected temperature The composite samples with increasing concentrations of QDs (12.5 to 250 ng/mL) with apoferritin (100 µg/mL) were biophysically characterized using absorbance (280 nm), intrinsic tryptophan fluorescence (Ex: 280 nm, Em: 320 nm), quantum dot fluorescence (Ex: 350 nm, Em: 590 nm), and far UV circular dichroism (250 − 200 nm) after overnight incubation at selected temperatures 25 ℃, 37 ℃ and 55 ℃. Absorbance The absorbance scans from 200–700 nm of QDs-apoferritin composite with variable QDs concentrations after overnight incubation at each selected temperature were taken using the Evolution 220 spectrophotometer (Thermo Scientific). Pure quantum dots (without apoferritin) of the same concentration were taken as blanks for each set of experiments. Fluorescence The intrinsic tryptophan fluorescence spectra (using Carry Eclipse 100; Agilent Technologies) of QDs-apoferritin composite with variable QDs concentrations at selected temperatures were taken using excitation at 280 nm and an emission scan between 300–400 nm. For the quantum dot fluorescence measurements, the same samples were excited at 350 nm and emission spectra from 400–600 nm were recorded. The slit width for excitation and emission was 5 and 10 nm, respectively. Circular Dichroism Far UV CD spectra (250 − 200 nm) of a QDs-apoferritin composite with variable QDs concentrations were taken on a Jasco A-720 spectropolarimeter. The equipment was pre-calibrated with a 0.1% d-10-camphor sulfonic acid solution. The scanning speed was 100 nm/min with a 1.0-sec response time. The measurements were carried out at 25°C, 37 °C and 55 °C using a cuvette with a path length of 0.1 cm. TEM imaging of QDs-apoferritin composite at the selected temperatures The actual size and morphology of the QDs-apoferritin composite were analyzed using negative staining TEM. These composites were deposited onto carbon-coated 3-mm copper grids using a drop-casting technique and stained with 1% uranyl acetate to enhance the contrast between the intense central quantum dot core and the lighter apoferritin shell. Images were captured on Talos S (Thermo Fisher Scientific) transmission electron microscopy operated at 200 kV. Image acquisition was done with a bottom-mounted 4 × 4 k CMOS camera. All the digital TEM micrographs were analyzed for size determination using ImageJ. Briefly, the measurement scale was set using the image scale bar, and the images were converted to grayscale. 250 individual particles were analyzed using a line descriptor extending across the diameter of the particles. EDS analysis was performed to detect quantum dots elements Cd, Se, S, and Zn. The average number of hollow apoferritin and composite were manually counted with 10 random images to report the % release of QDs at 37 °C and 55 °C compared to 25 °C. HR imaging of QDs-apoferritin composite at the selected temperatures A TEM specimen was prepared by drying each quantum dot-apoferritin composite solution on a carbon-coated copper (300 mesh) grid. The prepared TEM specimen was imaged using a Talos S (Thermo Fisher Scientific USA) microscope equipped with 200 kV. A fast Fourier transform (FFT) simulation and a strain analysis with the HR-TEM images were performed in a digital micrograph (Ceta, Thermo Fisher). This method involves filtering the image with an asymmetric filter at a Bragg spot of the Fourier transform simulation of the HR-TEM lattice image and performing an inverse Fourier transform simulation. This method can provide useful information on the local displacement of interesting regions in a given direction. Single particle analysis of QDs-apoferritin composite at selected temperature using the negatively stained sample Talos S 200 transmission electron microscope alignments were performed using previously standard methodologies, including determining parallel illumination using a long diffraction camera length. 2D images of negatively stained quantum dot-apoferritin composite were collected using a Talos S 200 transmission electron microscope (Thermo Fisher Scientific) with a field emission gun operating at an accelerating voltage of 200 kV and equipped with a 4Kx4K CMOS detector (Ceta, Thermo Fisher Scientific). All EM data were acquired using the EPU software (EPU 4.0, Thermo Fisher Scientific). Images of quantum dot-apoferritin composite were collected in counting mode (0.117 Å/pixel) and super-resolution mode (0.059 Å/super-resolution pixel), respectively, at a magnification of 120,000x over a defocus range of − 0.5 µm to − 1.0 µm. Images were obtained using an exposure rate of 577 e − /pixel/s for 1.0 s, resulting in a total exposure of ∼71333.84 e/nm 2 . Further details can be found in Table 01 . Single-particle image processing and 3D reconstruction The single particle image processing was done using cryoSPARC ver 4.5.3. A total of 1464 (25 °C), 1278 (37 °C), and 1778 (55 °C) micrographs were imported, and contrast transfer function (CTF) was estimated using patch CTF estimation followed by automatic particle picking using a Blob picker. The coordinates were extracted with a 200-pixel box size (2x binning) and subject to several rounds of 2D classification to remove junk particles. The clean particles were reextracted without binning and subjected to ab initio reconstruction followed by non-uniform refinement. The initial models for all three conditions (QDs-apoferritin composite at 27 °C, 37 °C, and 55 °C) were generated using Chimera software. Results TEM imaging of apoferritin, quantum dot and QDs-apoferritin composite Apoferritin exhibited a well-defined distribution and a hollow core structure with a non-crystalline fast Fourier transform (FFT) pattern in TEM imaging when not encapsulated with QDs (Fig. 1 A). The CdSe:CdS:ZnS hydrophilic quantum dots were uniformly distributed in negative staining, with peaks for Cd and Zn detected in the EDS analysis ( Fig. 1 B ). In contrast, the CdSe:CdS QD-apoferritin composite displayed higher contrast within the core. HR- TEM images of the core revealed the atomic arrangement of the quantum dots' crystallinity, which was also confirmed by selected area electron diffraction (SAED) ( Fig. 1 C ). These observations suggest successful encapsulation of the quantum dots within the apoferritin, while the crystalline structure of the quantum dots remains intact. Apoferritin was amorphous, as indicated by the lack of crystalline patterns in the SAED analysis. Biophysical characterization of QDs-apoferritin composite at various temperature The absorbance of the apoferritin-QD composite at 280 nm increased with higher concentrations of quantum dots at both 25°C and 55°C, using similar QD concentrations as a blank. However, at 37°C, the composite's absorbance was lower compared to 55°C at higher QD concentrations ( Fig. 2 A ). The intrinsic fluorescence emission spectra of the apoferritin-QD composite showed higher intensity at 25°C (220–250) and lowest intensity at 37°C (100–120). Fluorescence intensity at 55°C was intermediate between that observed at 25°C and 37°C. Variations in QD concentration did not significantly affect fluorescence intensity. Additionally, wavelength shifts were negligible across all temperatures and QD concentrations ( Fig. 2 B ). The fluorescence intensity of QDs (excitation: 350 nm, emission: 550–650 nm, peak maximum around 590 nm) increased with higher QD concentrations at both 25°C and 37°C. Conversely, fluorescence intensity decreased at 37°C and 55°C compared to 25°C ( Fig. 2 B & C). The CD of the QDs-apoferritin composite at 25 °C, and all the concentrations of QDs showed a similar α-helical structure with two prominent negative peaks at 222 and 208 nm. The ellipticity was around the − 20 to -22 deg.cm.deci.mole at 222nm, and − 18 to -19 deg.cm.deci.mole at 208 nm. However, the shape of the peaks was distorted, and 208 nm peaks disappeared at 37 °C with all the concentrations of the QDs. This showed the appearance of peaks at 215 nm, while the 222 nm peaks remained intact with ellipticity around − 18 to -20 deg.cm.deci.mole. The heating of the composite at 55 °C revealed a reduction in the ellipticity of around − 15 to -18 deg.cm.deci.mole at 222 nm with a similar shape as 37 °C. The 208 nm peaks disappeared at 37 °C and 55 °C ( Fig. 2 C ). CD and fluorescent analyses revealed that the secondary structures were kept almost unchanged upon thermal treatment of QD-apoferritin composite between 25 °C and 37 °C but showed molten globule-like structure at 55 °C ( Fig. 2 ) . TEM imaging of quantum dot-apoferritin composite at the selected temperatures TEM image at a higher resolution of the QDs-apoferritin composite showed structured QD core in nearly all the composite particles (~ 100%) at 25 °C but showed the slight release of QDs from the composite (6% QDs release from core) to form a hollow core at 37 °C ( Fig. 3 A ) . The composite at 55 °C treated sample showed the release of most of the QDs from the core (68% QDs release from core) ( Fig. 3 B ) . The average size of the composite was 12.0±1.0 nm and 12.5±1.0 nm, and 15.0±1.3 nm at 25 °C, 37 °C, and 55 °C treated samples respectively ( Fig. 3 C ) . EDS and SAED imaging also confirm the gradual reduction of the QDs in the core with increasing temperature ( Fig. 3 ) . HR imaging of these particles also showed the presence of QDs incubated overnight at 25 °C, while they were irregular at 37 °C and very rare at 55 °C temperature samples ( Fig. 4 A-C ) . Structural analysis of QDs-apoferritin composite at different temperatures using negative stain single-particle analysis To further investigate the quantum dot-apoferritin interaction at high resolution, we conducted single particle analysis of negatively stained samples containing 100 µg/mL apoferritin and 250 ng/mL QDs. This provided a high-contrast visualization of QDs-apoferritin composite structures to explore the structural effects of encapsulating quantum dots (QDs) within apoferritin shells under varying thermal conditions. The analysis was performed under three temperature conditions: 25°C, 37°C, and 55°C, with overnight incubation followed by quick drop-casting onto a grid for negative staining. This analysis revealed a clear temperature-dependent effect on the structural integrity of the QD-apoferritin composite. This technique, which provides high-contrast visualization of protein structures, was employed to assess the structural changes of quantum dots encapsulated within apoferritin shells under different thermal conditions ( Fig. 5 ) . 25°C The QD-apoferritin conjugates exhibit notable structural integrity at this temperature, as evidenced by the 2D class averages and 3D models. The single-particle analysis reveals that the apoferritin structure remains largely intact at this lower temperature. The encapsulated CdSe QD is observed to be well-enclosed within the protein shell. The 2D class averages and 3D reconstructions indicate minimal distortion or unfolding in the protein cage. The apoferritin-QD complexes exhibit a homogeneous and symmetrical appearance, suggesting that the structural stability of the complex was preserved at this temperature. The conjugates maintain a defined and stable structure with minimal distortion. The analysis at this temperature provides a baseline for comparing structural changes at elevated temperatures ( Fig. 5 A ) . 37°C There were noticeable structural changes observed in the conjugates and partial deformation of the apoferritin shell at their physiological temperature (37°C). The 2D and 3D models reveal a partial reorganization or movement of the apoferritin subunits. Despite these alterations, the structural integrity of the composite was only slightly compromised, with the quantum dot core remaining shielded by the apoferritin shell. This indicates a moderate tolerance of the complex to temperature at this level ( Fig. 5 B ) . 55°C At 55°C, significant structural alterations in the QD-apoferritin conjugates were detected. The 3D model shows substantial disassembly of the apoferritin structure, which could be attributed to denaturation or thermal unfolding. The 3D reconstructions of these particles reveal disordered and fragmented protein cages. The high temperature impacts the apoferritin shell and the QD core, leading to an unstable conjugate structure. Encapsulated QDs are less uniformly enclosed and, in some cases, appear to be exposed due to the disassembly of the protein shell. This suggests the encapsulation system is compromised at higher temperatures, leading to potential instability in functional applications ( Fig. 5 C ) . Discussion The use of quantum dot nanoparticles (QDs) in medicine, drug delivery systems, biosensors and diagnostic devices is increasing exponentially [ 17 – 19 ]. The compositions and surface chemistry of QDs play a key role in a physiological environment by interacting with nucleic acids, lipids, and protein molecules [ 5 ]. Standard techniques such as absorbance, fluorescence, and circular dichroism (CD) provide some insights into QD-protein interactions but may not fully capture the molecular-level details. However, by combining these techniques with high-resolution transmission electron microscopy (HR-TEM) and single-particle analysis, we can better understand the interactions between QDs and apoferritin nanoparticles. Apoferritin was selected for this study due to its known capacity to encapsulate various nanoparticles and its established use as a model for structural evaluation via single-particle analysis [ 10 ]. The CdSe:CdS:ZnS core-shell quantum dots used in this study are highly fluorescent and are commonly employed in biological systems [ 20 ]. Being hydrophilic, these QDs were encapsulated by apoferritin through simple overnight incubation at room temperature (RT), as confirmed by TEM and HR-TEM analyses ( Fig. 1 ). TEM images of hollow apoferritin revealed an empty, low-contrast core, showed no atomic lattice of QDs under HR-TEM and selected area electron diffraction (SAED) pattern. These results also confirm the absence of crystalline material in the core ( Fig. 1 A ) . In contrast, the TEM images of QDs alone displayed sizes ranging from 5 to 8 nm with well-defined crystalline atomic arrangements, confirmed by HR-TEM and SAED analysis ( Fig. 1 B ) . Overnight incubation validated QD encapsulation, revealing a more contrast-rich core with crystalline QDs, a non-crystalline apoferritin shell under HR-TEM, and a diversified SAED pattern. ( Fig. 1 C ) . This encapsulation process is unique to CdSe:CdS:ZnS QDs because the encapsulation of other QDs (e.g. PbS QDs, etc.) and nanoparticles typically requires apoferritin disassembly via acidic pH or chemical conjugation [ 12 , 21 – 23 ]. For optimal composite formation, equimolar concentrations of apoferritin (100 µg/mL) and QDs (ranging from 12.5 ng to 250 ng/mL) were tested, with 250 ng of QDs for efficient encapsulation. It was found that the 250 ng QDs showed the equivalent encapsulation of 100 µg of apoferritin ( Fig. 1 ) . Temperature is well-known to affect protein structure, potentially leading to aggregation via misfolding or unfolding [ 10 ]. Comparing the impact of thermal treatment on QD-apoferritin composites across different temperatures (25°C, 37°C, and 55°C) provides insight into how QD encapsulation influences the stability of apoferritin. We applied the thermal treatment overnight such as 25°C (for native room temperature study), 37°C (for physiological temperature study) and 55°C (for extreme temperature study), to study and compare the impact of these temperatures on QDs-apoferritin composite. Absorbance measurements (280 nm) revealed a gradual increase in absorbance with higher QD concentrations at both 25°C and 55°C. At the same time, it decreased at an initial concentration of QDs (in between 0–50 ng/mL QDs) and was stable at the higher concentration of QDs at 37°C ( Fig. 2 A ) . Prior studies have shown that apoferritin’s absorbance remains unchanged below 65°C [ 24 – 26 ]. This suggests that changes in absorbance in this study may be due to QD-induced structural alterations in apoferritin. The strong absorption of radiation by QDs at 280 nm likely leads to localized heating, resulting in elevated temperatures for individual composite molecules (Supplementary Fig. 1). Intrinsic fluorescence emission from tryptophan (Trp) residues in apoferritin is sensitive to the microenvironment around the fluorophore. Trp exhibits a strong fluorescence peak at 320 nm when excited at 280 nm. The fluorescence intensity was unaffected by QD concentration but varied significantly with temperature. It was highest at 25°C (~ 240 U), decreased at 55°C (~ 190 U), and was lowest at 37°C (~ 110 U) ( Fig. 2 B ) . This suggests that while QDs did not affect the intrinsic tryptophan fluorescence (ITF) spectra, temperature had a significant impact. Meanwhile, quantum dot fluorescence (Ex 350 nm, Em 590 nm) decreased progressively with increasing temperature (Fig. 2 C), consistent with previous reports showing reduced fluorescence of hydrophilic QDs at higher temperatures ( Fig. 2 C ) [ 20 ]. Circular dichroism (CD) measurements revealed a slight decrease in ellipticity with increasing temperature (~-22 deg.cm.deci.mole at 25°C, ~-20 deg.cm.deci.mole at 37°C, and ~ 18 deg.cm.deci.mole at 55°C). Far-UV CD spectra at 25°C displayed typical alpha-helical features with prominent peaks at 222 and 208 nm, but the shape became more symmetrical at 37°C and 55°C, with the 208 nm peak diminishing ( Fig. 2 D ) . This finding is notable, as native apoferritin’s Far-UV CD spectra are known to remain stable up to 80°C [ 26 ]. These results indicate that QD encapsulation promotes unfolding at lower temperatures, leading to molten globule-like structures in the QD-apoferritin composite. TEM analysis after overnight incubation at 25°C, 37°C, and 55°C showed that the composite maintained a discrete protein cage morphology at 25°C and 37°C, with exterior diameters of ~ 12.0 ± 1.0 nm and ~ 12.5 ± 1.0 nm, respectively ( Fig. 3 A and B) . These sizes are consistent with previously reported ferritin nanocages [ 25 , 27 ]. However, at 55°C, the composite size increased by 25% (15.0 ± 1.3 nm), as confirmed by TEM analysis ( Fig. 3 C ) . DLS analyses also support the finding about the increase in the size of these particles at 55°C ( Fig. 3 inset) . Encapsulation efficiency was approximately 100% at 25°C but dropped to 94% at 37°C due to the temperature-induced release of QDs. At 55°C, encapsulation efficiency plummeted to 32%, similar to iron release from ferritin at elevated temperatures [ 28 ]. Single-particle negative stain analysis provided further evidence of the thermal sensitivity of QD-encapsulated apoferritin. The data indicate that while apoferritin remains stable at moderate temperatures, it undergoes significant structural degradation at higher temperatures. This denaturation is critical for the potential use of QD-apoferritin composites in biological or industrial applications. At 25°C, the particles are well-encapsulated and spherical, but at 55°C, many particles exhibit irregular shapes or significant structural disruptions. The 2D class averages for each temperature condition reflect these changes, showing progressively disordered structures as temperature increases ( Fig. 4 A-C ) . The marked structural disintegration observed at 55°C underscores a limitation of the QD-apoferritin conjugate system under thermal stress. The 3D models generated at different temperatures reveal a loss of structural rigidity, with severe compromise at 55°C ( Fig. 5 A-C ) . The comparative analysis of the QDs-apoferritin conjugates at different temperatures indicates a strong correlation between temperature and structural stability. This thermal instability is an important consideration for future studies on the thermal management of quantum dot conjugates in various applications. Conclusions At lower temperatures (25°C and 37°C), apoferritin effectively encapsulates CdSe:CdS:ZnS core-shell quantum dots (QDs), maintaining structural integrity and protecting the QDs from external factors. The preservation of the spherical shape and encapsulated core under these conditions suggests that apoferritin can reliably function at room and physiological temperatures. However, at higher temperatures (55°C), thermal motion leads to significant unfolding and destabilization of the apoferritin structure. This exposes the encapsulated QDs, compromising the protective barrier function of the apoferritin shell. Such structural degradation could limit the performance of QD-apoferritin conjugates in applications that require thermal stability, such as in vivo biological systems or environments with fluctuating or elevated temperatures. These findings offer critical insights into the limitations and potential of QD-apoferritin conjugates, highlighting the need for further research to enhance their thermal stability. Declarations Conflict of Interest: All authors declare no conflict of interest. Credit authorship contribution statement SC performed the experiments for biophysical characterization, TEM imaging and single particle analysis. AM synthesized the hydrophilic QDs. UD analyzed the single particle analysis data. RMT provides suggestions for writing some parts of the manuscript. SCY designed the study, performed and supervised experiments, analyzed the data, generated the figures, wrote the manuscript, and directed the project. All authors read and approved the final draft of this manuscript. Funding Statement This work was supported by grants from the DBT ( BT/INF/22/SP44285/2021 ). Other funding from Indian Council of Medical Research (ICMR), New Delhi (EM/SG/Dev.Res/126/2842 − 2023 (E.O -169927) has been acknowledged. Author Contribution S.C. performed the experiments for biophysical characterization, TEM imaging and single particle analysis. A.M. synthesized the hydrophilic QDs. U.D. analyzed the single particle analysis data. R.M.T. provides suggestions for writing some parts of the manuscript. S.C.Y. designed the study, performed and supervised experiments, analyzed the data, generated the figures, wrote the manuscript, and directed the project. All authors read and approved the final draft of this manuscript. Acknowledgement We acknowledge the Electron Microscopy Facility, SAIF-AIIMS, for providing the TEM and Zeta Size facility analyzers. The Department of Biophysics AIIMS New Delhi and the CIF facility, School of Biotechnology JNU, were acknowledged for providing the CD facility. We acknowledge Mr. Sunil Kumar's help with fluorescence data collection. Funding from the DBT (BT/INF/22/SP44285/2021) and ICMR (EM/SG/Dev.Res/126/2842-2023 (E.O -169927) was acknowledged. Data Availability Datasets are available at a reasonable request to the corresponding author. References Kurylowicz, M.;Paulin, H.;Mogyoros, J.;Giuliani, M.; Dutcher, J. R. 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I.;Li, M.;Mann, S.;Patane, A.; Thomas, N. R. Aqueous Near-Infrared Fluorescent Composites Based on Apoferritin-Encapsulated PbS Quantum Dots. Adv. Mater. 2008, 20 , 3592–3596. Li, L.;Mu, Q.;Zhang, B.; Yan, B. Analytical strategies for detecting nanoparticle-protein interactions. Analyst 2010, 135 , 1519–1530. Zhu, C. Q.;Wang, P.;Wang, X.; Li, Y. Facile Phosphine-Free Synthesis of CdSe/ZnS Core/Shell Nanocrystals Without Precursor Injection. Nanoscale Res Lett 2008, 3 , 213–220. Chen, D.;Zhao, F.;Qi, H.;Rutherford, M.; Peng, X. Bright and Stable Purple/Blue Emitting CdS/ZnS Core/Shell Nanocrystals Grown by Thermal Cycling Using a Single-Source Precurso. Chem. Mater. 2010, 22 , 1437–1444. Li, J. J.;Wang, Y. A.;Guo, W.;Keay, J. C.;Mishima, T. D.;Johnson, M. B.; Peng, X. Large-scale synthesis of nearly monodisperse CdSe/CdS core/shell nanocrystals using air-stable reagents via successive ion layer adsorption and reaction. J Am Chem Soc 2003, 125 , 12567–12575. Abdellatif, A. A. 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Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery. Nat Protoc 2021, 16 , 4878–4896. Wong, K. K. W.; Mann, S. Biomimetic synthesis of cadmium sulfide-ferritin nanocomposites. Advanced Materials 1996, 8 , 928. Stefanini, S.;Cavallo, S.;Wang, C. Q.;Tataseo, P.;Vecchini, P.;Giartosio, A.; Chiancone, E. Thermal stability of horse spleen apoferritin and human recombinant H apoferritin. Arch Biochem Biophys 1996, 325 , 58–64. Theil, E. C.;Liu, X. S.; Tosha, T. Gated Pores in the Ferritin Protein Nanocage. Inorganica Chim Acta 2008, 361 , 868–874. Yang, R.;Tian, J.;Liu, Y.;Yang, Z.;Wu, D.; Zhou, Z. Thermally Induced Encapsulation of Food Nutrients into Phytoferritin through the Flexible Channels without Additives. J Agric Food Chem 2017, 65 , 9950–9955. Lv, C.;Bai, Y.;Yang, S.;Zhao, G.; Chen, B. NADH induces iron release from pea seed ferritin: a model for interaction between coenzyme and protein components in foodstuffs. Food Chem 2013, 141 , 3851–3858. Hoppler, M.;Schonbachler, A.;Meile, L.;Hurrell, R. F.; Walczyk, T. Ferritin-iron is released during boiling and in vitro gastric digestion. J Nutr 2008, 138 , 878–884. Table 1 Table 1: Negative stain single-particle data collection and processing parameters of QD-apoferritin composite at different temperatures Data Collection Parameters 25 ℃ 37 ℃ 55 ℃ Voltage (kV) 200 (Talos S) 200 (Talos S) 200 (Talos S) TEM magnification 120000x 120000x 120000x Exposure navigation Stage Stage Stage Detector BM-Ceta BM-Ceta BM-Ceta Defocus range (µm) 1.0 1.0 1.0 Grid Type 300 mesh carbon coated copper grids 300 mesh carbon coated copper grids 300 mesh carbon coated copper grids Sample concentration 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL C2 aperture size (μm) 150 150 150 Objective aperture size (μm) 70 70 70 Data collection software EPU 4.0 EPU 2.12 EPU 2.12 Gun type 200 kV Schottky 200 kV Schottky 200 kV Schottky Cs (mm) 2.7 2.7 2.7 Energy filter None None None Specimen holder Single tilt Single tilt Single tilt Exposure time (s) 1 1 1 Dose per second (e - /Å 2 /s) 71 63 63 Cumulative Exposure (e − Å −2 ) 71.33 81 73 Pixel size (Å)* 0.8 0.7 0.88 Exposure per frame (s) 1 1 1 Particles per micrograph (avg.) 70 200 30 Acquired Micrographs 1464 1278 1778 Micrographs Used 1200 1185 1608 Total extracted particles (no.) 102338 237000 53340 Refined particles (no.) 53,891 167000 33340 Reconstruction Final particles (no.) 53,891 167000 33340 Symmetry imposed C1 C1 C1 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2025 Read the published version in Journal of Nanoparticle Research → Version 1 posted Reviewers agreed at journal 13 Nov, 2024 Reviewers invited by journal 12 Nov, 2024 Editor assigned by journal 26 Oct, 2024 Submission checks completed at journal 23 Oct, 2024 First submitted to journal 23 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5318412","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370746347,"identity":"487870fe-0ce2-46e0-b10a-1ad0c7987189","order_by":0,"name":"Shikha Chaudhary","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shikha","middleName":"","lastName":"Chaudhary","suffix":""},{"id":370746348,"identity":"7c5cd175-aca3-4b77-8869-ef261d1f24e6","order_by":1,"name":"Anjali Maurya","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anjali","middleName":"","lastName":"Maurya","suffix":""},{"id":370746349,"identity":"15da4846-d1e1-42c2-b867-7a25005d4e1d","order_by":2,"name":"Uddipan Das","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Uddipan","middleName":"","lastName":"Das","suffix":""},{"id":370746351,"identity":"06d715bb-0b5b-42db-97e9-3752cbde4427","order_by":3,"name":"Ravi Mani Tripathi","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Ravi","middleName":"Mani","lastName":"Tripathi","suffix":""},{"id":370746352,"identity":"033b31b2-ce5e-4e66-8a88-6636f18042b3","order_by":4,"name":"Subhash Chandra Yadav","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFACHjYgcYDBgIH5AEOCAUzUAI8OhBa2BJK18OBVhgDyM3KPPfjx5060uUTOxw8PCrbZM/AvPibBUHAHpxaDG3nphr1tz3J3zsjdLJFgcDuxQeJZmgSDwTPcWiRyzCR4Gw7nbriRuwGkJYFB4oyxAYPBYTwOyzGT/PMHpCXn8Q+gFnuCWhhu5JhJ87CBtbCBbGFs4O8xfIBPi8GZd2nSsm2Hc3f2PDOzAPmlTYIt8UECPoe15x6TfAN02Hb25Mc3f/y5bc/Pf/jAgQ9/8DgMAwCdx8CQQIIGIOA/QJr6UTAKRsEoGPYAACsSXEiq8HvUAAAAAElFTkSuQmCC","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Subhash","middleName":"Chandra","lastName":"Yadav","suffix":""}],"badges":[],"createdAt":"2024-10-23 11:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5318412/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5318412/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11051-025-06218-0","type":"published","date":"2025-01-22T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69079275,"identity":"6beda909-94c1-4f28-88e9-17820f6088f1","added_by":"auto","created_at":"2024-11-15 11:39:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1569879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy imaging of (A) hollow apoferritin, (B) hydrophilic CdSe:CdS:ZnS quantum dots and (C) CdSe:CdS:ZnS quantum dots-apoferritin composite.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eThe negative stained TEM image showed a hollow structure in the core, which is visible in HR-TEM imaging, and no crystalline pattern in SAED analysis. \u003cstrong\u003e(B)\u003c/strong\u003eThe TEM image showed well-distributed QDs, well-characterized EDS (inset), better HR TEM image and crystalline pattern in SAED analysis. \u003cstrong\u003e(C)\u003c/strong\u003e The core of the QD-apoferritin composite showed more contrast due to the encapsulation of CdSe:CdS:ZnS quantum dots in the core, which is visible in HR TEM imaging and SAED.\u003c/p\u003e","description":"","filename":"FIGURE1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/070bd2cadb520e6734456265.jpg"},{"id":69079276,"identity":"b533ff73-124e-4037-8c3a-7b23df6e18ac","added_by":"auto","created_at":"2024-11-15 11:39:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":356396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiophysical characterization of CdSe:CdS:ZnS quantum dots-apoferritin composite\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eAbsorbance at 280 nm \u003cstrong\u003e(B)\u003c/strong\u003e Intrinsic tryptophan fluorescence (excitation at 280 nm) \u003cstrong\u003e(C)\u003c/strong\u003e quantum dot emission spectra (excitation at 350 nm) and \u003cstrong\u003e(D)\u003c/strong\u003eFar UV CD spectra at 25 °C, 37 °C, and 55 °C. The apoferritin (100 µg) was incubated with various amounts (0-250 ng) of CdSe:CdS:ZnS quantum dots overnight to form the QDs-apoferritin composite. These composites were incubated at selected temperatures for two hours and biophysically characterized for absorbance, ITF, QD fluorescence, and circular dichroism at different temperatures.\u003c/p\u003e","description":"","filename":"FIGURE2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/c76d24253df05a55d1bedbb9.jpg"},{"id":69078538,"identity":"61bd8b5f-bc44-4f02-bcf6-e332ed6ab419","added_by":"auto","created_at":"2024-11-15 11:31:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1475368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM imaging of quantum dot-apoferritin composite at the (A) 25 °C, (B) 37 °C and (C) 55 °C.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Nearly 100 % apoferritin cage was encapsulated with the quantum dots at 25 °C, and the average size of the QD-apoferritin composite by TEM was around 12.0 nm. \u003cstrong\u003e(B)\u003c/strong\u003e The number of hollow cages (without QDs) increased to 6.0 %. The average size was increased up to 12.5 nm (~4.0 % increase) at 37 °C. \u003cstrong\u003e(C)\u003c/strong\u003e The number of hollow apoferritin was increased up to 68 %, and the average size was nearly 15.0 nm (~ 25 % in comparison to 25 °C) at 55 °C. SAED pattern also showed low crystallinity at 55 °C and 37 °C in comparison to 25 °C (inset). The zeta size (DLS) analysis also revealed the increase in size with higher temperatures.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/8e48d586473f465700b2f30e.jpg"},{"id":69079277,"identity":"f06c6108-6930-46fa-acf2-39cd41827ce0","added_by":"auto","created_at":"2024-11-15 11:39:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1114280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHR TEM imaging of representative single particle of negatively stained QDs-apoferritin composite incubated overnight at (A) 25 ℃, (B) 37 ℃ and (C) 55 ℃.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The HR-TEM image showed the atomic lattice of QDs at the core and better FFT, showing dots at 25 ℃. \u003cstrong\u003e(B)\u003c/strong\u003e HR-TEM image at 37 ℃ showed the atomic lattice at the core but was not as intense as the 25 ℃.\u003cstrong\u003e (C)\u003c/strong\u003e The atomic lattice and FFT of the core were absent, confirming the temperature-induced release of QDs. The single particle of QDs-apoferritin was encircled at each temperature.\u003c/p\u003e","description":"","filename":"FIGURE4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/eabed843ce3a396415e88e4f.jpg"},{"id":69078542,"identity":"edda6de4-b9a5-4f4d-8e52-aef4e4360bc1","added_by":"auto","created_at":"2024-11-15 11:31:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":524214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNegative staining single particle analysis of QDs-apoferritin composite treated overnight at (A) 27 ℃, (B) 37 ℃, and (C) 55 ℃.\u003c/strong\u003e The selected temperature-treated composite (100 µg of apoferritin and 250 ng QDs) was drop cast on a TEM grid and negatively stained with 1 % uranyl acetate. The sample was subjected to single particle analysis at 120,000 X magnification, and negative stain image was collected on EPU software (Thermo Fisher Scientific), and data were processed using cryoSPARC software. The result indicated that the apoferritin core contains at least 4 quantum dots at 25 °C. Still, these particles were not modeled at 37 °C and 55 °C due to the cohomogeneity of the apoferritin cage. \u003cstrong\u003e(i)\u003c/strong\u003e Particle picking, \u003cstrong\u003e(ii)\u003c/strong\u003e particle view, \u003cstrong\u003e(iii)\u003c/strong\u003e2D class average, \u003cstrong\u003e(iv)\u003c/strong\u003e selected class average, and \u003cstrong\u003e(v)\u003c/strong\u003e model building by Chimera software.\u003c/p\u003e","description":"","filename":"Figure5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/339f436d40944f399a35ba6f.jpg"},{"id":74858657,"identity":"d5bfb4ea-c8cc-467f-b6ec-37b9b9a6b142","added_by":"auto","created_at":"2025-01-27 16:12:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6060015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/3c862363-7f19-4f75-a18f-4078b04df9a9.pdf"},{"id":69078540,"identity":"97e97711-199a-4953-8aab-6dbf0c70bbd9","added_by":"auto","created_at":"2024-11-15 11:31:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1087077,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5318412/v1/fa7a27e2d5d138853f39e519.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular interaction and Temperature-induced Structural alteration of hydrophilic CdSe:CdS:ZnS Quantum Dots-Apoferritin composite","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStudying quantum dots (QDs) and protein interfaces is vital for understanding molecular-level interactions that significantly affect the conformations of secondary and tertiary protein structures and their structure-function relationships [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. QDs, nanoscale semiconductor particles, have garnered immense attention due to their exceptional optical properties, making them valuable for clinical diagnostics, biosensing, and imaging applications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the specific interactions between QDs and proteins, especially their impact on protein structure and function, remain less explored than other nanoparticles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This knowledge gap is critical because the efficacy of QDs in biological applications depends on their favourable interactions with biomolecules [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Reports suggest that QDs must be hydrophilic, appropriately sized for encapsulation, and compatible with physiological conditions to function effectively in biological systems [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein nanocages, such as those derived from ferritin and viruses, naturally self-assemble into stable structures, making them ideal for studying protein-nanoparticle interactions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Apoferritin, a 24-mer protein complex, has been widely investigated for its ability to encapsulate a variety of semiconductor-based QDs instead of its natural iron core [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Understanding the immobilization of QDs within apoferritin cages is essential for elucidating the molecular nature of these interactions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Prior research has shown that PbS quantum dots can be successfully encapsulated within apoferritin cages, creating stable fluorescent composites that also provide a protective protein coating for the QDs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Various analytical techniques, including transmission electron microscopy (TEM), fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS), have been used to investigate the structural and conformational changes of apoferritin upon interaction with QDs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These methodologies are crucial for understanding the biophysical and structural properties of QDs-treated apoferritin at specific temperatures.\u003c/p\u003e \u003cp\u003eApoferritin is known for its remarkable ability to withstand high temperatures (up to 80 \u0026deg;C) without significantly disrupting its quaternary structure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This study presents a detailed biophysical analysis of the interactions between hydrophilic core-shell CdSe:CdS:ZnS quantum dots and horse spleen apoferritin. Using a combination of spectroscopic and microscopic techniques\u0026mdash;including CD, fluorescence spectroscopy, DLS, and high-resolution-TEM, we investigate the structural and conformational changes in apoferritin upon QD encapsulation at various temperatures (25\u0026deg;C, 37\u0026deg;C, and 55\u0026deg;C). Additionally, single-particle analysis via negative staining was used to elucidate the encapsulation process and the structural heterogeneity of the resulting QD-apoferritin composites at elevated temperatures. Our findings reveal that temperature plays a significant role in modulating apoferritin's structural integrity and the encapsulated QDs' stability, with potential implications for designing QD-based nanocomposites for biological applications.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eMaterial\u003c/p\u003e \u003cp\u003eAll the chemicals for QDs synthesis and apoferritin (sigma cat no. (A-3660) were procured from Sigma USA. TEM grids were from Agar Scientific UK.\u003c/p\u003e \u003cp\u003ePreparation of apoferritin\u003c/p\u003e \u003cp\u003eThe native horse spleen apoferritin stock solution (25 mg/mL in 50% glycerol) was diluted by 1X phosphate buffer saline (pH 7.4) to make a working solution (1.0 mg/mL), and glycerol concentration was reduced by dialysis under stirring at 400 rpm in 1X phosphate buffer saline (pH 7.4) overnight.\u003c/p\u003e \u003cp\u003eSynthesis of CdSe:CdS:ZnS hydrophilic quantum dots\u003c/p\u003e \u003cp\u003eThe CdSe core and core-shell (CdSe:CdS:ZnS) QD were synthesized with little modification by Zhu et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Chen et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] using an indigenously designed low-cost degassing apparatus. Purified CdSe:CdS:ZnS QDs (400 \u0026micro;L) were suspended in 600 \u0026micro;L chloroform. A 50% solution of 2-MPA was added and allowed to stir for 2h at RT. The sample was centrifuged at high speed, and the supernatant was discarded. The pellet was dried in air and resuspended in 200 \u0026micro;L 0.1 M sodium bicarbonate solution. The bicarbonate solution containing shelled QDs was mixed with acetone (three times the volume) and pelleted by centrifugation at room temperature. The supernatant was carefully discarded, and the pellet was allowed to dry. The pellet was finally dissolved in 1.0 mL of deionized water.\u003c/p\u003e \u003cp\u003eThe absorption scan (200\u0026ndash;600 nm), visual fluorescence under UV light, quantum dot fluorescence (Ex:350 nm; Em:400\u0026ndash;600 nm), hydrodynamic diameter/zeta potential (dynamic light scattering DLS), TEM imaging along with EDS (energy dispersive X-ray spectroscopy) and crystallinity using selected area electron diffraction (SAED) were used to characterize the hydrophilic CdSe:CdS:ZnS core-shell quantum dots.\u003c/p\u003e \u003cp\u003eSynthesis of QDs-apoferritin composite\u003c/p\u003e \u003cp\u003eThe purified apoferritin (0.1 mg/mL) was incubated with various concentrations of hydrophilic CdSe:CdS:ZnS QDs (12.5 to 250 ng/mL) overnight to determine the formation of composite to QDs and apoferritin. These samples were characterized by TEM and HR-TEM to confirm the incubation-induced encapsulation of QDs on apoferritin to form the QDs-apoferritin composite.\u003c/p\u003e \u003cp\u003eBiophysical characterization of QDs-apoferritin composite at selected temperature\u003c/p\u003e \u003cp\u003eThe composite samples with increasing concentrations of QDs (12.5 to 250 ng/mL) with apoferritin (100 \u0026micro;g/mL) were biophysically characterized using absorbance (280 nm), intrinsic tryptophan fluorescence (Ex: 280 nm, Em: 320 nm), quantum dot fluorescence (Ex: 350 nm, Em: 590 nm), and far UV circular dichroism (250\u0026thinsp;\u0026minus;\u0026thinsp;200 nm) after overnight incubation at selected temperatures 25 ℃, 37 ℃ and 55 ℃.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAbsorbance\u003c/strong\u003e \u003cp\u003eThe absorbance scans from 200\u0026ndash;700 nm of QDs-apoferritin composite with variable QDs concentrations after overnight incubation at each selected temperature were taken using the Evolution 220 spectrophotometer (Thermo Scientific). Pure quantum dots (without apoferritin) of the same concentration were taken as blanks for each set of experiments.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFluorescence\u003c/strong\u003e \u003cp\u003eThe intrinsic tryptophan fluorescence spectra (using Carry Eclipse 100; Agilent Technologies) of QDs-apoferritin composite with variable QDs concentrations at selected temperatures were taken using excitation at 280 nm and an emission scan between 300\u0026ndash;400 nm. For the quantum dot fluorescence measurements, the same samples were excited at 350 nm and emission spectra from 400\u0026ndash;600 nm were recorded. The slit width for excitation and emission was 5 and 10 nm, respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCircular Dichroism\u003c/strong\u003e \u003cp\u003eFar UV CD spectra (250\u0026thinsp;\u0026minus;\u0026thinsp;200 nm) of a QDs-apoferritin composite with variable QDs concentrations were taken on a Jasco A-720 spectropolarimeter. The equipment was pre-calibrated with a 0.1% d-10-camphor sulfonic acid solution. The scanning speed was 100 nm/min with a 1.0-sec response time. The measurements were carried out at 25\u0026deg;C, 37 \u0026deg;C and 55 \u0026deg;C using a cuvette with a path length of 0.1 cm.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTEM imaging of QDs-apoferritin composite at the selected temperatures\u003c/p\u003e \u003cp\u003eThe actual size and morphology of the QDs-apoferritin composite were analyzed using negative staining TEM. These composites were deposited onto carbon-coated 3-mm copper grids using a drop-casting technique and stained with 1% uranyl acetate to enhance the contrast between the intense central quantum dot core and the lighter apoferritin shell. Images were captured on Talos S (Thermo Fisher Scientific) transmission electron microscopy operated at 200 kV. Image acquisition was done with a bottom-mounted 4 \u0026times; 4 k CMOS camera. All the digital TEM micrographs were analyzed for size determination using ImageJ. Briefly, the measurement scale was set using the image scale bar, and the images were converted to grayscale. 250 individual particles were analyzed using a line descriptor extending across the diameter of the particles. EDS analysis was performed to detect quantum dots elements Cd, Se, S, and Zn. The average number of hollow apoferritin and composite were manually counted with 10 random images to report the % release of QDs at 37 \u0026deg;C and 55 \u0026deg;C compared to 25 \u0026deg;C.\u003c/p\u003e \u003cp\u003eHR imaging of QDs-apoferritin composite at the selected temperatures\u003c/p\u003e \u003cp\u003eA TEM specimen was prepared by drying each quantum dot-apoferritin composite solution on a carbon-coated copper (300 mesh) grid. The prepared TEM specimen was imaged using a Talos S (Thermo Fisher Scientific USA) microscope equipped with 200 kV. A fast Fourier transform (FFT) simulation and a strain analysis with the HR-TEM images were performed in a digital micrograph (Ceta, Thermo Fisher). This method involves filtering the image with an asymmetric filter at a Bragg spot of the Fourier transform simulation of the HR-TEM lattice image and performing an inverse Fourier transform simulation. This method can provide useful information on the local displacement of interesting regions in a given direction.\u003c/p\u003e \u003cp\u003eSingle particle analysis of QDs-apoferritin composite at selected temperature using the negatively stained sample\u003c/p\u003e \u003cp\u003eTalos S 200 transmission electron microscope alignments were performed using previously standard methodologies, including determining parallel illumination using a long diffraction camera length. 2D images of negatively stained quantum dot-apoferritin composite were collected using a Talos S 200 transmission electron microscope (Thermo Fisher Scientific) with a field emission gun operating at an accelerating voltage of 200 kV and equipped with a 4Kx4K CMOS detector (Ceta, Thermo Fisher Scientific). All EM data were acquired using the EPU software (EPU 4.0, Thermo Fisher Scientific). Images of quantum dot-apoferritin composite were collected in counting mode (0.117 \u0026Aring;/pixel) and super-resolution mode (0.059 \u0026Aring;/super-resolution pixel), respectively, at a magnification of 120,000x over a defocus range of \u0026minus;\u0026thinsp;0.5 \u0026micro;m to \u0026minus;\u0026thinsp;1.0 \u0026micro;m. Images were obtained using an exposure rate of 577 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/pixel/s for 1.0 s, resulting in a total exposure of \u0026sim;71333.84 e/nm\u003csup\u003e2\u003c/sup\u003e. Further details can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eSingle-particle image processing and 3D reconstruction\u003c/p\u003e \u003cp\u003eThe single particle image processing was done using cryoSPARC ver 4.5.3. A total of 1464 (25 \u0026deg;C), 1278 (37 \u0026deg;C), and 1778 (55 \u0026deg;C) micrographs were imported, and contrast transfer function (CTF) was estimated using patch CTF estimation followed by automatic particle picking using a Blob picker. The coordinates were extracted with a 200-pixel box size (2x binning) and subject to several rounds of 2D classification to remove junk particles. The clean particles were reextracted without binning and subjected to ab initio reconstruction followed by non-uniform refinement. The initial models for all three conditions (QDs-apoferritin composite at 27 \u0026deg;C, 37 \u0026deg;C, and 55 \u0026deg;C) were generated using Chimera software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTEM imaging of apoferritin, quantum dot and QDs-apoferritin composite\u003c/p\u003e \u003cp\u003eApoferritin exhibited a well-defined distribution and a hollow core structure with a non-crystalline fast Fourier transform (FFT) pattern in TEM imaging when not encapsulated with QDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The CdSe:CdS:ZnS hydrophilic quantum dots were uniformly distributed in negative staining, with peaks for Cd and Zn detected in the EDS analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e In contrast, the CdSe:CdS QD-apoferritin composite displayed higher contrast within the core. HR- TEM images of the core revealed the atomic arrangement of the quantum dots' crystallinity, which was also confirmed by selected area electron diffraction (SAED) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e These observations suggest successful encapsulation of the quantum dots within the apoferritin, while the crystalline structure of the quantum dots remains intact. Apoferritin was amorphous, as indicated by the lack of crystalline patterns in the SAED analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiophysical characterization of QDs-apoferritin composite at various temperature\u003c/p\u003e \u003cp\u003eThe absorbance of the apoferritin-QD composite at 280 nm increased with higher concentrations of quantum dots at both 25\u0026deg;C and 55\u0026deg;C, using similar QD concentrations as a blank. However, at 37\u0026deg;C, the composite's absorbance was lower compared to 55\u0026deg;C at higher QD concentrations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e The intrinsic fluorescence emission spectra of the apoferritin-QD composite showed higher intensity at 25\u0026deg;C (220\u0026ndash;250) and lowest intensity at 37\u0026deg;C (100\u0026ndash;120). Fluorescence intensity at 55\u0026deg;C was intermediate between that observed at 25\u0026deg;C and 37\u0026deg;C. Variations in QD concentration did not significantly affect fluorescence intensity. Additionally, wavelength shifts were negligible across all temperatures and QD concentrations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e The fluorescence intensity of QDs (excitation: 350 nm, emission: 550\u0026ndash;650 nm, peak maximum around 590 nm) increased with higher QD concentrations at both 25\u0026deg;C and 37\u0026deg;C. Conversely, fluorescence intensity decreased at 37\u0026deg;C and 55\u0026deg;C compared to 25\u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u003cb\u003e\u0026amp; C).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CD of the QDs-apoferritin composite at 25 \u0026deg;C, and all the concentrations of QDs showed a similar α-helical structure with two prominent negative peaks at 222 and 208 nm. The ellipticity was around the \u0026minus;\u0026thinsp;20 to -22 deg.cm.deci.mole at 222nm, and \u0026minus;\u0026thinsp;18 to -19 deg.cm.deci.mole at 208 nm. However, the shape of the peaks was distorted, and 208 nm peaks disappeared at 37 \u0026deg;C with all the concentrations of the QDs. This showed the appearance of peaks at 215 nm, while the 222 nm peaks remained intact with ellipticity around \u0026minus;\u0026thinsp;18 to -20 deg.cm.deci.mole. The heating of the composite at 55 \u0026deg;C revealed a reduction in the ellipticity of around \u0026minus;\u0026thinsp;15 to -18 deg.cm.deci.mole at 222 nm with a similar shape as 37 \u0026deg;C. The 208 nm peaks disappeared at 37 \u0026deg;C and 55 \u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e CD and fluorescent analyses revealed that the secondary structures were kept almost unchanged upon thermal treatment of QD-apoferritin composite between 25 \u0026deg;C and 37 \u0026deg;C but showed molten globule-like structure at 55 \u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTEM imaging of quantum dot-apoferritin composite at the selected temperatures\u003c/p\u003e \u003cp\u003eTEM image at a higher resolution of the QDs-apoferritin composite showed structured QD core in nearly all the composite particles (~\u0026thinsp;100%) at 25 \u0026deg;C but showed the slight release of QDs from the composite (6% QDs release from core) to form a hollow core at 37 \u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The composite at 55 \u0026deg;C treated sample showed the release of most of the QDs from the core (68% QDs release from core) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The average size of the composite was 12.0\u0026plusmn;1.0 nm and 12.5\u0026plusmn;1.0 nm, and 15.0\u0026plusmn;1.3 nm at 25 \u0026deg;C, 37 \u0026deg;C, and 55 \u0026deg;C treated samples respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. EDS and SAED imaging also confirm the gradual reduction of the QDs in the core with increasing temperature \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. HR imaging of these particles also showed the presence of QDs incubated overnight at 25 \u0026deg;C, while they were irregular at 37 \u0026deg;C and very rare at 55 \u0026deg;C temperature samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStructural analysis of QDs-apoferritin composite at different temperatures using negative stain single-particle analysis\u003c/p\u003e \u003cp\u003eTo further investigate the quantum dot-apoferritin interaction at high resolution, we conducted single particle analysis of negatively stained samples containing 100 \u0026micro;g/mL apoferritin and 250 ng/mL QDs. This provided a high-contrast visualization of QDs-apoferritin composite structures to explore the structural effects of encapsulating quantum dots (QDs) within apoferritin shells under varying thermal conditions. The analysis was performed under three temperature conditions: 25\u0026deg;C, 37\u0026deg;C, and 55\u0026deg;C, with overnight incubation followed by quick drop-casting onto a grid for negative staining. This analysis revealed a clear temperature-dependent effect on the structural integrity of the QD-apoferritin composite. This technique, which provides high-contrast visualization of protein structures, was employed to assess the structural changes of quantum dots encapsulated within apoferritin shells under different thermal conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e25\u0026deg;C\u003c/strong\u003e \u003cp\u003eThe QD-apoferritin conjugates exhibit notable structural integrity at this temperature, as evidenced by the 2D class averages and 3D models. The single-particle analysis reveals that the apoferritin structure remains largely intact at this lower temperature. The encapsulated CdSe QD is observed to be well-enclosed within the protein shell. The 2D class averages and 3D reconstructions indicate minimal distortion or unfolding in the protein cage. The apoferritin-QD complexes exhibit a homogeneous and symmetrical appearance, suggesting that the structural stability of the complex was preserved at this temperature. The conjugates maintain a defined and stable structure with minimal distortion. The analysis at this temperature provides a baseline for comparing structural changes at elevated temperatures \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e37\u0026deg;C\u003c/strong\u003e \u003cp\u003eThere were noticeable structural changes observed in the conjugates and partial deformation of the apoferritin shell at their physiological temperature (37\u0026deg;C). The 2D and 3D models reveal a partial reorganization or movement of the apoferritin subunits. Despite these alterations, the structural integrity of the composite was only slightly compromised, with the quantum dot core remaining shielded by the apoferritin shell. This indicates a moderate tolerance of the complex to temperature at this level \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e55\u0026deg;C\u003c/strong\u003e \u003cp\u003eAt 55\u0026deg;C, significant structural alterations in the QD-apoferritin conjugates were detected. The 3D model shows substantial disassembly of the apoferritin structure, which could be attributed to denaturation or thermal unfolding. The 3D reconstructions of these particles reveal disordered and fragmented protein cages. The high temperature impacts the apoferritin shell and the QD core, leading to an unstable conjugate structure. Encapsulated QDs are less uniformly enclosed and, in some cases, appear to be exposed due to the disassembly of the protein shell. This suggests the encapsulation system is compromised at higher temperatures, leading to potential instability in functional applications \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of quantum dot nanoparticles (QDs) in medicine, drug delivery systems, biosensors and diagnostic devices is increasing exponentially [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The compositions and surface chemistry of QDs play a key role in a physiological environment by interacting with nucleic acids, lipids, and protein molecules [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Standard techniques such as absorbance, fluorescence, and circular dichroism (CD) provide some insights into QD-protein interactions but may not fully capture the molecular-level details. However, by combining these techniques with high-resolution transmission electron microscopy (HR-TEM) and single-particle analysis, we can better understand the interactions between QDs and apoferritin nanoparticles.\u003c/p\u003e \u003cp\u003eApoferritin was selected for this study due to its known capacity to encapsulate various nanoparticles and its established use as a model for structural evaluation via single-particle analysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The CdSe:CdS:ZnS core-shell quantum dots used in this study are highly fluorescent and are commonly employed in biological systems [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Being hydrophilic, these QDs were encapsulated by apoferritin through simple overnight incubation at room temperature (RT), as confirmed by TEM and HR-TEM analyses \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e TEM images of hollow apoferritin revealed an empty, low-contrast core, showed no atomic lattice of QDs under HR-TEM and selected area electron diffraction (SAED) pattern. These results also confirm the absence of crystalline material in the core \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. In contrast, the TEM images of QDs alone displayed sizes ranging from 5 to 8 nm with well-defined crystalline atomic arrangements, confirmed by HR-TEM and SAED analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Overnight incubation validated QD encapsulation, revealing a more contrast-rich core with crystalline QDs, a non-crystalline apoferritin shell under HR-TEM, and a diversified SAED pattern. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. This encapsulation process is unique to CdSe:CdS:ZnS QDs because the encapsulation of other QDs (e.g. PbS QDs, etc.) and nanoparticles typically requires apoferritin disassembly via acidic pH or chemical conjugation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For optimal composite formation, equimolar concentrations of apoferritin (100 \u0026micro;g/mL) and QDs (ranging from 12.5 ng to 250 ng/mL) were tested, with 250 ng of QDs for efficient encapsulation. It was found that the 250 ng QDs showed the equivalent encapsulation of 100 \u0026micro;g of apoferritin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTemperature is well-known to affect protein structure, potentially leading to aggregation via misfolding or unfolding [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Comparing the impact of thermal treatment on QD-apoferritin composites across different temperatures (25\u0026deg;C, 37\u0026deg;C, and 55\u0026deg;C) provides insight into how QD encapsulation influences the stability of apoferritin. We applied the thermal treatment overnight such as 25\u0026deg;C (for native room temperature study), 37\u0026deg;C (for physiological temperature study) and 55\u0026deg;C (for extreme temperature study), to study and compare the impact of these temperatures on QDs-apoferritin composite. Absorbance measurements (280 nm) revealed a gradual increase in absorbance with higher QD concentrations at both 25\u0026deg;C and 55\u0026deg;C. At the same time, it decreased at an initial concentration of QDs (in between 0\u0026ndash;50 ng/mL QDs) and was stable at the higher concentration of QDs at 37\u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Prior studies have shown that apoferritin\u0026rsquo;s absorbance remains unchanged below 65\u0026deg;C [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This suggests that changes in absorbance in this study may be due to QD-induced structural alterations in apoferritin. The strong absorption of radiation by QDs at 280 nm likely leads to localized heating, resulting in elevated temperatures for individual composite molecules \u003cb\u003e(Supplementary Fig.\u0026nbsp;1).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIntrinsic fluorescence emission from tryptophan (Trp) residues in apoferritin is sensitive to the microenvironment around the fluorophore. Trp exhibits a strong fluorescence peak at 320 nm when excited at 280 nm. The fluorescence intensity was unaffected by QD concentration but varied significantly with temperature. It was highest at 25\u0026deg;C (~\u0026thinsp;240 U), decreased at 55\u0026deg;C (~\u0026thinsp;190 U), and was lowest at 37\u0026deg;C (~\u0026thinsp;110 U) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This suggests that while QDs did not affect the intrinsic tryptophan fluorescence (ITF) spectra, temperature had a significant impact. Meanwhile, quantum dot fluorescence (Ex 350 nm, Em 590 nm) decreased progressively with increasing temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), consistent with previous reports showing reduced fluorescence of hydrophilic QDs at higher temperatures \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCircular dichroism (CD) measurements revealed a slight decrease in ellipticity with increasing temperature (~-22 deg.cm.deci.mole at 25\u0026deg;C, ~-20 deg.cm.deci.mole at 37\u0026deg;C, and ~\u0026thinsp;18 deg.cm.deci.mole at 55\u0026deg;C). Far-UV CD spectra at 25\u0026deg;C displayed typical alpha-helical features with prominent peaks at 222 and 208 nm, but the shape became more symmetrical at 37\u0026deg;C and 55\u0026deg;C, with the 208 nm peak diminishing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. This finding is notable, as native apoferritin\u0026rsquo;s Far-UV CD spectra are known to remain stable up to 80\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These results indicate that QD encapsulation promotes unfolding at lower temperatures, leading to molten globule-like structures in the QD-apoferritin composite.\u003c/p\u003e \u003cp\u003eTEM analysis after overnight incubation at 25\u0026deg;C, 37\u0026deg;C, and 55\u0026deg;C showed that the composite maintained a discrete protein cage morphology at 25\u0026deg;C and 37\u0026deg;C, with exterior diameters of ~\u0026thinsp;12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 nm and ~\u0026thinsp;12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 nm, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eand B)\u003c/b\u003e. These sizes are consistent with previously reported ferritin nanocages [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, at 55\u0026deg;C, the composite size increased by 25% (15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nm), as confirmed by TEM analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. DLS analyses also support the finding about the increase in the size of these particles at 55\u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003einset)\u003c/b\u003e. Encapsulation efficiency was approximately 100% at 25\u0026deg;C but dropped to 94% at 37\u0026deg;C due to the temperature-induced release of QDs. At 55\u0026deg;C, encapsulation efficiency plummeted to 32%, similar to iron release from ferritin at elevated temperatures [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSingle-particle negative stain analysis provided further evidence of the thermal sensitivity of QD-encapsulated apoferritin. The data indicate that while apoferritin remains stable at moderate temperatures, it undergoes significant structural degradation at higher temperatures. This denaturation is critical for the potential use of QD-apoferritin composites in biological or industrial applications. At 25\u0026deg;C, the particles are well-encapsulated and spherical, but at 55\u0026deg;C, many particles exhibit irregular shapes or significant structural disruptions. The 2D class averages for each temperature condition reflect these changes, showing progressively disordered structures as temperature increases \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe marked structural disintegration observed at 55\u0026deg;C underscores a limitation of the QD-apoferritin conjugate system under thermal stress. The 3D models generated at different temperatures reveal a loss of structural rigidity, with severe compromise at 55\u0026deg;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e. The comparative analysis of the QDs-apoferritin conjugates at different temperatures indicates a strong correlation between temperature and structural stability. This thermal instability is an important consideration for future studies on the thermal management of quantum dot conjugates in various applications.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAt lower temperatures (25\u0026deg;C and 37\u0026deg;C), apoferritin effectively encapsulates CdSe:CdS:ZnS core-shell quantum dots (QDs), maintaining structural integrity and protecting the QDs from external factors. The preservation of the spherical shape and encapsulated core under these conditions suggests that apoferritin can reliably function at room and physiological temperatures. However, at higher temperatures (55\u0026deg;C), thermal motion leads to significant unfolding and destabilization of the apoferritin structure. This exposes the encapsulated QDs, compromising the protective barrier function of the apoferritin shell. Such structural degradation could limit the performance of QD-apoferritin conjugates in applications that require thermal stability, such as in vivo biological systems or environments with fluctuating or elevated temperatures. These findings offer critical insights into the limitations and potential of QD-apoferritin conjugates, highlighting the need for further research to enhance their thermal stability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest:\u003c/h2\u003e \u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCredit authorship contribution statement\u003c/h2\u003e \u003cp\u003eSC performed the experiments for biophysical characterization, TEM imaging and single particle analysis. AM synthesized the hydrophilic QDs. UD analyzed the single particle analysis data. RMT provides suggestions for writing some parts of the manuscript. SCY designed the study, performed and supervised experiments, analyzed the data, generated the figures, wrote the manuscript, and directed the project. All authors read and approved the final draft of this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the DBT (\u003cb\u003eBT/INF/22/SP44285/2021\u003c/b\u003e). Other funding from Indian Council of Medical Research (ICMR), New Delhi \u003cb\u003e(EM/SG/Dev.Res/126/2842\u0026thinsp;\u0026minus;\u0026thinsp;2023 (E.O -169927)\u003c/b\u003e has been acknowledged.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.C. performed the experiments for biophysical characterization, TEM imaging and single particle analysis. A.M. synthesized the hydrophilic QDs. U.D. analyzed the single particle analysis data. R.M.T. provides suggestions for writing some parts of the manuscript. S.C.Y. designed the study, performed and supervised experiments, analyzed the data, generated the figures, wrote the manuscript, and directed the project. All authors read and approved the final draft of this manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the Electron Microscopy Facility, SAIF-AIIMS, for providing the TEM and Zeta Size facility analyzers. The Department of Biophysics AIIMS New Delhi and the CIF facility, School of Biotechnology JNU, were acknowledged for providing the CD facility. We acknowledge Mr. Sunil Kumar's help with fluorescence data collection. Funding from the DBT (BT/INF/22/SP44285/2021) and ICMR (EM/SG/Dev.Res/126/2842-2023 (E.O -169927) was acknowledged.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eDatasets are available at a reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKurylowicz, M.;Paulin, H.;Mogyoros, J.;Giuliani, M.; Dutcher, J. R. The effect of nanoscale surface curvature on the oligomerization of surface-bound proteins. J R Soc Interface 2014, \u003cem\u003e11\u003c/em\u003e, 20130818.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedintz, I. L.;Uyeda, H. T.;Goldman, E. R.; Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 2005, \u003cem\u003e4\u003c/em\u003e, 435\u0026ndash;446.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, W. Introduction: Nanoparticles in Medicine. 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J Nutr 2008, \u003cem\u003e138\u003c/em\u003e, 878\u0026ndash;884.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Negative stain single-particle data collection and processing parameters of QD-apoferritin composite at different temperatures\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"649\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eData Collection Parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25 ℃\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e37 ℃\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e55 ℃\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eVoltage (kV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e200 (Talos S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e200 (Talos S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e200 (Talos S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eTEM magnification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e120000x\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e120000x\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e120000x\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eExposure navigation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eStage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eStage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eStage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eDetector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eBM-Ceta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eBM-Ceta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eBM-Ceta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eDefocus range (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eGrid Type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e300 mesh carbon coated copper grids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e300 mesh carbon coated copper grids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e300 mesh carbon coated copper grids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eSample concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e0.1 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e0.1 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e0.1 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eC2 aperture size (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eObjective aperture size (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eData collection software\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eEPU 4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eEPU 2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eEPU 2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eGun type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e200 kV Schottky\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e200 kV Schottky\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e200 kV Schottky\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eCs (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eEnergy filter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eSpecimen holder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eSingle tilt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eSingle tilt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eSingle tilt\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eExposure time (s)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eDose per second (e\u003csup\u003e-\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e/s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eCumulative Exposure (e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026Aring;\u003csup\u003e\u0026minus;2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e71.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003ePixel size (\u0026Aring;)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eExposure per frame (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eParticles per micrograph (avg.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eAcquired Micrographs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e1464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eMicrographs Used\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e1200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e1608\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eTotal extracted particles (no.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e102338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e237000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e53340\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eRefined particles (no.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e53,891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e167000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e33340\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReconstruction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eFinal particles (no.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003e53,891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e167000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003e33340\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2851%;\"\u003e\n \u003cp\u003eSymmetry imposed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.188%;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2635%;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Apoferritin, CdSe:CdS:ZnS quantum dots, circular dichroism, negative stain single particle analysis, HR-TEM, QDs-apoferritin composite","lastPublishedDoi":"10.21203/rs.3.rs-5318412/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5318412/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe encapsulation of core-shell quantum dots (QDs) on apoferritin protein and the thermal stability of these composites have been sparingly reported. In this study, we created a quantum dot-apoferritin composite and investigated its interaction and temperature-induced structural changes. The encapsulation of mercaptopropionic acid functionalized CdSe:CdS:ZnS core-shell QDs in apoferritin was validated using a high-resolution transmission electron microscope. The increasing concentrations (0-250 ng/mL) of QDs in composite (using 0.1 mg/mL apoferritin) showed an increase in absorbance, a decrease in tryptophan fluorescence intensity, and a change in circular dichroism characteristic peaks with increasing temperatures (25 \u0026deg;C, 37 \u0026deg;C and 55 \u0026deg;C). HR-TEM image supports these findings, showing an increase in size (12.0\u0026plusmn;1.0 nm at 25 \u0026deg;C, 12.5\u0026plusmn;1.0 nm at 37 \u0026deg;C, and 15\u0026plusmn;1.3 nm at 55 \u0026deg;C) and gradual release of QDs from the core showing 6\u0026plusmn;1% (37 \u0026deg;C) and 68\u0026plusmn;5% (55 \u0026deg;C) hollow composite particles. The single particle analysis for molecular structural elucidation using the negative stain sample confirmed the encapsulation of four QD particles at 25 \u0026deg;C. However, it showed multiple 2D class averages at 37 \u0026deg;C and 55 \u0026deg;C. This heterogeneity in 2D class averages confirms the destabilization of this composite at 37 \u0026deg;C and 55 \u0026deg;C. The single particle analysis revealed the molten globule-like structure of the QD-apoferritin composite at 55 \u0026deg;C. This study revealed that QDs induced significant structural alteration in the apoferritin at a much lower temperature than its melting temperature (80 \u0026deg;C).\u003c/p\u003e","manuscriptTitle":"Molecular interaction and Temperature-induced Structural alteration of hydrophilic CdSe:CdS:ZnS Quantum Dots-Apoferritin composite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 11:31:07","doi":"10.21203/rs.3.rs-5318412/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"160549808815786611682119008626999696952","date":"2024-11-13T14:14:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-12T21:16:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-26T19:20:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-23T22:01:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanoparticle Research","date":"2024-10-23T11:06:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d6f85cf7-658e-4586-b954-fc08d39ecd04","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T16:07:04+00:00","versionOfRecord":{"articleIdentity":"rs-5318412","link":"https://doi.org/10.1007/s11051-025-06218-0","journal":{"identity":"journal-of-nanoparticle-research","isVorOnly":false,"title":"Journal of Nanoparticle Research"},"publishedOn":"2025-01-22 15:57:04","publishedOnDateReadable":"January 22nd, 2025"},"versionCreatedAt":"2024-11-15 11:31:07","video":"","vorDoi":"10.1007/s11051-025-06218-0","vorDoiUrl":"https://doi.org/10.1007/s11051-025-06218-0","workflowStages":[]},"version":"v1","identity":"rs-5318412","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5318412","identity":"rs-5318412","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}