of luminescent quantum dots coated with modified Towards new

A novel family of luminescent nanoparticles, CdTeQDs@MNs, made of quantum dots coated by mesoporous silica is reported and fully characterized. While the presence of the luminescent QDs can allow for the imaging of the nanoparticles, as for instance to track their cell internalization, the mesoporous character of the silicon oxide coating simultaneously allows for the encapsulation and controlled release of different active principles such small molecules (rhodamine B), drugs such as doxorubicin and and isolated proteins ( BSA, LYS, CA, OVA, LAC, Hb, Myb, CYT) . Finally, and as a proof-of-concept to demonstrate the efficacy of these novel platforms, they have been used to extract and recognize proteins in raw serum of arthritis and prosthesis patients, without any previous protein depletion.

Fluorescence quantum yields 39 for green (1) and orange (2) CdTe QDs were measured using as standards fluorescein (=0.79) 40 and rhodamine (=0.70) 41 in ethanol. The values of fluorescence quantum yields were corrected accordingly with the different refraction indexes.
The nanoparticle size distributions and zeta potential were measured using a dynamic light scattering, a Malvern Nano ZS instrument with a 633 nm laser diode. Transmission electron microscopy (TEM) images were obtained in a JEOL JEM 2010F operating at 200 kV, TEM images were collected using a multiscan camera and Digital Micrograph software from Gatan.
Energy-dispersive X-ray spectroscopy (EDS) data were obtained using an INCA 200 spectrometer from Oxford Instruments. Elemental maps were constructed combining the STEM unit with the EDS signal. Power X-ray diffraction (XRD) measurements were performed on a RIGAKU MiniFlex II using filtered CuK radiation at 30 KV, 15 mA. SEM images were obtained in a Quanta 650 FEG operating between 5-15 kV and 10 -5 Pa of vacuum in the chamber. The samples were prepared by drop casting of the corresponding dispersion on aluminium tape followed by evaporation of the solvent at room temperature and pressure. Before analysis the samples were metalized with a thin layer of platinum, using a sputter coater (Leica Microsystems EM ACE600). N 2 adsorption-desorption isotherms were recorded on a Micromeritics ASAP2010 automated sorption analyzer. The samples were degassed at 120⁰C in vacuum overnight. The specific surface areas were calculated from the adsorption data in the low pressures range using the BET model. Pore size was determined following the BJH method.

2.5.Doxorubicin loading and released studies.
To solution of Doxorubicin (0.5 mg/mL) previously prepared in phosphate buffer (PBS) buffer pH 7.4, was collected 0.5 mL, which were mixed with 0.5 mL of green CdTeQDs@MNs and kept under stirring for 2 and 20 hours. The samples were centrifuged and the supernatant quantified by absorption in the NANODROP ND-1000. The encapsulation efficiency (EE%) and the loading capacity (mg/g) were determined by the same equations reported in section 2.7.
A similar procedure was also performed with rhodamine B.
The in vitro doxorubicin release of the encapsulated CdTeQDs(1)@MNs@DOX was performed by suspending the washed nanoparticles in a 2mL solution of PBS 7.4 and PBS 5.0 (final nanoparticles concentration= 0.5 mg/mL). All suspensions were stirred and kept at 37ºC.
Aliquots at different times were collected and the free doxorubicin quantified by absorption in the NANODROP ND-1000.

Protein Quantification
The total protein content was determined using a Bradford protein assay, using BSA as the standard protein.

CdTeQDs@ mesoporous silica protein fractionation
After protein quantification, 20 µL of each pool (1 mg of total protein) was mixed with 20 µL of green CdTeQDs@ mesoporous silica nanoparticles and incubated at room temperature in a shaker for 2 h. The pellet was then harvested by centrifugation at 17200 g during 5 minutes.
Afterwards, the pellet was washed twice with 30 µL of milliQ water and harvested by centrifugation at 17200 g during 5 min.

Gel electrophoresis
Amounts of (i) 15 µg total protein for crude sample and for supernatant fraction and (ii) total pellet fraction were separately mixed with Laemmli Sample Buffer and loaded onto a 12.5% SDS-PAGE, of 1 mm of thickness. After electrophoresis at 200 V during 50 min, gels were rinsed with milli-Q water and then stained overnight with colloidal Coomassie Blue G-250. Gels were rinsed with milli-Q water until a clear background was observed. Gel imaging was carried out with a ProPicII-robot (Digilab-Genomic Solutions, USA) using an exposure time of 16 ms and resolution of 70 µm.

In-gel protein digestion
Protein bands were excised manually and transferred to 0.5 mL low adhesion tubes, and then (v/v) formic acid was added and the supernatants were transferred to new low adhesion tubes.
Peptides were further extracted from the gel with 50% (v/v) acetonitrile/0.1% TFA. The samples were dried-down and stored at -60ºC until MALDI-TOF MS analysis.

In-gel protein digestion
Prior to analysis, sample were re-suspended in 10 µL of 0.3% (v/v) formic acid and 0.5 µL of sample was hand-spotted onto a MALDI target plate (384-spot ground steel plate) then 1 µL of a ACN was added and allowed to air dry. Mass spectrometry data were acquired using an Ultraflex close-external calibration. The significance threshold was set to a minimum of 95% (p≤0.05). A match was considered successful when a protein identification score is located out of the random region, and the protein analysed scores first.

Synthesis and characterization of green (1) and orange (2) CdTe-QDs and
where  is the absorption maximum. The estimated diameters obtained for green (1) and orange (2) quantum dots were of 2.6 and 3.2 nm, respectively. These estimated values are mostly in agreement with those obtained by dynamic light scattering (DLS) of 2.8±0.4 for (1) and, 7.5±1.2 (2), which as expected are slightly as long as they consider the hydrodynamic ratio. Regarding the zeta potential, the green QDs (-50±5 mV) are more stable than the orange QDs (-31±2 mV). Luminescent mesoporous silica nanoparticles were obtained by encapsulation of CdTe QDs inside a silica matrix to generate mesoporous nanoparticles. Figure 1 illustrates the general synthetic process. In this synthesis, TEOS was used as a silica source, CTAB as cationic surfactant and template, ethylene glycol as a stabilizer and NaOH as a morphological catalyst.
The MNs synthesis was based on the synthesis of MCM-41 44,45 , but with the introduction of ethylene glycol. As expected the mesoporous nanoparticles obtained were of spherical shape (see figure 2) since the NaOH was used as the morphological agent. Moreover, the pore size of these hexagonal-symmetry nanoparticles showed to be higher than the common MCM-type materials, which have a pore size around 2-3 nm 1 fig. 3 B, D). The obtained elemental maps clearly show the relative distribution of silica and cadmium, as can be seen by the green color (silica) and the oranges dots (cadmium) confirming once more the encapsulation of QDs in the center of the nanoparticle ( fig. 3 B-D).
The presence of the elements silica, cadmium and tellurium were also demonstrated in the EDS spectrum shown in fig. 3E,

3.2.Doxorubicin loading/release and in vitro imaging studies in Hela cells.
As an exemplary study, the capability of the green luminescent mesoporous nanoparticles