Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.


Supplementary Tables
Supplementary

Synthesis of cubic phase α-NaYbF 4 : 10 % Er nanoparticles
The trifluoroacetates (TFA) of Y, Yb, Tm, and Er were prepared by the procedure reported by Roberts et al. 2 The syntheses of the core nanoparticles in this work were similar to that reported previously by Chen et al. 3 In a typical procedure (NaYbF 4 : 10 % Er, ~ 9 nm),

Calculate the amount of shell precursor for the growth of each monolayer
The spherical concentric shell model (CSM) was employed to calculate the amount of shell precursor necessary for the growth of each monolayer (ML). 3 Because of the highly symmetric structure of NaGdF 4 , the ML was inferred to a thickness equal to half the c-lattice parameter. Although the NaGdF 4 shell adopted here has also hexagonal structure (P6 3 /m, a = 6.02 Å, c = 3.60 Å), the cation sites are of three types. A one-fold site occupied by RE 3+ , another site occupied randomly by 1/2Na + and 1/2RE 3+ , and a twofold site occupied randomly by Na + and vacancies. 42 Therefore, referral to a NaGdF 4 , the ML could be taken to mean a thickness equal to the c-lattice parameter of the bulk material, 0.36 nm in the case of the hexagonal NaGdF 4 . The required Gd-OA precursor amount for every layer of one 4 3 layer n particle n particle n particle n particle n Density of the NaGdF 4 materials (ρ) is calculated as follows: Here M represents relative molecular mass of the material of NaGdF 4 , N´ means the number of NaGdF 4 units that one crystal cell contains and according to crystal structure of the hexagonal NaGdF 4 , N´ = 1.5. So, for NaGdF 4 , the cell parameter is a = 6.02 Å, c = 3.60 Å, Here, we supposed that the reactants were complete reacted after the reaction prolong 100 min (the synthesis of the core nanoparticles). So, in the above equation, r 0 = 5 nm. Because S24 the particle number is constant during the particle growth from smaller to bigger, so the particle number of the obtained initial seed, which was used for the synthesis of core/shell nanoparticles, is nearly the same as N. According to equation (1), (2), (3), the calculation of doses of the precursors for the core/shell nanoparticles growth with different ML are shown in Supplementary Table 1.

sb-UCNPs-Abs Conjugation
The conjugation was realized through active ester maleimide-mediated amine and sulfhydryl coupling. 4  were concentrated using ultrafiltration and purified by dialysis.

Laser-scanning confocal microscopy
Images of antibody conjugated sb-UCNPs were acquired using a confocal microscope Place cover slip cell layer down on a glass slide containing a drop of mounting medium. Then observe in microscopes.

Multiplexed immunohistochemical staining for tissue specimens with sb-UCNPs-Abs 5
All the waxed specimens were subjected to a 4 um thick serial sectioning for immunohistochemical staining. After xylene dewaxing, the slides were de-benzened using con-ventional gradient ethanol and immersed in water. After incubation for 10 min with 3 % H 2 O 2 , the slides were washed thoroughly using PBS and placed in the phosphate-EDTA buffer solution (50 %, pH 9.0). Antigens were retrieved in the 121 °C high-pressure cooker for 5 min, followed by cooling down of the slides to room tempera-ture. Approximately, 1 mL of the ER, PR and HER-2 monoclonal antibodies conjugated sb-UCNPs (20 nM in PBS with 5 % albumin) were added to each slide. The slides were incubated with antibodies for 2 h at room temperature and washed with PBS. The slides were then washed 3 times with PBS.
The slides were then washed with distilled water and sealed with neutral resin. The UCNPs staining slides were directly detected after incubation.
Immunohistochemical staining for tissue specimens 5 ER, PR and HER2 were evaluated by IHC on formalin-fixed paraffin-embedded tissue.