Sialyl-Tn Antigen-Imprinted Dual Fluorescent Core–Shell Nanoparticles for Ratiometric Sialyl-Tn Antigen Detection and Dual-Color Labeling of Cancer Cells

Sialyl-Tn (STn or sialyl-Thomsen-nouveau) is a carbohydrate antigen expressed by more than 80% of human carcinomas. We here report a strategy for ratiometric STn detection and dual-color cancer cell labeling, particularly, by molecularly imprinted polymers (MIPs). Imprinting was based on spectroscopic studies of a urea-containing green-fluorescent monomer 1 and STn-Thr-Na (sodium salt of Neu5Acα2-6GalNAcα-O-Thr). A few-nanometer-thin green-fluorescent polymer shell, in which STn-Thr-Na was imprinted with 1, other comonomers, and a cross-linker, was synthesized from the surface of red-emissive carbon nanodot (R-CND)-doped silica nanoparticles, resulting in dual fluorescent STn-MIPs. Dual-color labeling of cancer cells was achieved since both red and green emissions were detected in two separate channels of the microscope and an improved accuracy was obtained in comparison with single-signal MIPs. The flow cytometric cell analysis showed that the binding of STn-MIPs was significantly higher (p < 0.001) than that of non-imprinted polymer (NIP) control particles within the same cell line, allowing to distinguish populations. Based on the modularity of the luminescent core–fluorescent MIP shell architecture, the concept can be transferred in a straightforward manner to other target analytes.

S-3 was neutralized with acidic cation exchange resin. The resin was filtered off and the solvent removed in vacuo. The crude product was dialysed to obtain Neu5Ac-GalNAc-Thr (30 mg, 49 µmol) in 29 % yield over two synthetic steps. HR-ESI-MS: m/z: calcd for [M+H] + : 614.2403, found: 614.2409. NMR was in accordance with previous published data described from a synthesis of STn-Thr using an enzymatic approach. 2 1.4 Spectroscopic studies 1.4.1 Interaction studies with molecular components. In the spectroscopic studies of 1 with STn-Thr-Na and the other sodium salts (sodium benzoate, perfluorooctanoate, formate and acetate) in DMF, 15 µL of fluorescent monomer (1, 4 mmol L -1 ) was mixed with 45 µL of 0 -1.0 eq. of STn-Thr-Na (or other sodium salts) to obtain a 60 µL solution. Absorption and fluorescence spectra were acquired after pipetting 15 µL of as-prepared solutions to the center of a micro-cell cuvette. In case of the molecularly imprinted polymer (MIP) pre-polymerization mixture, 15 µL of each STn-Thr-Na (or other sodium salts, 5 mmol L -1 ), 1 (5 mmol L -1 ), VBA (5 mmol L -1 ), 2D (40 mmol L -1 ) and EGDMA (65 mmol L -1 ) were added to obtain a 75 µL solution. A ca. 5 min sonication was applied to dissolve STn-Thr-Na, sodium salts and monomers in DMF. Absorption and fluorescence spectra were acquired after pipetting 15 µL of as-prepared solution to the center of the micro-cell cuvette. In case of non-imprinted polymer (NIP), 15 µL solvent was added instead of the template solution.
The equilibrium constants (Keq) were determined via fitting the plot of the equivalency between 1 and sodium salts vs. ∆A500/A500initial=(A500x-A500initial)/A500initial, which was calculated for each absorption spectrum (where A500x is the absorbance at 500 nm for each spectrum after salt addition, while A500initial is the absorbance at 500 nm before addition of salt) with Bindfit (1:1 model, 3,4 ). Calculated parameters of sodium salts were obtained from optimized S0 ground-state geometries in the gas phase, using the density functional theory (DFT) method employing the hybrid functional B3LYP with a 6-31G basis set and energy minimized as implemented in Gaussian 16W 5 and linear regressions of Keqs and the calculated parameters were performed via Microsoft Excel.

Studies with STn-MIPs and NIPs in solution.
To perform fluorescence titrations of the STn-MIPs and NIPs with the template as the analyte and potential competitors, 0.5 mg mL -1 suspensions of the STn-MIP and NIP particles were prepared in DMF; a 6.5 mmol L -1 solution of STn-Thr-Na in DMF was also prepared. Increasing volumes of the 6.5 mmol L -1 STn-Thr-Na solution were added to 2 mL suspensions of the STn-MIP and NIP particles, and after 2 min of stirring for each addition step the resultant absorption and fluorescence spectra recorded. ∆F/F0 = (Fx-F0)/F0 was calculated for each fluorescence spectrum of the STn-MIP and NIP particles (where Fx is the fluorescence intensity at 510 nm for each spectrum after template addition, while F0 is the fluorescence intensity at 510 nm before addition of STn-Thr-Na). The S-4 imprinting factor (IFfl) was determined from the MIP:NIP ratio of ∆F/F0 at the saturation point of the titration. The discrimination factor (DFfl) was determined from the ratio of ∆F/F0 at the saturation point (0.22 mmol L -1 ) of the STn-MIPs titrated with STn-Thr-Na and with the competitors (NaOAc, NaOBz, Glc, Gal, Man, GlcNAc and GalNAc).

Immunofluorescence (IF) staining
Cells were seeded on glass coverslips for overnight culture, followed by fixation with 4 % paraformaldehyde. The cells were permeabilized in 0.1 % Triton X-100 (Sigma-Aldrich, Cat# T8787) for 10 min at RT, which were then blocked in 1 % FBS in 1x PBS for 1 h. Afterwards, the cells were stained overnight with primary antibodies (1:50 dilution) at 4 °C. The STn antibody TKH2 6 and the Tn antibody

STn-MIPs and NIPs staining for microscopy analysis
Cells were seeded on coverslips for overnight culture, followed by fixation with 4 % paraformaldehyde.
The cells were then stained in 2 mL STn-MIPs solution (0.02 mg mL -1 STn-MIPs in PBS) in a 6-well plate. The staining was performed on an orbital shaker at 120 rpm under 37 °C for 1 h. After that, the cells were washed three times using 2 mL PBS and stained by DAPI to highlight the nucleus.  A flow cytometer LSR II (BD Biosciences) was applied to analyze the antibody/lectin/nanoparticlestained samples. The data were analyzed with the software FCS Express 6 (DeNovo Software).

Procedure of the determination of equilibrium constants (Keq) in solution
1. Measure the absorption spectrum of 1 (1 mmol L -1 ) in DMF. The absorbance at 500 nm is set as 500initial .
2. Measure the absorption spectra of 1 (1 mmol L -1 ) with varying equivalents of sodium salts. The absorbance at 500 nm of each spectrum after salt addition is set as 500 .

Statistical analyses
For in vitro cell experiments, at least two independent experiments were performed. The student t-test was used. The statistical tests were two-sided, and p values less than 0.05 were considered to be statistically significant. The mean is the average value of all samples. The standard deviation (SD) is an indication of the variability of all samples.
S-9 Table S1. Correlations between lowest-energy absorption maxima of 1 in various organic solvents and theoretical as well as empirical solvent polarity scales.

Measurement Uncertainties
The overall uncertainty budget of the fluorescence measurements was derived as detailed below.
Such an uncertainty budget is always helpful to get an overview of the contributions of the single steps of the protocol to the overall measurement uncertainty. Because of the multiplicative and quotient forms of the respective equations and because correlations between the quantities are assumed to be negligible, a summation of the squares of the relative uncertainties was performed. 42,

Fluorescence measurements
a) The relative uncertainty of the emission spectrum across the respective wavelength range: