Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining

The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self‐quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye‐loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA‐ZI‐biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye‐streptavidin conjugate. Single‐particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21‐fold higher than quantum dot‐585 (QD‐585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye‐based labeling. Importantly, cetuximab labeled with PEMA‐ZI‐biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high‐sensitivity detection of disease biomarkers.


DOI: 10.1002/smtd.202201452
molecules. [1][2][3] Due to their specific and selective epitope recognition, fluorescent antibodies light up any desired target in biological samples, and thus found a great variety of applications in biology for protein analysis using microarrays, [4] detection of antigens in lateral flow immunoassay, [5][6][7] single-cell analysis by flow cytometry, [8] subcellular proteome analysis by fluorescence microscopy, [9] cell sorting in complex systems using flow cytometry, [10] super-resolution cellular imaging, [11][12][13] and for in vivo imaging. [14] Fluorescent antibodies were also translated to medical applications, especially for diagnostic imaging, [15] flow-cytometry based diagnosis, [16,17] intraoperative detection of cancer cells, [18] and image-guided surgery. [19,20] In cancer diagnostics, molecular imaging of cancer cell surface markers is very attractive. [15] The epidermal growth factor receptor (EGFR) is a cell surface receptor often overexpressed in a number of different cancers, being involved in tumor cell proliferation, differentiation, and survival. [21] Molecular imaging of EGFR is often used in tumor diagnosis and patient stratification before the decision on the therapy. [22][23][24][25] The selectivity of therapeutic antibodies for tumor cells makes them optimal platforms for imaging. [26] Cetuximab (Erbitux) is a therapeutic chimeric antibody directed against EGFR and approved for the treatment of head and neck squamous cell (HN-SCC) and colorectal cancer. [27,28] Application of cetuximab labeled with near-infrared dye IRDye800 reached clinical trials on humans for intraoperative pancreatic cancer detection, [23] imaging of glioblastoma, [29] fluorescence-guided surgical navigation in head and neck cancers, [30] and intraoperative visualization of clear cell renal cell carcinoma. [31] The use of fluorescent dyes for antibody labeling has several limitations. First, the heterogeneity of labeling. Indeed, the difficulty to control the degree of conjugation and the stochastic positioning of fluorophores result in a heterogeneously labeled antibody batch and difficulties to quantify the signal. [32,33] Second, the brightness of a single dye is physically limited, therefore, one antibody is usually labeled by multiple dye copies. [34] However, high fluorophore density per antibody can lead to self-quenching effects, and thus reduce brightness due to interactions between neighboring dyes and between dyes and the antibody. [32,35,36] The quantum yield (QY) of dye-conjugates is influenced by the fluorophore nature and conjugation itself. [37,38] Finally, the positioning of the fluorophores, mainly close to the antigen-binding region, and the multiple fluorophore conjugation can negatively influence the antibody avidity and affinity. [3,32] The use of nanomaterials can drastically improve the performance of immunoassays, because they can enhance the signal of different analytical techniques. [39][40][41] Fluorescent nanoparticles (NPs) are an attractive alternative to conventional fluorescent dyes for labeling of antibodies, due to their higher brightness, stability, and multivalency. [42][43][44] Semiconductor quantum dots (QDs) have been the earliest and the most popular examples of nanoscale luminescent labels for antibodies, giving a large variety of examples of application in vitro and in vivo. [45][46][47][48][49] For example, antibody-conjugated QDs allowed rapid tumor targeting and real-time imaging of human prostate cancer cells growing in mice compared to passive tumor targeting. [46] However, their limitations related to the presence of toxic elements and intrinsic blinking, stimulated the search for other luminescent NPs for antibody labeling. [43] Thus, ultrasmall dye-doped aluminosilicate NPs functionalized with antibodies allowed quantitative live-cell super-resolution imaging of intracellular vesicles. [50] Dye-doped silica NPs bearing a single-chain antibody fragment against HER2 were used to enhance the imaging of breast cancer tissues. [51] Their small size not only enhanced accumulation and retention at the tumor site but also improved tissue penetration and diffusion. Polymer-coated gold nanorods could drastically enhance the sensitivity of immunoassay and antigen detection on the cell surface. [41] Moreover, all organic NPs, such as conjugated polymer NPs [52][53][54] and aggregation-induced emission (AIE) NPs [55][56][57] were successfully used with antibodies for targetspecific imaging of cells and tissues.
Previously, we developed dye-loaded polymeric nanoparticles based on a concept of ionic dye insulation by bulky hydrophobic counterions. [44,58] Their size can be tuned over a broad range down to 9 nm through a controlled number of charges in the hydrophobic polymer. [59,60] The use of bulky counterions allowed strong dye loading with minimized aggregation-caused quenching, [61] yielding NPs that feature 100-fold higher brightness compared to corresponding QDs, [62,63] and unprecedented light-harvesting properties. [64] Different dyes can be encapsulated to prepare NPs of any desired color, which was applied for multicolor barcoding of live cells and for long-term tracking in vitro and in vivo. [65] Their functionalization with PEG [66] or zwitterionic [67] groups provides them with "stealth" properties with minimized nonspecific interactions. Moreover, zwitterionic groups stabilize NPs in physiological conditions. [67] They were functionalized with nucleic acids by a strain-promoted cycloaddition reaction with azide groups effectively exposed on the NPs surface. [62] The obtained DNA-functionalized NPs were applied for Förster resonance energy transfer (FRET)-based detection of nucleic acid targets [62,68,69] as well as amplified fluorescence in situ hybridization (AmpliFISH) in multiple colors using fixed and permeabilized cells. [70] However, these NPs have not been explored to date for conjugation with antibodies, and this approach could drastically enhance the fluorescence signal in immunoassays.
Several methods exist to functionalize NPs with antibodies. [71,72] Bioconjugation can take place by adsorption via electrostatic interactions, direct covalent conjugation, or through a strong molecular interaction, such as streptavidinbiotin. The latter approach is often used for immunostaining and biosensing [73,74] and the vast number of biotinylated antibodies are commercially available. Fluorescent immunolabeling of extracellular vesicles using biotin-streptavidin quantum-dot labeling allowed a rigorous and reproducible immunophenotypic characterization. [75] Indirect labeling using biotin also overcomes the challenges of controlled antibody bioconjugation, where unclear orientation of the target binding site and diminished reactivity of antibodies occurs upon conjugation with NPs. [76] Therefore, we considered that biotinylation of dye-loaded polymeric NPs could open the way to effective and versatile labeling of antibodies, which can greatly enhance the fluorescence performance of immunostaining assays.
In the present work, we developed a methodology of antibody labeling based on biotinylated zwitterionic dye-loaded polymeric NPs. A rationally designed hydrophobic polymer bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enabled preparation of small and bright fluorescent biotinylated NPs. The exposure of biotin was confirmed by FRET studies with fluorescent streptavidin and microscopy observations with biotinylated surfaces. In this method, labeling biotinylated antibody (cetuximab) with bright biotinylated NPs through a streptavidin bridge significantly improved the signal compared to commercial dye-labeled streptavidin on cells expressing EGFR. Importantly, PEMA-ZI-biotin-NPs-based antibody labeling could differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal of biotinylated antibodies labeling, and thus become a useful tool in the detection of disease biomarkers.

Design and Characterization of Biotin NPs
In our design, biotinylated NPs and antibodies are bridged by streptavidin yielding antibody-functionalized NPs. To this end, dye-loaded polymeric NPs need to be decorated with a biotin motif exposed at the surface (Figure 1). Our approach was to assemble the nanoparticles from the dye salt and polymers bearing biotin groups through nanoprecipitation. For this, we first aimed to functionalize the polymers with an amino acid derivative, bearing a biotin motif and a negatively charged carboxylate group. According to our previous works, nanoprecipitation of polymers bearing negative charge and functional group (azide) ensure both formation of small NPs [60] and exposure of the reactive group at the NPs surface. [62,77] Here, we expected that the carboxylate group in these polymers would favor exposure of biotin at the NP surface. For polymer functionalization, we synthesized an amino acid derivative 4, bearing biotin unit, tert-butyl protected carboxylate, and amino group for conjugation with the polymer. First, biotin was functionalized with Boc-protected ethylene diamine (1), which after Boc removal in TFA yielded amino group bearing biotin 2. It was further reacted with bi-protected aspartic acid, commonly used for solid-phase peptide synthesis, Fmoc-Asp(OtBu)-OH, resulting in product 3. Then, the Fmoc group of 3 was removed in mild conditions using sodium azide [62] and the obtained amino acid derivative 4 was coupled with COOH bearing polymers through an amide bond, yielding the final conjugates after hydrolysis of the t-butyl ester.
Three types of carboxylic acid bearing polymers were modified in this way: a commercial poly(methyl methacrylate) bearing 1.6 mol% COOH groups (PMMA), being a standard polymer used in assembly of dye-loaded NPs; a poly(ethyl methacrylate) bearing 5 mol% COOH groups (PEMA), which has proven to give small particles with better fluorescence brightness; and a poly(ethyl methacrylate) bearing 5 mol% COOH groups and 10 mol% of zwitterionic sulfobetiane groups (PEMA-ZI). The latter copolymer was expected to yield NPs decorated with zwitterionic groups, which was shown to prevent nonspecific interactions of NPs in a biological environment while keeping small particle size and good stability in physiological media. [67] NMR spectroscopy allowed to follow the modification of the three polymers and yielded modification yields of 85% for the first two and 43% for the zwitterionic polymer, resulting in 1.4, 4, and 2 mol% of biotin groups on PMMA-biotin, PEMA-biotin, and PEMA-ZIbiotin, respectively.
NPs were formulated by nanoprecipitation [60] by adding a solution of the polymer and, where needed, a hydrophobic dye salt (R18/F5-TPB, 30 wt% with respect to the polymer) to a 10-fold excess of phosphate buffer. The bulky hydrophobic counterion F5-TPB minimizes dye aggregation-caused quenching (ACQ) while ensuring effective encapsulation without dye leakage. [58,61] According to dynamic light scattering (DLS), NPs made of PMMA-biotin gave very large NPs without and with the dye (Figure 2a). This was unexpected given that nonfunctionalized PMMA polymer yields NPs of 50 nm size in the same  Errors are standard deviation of the mean (n = 3).
conditions. [63,64] In the case of PEMA-biotin with and without the dye, the NPs were smaller but still close to 100 nm in size (Figure 2a), which was again larger than those observed for nonfunctionalized PEMA polymer (30 nm). [78] We suspect that biotin, containing H-bonding and hydrophobic fragments, interfered with the nanoparticle formation process for both PMMA and PEMA-based polymers, leading to (at least partial) aggregation.
In the case of the PEMA-ZI-biotin polymer, no NPs detectable by DLS were formed in the absence of the dye salt. However, in the presence of the dye NPs with a hydrodynamic radius of about 15 nm were obtained. Remarkably, the hydrodynamic size of NPs made of PEMA-ZI without biotin and 30 wt% of R18/F5-TPB was also 15 ±1 nm, indicating no effect of biotin on NPs formation in case of zwitterionic polymer. Transmission electron microscopy (TEM) imaging confirmed spherical shape and small size (Figure 2b) of PEMA-ZI-biotin NPs with a mean size of 14.4 ± 3.0 nm, as well as narrow size-distribution ( Figure 2c). Thus, zwitterionic groups on the polymer play a crucial role in the formation and stabilization of biotinylated NPs. Different dye concentrations (10-30 wt% versus polymer) had only a minor influence on the obtained particle size ( Table 1). All formulations of zwitterionic biotin bearing nanoparticles showed good fluorescence QYs above 30%, in good agreement with previous results. [67] Based on these results, we chose the maximum (30 wt% versus polymer) dye loading for the next experiments, which corresponded to 157 dyes (R18/F5-TPB) loaded per 14.4-nm particle.
To determine the active biotin units at the surface of the PEMA-ZI-biotin NPs, we incubated them with streptavidin labeled with Cyanine 5 (streptavidin-Cy5). The streptavidin-Cy5 was expected to serve as an acceptor for energy transfer (FRET) upon excitation of the donor NPs loaded with R18/F5-TPB.
Previous works showed that this type of dye-loaded NPs are efficient FRET donors and behave like giant light-harvesting nanoantenna, [64] allowing efficient FRET to the acceptors located at the NP surface. [62,69] In order to remove streptavidin-Cy5 excess, we used ultracentrifugation method with 100 kDa filter. In the absorption spectra, the long-wavelength band corresponding to streptavidin-Cy5 showed a rapid drop after ultrafiltration steps and the drop was clearly faster than that corresponding to biotin-NPs (Figure 3a). The ratio of absorbance A(acceptor)/A(donor) decreased rapidly after the first 3 ultrafiltration steps and then stabilized at a value of ≈0.053 (Figure 3b). These observations implied that the unbound streptavidin-Cy5 was removed by ultrafiltration, while a small fraction remained grafted to biotin-NPs. Based on the concentration of donor and acceptor, estimated from the absorption spectra, and in the calculated number of dyes per particle (157 for NPs of 14 nm size according to TEM), we found that ≈4 acceptor molecules (Cy5 dyes) were present per NP after 3-5 ultrafiltration steps ( Figure 3b). As the labeling of streptavidin with Cy5 was ≈0.5 Cy5 per streptavidin (defined as the absorption spectrum), the number of streptavidin-Cy5 proteins per NP was ≈8. In the emission spectrum of the purified complex that excited at the donor, we observed a dual emission with a long-wavelength band corresponding to streptavidin-Cy5 (acceptor). Importantly, the emission profile did not change upon purification (Figure 3c), which showed that the direct excitation of the acceptor was minimal even in the presence of an excess of streptavidin-Cy5 so that we systematically observed the emission of Cy5 due to FRET from NPs.
To check the capacity of our biotin-NPs to detect protein binding by FRET, we titrated the biotin-NPs with an increasing concentration of streptavidin-Cy5. The measured emission spectra showed a progressive growth of the acceptor band with the increase in streptavidin-Cy5 concentration (Figure 4a). The acceptor/donor emission ratio displayed a rapid growth until 200 × 10 −12 m streptavidin-Cy5, followed by a plateau, indicating that saturation was reached. Using this titration curve (Figure 4b), we could determine the limit of the detection of streptavidin-Cy5 by our system at 3.4 × 10 −12 m.
Next, we characterized the PEMA-ZI-biotin-NPs at the singleparticle level using epifluorescence microscopy ( Figure 5). To this end, we modified the glass surface with a BSA-biotin-neutravidin complex, similar to previous reports. [62] After incubation of this functionalized surface with biotin-NPs, we observed fluorescent dots with quite homogenous intensity. In contrast, control   PEMA-ZI NPs without grafted biotin did not show any fluorescence, indicating low nonspecific binding of control NPs to the streptavidin surface. Thus, biotin-NPs bound to the functionalized surface through specific interactions of the grafted biotin with the streptavidin. In a separate test, the biotin-NPs were compared with QDs-585, characterized by a similar emission wavelength. PEMA-ZI-biotin-NPs were significantly brighter than QDs-585, which needed 7.5 times more laser power to present de-tectable fluorescence. Based on quantitative image analysis, the single-particle brightness of PEMA-ZI-biotin-NPs was 2.9-fold higher than that of QD-585 ( Figure S1, Supporting Information). Overall, PEMA-ZI-biotin-NPs are 2.8×7.5 = 21-fold brighter than QD-585 at 550 nm excitation. To explain this result, we estimated the brightness of NPs as N × × QY, where N is the number of dyes per NP, is the extinction coefficient at 550 nm, and QY is the fluorescence QY of the NPs, yielding the following brightness value: 157 × 10 5 m −1 × cm −1 × 0.31 = 4.9 × 10 6 m −1 × cm −1 . Theoretical brightness of QD-585 at 550 nm excitation is 2.5 × 10 5 m −1 × cm −1 , which is ≈20-fold lower compared to our NPs. One should note that QDs are brighter when excited in the violet region (e.g., brightness at 405 nm excitation of QD-585 is 2.2 × 10 6 m −1 × cm −1 ). Overall, our experimental data are in line with theoretical estimation, showing high brightness of PEMA-ZI-biotin-NPs.

Validation of PEMA-ZI-Biotin NPs for Amplified Immunostaining
Encouraged by these results, we then wanted to apply our PEMA-ZI-biotin-NPs to specifically label biomolecules in cellular imaging applications. For this purpose, we conjugated biotin to ce- tuximab, which is a clinically used antibody against EGFR, expressed on the cell surface. [29,30,56,79] The conjugation was done using biotin-NHS ester that reacts with amino groups of the antibody at physiological pH. The labeling was confirmed through absorption measurements after treatment with streptavidin-Cy5 and purification by ultrafiltration through 100 kDa filters ( Figure  S2, Supporting Information).
The next step was to target EGFR on the cell surface with biotinylated cetuximab and then label the antibody with the biotin-NPs through a streptavidin bridge (Figure 6a). To benchmark the performance of our PEMA-ZI-biotin-NPs, we compared them to streptavidin bearing a commercially available Cy3 dye to label the biotinylated cetuximab at the cell surface. To avoid antibody internalization, we incubated the antibody at 4°C. After fixation and incubation with streptavidin, the cells were washed and incubated with PEMA-ZI-biotin-NPs for 30 min, then washed and imaged using fluorescence microscopy ( Figure 6a). We found that harsh fixation conditions (4% PFA) impacted the integrity of the plasma membrane because NPs and labeled streptavidin were able to enter the cytosol of the fixed cells (data not shown). In contrast, fixation with 1% PFA showed membrane fluorescence labeling using both biotinylated NPs and labeled streptavidin (Figure 6b,c and Figure S3, Supporting Information). Using the same acquisition settings, dye-loaded biotinylated NPs provided much brighter membrane labeling than labeled streptavidin. Interestingly, using NPs all cells were labeled while using streptavidin-Cy3 we obtained more inconsistent labeling, represented by white arrows in Figure 6. Then, to confirm the specificity of the labeling, cells were incubated only with NPs or labeled streptavidin without the biotinylated antibody (Figure 6d,e and Figure S3, Supporting Information). Importantly, nonspecific labeling of the cell surface was not observed, demonstrating that membrane labeling comes from recognition of the biotinylated antibody. Quantitative analysis confirmed these observations, showing that the average signal of biotinylated-NPs was >4-fold higher than that for streptavidin-Cy3 labeling, and a negligible signal for controls without the antibody (Figure 6f).
The remaining question was whether our method of receptor detection using PEMA-ZI-biotin-NPs can differentiate cancerrelevant cell lines according to their EGFR expression level: high EGFR expressing U87 cells, and EGFR-negative LN319 and MCF-7 cells. [80,81] To this end, the cells were incubated with biotinylated cetuximab, followed by fixation and then addition of streptavidin and finally biotinylated NPs (Figure 7a and Figure  S4, Supporting Information). For comparison, analogous staining was done using Cy3-labeled streptavidin. U87 cells overexpressing EGFR, displayed strong fluorescence of PEMA-ZIbiotin-NPs, while LN319 and MCF-7 lines, which do not express EGFR, showed practically no signal, close to autofluorescence of nontreated cells (Figure 7). The signal from EGFRpositive U87 cells was 10-fold higher compared to the control cell lines LN319 and MCF-7 ( Figure 7). Thus, PEMA-ZI-biotin-NPs can clearly distinguish cells according to their level of expression of EGFR. Cy3-labeled streptavidin also allowed distinct labeling of the cells, but in this case, the signal in EGFRpositive cells was much lower. As a result, the difference between EGFR-positive U87 cells and negative controls was only ≈4-fold.
We also tested the specificity of our biotinylated NPs to target the surface of cells overexpressing EGFR by flow cytometry. The shift in fluorescence intensity distribution to higher values corresponded well to the higher expression level of EGFR in U87 cells compared with MCF7 cells ( Figure S5, Supporting Information). Moreover, the mean fluorescence intensity of our PEMA-ZI-biotin-NPs was ≈10-fold higher than the one of Cy3-labeled streptavidin, which confirmed much higher performance of the NPs compared to the dyes as labels.
Thus, we can conclude that biotinylated NPs can be used to label membrane-bound antibodies in different cancer cell lines with distinct expression profiles of cell membrane receptors. In comparison to streptavidin labeled with an organic dye (Cy3), the use of NPs enables a clear improvement in the fluorescence signal and, inconsequence, in the capacity to distinguish EGFR expression levels.

Conclusions
In this study, we aimed to functionalize dye-loaded polymeric NPs to significantly enhance antigen detection in cell imaging. For this, we introduced a new method to assemble fluorescent NPs bearing biotin groups, by modifying various polymers with biotin groups next to charged groups, followed by nanoprecipitation.
We found that polymers bearing zwitterionic groups produce homogeneous spherical NPs with a size under 20 nm. The effective exposure of biotin units at the NP surface could be shown by their interaction with a streptavidin-bearing acceptor dye undergoing efficient FRET, and their specific binding to streptavidincoated glass surfaces. Single-molecule microscopy of the immobilized NPs revealed 21-fold higher brightness of these 14-nm NPs compared to QD-585 of similar color and size excited at the same wavelengths (550 nm). The obtained PEMA-ZI-biotin-NPs could be used for detection and imaging of EGFR on the cell surface, based on a simple protocol of cell preparation and incubation with NPs. Remarkably, the method allows a distinction of cell lines according to their receptor expression level with a much better dynamic range compared to conventional immunolabeling approaches. Comparison with commercially labeled streptavidin using both cellular imaging and flow cytometry showed that our nanoprobes are brighter and present less unspecific labeling in EGFR-negative cell lines. We expect that the developed NP-antibody conjugates will find multiple biological applications. First, we expect that the brightness and multivalency of our system, together with its simple realization, will greatly facilitate and increase the specific detection of cancer biomarkers at the cell surface, even at low expression levels. Indeed, simple epifluorescence microscopy enables the visualization of single nanoparticles, which will allow detection of less expressed antigens with sensitivity down to a single molecule. Second, they can be applied to study cell surface receptor internalization and tracking receptor-antibody complex in the intracellular compartments. Third, these NPs could be compatible with advanced microscopy techniques, which include singleparticle tracking and super-resolution imaging, especially ON-OFF switching NPs [58,82] could be applied for single-molecule localization microscopy. Fourth, owing to the capacity of these NPs to encapsulate dyes of different color, [65,70] multiplexed imaging of different antigens becomes possible. Fifth, given the stealth character of the zwitterion shell, which was confirmed by cellular imaging, they could be potentially applied in vivo for the detection of target cancer tissues overexpressing a given antigen (e.g., EGFR, HER2, etc.). Finally, biotinylated NPs developed here could be compatible with multiple labeling technologies, which exploit streptavidin-biotin conjugation. [83]
Preparation of Nanoparticles: The protocol is based on that reported previously. [60] Stock solutions of corresponding polymers were prepared at a concentration of 10 g L −1 in acetonitrile with 20 vol% methanol. These solutions were then diluted to 2 g L −1 and the corresponding amount of dye salt was added. A total of 30 μL of the polymer solution with dye salt in acetonitrile (2 mg mL −1 with 30 wt% of R18/F5-TPB relative to the polymer) were added quickly using a micropipette to 270 μL of phosphate buffer (20 × 10 −3 m, pH 7.4) under shaking (Thermomixer comfort, Eppendorf, 1100 rpm). Then, 200 μL of this intermediate solution was added quickly using a micropipette to 800 μL of the same buffer under shaking.
Nanoparticle Characterization: Measurements for the determination of the size of nanoparticles were performed on a Zetasizer Nano ZSP (Malvern Instruments S.A.). The mean value of the diameter of NPs was obtained based on analysis by volume. Absorption spectra were recorded on a Cary 5000 Series UV-Vis-NIR Spectrophotometer (Agilent). Excitation and emission spectra were recorded on an FS5 Spectrofluorometer (Edinburg Instruments). For the standard recording of fluorescence spectra, the excitation wavelength was set to 530 nm. The fluorescence spectra were corrected for detector response and lamp fluctuations. QYs of donor dye in NPs were calculated using Rhodamine 101 in methanol as a reference (QY = 1.0) with an absorbance of 0.01 at 530 nm. [84] Transmission Electron Microscopy: Carbon-coated copper-rhodium electron microscopy grids with a 300 mesh (Euromedex, France) were surface treated with a glow discharge in an amylamine atmosphere (0.45 mbar, 4-4.5 mA, 22 s) in an Elmo glow discharge system (Cordouan Technologies, France). Then, 5 μL of the solution of NPs at 0.04 g L −1 was deposited onto the grids and left for 2 min. The grids were then treated for 1 min with a 2% uranyl acetate solution for staining. They were observed with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Areas covered with nanoparticles of interest were recorded at different magnifications on a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, CA). Image analysis was performed using the Fiji software.
Estimation of the Number of Dyes Per Particle and the Molar Extinction Coefficient of NP Probe: Using the diameter of NPs measured by TEM (14.4 nm), the volume of a nanoparticle in form of a sphere was calculated. Then, assuming a density of 1 g mL −1 and based on the loading of the donor dye into the polymer matrix of NPs (30 wt% with respect to the polymer), the number of moles of the dye per mass of the single particle was calculated. The latter value was multiplied by the Avogadro constant giving the estimated number of donor dye molecules per particle (≈157). Then, based on the absorption spectrum of NPs and the known molar extinction coefficient of R18 dye (125 000 m −1 cm −1 ), the extinction coefficient of the dye was estimated at 550 nm (100 000 m −1 cm −1 ), corresponding to the excitation wavelength used in microscopy. Then, the total molar extinction coefficient of NP at 550 nm was estimated as 157 × 100 000 = 1.57 × 10 7 m −1 cm −1 , while at the maximum (560 nm) it was 157 × 125 000 = 1.96 × 10 7 m −1 cm −1 . To calculate the number of Streptavidin-www.advancedsciencenews.com www.small-methods.com Cy5 per particles, the molar concentration of the donor NPs (C(donor)) and the acceptor Streptavidin-Cy5 (C(acceptor)) were estimated based on the absorbance of streptavidin-functionalized NPs at 560 and 650 nm, respectively. Then, the number of streptavidin-Cy5 (based on Cy5 molecules) per particle was estimated as C(acceptor)/C(donor). To estimate the ratio of streptavidin protein per particle, the obtained ratio was divided by the number of Cy5 dyes per streptavidin (0.5, estimated according to the absorption spectrum of the commercial streptavidin-Cy5 sample).
Single-Particle Fluorescence Microscopy: Immobilization of nanoparticles and QD-585 in the LabTek chamber was performed according to previously described protocols. [62] The LabTek chamber was washed three times with PBS followed by incubation with 200 μL of BSA-Biotin (0.5 mg mL −1 in PBS) for 5 min. Then, the BSA-biotin solution was removed, and the chamber was washed three times with 500 μL of PBS. In the case of PEMA-ZI-biotin-NPs immobilization, the chamber was incubated with 200 μL of neutravidin solution (0.5 mg mL −1 in PBS) for 5 min and washed three times with 500 μL of PBS. Then, the chamber was incubated with 200 μL of the PEMA-ZI-biotin-NPs solution with proper concentration to achieve desired density and incubated for 30 min at room temperature in the dark. Before measurements, the chamber was washed two times with PBS and covered with 200 μL of the same buffer. In the case of QD-585 immobilization, QD-585 Streptavidin Conjugate (ThermoFisher Scientific) was diluted to 100 × 10 −12 m in PBS and 300 μL was added to the chamber. After 30 min of incubation, the chamber was washed three times with 300 μL of PBS and filled with 200 μL of PBS. Single-particle measurements were performed in the epifluorescence mode using Nikon Ti-E inverted microscope with a Nikon CFI Plan Apo 60× oil objective (NA = 1.4). The excitation was provided by light emitting diodes (SpectraX, Lumencor) at 550 nm. The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 camera. The exposure time was set to 100 ms per image frame.
Single-particle analysis was performed using the Fiji software. Particle locations were detected through a Fiji routine applied to a projection (maximum intensity) of all obtained frames per experiment. After the automatic background subtraction, the mean intensities of circular regions of interest with a diameter of 50 pixels around the found particle locations were then measured. At least three image sequences (500 × 500 pixels) per condition were analyzed.
Biotinylation of Antibodies: Conjugation experiments were carried out in Eppendorf DNA low-bind tube 1.5 mL at atmospheric pressure at room temperature unless otherwise stated. All buffer solutions were prepared with deionized water and filter-sterilized prior to use. Borate buffer was prepared from 25 × 10 −3 m sodium tetraborate, 25 × 10 −3 m NaCl, and 1 × 10 −3 m EDTA at pH 8.1 or 8.4. PBS was 140 × 10 −3 m NaCl and 12 × 10 −3 m sodium phosphate at pH 7.4. Ultrapure DMF (analytical reagent grade) was purchased from Fischer Chemical. Solutions of tris(2carboxyethyl)phosphine hydrochloride (TCEP) 10 × 10 −3 m (2.87 g mL −1 ) were prepared in borate buffer. Ultrafiltration was carried out in a vivaspin 500 polyethersulfone (PES) membrane concentrator with a molecular weight cut-off (MWCO) of 50 kDa. Centrifugation was carried out on an Eppendorf mini spin operating at 12 000 RPM at room temperature. Cetuximab/Erbitux is a chimeric IgG1 full-length antibody directed against EGFR. The antibody was obtained in its clinical formulation (Eli Lilly). The buffer exchange of antibodies was carried for borate buffer pH 8.14 via ultrafiltration (MWCO 50 kDa, Vivaspin). The concentration of antibody was determined by UV-Vis absorbance (ɛ 280 = 210 000 m −1 cm −1 for cetuximab mAb), adjusted to 32 × 10 −6 m (5.0 mg mL −1 ), and was stored as an aliquot at −20°C. For experiments, an aliquot was thawed and used immediately.
The conjugate of cetuximab with biotin was prepared through a modification of a reported protocol). [79,80] Cetuximab (32 × 10 −6 m, 500 μL, 0.016 μmol) was prepared in the borate buffer pH 8.4. Next, biotin-NHS ester was prepared in DMF (100 × 10 −3 m) and added to cetuximab (5 μL, 30 eq). The reaction was incubated at 24°C overnight with rotary shaking at 240 rpm. Afterward, excess of the reagent was removed by ultrafiltration (50 kDa MWCO) with PBS (pH 7.4) to afford the modified antibody-biotin in PBS.
Complex of Cetuximab-Biotin with Streptavidin-Cy5: The cetuximabbiotin (6.4 × 10 −6 m, 10 μL, 0.064 pmol) in PBS buffer was mixed with Cy5-streptavidin (16 × 10 −6 m, 40 μL, 0.64 pmol) in PBS buffer. The mixture was incubated at 24°C for 2 h with rotary shaking at 450 rpm. An excess of Cy5-streptavidin was removed by ultrafiltration (MWCO 100 kDa, Vivaspin) with PBS. Absorbance was measured before and after the process of ultrafiltration. Fluorophore to protein ratio (FRP) value between the antibody-biotin and Cy5 dye of streptavidin-Cy5 was 0.4. For calculation of Cy5 fluorophore to protein ratio (FPR), the following formula was used: where ɛ protein = 210.000 m −1 cm −1 for cetuximab mAb and ɛ Cy5 = 250 000 m −1 cm −1 for Cy5 and 0.59 as a correction factor of the streptavidin-Cy5 conjugate absorption at 280 nm (Cf 280 ). The obtained FPR was 0.59. Then, taking into account that the number of Cy5 dyes per streptavidin was 0.40, the number of biotin units per antibody was 0.59/0.40 = 1.5.
Cell Imaging: For imaging purposes, 8000 cells were seeded per well on an 8-well LabTek (Thermo Scientific NUNC). Cells were first washed with ice-cold OptiMEM (Gibco) and then incubated on ice with 10 μg mL −1 of biotinylated cetuximab. After incubation on ice for 30 min, cells were briefly washed with ice-cold OptiMEM. Cells were fixed with 1% paraformaldehyde (Electron Microscopy Sciences) for 5 min. After washing with DPBS, cells were blocked with 3% bovine serum albumin (BSA, Sigma)/DPBS for 1 h at room temperature. A total of 20 μg mL −1 of neutravidin or streptavidin-Cy3 (Amersham) in DPBS was added for 20 min. Cells were washed with 0.1% BSA/DPBS and incubated with 1 × 10 −9 m of PEMA-ZI-biotin-NPs in 0.1% BSA/DPBS for 20 min at room temperature. Cells were washed two times with 0.1% BSA/DPBS and imaged in the epifluorescence mode using Nikon Ti-E inverted microscope with a Nikon CFI Plan Apo 60× oil objective (NA = 1.4). The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 camera. The excitation was provided by light emitting diodes (LED, SpectraX, Lumencor). Acquisition settings for Rhodamine B were: excitation at 550 nm; emission filter was 600/50 nm; the excitation power of LED was 8% and exposure time of 500 ms. Bright-field images were recorded simultaneously. The images were recorded using NIS Elements and then processed with Fiji software. All images are presented with the same brightness and contrast. For quantitative cellular imaging, mean fluorescence intensity was measured on around 20-40 cells per condition. Statistical analysis was done with the ANOVA algorithm.
Flow Cytometry Analysis: After detachment with 0.2 m EDTA, 500 000 cells were incubated for 30 min with 10 μg mL −1 biotinylated-cetuximab at 4°C under gentle agitation to avoid cell sedimentation. Upon washing, cells were incubated for 20 min at 4°C with 20 μg mL −1 of streptavidin (unlabeled or Cy3-labeled). Finally, cells were washed and incubated at 4°C for 20 min with 1 × 10 −9 m of biotinylated NPs, and excess of particles were washed out. Cells were finally analyzed (counting 20 000 events) using an MACSQuant flow cytometer (Miltenyl Biotec, Saint Jose, CA, USA). Flowing software (version 2.5.1, Turku Bioscience, Turku, Finland) was used to analyze data.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.