Multiplexed immunolabelling of cancer using bioconjugated plasmonic gold–silver alloy nanoparticles

Reliable protein detection methods are vital for advancing biological research and medical diagnostics. While immunohistochemistry and immunofluorescence are commonly employed, their limitations underscore the necessity for alternative approaches. This study introduces immunoplasmonic labelling, utilizing plasmonic nanoparticles (NPs), specifically designed gold and gold–silver alloy NPs (Au:Ag NPs), for multiplexed and quantitative protein detection. These NPs, when coupled with antibodies targeting proteins of interest, enable accurate counting and evaluation of protein expression levels while overcoming issues such as autofluorescence. In this study, we compare two nanoparticle functionalization strategies—one coating based on thiolated PEG and one coating based on calix[4]arenes—on gold and gold–silver alloy nanoparticles of varying sizes. Overall results tend to demonstrate a greater versatility for the calix[4]arene-based coating. With this coating and using the classical EDC/sulfo-NHS cross-linking procedure, we also demonstrate the successful multiplexed immunolabelling of Her2, CD44, and EpCAM in breast cancer cell lines (SK-BR-3 and MDA-MB-231). Furthermore, we introduce a user-friendly software for automatic NP detection and classification by colour, providing a promising proof-of-concept for the practical application of immunoplasmonic techniques in the quantitative analysis of biopsies in the clinical setting.


Introduction
Reliable protein detection methods are crucial for advancing biological research and medical diagnostics.In particular, quantication of protein expression levels in biopsies is usually performed with immunohistochemistry (IHC), a semiquantitative technique that oen lacks the desired accuracy. 1hile immunouorescence (IF) stands out as a promising alternative in research due to its impressive multiplexing capability and resolution, 2 precise quantication remains challenging.Furthermore, the overall process is time-consuming, and the presence of autouorescence in certain tissues limits the widespread adoption of IF in clinical facilities. 3To address this challenge, researchers have been investigating the use of plasmonic nanoparticles (NPs) as protein tags, enabling multiplexed and quantitative detection of analytes in biological samples. 4,5ompared to uorophores, individual plasmonic NPs exhibit a strong signal through light scattering, which allows detection at the single NP level.Moreover, due to their excellent stability, plasmonic NPs do not suffer from photo-bleaching.In particular, we have reported specically designed gold-silver alloy NPs (Au:Ag NPs) displaying optimal optical properties for such use. 6hen coupled with antibodies (Abs) targeting proteins of interest, these NPs can be used to target cells expressing these antigens.Using darkeld microscopy, the number of plasmonic particles bound to the cells can be precisely counted, allowing the evaluation of protein expression levels. 4Termed immunoplasmonic (IP) labeling, this method provides reliable results unaffected by autouorescence and does not require extensive intensity calibration, offering a promising avenue for accurate protein expression level quantication.
Earlier research has included rening a darkeld microscopy approach to optimize the contrast of nanoparticles on cell membranes.This method, entitled side-illumination microscopy, relies on the lateral illumination of the sample with Red-Green-Blue (RGB) Light Emitting Diodes (LEDs).Collecting the signal in inverted or upright mode produces the expected darkeld effect while reducing the signal from the cell membranes and can be performed with a simple RGB camera. 7Notably, we have pinpointed the most effective NPs for protein detection by selecting nanoparticles with Localized Surface Plasmon Resonance (LSPR) bands that align with our lateral illumination diode wavelengths and present high scattering intensity.Achieving such combinations requires the use of NPs with diverse sizes and compositions.
One key challenge in using NPs as protein reporters is to ensure precise control over the graing of antibodies, which is critical to achieving high specicity of interaction with the target cells.This step should, however, not lead to particle aggregation, which would alter the NP plasmonic properties.Typically, bioconjugated nanoparticles nd application in lateral ow assays, [8][9][10] immunoblotting, 11 or immunoprecipitation assays, [12][13][14][15] where stringent control over the level of aggregation is not imperative.However, in IP labelling, where each individual NP is detected under the microscope, maintaining individual nanoparticles is of utmost importance to retain their original colour.Moreover, as our technique involves a variety of NPs, selecting a bioconjugation method that performs uniformly on all the NPs is essential to avoid biases arising from differences in functionalization densities.A common way to conjugate proteins to NPs involves the EDC/sulfo-NHS crosslinking procedure, which requires the presence of carboxyl groups on the surface of the NPs.The use of thiolated PEG-COOH on gold [16][17][18] and silver 19,20 NPs as a precursor for antibody graing has been extensively studied and documented.However, some studies indicate that the thiolation of silver NPs might be challenging for some applications. 21Given our approach involves gold-silver alloy NPs as well as gold NPs, we have also investigated the use of a calix [4]arene-based coating obtained through the reduction of a calix [4]arenetetradiazonium salt. 22,23Indeed, due to its extreme stability, such coating has shown superior performance in protein bioconjugation on silver surfaces compared to traditional thiolation methods, 24 suggesting that this strategy would be efficient for gold-silver alloy nanoparticles.
Our study aims to explore and compare two identied strategies, one involving a thiolated PEG 5kDa coating and the other a calix [4]arene-based coating, for bioconjugation of nanoparticles of various sizes and compositions and their use as protein reporters.We employed 100 nm AuNPs, 50 : 50 Au : Ag alloy NPs measuring 63 nm, and 10 : 90 Au : Ag alloy NPs measuring 50 nm.These specic NP sizes and compositions were selected based on the position of their scattering peaks and to ensure similar light scattering intensities.For conciseness purposes, we will refer to the NPs using their corresponding scattered colours: the 100 nm AuNPs as yellow NPs (yNPs), the 63 nm 50 : 50 Au : Ag NPs as green NPs (gNPs) and the 50 nm 10 : 90 Au : Ag NPs as blue NPs (bNPs).Our research outcomes encompass successful multiplexed immunolabelling of Her2, CD44, and EpCAM in the SK-BR-3 and MDA-MB-231 breast cancer cell lines.Additionally, we have developed a user-friendly soware capable of automatically detecting and classifying nanoparticles based on their distinct colours.These advancements serve as a promising proof-of-concept for the practical application of the immunoplasmonic technique in quantitative biopsy analysis.

Nanomaterials and supplies
The 100 nm, 60 nm gold and 60 nm silver nanoparticles were purchased at Nanocomposix, San Diego.The alloy nanoparticles of various sizes and compositions were synthesized according to the previously reported method. 6First, gold seeds of about 13 nm in diameter are synthesized using the Turkevich method.Then, 2 to 3 growth steps are performed, using the method described at length in the ESI.† The HS-PEG 5kDa -COOH was purchased at Nanocs.The X 4 C 4 was synthesized according to the previously reported method. 25The synthesis of calix [4]arene-tetraacid tetradiazonium salt X 4 C 4 was achieved according to the literature 26 in four steps (i.e.ipso-tetra-nitration, reduction of the four nitro groups, ester hydrolysis and diazotation in 57% overall yield) from commercially available 4-t-butylcalix [4]arene-tetraacetic acid tetraethyl ester (Eburon Organics).Note however that the reduction of the nitro groups of the intermediate tetra-nitro derivative was achieved in 94% yield through hydrogenation (H 2 , Pd/C) and not by using SnCl 2 , as it was previously described.The NPs were functionalized with aCD44 antibodies (Hermes1, MA4400, ThermoFisher), aHer2 antibodies (ICR12, ab11710 and EPR19547-12, ab214275 from Abcam), and aEpCAM antibodies (ab71916, Abcam).

Nanoparticles characterization
Transmission Electron Microscopy images of citrate nanoparticles were taken with a JEM-2100 instrument from JEOL.Briey, the NPs were centrifugated and resuspended in ethanol.The NPs were then placed onto a copper grid coated with a thin carbon lm for electron microscopy observation.All obtained NPs were also characterized with the UV-visible spectrometer Epoch and the hydrodynamic diameters were measured using the Zetasizer Pro Blue instrument from Malvern Panalytical (Malvern, U.K.) in an Ultra-Micro Quartz Cell (ZEN2112).Attenuated total reection Fourier-transform infrared (ATR-FTIR) spectra were recorded at 20 °C on a Shimadzu QATR-S FTIR spectrophotometer.The nanoparticles were centrifugated, and 2 mL of the pellet were deposited on the diamond.Water was removed with a ow of nitrogen gas.Data were processed and analyzed using the instrument soware by correcting the baseline, setting apodization at 10 cm −1 , and normalizing on the most intense signal.The bioconjugation of NPs was veried using a homemade lateral ow assay.Briey, protein A/G at 1 mg mL −1 was deposited with a syringe on a nitrocellulose membrane and dried at 40 °C for a minimum of 2 h.Then the absorbent pad was affixed to the membrane and the membrane was cut into 4 mm strips.The conjugation test is performed by diluting 1-2 mL of NPs (∼5 × 10 10 NPs/mL) in 40 mL of PBST in a well from a 96-well plate.The test is then immersed vertically in the well, allowing the nanoparticles to migrate along the membrane by capillary action.Aer all the liquid has passed through the membrane (approximately 5-10 min), the test is ready for interpretation.

Nanoparticles functionalization
For the PEGylation of NPs, the HS-PEG 5kDa -COOH was rst reduced using TCEP by adding 10% of TCEP$HCl (0.5 M in dH 2 O) in a solution of SH-PEG 5kDa -COOH (10 mg mL −1 in dH 2 O) for 15 min.The TCEP was then ltered twice using 3 kDa centrifugal lters.300 mL of the activated SH-PEG 5kDa -COOH

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was then added to 10 mL of citrate-capped NPs in glass vials and le overnight at room temperature (RT), under stirring.The next day, excess PEG was removed through 3 centrifugation cycles and the PEGylated NPs were resuspended in milliQ water.
For the functionalization of gNPs, yNPs (or bNPs) with X 4 C 4 , 150 mL (100 mL in the case of bNPs) of NaBH 4 at 100 mM was added to 10 mL of citrate-capped NPs in glass vials.Then, 2 × 500 mL (1 × 500 mL in the case of bNPs) of X 4 C 4 at 5 mM were added drop by drop in the solution under stirring.The reaction mixture was stirred overnight at RT and then 20 mL of NaOH at 1 M was added under stirring.The NPs were then rinsed through three centrifugation cycles with NaOH 5 mM.At the last cycle, the NPs-X 4 C 4 were resuspended in Milli-Q water.

Nanoparticles bioconjugation
Two slightly different procedures were followed for the NPs-X 4 C 4 or the NPs-PEG 5kDa -COOH as the attempts to homogenize the protocols were leading to aggregations of NPs or inefficient antibody graing.In a 1.5 mL VWR centrifuge tube, 150 mL of NPs-X 4 C 4 (∼5 × 10 10 NPs/mL) was added.Then, 15 mL MES (100 mM, pH = 5.8), 15 mL EDC$HCl (6 mM, 1.15 mg mL −1 in milliQ H 2 O) and 15 mL Sulfo-NHS (10 mM, 2.17 mg mL −1 ).The activation step was carried out for 1 hour and then, the centrifuge tube was lled to 0.75 mL with MilliQ H 2 O.The centrifuge tube was centrifugated once (12 min, 3000 g) at room temperature.The supernatant was discarded, and the pellet was resuspended in 150 mL phosphate buffer (5 mM, pH = 7 or pH = 8).Then, 10 mL of antibodies (0.1 mg mL −1 in 5 mM PB pH 7 or 8) were added and the reaction mixture was stirred for 1 hour at room temperature.Aer 1 hour, 600 mL of a solution of 1% BSA, 0.1% Tween 20 in 5 mM PB pH 8.The centrifuge tube was stirred for 3-5 minutes and then, centrifuged for 12 min at 3000 g.Aerward, the supernatant was discarded, and NPs were resuspended in 1 mL of the solution 1% BSA, 0.1% Tween 20 in 5 mM PB pH = 8 and once again, centrifuged as before.At the last cycle, NPs were re-suspended in 150 mL of 0.1% Tween 20 in 5 mM phosphate buffer (pH = 7 or pH = 8).The resulting particles were stored at 4 °C.
The protocol for the PEGylated NPs differs slightly but shares the key steps.In a 1.5 mL centrifuge tube, 150 mL of NPs-PEG-COOH (∼5 × 10 10 NPs/mL) were added.MilliQ was replaced by MES buffer (0.5 M) through the centrifugation cycle.The activation step was then carried out for 30 min by adding 13 mL of EDC$HCl (6 mM) and 7.4 mL of sulfo-NHS (10 mM).The excess EDC and sulfo-NHS were washed through a centrifugation cycle and the NPs were resuspended in 5 mM potassium phosphate (pH = 7) and 0.1% Tween 20.Then, 10 mL of antibodies (0.1 mg mL −1 ) were added and the reaction was carried out for one hour.The reaction was then quenched by adding 2 mL of hydroxylamine (50%) for 10 min.The NPs-PEG-Abs were then washed through 3 centrifugation cycles and resuspended in the 5 mM potassium phosphate (pH = 7) and 0.1% Tween 20.The resulting NPs-PEG-Ab were stored at 4 °C.

Cell culture and immunolabelling
The breast cancer cells MDA-MB-231 (ATCC, HTB-26) were cultured in Dulbecco's Modied Eagle Medium (DMEM) while the SK-BR-3 (ATCC, HTB-30) cells were cultured in the recommended McCoy culture medium.Following standard practices, both media were supplemented with 10% FBS, and 1% P/S.We prepared the samples by seeding cells in 8 well plates with removable chambers (Ibidi) and letting the cells grow for 24 to 48 hours.Before immunolabelling, the cells were xed with cold methanol/acetone (70/30%) for 10 min and rinsed with PBS.A one-hour blocking step was performed in (PBS, Tween 20 0.1%, 1% BSA) and the samples were rinsed with PBS before protein labelling with the method of interest.For immunouorescence, two times 1 h incubations were performed with a dilution of 1 : 200 and 1 : 500 respectively for the primary and secondary antibodies.For the immunoplasmonics method, the NPs were incubated for one hour with the cells.Aer immunolabelling, the cells were rinsed 3 times with PBS and 0.1% Tween 20.

Cell imaging
The samples were imaged on an inverted Ti-Eclipse microscope (Nikon) equipped with a lateral illumination adapter for the darkeld mode, as reported previously. 7The images were taken with a colour camera (pandas4.2,PCO) with xed parameters.The images were taken using alternatively a 40X-0.6 air objective or a 60X-1.1 N.A. immersion oil objective, and the sample was scanned along the z-axis to capture all the NPs present on the cell membranes.The acquired slices were then stacked and projected onto a single picture that was later used for the analysis.

Results
Nanoparticles coating with either a thiolated PEG 5kDa or a calix [4]arene The citrate-stabilized alloy NPs were rst synthesized using a previously reported procedure. 6TEM and EDS measurements showed that we achieved highly monodisperse 50 nm 15 : 85 Au : Ag NPs displaying a blue colour, and 63 nm 43 : 57 Au : Ag NPs displaying a green colour as shown in Fig. S1, † displaying the TEM and UV-vis characterization of these NPs.They were then functionalized either through chemisorption of a thiolated PEG, i.e.HS-PEG 5kDa -COOH, or reductive graing of calix [4]  tetra-diazonium salt X 4 C 4 (N 2 + ) 4 (Scheme 1).For conciseness, in the rest of the paper, we will refer to the two types of coating we observed a mean increase in the hydrodynamic diameter of 15 nm (Table 1).For the PEG 5kDa coating, we measured an increase in the hydrodynamic diameter of 37 nm for the yNPs and 20 nm for the bNPs and gNPs.
In addition, the functionalization led to a red shi of the plasmon peak position of 3, 9, and 12 nm for the X 4 C 4 and 2, 5, and 7 nm for the PEG 5kDa coatings of yNPs, gNPs, and bNPs, respectively (Fig. 1A).These shis in the extinction spectra can be attributed to changes in the surrounding environment of the nanoparticles, indicating a modication of their organic coating.The extent of the shi correlates with the intrinsic properties of the NPs, the thickness of the coating corona and the effective refractive index of the coating.The PEG 5kDa a layer is mostly swollen by water and, therefore, its effective refractive index is relatively close to that of water.In contrast, the X 4 C 4 coating should lead to a signicantly higher refractive index than water due to the presence of the hydrophobic polyaromatic macrocycles.Therefore, despite being thinner, the X 4 C 4 coating induces a much greater peak shi than the PEG 5kDa layer.A certain broadening of the LSPR band was also observed for all NPs-PEG 5kDa , indicating a higher aggregation level or a higher disparity in surface coating with this ligand (Fig. 1A).
The characterization of the resulting NPs-X 4 C 4 using FTIR spectroscopy conrmed the graing of calix [4]arene X 4 C 4 , with characteristic bands at 1600 and 1450 cm −1 corresponding to COO − stretching and aromatic ring stretching, respectively. 27he FTIR spectrum of NPs-PEG 5kDa is dominated by bands attributed to C-O-C asymmetrical stretching (1110 cm −1 ) and C-H(CH 2 ) stretching (2840-3000 cm −1 ) from the long polymer chain.The spectra obtained for yNPs are shown in Fig. 1B, while comparisons for all types of NPs are available in the ESI (Fig. S2).† It is noteworthy that characteristic bands originating from citrate, such as those at 1590 and 1400 cm −1 for COO − asymmetrical and symmetrical stretching, were no longer observed aer functionalization, conrming the efficiency of the ligands exchange process.
The colloidal stability of NPs-PEG 5kDa and NPs-X 4 C 4 was evaluated in saline buffer (PBS 1×) and compared to that of NPscitrate.The results obtained for the bNPs are presented in Fig. 2 (see Fig. S3 † for gNPs and yNPs).As shown in Fig. 2A, a signicant modication of the UV-vis spectrum was observed for citrate-capped NPs, indicating NPs aggregation and dissolution.Indeed, citrate-capped NPs are only stabilized through electrostatic repulsion between the negatively charged citrate molecules.When the ionic strength is increased by the addition of PBS, the electrostatic repulsion vanishes leading to the aggregation of the NPs driven by the attractive van der Waals forces.In contrast, the coatings based on calix [4]arene and thiolated PEG ligands led to stable colloids in PBS, validating these functionalization strategies (Fig. 2B and C).Regarding the PEGylated NPs, although the yNPs-PEG 5kDa remained stable for months (at RT in the dark), both bNPs-PEG 5kDa and gNPs-PEG 5kDa started to show signs of instability aer a few weeks (decreasing OD, adsorption on tubes).Conversely, no signicant loss was observed for all NPs-X 4 C 4 , highlighting their longterm storage stability which stands as a clear advantage for practical applications in biopsy diagnostics in clinical settings.

Bioconjugation using covalent bonding or adsorption
The bNPs, gNPs and yNPs either coated by X 4 C 4 or the thiolated PEG were bioconjugated with aCD44 or aHer2 antibodies (Ab) through classical peptide coupling chemistry using EDC and sulfo-NHS.The pH of the peptide coupling protocol had to be optimized for each antibody in order to ensure colloidal stability throughout the process: aHer2 Ab coupling was achieved at pH 8, while aCD44 Ab bio-conjugation was performed at pH 7. To ascertain the necessity of covalently attaching the biomolecules to the NPs, we also explored a faster and simpler alternative for bioconjugation with bNPs by performing onestep antibody adsorption.All batches of bioconjugated NPs presented sharp and intense extinction spectra, indicating no signicant aggregation of the NPs upon bioconjugation (see Fig. 3 for bNPs).In all cases, the presence of antibodies was assessed using a protein A/G lateral ow assay (LFA), which relies on the high affinity of protein A/G for the heavy chains of antibodies.The interaction between the antibodies bound to   the NP surface and the protein A/G deposited on the test line, leads to the immobilization of the nanoparticles on the test line, resulting in a positive, coloured line that displays the transmitted colour of the NPs (see the ESI † for more details).Bioconjugation of the various Abs was successfully achieved on both yNPs-X 4 C 4 and yNPs-PEG 5kDa , as attested by the positive red line visible on the LFA strips (Fig. S4 †).However, in the case of bNPs and gNPs, the protein A/G LFA yielded a positive result only when the antibody was conjugated to the X 4 C 4 coating, either via covalent bonding or adsorption (Fig. 3 and S4 †).The absence of a coloured line with the bNPs and gNPs PEG 5kDa suggested the absence of conjugated antibodies.Additionally, in contrast to NPs-X 4 C 4 , no redshi of the plasmon peak of these colloids was observed aer the antibody bioconjugation step with NPs-PEG 5kDa .To investigate whether this differential behaviour between the 100 nm AuNPs (yNPs) and the smaller Au:Ag alloy NPs (gNPs and bNPs) was due to the size or the intrinsic composition of the NPs, 60 nm AuNPs and AgNPs were also coated with the thiolated PEG 5kDa ligand and then bioconjugated as described above (Fig. S5 and Table S2

†).
A low mAb conjugation rate was also observed for 60 nm AuNPs and AgNPs, as indicated by the absence of a line on the LFA strip.This additional result suggests that the size and curvature of the NPs might affect the low accessibility of the carboxyl groups from the PEG chains, resulting in low Ab graing efficiency.

Protein detection in cells
To assess the effectiveness of the NPs-X 4 C 4 -Ab and NPs-PEG 5kDa -Ab as immunoplasmonics protein tags in biological samples, we performed a one-step immunolabelling procedure with two breast cancer cell lines, MDA-MB-231 and SK-BR-3, which exhibit different levels of CD44, Her2 and EpCAM protein expressions.The protocol was adapted from the standard direct immunouorescence method and involved a blocking step, followed by a one-hour incubation with NPs-Ab, and a washing step.As a control for the quantication of protein expression in the chosen cell lines, we employed a standard indirect IF procedure using the Abs that were used for bioconjugation and secondary antibodies labelled with AF488 (green uorescence).The results from IF analysis revealed distinct patterns of protein expression in the SK-BR-3 and MDA-MB-231 cells for the three markers of interest (Fig. 4).Specically, the SK-BR-3 cells displayed a strong expression of the Her2 protein whereas the MDA-MB-231 cells showed minimal expression.Additionally, our control experiments highlighted a unique behaviour within the SK-BR-3 cell population.Although the majority of these cells showed negligible expression of the CD44 protein, a small fraction demonstrated pronounced overexpression.In contrast, the MDA-MB-231 cells exhibited a strong homogeneous expression of the CD44 protein.Both cell lines weakly express the EpCAM protein, with a slightly higher expression level for the SK-BR-3 cell line.The full immunouorescence data, including the negative controls with the secondary antibodies only, are included in the ESI (Fig. S6).† First, using yNPs-PEG 5kDa -Ab as protein labels, a strong correlation was observed between IP and indirect IF for both cell lines(Fig.4).The staining of the cellular cytoplasm with Wheat Germ Agglutinin (WGA), along with the negligible binding of NPs to the glass substrate conrmed the co-localization of the NPs with the cell membrane, indicating a good specicity of the obtained yNPs-PEG 5kDa -Ab (Fig. S7 †).For further validation of the specicity of yNPs-PEG 5kDa -aCD44, we conducted a dualmode immunolabelling by applying both immunoplasmonics and immunouorescence methods to the same sample of SK-BR-3 cells (Fig. S8 †).This allowed us to conrm the equivalence of the results obtained with the two staining techniques, providing additional evidence for the reliability of immunoplasmonics.Similar results were obtained with yNPs-X 4 C 4 -Ab.
Aer conrming the potential of functionalized NPs as protein reporters, we applied the same procedure to bNPs coated with either PEG 5kDa or X 4 C 4 and further bioconjugated to antibodies through EDC/sulfo-NHS chemistry (NPs-PEG 5kDa -Ab and NPs-X 4 C 4 -Ab cov ) or adsorption (NPs-X 4 C 4 -Ab ads ).As

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Nanoscale Advances expected, no signicant cell or substrate binding was observed for bNPs-PEG 5kDa -aCD44 (Fig. S9A †), in line with the LFA results (vide supra).For bNPs-X 4 C 4 -aCD44 ads , we mostly observed non-specic attachment on the substrate, and few NPs on cells (Fig. S9B †), which was unexpected given the positive result from the LFA.This discrepancy might be due to an unfavorable orientation of the adsorbed Ab on the surface; however, further investigation is warranted to better understand this result.In contrast, bNPs-X 4 C 4 -aCD44 cov led to the expected heterogeneous binding with the SK-BR-3 cells (Fig. S9C †).Covalent graing of antibodies on the NPs-X 4 C 4 was therefore chosen as the preferred technique for NPs bioconjugation and was used for the rest of the study.
Next, we tested all types of NP-X 4 C 4 -Abs cov on the two cell lines to validate their specicity.Fig. 5 shows the CD44 staining results on both cell lines using different colours of NPs, illustrating the tunability of the technique and the reliability of the results.Consistent results were also obtained with aHer2 antibodies (Fig. S10 †).Upon initial examination, the optical properties of the various NP types were largely preserved during incubation with cells, and the samples prepared with a single NP type were readily distinguishable.These single-NP-type samples were later used as controls for the training of the automated classication soware.

Application to multiplexed immunolabelling on breast cancer cell lines
Aer conrming the feasibility of antibody graing on each type of NPs and the ability of these hybrid nanoconjugates to target specic receptors at the surface of cells, we used various combinations of these bioconjugated NPs for dual immunolabelling on both cell lines.The results for the SK-BR-3 cell line are depicted in Fig. 6A.For clarity, the images were cropped to emphasize cases where a cell over-expresses CD44 and underexpresses Her2 compared to its neighboring cells.This phenomenon was corroborated through dual immunouorescence, even though some cells expressed both receptor types in similar proportions, as observed through both techniques.Examples of dual immunouorescence assays are available in the ESI (Fig. S11).† Additionally, we achieved consistent plasmonic immunolabelling on the MDA-MB-231 cell line, as shown in the ESI (Fig. S12).† Similarly, immunolabelling of three markers was achieved on both cell lines, using bNPs, gNPs, and yNPs to target Her2, CD44, and EpCAM proteins, respectively.

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The comparison between the two cell lines is provided in the ESI (Fig. S13), † validating the possibility of performing multiplexed detection of up to three membrane receptors (Fig. 6B).

Image analysis and automated classication of NPs
To advance the use of bio-conjugated NPs as protein reporters, we developed an automated classication and tested various standard supervised classication techniques.The soware is publicly available on GitHub.§ The best results were obtained using a Support Vector Machine (SVM) with a Radial Basis Function (RBF) kernel on normalized Hue, Saturation and Value (HSV) signals extracted from the NPs, resulting in three features per NP.First, each RGB image is transferred to the HSV colour space.The HSV colour space was selected as it enables a better separation of the colour and saturation from the brightness, making the colour identication more resilient to variations in lighting and illumination.Then, an algorithm based on local maxima detection locates each NP present in the image.The average signals from a 5 × 5 pixels region of interest (ROI) around each NP location are extracted, resulting in a feature vector composed of the mean H, S and V values associated with each NP detected.Fig. S14 † illustrates the different representations of the NPs signals in RGB or HSV colour space.A dataset composed of ten thousand labelled NPs was normalized, split into a training set, used to train the model, and a validation set, used to assess the algorithmic performances.Fig. 7 shows the input data (Fig. 7A), considered as the ground truth, the classication of these data using a Naïve Gaussian Bayes classier (Fig. 7B), and the classication using the supervised SVM with an RBF kernel (Fig. 7C).A way to better encompass the performances of classication algorithms is to use the confusion matrix, which compares the ground truth (true label) to the classication output (predicted label).The confusion matrixes for the two classiers are also depicted in Fig. 7D and E.
Then, we used the trained classier to count the number of nanoparticles of each type in the various multiplexed samples, as shown in Fig. 8A shows the results of the classication performed on a new sample prepared with bNPs-X 4 C 4 -aHer2 only.The algorithm classied 98% of the NPs as blue, which is the expected answer given that we have incubated the cells with blue NPs only.We can see that among the NPs that were mis-classied, some were considered green (1.5%) or yellow (0.5%).For each class k, the error in classication can be measured as follows: where the rst terms represent NPs from other classes classied as class k (false positive) and the second term NPs from class k that were classied as other NP types (false negative).In the eqn (1), c i,j are the coefficients from the confusion matrix, and n i the number of particles from class i.To calculate the total classication error, we used the confusion matrix that was obtained aer the training (Fig. 7E).For instance, in the entire image from which we extracted Fig. 8C, we counted 3107 ± 42 bNPs, 783 ± 29 gNPs, and 553 ± 31 yNPs, which is consistent with the expression levels of the proteins in the cell line, as validated with IF.

Discussion
We have successfully conrmed the potential of plasmonic nanoparticles as protein reporters.While the use of HS-PEG 5kDa -COOH as a surface ligand appeared suitable for the bioconjugation of antibodies with 100 nm AuNPs, the NPs coating with calix [4]arene X 4 C 4 proved to be a much more versatile strategy for the bioconjugation of antibodies with nanoparticles of various compositions and sizes.The stability of

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NPs-PEG 5kDa in PBS clearly indicates the presence of the PEG layer at their surface for all types of NPs.However, both LFAs and incubation with cells have shown poor antibody coupling efficiency to our smaller NPs using the PEG 5kDa linker.The absence of reactivity of the terminal COOH groups with amino groups upon peptide coupling conditions could be due to several reasons.It is known that for a given molecular weight, the graing density achieved is lower for smaller nanoparticles. 28Hence, for the smaller nanoparticles, the greater radius of curvature could lead to an increasing distance between two PEG strands, allowing the bending of their tips in a mushroom-like conguration, as reported previously for PEG 5kDa on 60 nm AuNPs. 29This arrangement could potentially heighten steric hindrance around the carboxyl group, preventing an efficient bioconjugation.Another issue regarding the PEGylation of NPs containing silver was the observed desorption with time, leading to a signicant loss in colloidal stability.While the strategy adopted for the PEGylation leverages the adsorption of thiols on gold and silver surfaces, the mechanism of graing of X 4 C 4 on metal surfaces involves the reduction of its aryl diazonium units into the corresponding aryl radicals, which subsequently react with the metal surface to form strong and irreversible covalent metal-C bonds. 26X 4 C 4 being equipped with four diazonium groups, each macrocycle can form up to four metal-C bonds with the surface, resulting in an increased density of anchoring points and much more robust coating compared to the use of simple aryl diazonium salts or thiolated ligands. 23The latter is generally considered as covalent bond but, however, is still subject to debate in literature.Previous studies determined a density of 1.5 X 4 C 4 /nm 2 for AuNPs of 15 nm and 0.53 X 4 C 4 /nm 2 for at surfaces. 30,31Therefore, we can assume an intermediate value of graing density for NPs size ranging from 60 to 100 nm.Overall, the X 4 C 4 coating has shown superior performances in terms of stability and bioconjugation than the PEG 5kDa coating.The preparation of the samples containing a single NP type also gives an insight into the maximal precision that could be reached with the technique, considering the experimental conditions studied in this work.Indeed, although they represented less than 5% of the NPs detected, few NPs did not display the expected colour.Various factors could explain the discrepancies, ranging from disparity in the synthesis to the formation of clusters during the bioconjugation process or the incubation with the cells.This issue could be mitigated by investigating ltering steps using either a mechanical lter or centrifugation steps prior incubation with the cells.Regarding the multiplexed detection of proteins, it is noteworthy that the incubation of the multiple NP types was conducted in one step, resulting in a signicant gain of time as compared to subsequent incubations.Importantly, mixing the various NP types did not increase the aggregation levels of the colloids, leading to highly distinguishable labelling of each protein type.Once again, the comparison with immunouorescence has reaffirmed that the observed NP attachment aligns with the protein expressions of the studied cell lines.Yet, the immunoplasmonic technique exhibits greater versatility compared to multiplexed immuno-uorescence, as it is unaffected by antibody host constraints which requires making some compromises when elaborating a multiplexed antibody panel.Moreover, it is not sensitive to channel bleed-through, which typically necessitates extensive calibration for intensity quantication.Although all the prepared NPs-X 4 C 4 -Ab have shown the expected behaviour, it was noted that direct visual examination of the samples was challenging for some NPs combinations.For instance, the chosen yNPs display a much higher scattering level than the bNPs.In addition, within the MDA-MB-231 cell samples, discerning the sparse bNPs-X 4 C 4 -aHer2 among the numerous yNPs-X 4 C 4 -aCD44 was particularly challenging without image analysis.Conversely, one can leverage the great brightness of yNPs to detect rare antigens, as demonstrated in our ability to detect the EpCAM protein in both cell lines and the Her2 protein in MDA-MB-231 cells.Compared to more complex techniques, such as immunouorescence or Raman spectroscopy, a very simple apparatus can be affixed to any microscope and a standard RGB image is only needed to enable the automated analysis.As for the automated detection of NPs, the use of a simple supervised training algorithm for classication such as a Support Vector Machine has helped to gain in accuracy compared to naïve classication based on Bayes ltering.The soware developed in this study will prove invaluable for future research endeavours, particularly in the calibration of the developed technique, with a notable focus on advancing protein quantication using these nanoparticles.

Conclusions
We have developed an effective methodology for graing antibodies onto a wide range of plasmonic nanoparticles, which offers essential advantages for immunolabeling while preserving their distinct optical properties.Furthermore, in comparison with the classical coating approach using thiolated PEG ligands, we have demonstrated the superiority of the calix [4]arene coating for the formation of ultrastable and bioconjugable plasmonic NPs of various sizes and compositions.The utility of these calix [4]arene-based nanoparticles has been corroborated through their efficient use as multiplexed optical tags for the detection of a variety of membrane proteins.Additionally, a sophisticated soware tool has been developed for automated nanoparticle quantication in biological samples based on their class, further emphasizing the practical applications of immunoplasmonic in clinical settings.While triple multiplexing has been shown in this work, using NPs presenting other plasmonic peaks coupled with potentially hyperspectral imaging may open up the possibility of offering a higher level of multiplexing to biologists and pathologists.

Fig. 1 (
Fig. 1 (A) UV-vis spectra of the various NPs before and after functionalization with HS-PEG 5kDa -COOH or X 4 C 4 ; (B) FTIR spectra for yNPs with various surfactants.

Fig. 3
Fig.3UV-vis spectra of bNPs before and after antibody bioconjugation and picture of the protein A/G strip used to assess the presence of antibodies for (A) aCD44 covalently grafted on bNPs-PEG 5kDa , (B) aCD44 covalently grafted on bNPs-X 4 C 4 and (C) aCD44 adsorbed on bNPs-X 4 C 4 .

Fig. 4
Fig. 4 Immunofluorescence (protein of interest in green AF488, nuclei in blue, DAPI) and immunoplasmonics (yellow anti-protein NPs, yNPs-PEG 5kDa -Ab) images of proteins Her2, CD44 and EpCAM in MDA-MB-231 and SK-BR-3 cell lines showing the equivalence between the results obtained with these two techniques.The images were acquired on an inverted Ti-Eclipse microscope (Nikon), a 40X-0.6NAair objective and using the epi-illumination arm (IF) or custom lateral illumination (IP).

Fig. 5
Fig. 5 Immunoplasmonics images showing NPs-X 4 C 4 -aCD44 cov of various types used for CD44 protein detection on SK-BR-3 (top row) and MDA-MB-231 (bottom row) cell lines.For the SK-BR-3 cell line (top row), the insets show the heterogeneous attachment of NPs-X 4 C 4 -aCD44 cov , which correlates with the CD44 expression found with immunofluorescence.

Fig. 6 (
Fig. 6 (A) Immunoplasmonics images showing the double detection of Her2 and CD44 proteins on the SK-BR-3 cell line using various NP types.The images on the diagonal display the corresponding NPs on a glass substrate.Scale bar is 25 mm in all images.(B) Triple detection of Her2, CD44 and EpCAM on the SK-BR-3 cell line using respectively bNPs-X 4 C 4 -aHer2, gNPs-X 4 C 4 -aCD44 and yNPs-X 4 C 4 -aEpCAM.Scale bar is 50 mm.

Fig. 7 (
Fig. 7 (A) -Input data.Each dot represents a single NP in the HSV color space.The color of the dot represents their class.(B)result of the classification on the validation dataset using a na ïve Bayes classifier.The red dots correspond to misclassified NPs.(C)result of the classification using a Support Vector Machine (SVM) with a Radial Basis Function (RBF) as a kernel.(D and E)are the corresponding confusion matrixes, normalized according to the real number of NPs of each class in the testing set.