Results and discussion

Synthesis and biofunctionalization of AuNPs to achieve specificic cell labeling

Since the CTCs detach from a primary tumor we chose an adherent tumor cell line, Human Colon Adenocarcinoma Cell line (Caco2), as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM (close to 100%) and for this reason this glycoprotein was used as target.^[11] Several commercial antibodies were tested by flow cytometry in order to choose the one that allows a better labeling of cells, and that would later be conjugated to the AuNPs forming a biofunctionalized specific label for the electrochemical detection of Caco2.

The biofunctionalized electrochemical labels were prepared by conjugation of AuNPs (20nm prepared using Turkevich’s citrate capped modified synthesis^[23]) with anti-EpCAM antibody following a previously optimized protocol.^[15] The nanoparticles were characterized by Transmission Electron Microscopy (TEM) and also UV/Vis Absorbance spectroscopy, to check both the size distribution, and the presence of the antibody layer around them after biofunctionalization (Figure 2). We observed a size distribution of 19.2 ± 1.37 nm and a typical absorbance maximum at 520 nm that shifts to 529 nm after biofunctionalization. This red-shift in the absorbance is explained by the changes in the AuNPs-surface plasmon resonance, indicative of a different composition of the surface and evidencing the formation of the conjugate.

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Microscopy images and cytometry analysis of cell interaction with biofunctionalized AuNPs

To assess the effectiveness of AuNPs/anti-EpCAM-conjugate labels their specific interaction with cells was evaluated. Caco2 cells were used in suspension.

The free anti-EpCAM antibody proved to have high affinity for EpCAM at Caco2 surface, but it was necessary to verify that after conjugation with AuNPs the antibody maintains the ability to recognize the target protein. Therefore, fluorescence microscopy imaging of cell samples before and after incubation with biofunctionalized AuNPs, using a fluorescent-tagged secondary antibody, was performed. Prior to the incubation, cells (10⁶ cells/mL) were centrifuged (1000rpm, 5min) and the pellet was re-suspended in PBS-BSA 0.1%. Afterwards, cells (200 x10³ cells) were incubated with 50µL of AuNPs/anti-EpCAM-conjugate, as prepared solution. After incubation (30 minutes/ 25ºC, under agitation) labeled cells were centrifuged, washed two times to eliminate the excess of AuNPs/anti-EpCAM-conjugate, resuspended in PBS-BSA 0.1% and incubated with FITC-conjugated anti-rabbit antibody used as a label for fluorescence analysis.

As shown in Figure 3, fluorescence at the cell membrane allows assuring the specific biorecognition of the Caco2 cells with the anti-EpCAM antibody functionalized AuNPs. This fact is also evidenced by flow cytometry analysis of cell samples. This method is well suited to check the affinity of different antibodies to several cell proteins and by using the proper controls it can also be used to quantify both labeled and unlabeled cells. Using the same protocol of sample preparation as for optical microscopy, cell samples were analyzed using a flow cytometry. Similarly to the previous method, only when the cells were labeled with AuNPs/anti-EpCAM-conjugate, besides the staining with fluorescent secondary antibody, a strong increase in cell fluorescence was observed (Figure 3c). Several controls were

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performed for both methods. Caco2 cells were incubated with anti-EpCAM antibody both free and conjugated to AuNPs. Controls were also performed with citrate modified AuNPs without anti-EpCAM, and with AuNPs conjugated to another polyclonal anti-EpCAM antibody which proved to be non-specific to Caco2 cells.

Electrochemical detection of AuNPs labeled Caco2 cells

After the optimization of several incubation related steps (time, temperature, agitation, etc.) the cell samples were analyzed by the electrochemical method. After the incubation protocol (detailed in experimental section), Caco2 cells were detected through the chronoamperometric measurement of the HER in 1M HCl that was electrocatalyzed by AuNPs labels. Figure 4a displays the relation between the analytical signal and the concentration of Caco2 cells, in the range between 0 and 1.5 x10⁵cells. A linear relation was observed between 1 x10³and 5 x10⁴ cells with a limit of detection (LOD) of 4.41 x10³ cells (calculated as the amount corresponding to three times the standard deviation of the estimate) with a correlation coefficient of 0.9902 and a RSD of around 4% for three repetitive assays performed with 5 x10⁴ cells.

To demonstrate the specificity of the electrochemical detection a selectivity test was devised.

CTCs circulate in the blood flow among thousands of other human cells and their detection must be selective enough to avoid false positive results. Thus we chose a circulating blood cell line (monocytes) to simulate the possible interference caused by other cells in our detection. Samples were prepared by mixing in suspension the Caco2 cells with monocytes (THP-1 cells) in different proportions. The cell samples were then incubated with the AuNPs/anti-EpCAM-conjugate (50µL). After removing the excess of conjugate by

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centrifugation, samples were analyzed by the electrochemical method described above. The total quantity of cells was fixed at 5x10⁴, and different ratios between Caco2 cells and monocytes were evaluated. No analytical signal was obtained from the sample containing 100% of monocytes. Furthermore, the increasing percentage of Caco2 cells assayed in the presence of decreasing quantities of monocytes resulted in an increase in the analytical signal independent of the monocytes quantity, demonstrating the specificity of the assay.

The statistical analysis reported an LOD of 5.42 x10³ Caco2 cells, with a correlation coefficient of 0.9928 in a linear range from 1 x10³ to 5 x10⁴ cells, with an RSD = 2% for 5 x10⁴ cells (3 replicates). The results demonstrate that this method is selective for the target cells and that the electrochemical signal is not affected by the presence of other circulating cells.

Scanning Electron Microscopy (SEM) images of cel interaction with biofunctionalized AuNPs

Although Scanning Electron Microscopy (SEM) is a well-known cell characterization technique, its use for liquid suspensions that involve interaction of cells with small nanometer-sized materials is rather difficult. Due to the requirements of structure stability and electron conductivity necessary for high magnification SEM images, it is often necessary to perform sample metallization that would hide the low nanometer nanoparticles interacting with the cell surface, in addition to changing its outer-layer chemical composition. To perform cell analysis, after their incubation with AuNPs/anti-EpCAM-conjugate, the cells were kept in suspension and treated with glutaraldehyde solution followed by sequential dehydration with ethanol and resuspended in hexamethyldisilazane solution. This protocol allowed a good fixation of cells (from suspension) while maintaining cell shape (Figure 5) and the membrane

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outer structure intact. The obtained results proved this protocol to be well suited for the observation of cells without the need of metallization or any other procedure that would change or mask the nanosized conjugate (AuNPs/anti-EpCAM) used to label the cell membrane. Figure 5a shows SEM-Backscattered images of CaCo2 cells while figure 5b and 5c show the SEM-Backscattered images of both Caco2 and THP-1 cells contained in the

mixture 70%-Caco2/30%-THP1 after their incubation with AuNPs/anti-EpCAM-conjugate. In figure 5b it is possible to observe the cell membrane with enough detail to discriminate the small nanoparticles attached. We used the Backscattered Electrons mode to be sure that these small structures are indeed the specifically attached AuNPs. Since heavy elements backscatter electrons more strongly than light elements, they appear brighter in the image enhancing the contrast between different chemical compositions.

Both Caco2 cells in suspension and monocytes have a round shape and it is difficult to differentiate them by optical microscopy techniques. But with the optimized SEM preparation protocol we obtained high quality images where we can clearly observe the detail of Caco2 plasma membrane and perceive the numerous particles all around the cell surface.

3. Conclusions

In conclusion, a novel electrochemical strategy to detect and quantify CTCs based on the selective labeling with biofunctionalized AuNPs has been achieved and its efficiency followed by flow cytometry and SEM-Backscattering imaging. The proposed sensor is a rapid and simple CTC detection device that uses specific antibody/AuNPs conjugate to recognize tumor cells in suspension followed by detection in a user-friendly platform.

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In our particular example, the observations proved that the labeling with anti-EpCAM-functionalized AuNPs is selective for Caco2 cells and therefore, the catalytic electrochemical detection method developed is specific for the target cells despite the presence of other circulating cells. The electrocatalytic detection of AuNPs/anti-EpCAM labeled Caco2 cells resulted in a limit of detection near 4 x10³ cells.

Our strategy can be adapted for the detection of other tumor cells that also overexpress Ep-CAM, or to other cancer cell receptors by redesigning the AuNPs conjugate. We believe this is a big input in the CTC quantification state of the art techniques that struggle to achieve novel tools for “liquid biopsies” in order to perform patient prognosis, predict metastasis formation and monitor the therapeutic outcomes of cancer. In addition we expect that this method can be combined with cell separation/filtration fluidic platforms, in order to obtain portable and cost-effective alternative CTC quantification devices in the optic of point-of-care sensing systems.

4. Experimental section