Gold nanoflower‐based surface‐enhanced Raman probes for pH mapping of tumor cell microenviroment

Abstract Objectives Early diagnosis of tumour cells is critically important for cancer treatment. Given that the tumour environment is slightly acidic, the pH value of the cell environment can be used as a criterion for tumour diagnosis. However, mapping pH in the cell environment with high resolution, high sensitivity and accuracy remains challenging. Materials and Methods Based on gold nanoflower as surface‐enhanced Raman scattering (SERS) substrate loading with p‐mercaptobenzoic acid (MPA) as pH‐responsive Raman reporter, a new SERS nanoprobe for pH mapping was developed. Results This probe showed a characteristic Raman spectrum signal in response to the different pH in solutions or cells. The signal intensity is positively correlated to the pH value. Moreover, this probe is self‐correctable, which can help eliminate the influence of probe concentration on the accuracy of pH measuring. Conclusions We demonstrate the pH mapping of cell environment using the probe, which can be used to distinguish normal cells and tumour cells. This method may provide a new imaging tool for early diagnosis of cancer.

as X-ray computed tomography (CT), 13,14 magnetic resonance spectroscopy (MRS), 15 magnetic resonance image (MRI), [16][17][18] positron emission tomography (PET) 19 and other technologies. 20,21 However, it is difficult to find the small tumour cell clusters and metastases with these techniques for low spatial resolution and low sensitivity.
Recent studies have indicated that the microenvironment of the tumour is weakly acidic, while the normal tissue is neutral/weakly alkaline. [22][23][24] Therefore, early diagnosis of tumour could be achieved with monitoring the pH microenvironment of tissues. [25][26][27][28] For this point, a variety of fluorescent pH probes have been used for the development of tumour imaging methods. [29][30][31] Fluorescence imaging methods possess higher resolution and sensitivity compared with other imaging ways. 32,33 However, there are some disadvantages for this kind of imaging. For example, the fluorescence molecules were poor in photostability and anti-interference ability. 34 In addition, the signal intensity of the pH-responsive fluorescent probe is difficult to achieve quantitative analysis. Therefore, it is important to develop a pH imaging method that combines high sensitivity, stability and quantitative analysis.
Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive non-destructive detection technology. 35,36 Based on this technology, detection sensitivity could be improved to reach 14 orders of magnitude higher than conventional Raman spectroscopy, 37 which is comparable to fluorescence detection. The extremely short fluorescence lifetime of SERS reduces the photobleaching effect and the half-peak width of the scattering peak compared to the fluorescent method. 38 At present, various metals (such as Au, Ag, Co, Ni) can be used as the base material of SERS. [39][40][41][42] Moreover, previous studies have shown that the SERS effect is related to the surface roughness of nanomaterials. 43,44 In this paper, gold nanoflowers (AuNPs) with spiny protrusions on the surface were used as the SERS substrate, and p-mercaptobenzoic acid (MBA) working as the Raman reporter was modified on the surface of AuNFs. In addition, MBA has the advantages of simple structure, easy bonding with gold surface, sensitivity to pH and high photochemical stability. 45,46 Base on that, MBA-functioned AuNFs SERS nanoprobes can respond to different pH conditions with the SERS technology. At the same time, based on the fingerprint effect of the Raman scattering signal, the self-calibration of the signal can be achieved by using the Raman peak which is less sensitive to pH change as the reference. In this way, the influence of the probe concentration is eliminated. Moreover, we propose that the SERS pH nanoprobes can be used to detect the acidity and alkalinity of the cell microenvironment, which would improve the development of early diagnosis methods of tumours.

| Experimental materials and apparatus
All the chemicals and reagents were purchased from Sigma-Aldrich without any further purification unless otherwise stated.
Transmission electron microscopy (TEM) images were taken with a Tecnai instrument (FEI). Raman measurement was performed on the XPLORA (Horiba) Raman microscope system. The dark-field measurements were carried out on an inverted microscope (Olympus IX71). UV-vis absorption obtained with a UV-3100 (Hitachi) UV-vis spectrophotometer.

| Preparation of AuNFs SERS pH Nanoprobes
For synthesis SERS pH nanoprobes, 2 µL of Raman reporter MBA (0.1 mol/L) solution was added to the 100 µL gold nanoflower solution (2 nmol/L). The mixture was incubated at room temperature overnight, and then, the excessive Raman reporters were removed by washing it three times.

| Cellular imaging under Dark-Field Microscope (DFM)
HEK 293 and Hela cells were incubated, respectively, with AuNFs SERS pH nanoprobes for 4 hours. They were then washed for three times with phosphate-buffered saline (PBS). After that, cells were fixed by 4% formaldehyde and imaged under a dark-field microscopy.

| Cellular imaging under Raman confocal microscope
HEK 293 and Hela cells were incubated, respectively, with AuNFs SERS pH nanoprobes for 4 hours. The final concentrations of them were 1 nmol/L. After washing with phosphate-buffered saline (PBS) for three times, cells were fixed by 2.5% glutaraldehyde and imaged under a Raman confocal microscope listed above.

| Synthesis and characterization of AuNFs SERS pH nanoprobes
As depicted in Figure 1A 45 Besides, the peak intensity would increase with pH increasing. More importantly, the SERS pH nanoprobe could be taken up via endocytosis. 47 Based on that, MBA-functioned AuNFs could be used as a sensitive SERS pH nanoprobe, which further used to pH mapping of tumour cell microenvironment as shown in Figure 1B.
Following the above-described synthesis way, uniform AuNFs with almost 50 nm size were obtained as shown in Figure 2A. As reported in previous literature of our group, the AuNFs have uniform surface spines and interior nanogaps (yellow segment) as shown in the TEM images in Figure 2B, and the gap size was almost 1 nm. Former work has indicated that polyA used in the synthetic process played a key role with blocking the direct deposition of gold onto the gold seed surface in forming the nanogap-containing particles. 48 With the formation of spines and nanoshell, the absorbance peak of nanoparticles red shifts from 520 to 608 nm as shown in Figure 2C. 49,50 The UV-vis spectrum of AuNFs in our research was consistent with results published previously. The near-field electromagnetic field distribution of the nanostructure was calculated using the finite-difference time-domain (FDTD) method with the acquired structural parameters ( Figure 2D). 51,52 The FDTD simulation confirmed that the incident electric field would form a localized electric field (hot spot) in the tip of the surface spines and the nanocavity. The modified MBA Raman signal on the spines would be greatly enhanced due to the surface-enhanced Raman effect.

| Analytical performance evaluation of the SERS pH nanoprobe
To bring these SERS pH nanoprobes into practical applications, the pH nanoprobe was evaluated in standard H 2 O solution with different pH situations. As shown in Figure 3A, AuNFs SERS pH nanoprobes showed strong Raman signals at different pH conditions. There were two absorption peaks at 1079.4 and 1587.1 cm −1 correspond to the vibrational peaks of ν8a and ν12 benzene rings, respectively. In the acidic environment, the carboxyl group exists in protonated form.
But in the neutral and alkaline environment, the carboxyl group was deprotonated with COO − form, and the Raman spectrum showed a weak absorption peak near 1428.2 cm −1 , which corresponded to COO − groups. At the same time, the intensity of the absorption peak near 1428.2 cm −1 was positively correlated with pH ( Figure 3C). This result indicates that the prepared SERS pH nanoprobe could indicate the pH change of the solution well. We also noticed that there was a sudden change of Raman intensity around pH 7 which be the isoelectric point of SERS pH nanoprobe.
Encouraged by the above investigations, the SERS pH nanoprobes were also evaluated in cell culture medium. As shown in Figure 3B,D, the change in SERS signal in the cell culture medium was similar to the change in the aqueous phase. It is indicated that the stability of the nanoprobe was not affected in the cell culture medium environment, and it still possesses good response ability to pH.

| Cell uptake efficiency and biocompatibility evaluation based on Dark-Field Image (DFI)
The cell uptake efficiency of SERS pH nanoprobes was evaluated with dark-field microscopy. The nanoprobes were incubated with normal human cells (HEK 293) and tumour cells (Hela) for 4 hours, and then, dark-field imaging was performed. As shown in Figure 4, the nanoprobes showed high cell uptake efficiency for normal cells and tumour cells. At the same time, both cell morphologies were normal, which indicated good biocompatibility of the SERS pH nanoprobe.

| pH mapping for cell analysis based on SERS pH nanoprobes
To validate the practicability of SERS pH nanoprobes, two different kinds of cells were selected for Raman imaging, which were normal HEK 293 cells and tumour Hela cells. The performance of SERS pH nanoprobes for intercellular pH mapping was investigated by using a streamline Raman mapping system. After

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.