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In the present work, we report a dry-based application technique of Au/SiO2 clouds in powder for rapid ex vivo adenocarcinoma diagnosis through surface-enhanced Raman scattering (SERS); using low laser power and an integration time of one second. Several characteristic Raman peaks frequently used for the diagnosis of breast adenocarcinoma in the range of the amide III are successfully enhanced by breading the tissue with Au/SiO2 powder. The SERS activity of these Au/SiO2 powders is attributed to their rapid rehydration upon contact with the wet tissues, which promotes the formation of gold nanoparticle aggregates. The propensity of the Au/SiO2 cloud structures to adsorb biomolecules in the vicinity of the gold nanoparticle clusters promotes the necessary conditions for SERS detection. In addition, electron microscopy, together with elemental analysis, have been used to confirm the structure of the new Au/SiO2 cloud material and to investigate its distribution in breast tissues.
© 2016 Optical Society of America
1. Introduction
One of the most famous lectures given by Richard Feynman was in 1959 at the annual meeting of the American Physical Society “There’s plenty of room at the bottom” in which Feynman stated: As we go down in size, there are a number of interesting problems that arise. All things do not simply scale down in proportion. The qualitative and quantitative analysis of biochemical and structural changes due to the interactions of materials with tissues, at the nanoscale, plays a central role in modern biomedicine. Some of these changes are highly related to the development of diseases, and may provide important clues for diagnosis [1]. There are several imaging techniques that can provide diagnosis for cancerous specimens, such as targeted molecular imaging, ultrasound with magnetic nanoparticles, and nanoparticle-enhanced magnetic resonance [2–4]. Moreover, high spatial resolution microscopy techniques, such as confocal, scanning and transmission electron methods, can characterize the interaction and structures of biospecimens on the nanoscale size regime [5,6]. However, despite all the outstanding progress that biomedicine has achieved using these tools, there are still several hurdles that need to be resolved, such as improving the poor spatial resolution at molecular level and optimizing methods of sample preparation.
Raman spectroscopy is a powerful technique used to identify the energy levels of the bonds present in a molecule, which are associated with the vibrational modes of specific chemical interactions [7, 8]. Recent uses of Raman spectroscopy in biomedicine have been specifically aimed toward clinical examinations related to the detection of malignancies and cancers in tissues [9]. Raman spectroscopy additionally provides substantial information about the sample identity. However, for biological samples, it is necessary to decrease the power of the laser source, which results in a reduction of the signal used to extract information from the samples [10]. To overcome this issue, surface-enhanced Raman spectroscopy (SERS) has been used as an ultrasensitive and non-destructive spectroscopic technique that can provide specific molecular information of analytes present in concentrations relevant to medical applications [11, 12]. SERS increases the Raman signals of molecules near metal nanostructures, which are typically composed of noble metals such as gold and silver [13]. Effective SERS depends on the increase in the Raman cross-section as a result of the excitation of surface plasmons, which are collective oscillations of the conduction band electrons. When the excitation photon is resonant with the dipolar plasmon of a metallic nanostructure, the nanoparticle will emit light characteristic of dipolar radiation. Due to the relationship between this radiation and the incident light, certain areas near the metallic nanoparticles will show an enhancement in the local electromagnetic field [14].
The use of gold-modified silica particles for SERS has been widely reported in biological applications due to their strong adsorption of biomolecules. This adsorption promotes the specific interaction and localization of the metallic nanoparticles at suitable distances from target biomolecules, generating the optimal conditions for SERS [11, 12, 15, 16].
Mortality rates of breast cancer have increased in the last 40 years in most Latin American countries [17]. Adenocarcinoma starts in glandular tissue and is the most common type of breast carcinoma. The development of appropriate diagnostic tools is critical to overcome this disease [18]. Even in developed countries like the United States, the breast cancer claims the lives of 40,000 women per year [10]. Minimally invasive optical imaging and spectroscopic techniques have the potential to improve the early diagnosis of breast cancer. In particular, Raman spectroscopy has a high potential in this field due to the large number of Raman active molecules present in breast cancer tissue [10]. Currently, in most Latin American countries, tissue fixation in formalin immediately after the biopsy is the common practice. This process is known to alter the Raman spectral signature of the tissue by cross-linking the collagen proteins [19].
In this paper, we present a systematic study of the SERS activity of Au/SiO2 clouds in powder on normal glandular and adenocarcinoma tissue after an incubation time of five minutes. The SERS activity of Au/SiO2 is compared against the SERS obtained by incubating the tissues for one hour with standard naked colloidal gold nanoparticles (AuNPs) in the same size range. A low laser power of ~5 mW and an integration time of one second were used, thus avoiding damage in the samples. This work is not aimed at the differentiation between types of adenocarcinoma in breast tissue; however, this technique could also be used for this purpose. Enhanced bands that allow us to note the characteristics of each tissue are found in the amide III region of the Raman spectrum after inoculating the tissues with Au/SiO2 material. The SERS effects resulting from the interaction of the tissues with powdered Au/SiO2 show the potential of this material to serve as a tool for rapid tissue diagnosis.
2. Experimental
Materials. Reagent grade and highest purity Sigma-Aldrich samples of tetrachloroauric acid (HAuCl4), cetyltrimethylammonium bromide (CTAB), ammonium hydroxide (NH4OH), and tetraethyl orthosilicate (TEOS) were used. Absolute ethanol was purchased from Jalmek. Sodium borohydride (NaBH4) and deionized water were purchased from Fluka and Quimicurt, respectively.
Preparation of AuNPs and Au/SiO2 powder. The Au/SiO2 powder is a mixture of AuNPs embedded into SiO2 rod-shaped structures (Au/SiO2 clouds) and SiO2 spheres. AuNPs embedded into SiO2 clouds were synthesized following the Turkevich method. Gold reduction occurs by addition of a 1% citrate solution to a boiling solution of 1.0 mM HAuCl4 under vigorous stirring. After the reduction, a red wine color, characteristic of AuNPs, was obtained [20]. Separately, 0.48 g of CTAB were dissolved in 15 mL of ethanol and 6 mL of NH4OH. To this solution, the as-prepared AuNPs seeds (0.25 mL) were then added, under vigorous stirring, to 10 mL of 0.5 mM HAuCl4 followed by 25 mL of H2O. Finally, this solution was mixed with the previously prepared solution containing CTAB. The mixture was stirred for 15 minutes and then TEOS was dropwise added inducing the formation of Au/SiO2 clouds and SiO2 nanoparticles according to the well-known Stöber method by hydrolysis and condensation of TEOS in the presence of a surfactant [21]. At first, to obtain the Au/SiO2 rod-shaped clouds, the molar ratio of the reaction mixture was within the range of 200-400 (CTAB): 1000 (H2O): 20 (NH4OH): 1.4 −2.8 (TEOS) [22]. The colloidal solution was heated at 100 °C for 24 hours. For the elimination of excess CTAB, the nanoparticles were calcined at 500 °C, thereby obtaining a mixture of semitransparent and flocculent SiO2 matrix embedded with gold nanoparticles (Au/SiO2 clouds) and SiO2 nanoparticles. Thus, the Au/SiO2 product was obtained with a concentration of approximately fifteen SiO2 nanoparticles per Au/SiO2 cloud structure. Each Au/SiO2 cloud contained an average of ~750 AuNPs.
The preparation of naked colloidal gold nanoparticles (AuNPs) is based on a modified method reported by Martin et al. [23] First, 1.0 mL of a 1.0 M HAuCl4 aqueous solution is added to 90 mL of distilled water under vigorous stirring. After 1 minute, 1.0 mL of an ice-cooled (5 °C) 0.1 M NaBH4 solution is added [24].
Breast tissue samples. Normal and breast adenocarcinoma tissues were obtained from patients who were undergoing surgical breast biopsy mammoplasties and mastectomies. The tissues were routinely processed and examined by an experienced breast pathologist from the Pathology Department of the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE) in Guanajuato State (Mexico). Upon removal, the samples were fixed in formalin. To reduce the formalin fixation artifacts in the Raman spectra, the specimens were rinsed in phosphate buffered solution (PBS) before the Raman measurements [25]. Each tissue sample was divided into two parts, one being incubated with naked AuNPs and the other was breaded with Au/SiO2 for sixty and five minutes, respectively. A total of 150 spectra were examined using Raman spectroscopy: 70 spectra of normal tissue and 80 of malignant lesions diagnosed as adenocarcinoma (with a mean patient age of 56 years, reflecting the natural age incidence of this lesion) [10].
Equipment and methods. Field emission scanning electron microscopy (FE-SEM) was performed in a JEOL JSM-7800F microscope coupled with X-ray energy dispersive spectroscopy (EDS). The UV-Vis absorption spectra of colloidal AuNPs and Au/SiO2 have been obtained by transmittance using an Agilent Technologies Cary Series UV-Vis-NIR spectrophotometer (Cary 5000). Powder XRD patterns were obtained using a Bruker D2 Phaser with a Bragg-Brentano array. A Renishaw Raman System (Via Raman microscopy) with an objective lens of 20 X magnification and a spot size of 5 µm was used. The excitation laser was operated at 785 nm using a power of ~5 mW. The integration time for each Raman measurement was one second to avoid sample damage, as reported by Kneipp et al. [26]. The Raman spectra were acquired in the Amide III region, this region does not present interfering OH vibrations from H2O and has been used as a direct qualitative indicator for conformational change in proteins [27]. For the Raman signal enhancement, 0.1 mg of Au/SiO2 powder was spread on the surface of a wet tissue sample in an area of approximately 8 square millimeters and, after an incubation time of five minutes, the sample was ready for Raman spectroscopy measurements. By following this procedure, between 6 and 7 Au/SiO2 clouds could be found in the irradiation spot. In the case of colloidal AuNPs, the tissues were incubated for sixty min in 0.5 mL of AuNPs. Around 150 spectra of selected areas (~22500 µm2) of the tissue samples were obtained. These areas were selected using standard tissue pathology criteria [28]. The Raman spectra were averaged to obtain the representative data for each type of tissue sample. Different areas were also measured in duplicate on the same tissue sample to identify characteristic Raman signals and evaluate their reproducibility. The differences found in the Raman signal between points in the mapped areas, attributed to the natural irregularities of the tissue, were not noticeable when considering the overall averages.
3. Results
UV-Vis absorption spectroscopy. Fig. 1 shows the UV-Vis spectra of (a) the Au/SiO2 powder, (b) the colloidal AuNPs seeds used to prepare Au/SiO2, (c) the naked colloidal AuNPs, and (d) the SiO2 powder. For Au/SiO2 and the colloidal AuNP seeds, the localized surface plasmon resonance (LSPR) was centered at 514 nm. For the naked colloidal AuNPs, the LSPR was centered at 524 nm, and the wide band was indicative of some aggregation of the colloidal nanoparticles due to the lack of surfactant [24]. The absorption spectrum of the Au/SiO2 samples showed wider bands, which is consistent with the existence of various particle sizes and some degree of agglomeration of the particles. But, this effect is mainly due to the interplay of AuNPs with the silica framework, as has been reported in the literature [12]. UV-Vis absorbance was used to calculate the concentration of naked AuNPs in colloidal solution, which is estimated to be ~ particles/mL and is in good accordance with previous reports [29, 30].
Fig. 1 UV-Vis absorption spectroscopy. (a) Au/SiO2 material powder. (b) Colloidal AuNP seeds used to prepare Au/SiO2. (c) Naked AuNPs. (d) SiO2 nanoparticles.
Electron microscopy. The size and morphology of the SiO2 nanoparticles, the as-prepared Au/SiO2 powder, and the naked AuNPs are shown in Fig. 2. SiO2 nanoparticles were obtained with an overall diameter of 400 nm, were highly homogeneous and exhibited a narrow size distribution (Fig. 2(a)). The AuNPs were found embedded into the SiO2 clouds with a size distribution of 3-20 nm (Fig. 2(b)) and were evenly distributed over all of the silicon oxide structural matrix. The size of the naked AuNPs was in the range of 6-20 nm, as shown in Fig. 2(c).
Fig. 2 Electron microscopy micrographs. (a) SEM image of SiO2 nanoparticles with an average size of 400 nm and (b) BF-STEM imaging of gold nanoparticles, with average diameter of 20 nm embedded into an Au/SiO2 cloud. (c) BF-STEM imaging of naked AuNPs.
For the Au/SiO2 cloud structures, the presence and location of AuNPs embedded into the SiO2 matrix was confirmed by the elemental mapping through EDS analysis (Fig. 3). The embedded AuNPs were visualized to be evenly distributed throughout the SiO2 framework. A hybrid nanostructure with 200 and 500 nm width and length, respectively, can be seen in the SEM images (Fig. 3(a)). The results obtained by UV-Vis absorption indicate the presence of AuNPs in the Au/SiO2 material, as corroborated by the LSPR at 514 nm, and their presence was further confirmed by SEM, as shown in Figs. 2(b) and 3(a). Both SEM images clearly demonstrate the presence of AuNPs embedded in the framework of the SiO2 material.
Fig. 3 (a) SEM image of an Au/SiO2 cloud surrounded by SiO2 NPs and its elemental EDS mapping: (b) oxygen (red), (c) silicon (blue) and (d) gold (purple). Scale bar 500 nm.
Representative XRD patterns of the Au/SiO2 powder and SiO2 are presented in Fig. 4. The crystallinity of the AuNPs embedded in the SiO2 material was verified by this technique. The patterns of the as-prepared Au/SiO2 and SiO2 samples are presented in Fig. 4. Peaks at 2θ = 38.3°, 44.5°, 64.8°, and 77.7° were assigned to (111), (200), (220), and (311) reflection lines, respectively, indicating the formation of a face-centered cubic phase of metallic gold (Figs. 4(a) and 4(b)) [31]. No impurities were observed and the absence of any diffraction peaks for SiO2 nanoparticles indicates its amorphous nature (Fig. 4(c)). The transition temperature to form a crystalline phase from amorphous silica occurs at around 1300 °C [32].
Fig. 4 Representative XRD patterns of (a) the Au/SiO2 clouds mixed with SiO2 spheres in a proportion of 1:15, (b) the Au/SiO2 clouds mixed with SiO2 spheres in a proportion of 1:40 and (c) SiO2. The patterns (a) and (b) can be indexed as a gold FCC structure [31].
To observe the distribution of gold in the tissue samples after having been breaded with Au/SiO2 powder or incubated with naked AuNPs, EDS analysis was performed. As shown in Fig. 5, a greater dispersion of AuNP cluster structures (shown in green Fig. 5(a)) is observed for the Au/SiO2 breaded sample, while, in the sample incubated with naked AuNPs, the tissue sample appears to have bigger aggregates (shown in yellow Fig. 5(a)).
Fig. 5 EDS analysis showing the distribution of gold in breast glandular tissue inoculated with (a) Au/SiO2 powder (shown in green) and (b) naked AuNPs (shown in yellow). The scale bar is 2.5 µm.
Raman spectroscopy. Both Au/SiO2 powder and naked AuNPs samples do not exhibit Raman peaks, as shown in Figs. 6(A) and 6(B), of spectra a and b, respectively. The Raman spectra of normal and adenocarcinoma tissue samples show similar characteristics, which makes difficult to differentiate from one another (Figs. 6(A) and 6(B), spectra c). After incubation of both tissue samples with naked AuNPs, an enhancement in the signal is obtained. Interestingly, differences in the Raman signals can be observed between the two tissues. In normal tissue, the peak at 1300 cm−1 corresponding to fatty acids is marked with a black circle. This signal continues to be the most intense in the region near the amide III, allowing the detection of a shift at 1260 cm−1 (Fig. 6(A), spectrum d). The ubiquitous presence of peaks at 1300 and 1440 cm−1 in all spectra is one of the characteristics of normal glandular tissue. Spectral characteristics of adenocarcinoma tissue in the presence of naked AuNPs reveal an enhancement in the peak at 1367 cm−1 (tryptophan, α-helix), which is marked with a black circle in Fig. 6(B), spectrum d. However, the strong presence of the peak at 1300 cm−1 limits a possible diagnosis based solely on this spectrum. As a comparison, the spectra obtained using Au/SiO2 powder are shown in Figs. 6(A) and 6(B) (spectrum e) for the normal and adenocarcinoma tissue samples, respectively.
Fig. 6 Raman signals of (A): (a) Au/SiO2 powder and (b) AuNPs respectively. (c) Raman signal of normal breast glandular tissue. SERS spectra obtained from normal tissue (d) incubated with AuNPs, (e) breaded with Au/SiO2 powder. (B): (a) and (b) show the Raman signals of Au/SiO2 powder and AuNPs respectively. (c) Raman signal of glandular breast adenocarcinoma tissue. SERS spectra obtained from adenocarcinoma tissue (d) incubated with AuNPs, (e) breaded with Au/SiO2 powder. All spectra were acquired following an excitation at 785 nm.
The Raman signal obtained from normal glandular tissue breaded with Au/SiO2 powder (Fig. 7(a)) yields peaks at 548 (cholesterol), 886 (proteins), 932 (collagen/ Si–O−), 963 (lipids), 1076 (lipids in normal tissues), 1139 (fatty acids/ Si–O−), 1263 (amide III, protein band), 1300 (fatty acids), 1367 (tryptophan, α-helix), 1440 (collagen), and 1482 cm−1 (amide II, formalin). All these Raman signals are in agreement with previous reports [9, 10, 33]. Some of the peaks described above are absent or unclear in the spectra obtained using naked AuNPs (Fig. 6(A), spectrum d). The Raman signals of adenocarcinoma tissue breaded with Au/SiO2 powder are displayed in Fig. 7(b). The Raman spectrum shows peaks at 548 (cholesterol), 863 (collagen), 892 (protein), 931(collagen/Si–O−), 958 (cholesterol), 988 (proteins), 1070 (proline-collagen), 1138 (fatty acids/ Si–O−), 1260 (amide III), 1367 (tryptophan, α-helix), 1440 (collagen) and 1484 cm−1 (amide II, formalin). In this case, the characteristic peak at 1300 cm−1 corresponding to fatty acids is practically absent. Moreover, the intensity of the protein signal at 1260 cm−1 increased with respect to the intensity of the peak at 1440 cm−1 [9, 19, 33]. For both tissues, the peaks around 1480 cm−1 are assigned to formalin [9, 25]. The intensity of this band is higher for adenocarcinoma tissue. This may be due to differences in molecular content between the two tissues.
Fig. 7 (a) Representative SERS spectra obtained from normal tissue breaded with Au/SiO2 powder and (b) SERS spectra obtained from adenocarcinoma tissue breaded with Au/SiO2 powder. All spectra were acquired following an excitation at 785 nm.
Figure 8(a) shows a plot of the integrated Raman signal for the tissues at different regions of the Raman spectrum in the presence of naked AuNPs and Au/SiO2 powder. Figure 8(b) shows the average values of the enhancement factor in the Raman signal versus different regions of the spectrum in the presence of naked AuNPs and Au/SiO2 powder. Each plot represents the average values from 50 spectra. The presence of the Au/SiO2 powder resulted in a clear enhancement of the Raman signals, as shown in Fig. 8(b). The peak of maximum SERS activity is observed in the middle region of the spectrum (1200~1370 cm−1) when using Au/SiO2 powder. The enhancement factor was calculated using the formula E = (ASERS) / (AREF); where ASERS is the area of SERS peaks in the region of interest and AREF corresponds to the calculated area obtained for the same integration interval from the Raman spectra of the tissue samples.
Fig. 8 (a) Plot of the integrated Raman signal vs. different regions of the spectrum in the presence of naked AuNPs and Au/SiO2 powder. (b) Plot of enhancement factor in the Raman signal vs. different regions of the spectrum in the presence of Au/SiO2 powder and AuNPs. Error bars show the standard deviations. Each graph represents the average from 50 SERS spectra.
4. Discussion
The LSPR of AuNPs at 514 nm is consistent with the overall size distribution (~20 nm) and the red color of AuNPs fabricated by the traditional Turkevich method [34]. The formation of the Au/SiO2 powder can be rationalized based on the surfactant-templated sol-gel method. When the silica precursor, TEOS, was injected into the gold seeds solution, it hydrolyzed and condensed into silicate polyanions. These negatively charged polyanions interacted via electrostatic interactions with the positively charged groups of the surfactant CTAB, which was surrounding the AuNPs. This process promoted the growth of the silica structure around the AuNPs, while inhibiting the TEOS homogeneous nucleation process [35, 36]. While TEOS was being added dropwise, depending on the availability of AuNPs and the changes in the molar ratio of CTAB/H2O/NH4OH/TEOS, rod-shaped Au/SiO2 clouds were first formed and later the SiO2 spherical nanoparticles [22]. The number of AuNPs per unit volume in the Au/SiO2 clouds is limited by interparticle Coulombic repulsion, as in the case of the attachment of gold colloids on planar silica surfaces [35]. One of the main characteristics of this powdered Au/SiO2 material is its hydrophilic behavior due to hydroxyl groups (-OH). On the other hand, the support constituted by SiO2 nanoparticles served to create a diluent and stable environment for the Au/SiO2 clouds. In this work, the presence of embedded AuNPs was confirmed by SEM-EDS and powder XRD. SEM-EDS showed clearly the presence of elemental Au in the framework of the Au/SiO2 clouds. Moreover, the powder XRD patterns obtained from this material corroborated the presence and crystallinity of the AuNPs.
As has been reported in the literature, the incubation of cells and tissues with naked AuNPs affords the formation of gold nano-clusters, which increase the Raman signals due to the production of hot-spots (enhanced localized electromagnetic fields) [13, 26, 27, 37]. Incubation times to obtain enhanced Raman signals by using gold colloidal nanoparticles have been reported in a range from half an hour to 24 hours [37, 38]. The results obtained in this work show that, while the interaction of both normal and adenocarcinoma tissues with naked AuNPs resulted in an increase of the Raman signal intensity, this overall increase does not have a practical use for an effective diagnostic of adenocarcinoma tissue. We hypothesize that this is due to a lack of specific affinity between naked AuNPs and the biomolecules in these tissues [26, 38, 39]. On the contrary, when Au/SiO2 powders are used to enhance the Raman signal, the characteristic Raman peaks of normal and adenocarcinoma tissue were clearly different from one another after an incubation with the material for five minutes. Previously reported spectral features due to Si–O− stretching bonds were found at 930 and 1140 cm−1 in tissue samples inoculated with the Au/SiO2 powder. Also, low-frequency oligomeric features, probably due to ring breathing modes, were found at 500–550 cm−1 in the same tissue samples in the presence of Au/SiO2 [40]. The enhancement of the Raman signal in the presence of the Au/SiO2 powder is the result of the electromagnetic field improvement produced by the formation of gold cluster nano-structures during the hydration of the Au/SiO2 clouds in the wet tissue and by the adsorption of biomolecules into the SiO2 matrix [15]. Interestingly, in this work, a greater enhancement of the Raman signal was observed in the amide III region of the Raman spectrum. This is a strong indication of higher adsorption of protein molecules in the vicinity of the hot-spots due to the presence of the SiO2 structural matrix. The surface chemistry of the Au/SiO2 clouds, and specifically the presence of OH-silanol groups, ensures the interaction with proteins, such as with collagen in the case of glandular breast tissues [41]. This is why the use of gold decorated SiO2 nanoparticles has been widely applied to SERS studies for biomedical purposes. In fact, this Au/SiO2 powder is especially useful in the case of breast cancer tissue, where the expression of proteins plays an important role [42, 43]. Moreover carboxylic groups of fatty acids may be covalently incorporated into silanol groups present on the surface of the Au/SiO2 structures by Si-O-Si bonds [16, 31, 32, 38, 40, 44]. In this work, a significant increase in the Raman signal in adenocarcinoma tissue was observed with the use of Au/SiO2 powders. The detection is up to three times more sensitive in the presence of Au/SiO2 powder compared to the use of naked AuNPs. Thereby, the importance of the presence of the SiO2 framework for detection of breast cancer tissue is corroborated. The ability to diagnose different types of adenocarcinoma tissues is certainly an interesting challenge, but it was not examined as part of this study. The results obtained in this work confirm that Au/SiO2 powder is a promising material for cancer tissue diagnosis. In particular, we have shown that, by using the outstanding SERS properties of the Au/SiO2 powder, adenocarcinoma tissue can be detected. The methodology proposed here for the use of Au/SiO2 powder for diagnosis is simple, reliable and does not require additional instrumentation other than the Raman spectrometer.
5. Conclusions
In this work, a SERS-active Au/SiO2 powder for ex-vivo breast tissue diagnosis was synthesized and characterized. In this material, AuNPs were embedded in the SiO2 framework. Spectroscopic and microscopic analyses such as UV-Vis, SEM and SEM-EDS clearly demonstrated that the AuNPs have been incorporated into the framework of rod-shaped SiO2 structures without losing their optical properties (e.g. LSPR). After incubation with normal and adenocarcinoma tissues, the Au/SiO2 clouds are dispersed all over the tissue. Raman spectroscopy shows that Au/SiO2 clouds are highly SERS-active when applied to wet tissue, allowing rapid distinction between normal glandular breast and adenocarcinoma tissue in the amide III region of the Raman spectrum. On the contrary, the same tissue samples incubated in the presence of naked AuNPs do not show such dramatic enhancement in the Raman signal. We hypothesize that the tendency of the Au/SiO2 clouds to adhere to wet tissues promotes the formation of AuNPs aggregates in the vicinity of the biomolecules located on the tissue surface. This aggregation affords the enhancement of the Raman signal due to the formation of hot-spots in the vicinity of adsorbed biomolecules. The promising results obtained in this work demonstrate that Au/SiO2 clouds have the potential to be used as a diagnostic tool to distinguish between normal and adenocarcinoma tissues. Future endeavors will center on the toxicological analysis, the diagnosis of different types of adenocarcinoma tissues, and the possibility of using this Au/SiO2 material for in vivo diagnosis.
Acknowledgments
The authors would like to thank M.D. Pablo Medina-Sánchez, M.D. Emilio Lemus-Bedolla and Hilda Lissette López-Lemus (Pathology Department of the ISSSTE regional hospital in León Guanajuato) for their invaluable help in the selection of tissues and during the development and acceptance of the research protocol. The microscopy work was supported by the National Institute on Minority Health and Health Disparities (NIMHD) in the program Research Centers in Minority Institutions Program (RCMI) Nanotechnology and Human Health Core (G12MD007591). The authors would also like to acknowledge the Mexican Council for Science and Technology, CONACYT # 250836 (Mexico) through the national scholarship and grant # 252054 for ECP and the International Center for Nanotechnology and Advanced Materials (ICNAM). J. V.-E. would like to acknowledge financial support from UNC Charlotte. The authors are grateful to Dr. Richard Jew (UNC Charlotte) for critical reading of the manuscript and helpful suggestions. The authors would also like to thank Christian Albor for assistance with electron microscopy and Eduardo Hernández for the valuable help in the preparation of samples.
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