2.1. UV-Vis Spectroscopy Measurements
UV-Vis spectra were taken to analyze the nanoparticles absorption characteristics and interaction with analytes. The pharmaceutical-grade synthesized nanoparticles show two absorption peaks, one at 532 nm and another one at 824 nm, which means that such products are anisotropic, having a longitudinal as well as a transverse mode (
Figure 1a). This can be verified by the scanning electron microscope images. The SEM micrograph inset in
Figure 1a shows nanoparticles with either a quasispherical shape, or triangular and trapezoidal plates. Sizes vary from 45 to 60 nm for the quasispherical products while triangles are about 45–75 nm. The situation is completely different, however, for the reactive-grade heparin nanoparticles. The UV-Vis spectra shows one band at 536 nm and a bump to its right side at 630 nm which indicates aggregation in the nanoparticles, see
Figure 1b. Quasispherical particles with sizes in the 30–35 nm range are observed in SEM micrograph inset in
Figure 1b. The distribution is more monodisperse than the one shown by pharmaceutical-grade heparin synthesized nanoparticles. No other morphology is observed, but it is clear that aggregates are formed.
UV-Vis spectra (
Figure 2a,b) were measured to monitor how the nanoparticles interact with the studied analytes; however there are no dramatic changes in absorption spectra of the mixed solution of colloidal nanoparticles and dyes. In the case of the pharmaceutical-grade nanoparticles (
Figure 2a), an almost negligible red-shift is observed, whereas for the reactive-grade heparin nanoparticles a small red-shift is observed (
Figure 2b). Though these nanoparticles were already aggregated before analyte addition, analyte addition was found to result in an almost negligible change in absorption spectra profile. So it is not possible to discern form absorption spectra the degree of interaction between the nanoparticles and dyes.
Figure 1.
(a) UV-Vis spectra of pharmaceutical-grade heparin nanoparticle synthesis; (b) UV-Vis spectra of reactive-grade heparin nanoparticle synthesis.
Figure 1.
(a) UV-Vis spectra of pharmaceutical-grade heparin nanoparticle synthesis; (b) UV-Vis spectra of reactive-grade heparin nanoparticle synthesis.
Figure 2.
(a) UV-Vis spectra of pharmaceutical-grade heparin nanoparticles synthesis and its mix with the differently-charged dyes; (b) UV-Vis spectra of reactive-grade heparin nanoparticles synthesis and its mix with the differently-charged dyes.
Figure 2.
(a) UV-Vis spectra of pharmaceutical-grade heparin nanoparticles synthesis and its mix with the differently-charged dyes; (b) UV-Vis spectra of reactive-grade heparin nanoparticles synthesis and its mix with the differently-charged dyes.
2.2. Raman Measurements
The Normal Raman and SERS spectra, peak assignment and structure are shown for each dye in
Figure 3,
Figure 4 and
Figure 5. When it comes to metal nanoparticles, spontaneous adsorption occurs if the analyte possesses a charge opposite to that of the particles. Most colloidal metal particles prepared by the reduction of metal salts (in our case, photochemically) carry a negative charge as a result of an adsorbed layer of stabilizing anions, so they will be electrostatically attracted to the nanoparticles surface. If the target molecule carries the same charge as the nanoparticle, it will not interact electrostatically with the metal surface, but still, it can make contact with it by displacing an existing ion from a site on the surface [
27].
Table 1,
Table 2 and
Table 3 indicate the corresponding peak assignments; we determined non-referenced peaks.
Figure 3.
Normal Raman and SERS spectra of Methylene Blue.
Figure 3.
Normal Raman and SERS spectra of Methylene Blue.
Table 1.
Methylene Blue Raman peak assignment.
Table 1.
Methylene Blue Raman peak assignment.
| Reported Powder Peaks (cm−1) [28] | 1 mM Aqueous Solution (cm−1) | PG-Heparin AuNPs (cm−1) | RG-Heparin AuNPs (cm−1) | Band Assignment |
|---|
| 1067 (w) | 1038 (w) | 1033 (w) | 1037 (m) | C‑H in-plane bending |
| 1121 (w) | - | - | 1122 (w) | C‑H out-of-plane-bending |
| 1181 (m) | 1185 (w) | 1181(w) | 1178 (m) | C‑N stretching |
| 1272 (w) | 1299 (w) | 1293 (w) | 1299 (m) | - |
| 1396 (m) | 1393 (m) | 1386 (m) | 1393 (s) | C‑H in-plane ring deformation |
| 1441 (w) | - | - | 1464 (w) | C‑N asymmetric stretching |
| 1544 (w) | 1541 (w) | 1537 (w) | 1541 (w) | C‑C ring stretching |
| 1618 (s) | 1620 (m) | 1607 (m) | 1619 (s) | C‑C ring stretching |
Figure 4.
Normal Raman and SERS spectra of Rose Bengal.
Figure 4.
Normal Raman and SERS spectra of Rose Bengal.
Table 2.
Rose Bengal Raman peak assignment.
Table 2.
Rose Bengal Raman peak assignment.
| Reported Peaks (cm−1) [29] | 1 mM Solution (cm−1) | PhG Heparin Au NPs (cm−1) | RG Heparin Au NPs (cm−1) | Band Assignment |
|---|
| 616 | - | - | - | - |
| 761 | - | 787 (w) | 793 (w) | C‑Cl stretching |
| 958 | - | 941 (vw) | 947 (w) | - |
| 1012 | - | 997 (vw) | 1000 (w) | C‑OH stretching |
| 1166 | - | 1170 (vw) | 1172 (w) | C‑O and C‑C stretching, C‑H skeletal deformation |
| 1270 | - | 1280 (vw) | 1274 (w) | CCC skeletal deformation in ring, C‑H skeletal deformation |
| 1297 | 1299 (vw) | - | 1291 (w) | CCC skeletal deformation in ring, C‑H skeletal deformation |
| 1340 | 1343 (m) | 1324 (m) | 1322 (m) | C‑C stretching in ring |
| 1491 | 1484 (w) | 1478 (vw) | 1489 (m) | C=C asymmetric stretching in ring |
| 1553 | 1551 (vw) | 1547 (vw) | 1537 (w) | C‑C stretching in ring |
| 1615 | 1613 (vw) | 1603 (w) | 1605 (s) | C=C symmetric stretching in ring |
Figure 5.
Normal Raman and SERS spectra of Neutral Red.
Figure 5.
Normal Raman and SERS spectra of Neutral Red.
Table 3.
Neutral Red Raman peak assignment.
Table 3.
Neutral Red Raman peak assignment.
| 1 mM Solution (cm−1) | PG Heparin Au NPs (cm−1) | RG Heparin Au NPs (cm−1) | Band Assignment |
|---|
| 1605 (w) | 1618 (w) | 1609 (m) | Scissor vibration of primary amines |
| 1341 (vw) | 1336 (w) | 1374 (m) | C‑N stretching vibration. |
| 1370 (w) | 1370 (w) | 1403 (w) | C‑N stretching vibration |
| 1459 (vw) | 1470 (w) | 1472 (w) | CH3 deformation vibration |
The SERS spectra show that reactive-grade heparin prepared nanoparticles (RGHep lines) lead to better SERS signals than the pharmaceutical-grade heparin products (PhGHep lines), the only differences arising from the different charges of the analytes, that is, the best interactions were obtained from the oppositely charged one (Methylene Blue) while not being the case with same-charged (Bengal) and neutral (Neutral Red) molecules. Even though some enhancement is observed, which indicates that nanoparticles have made some contact with the metallic surface, it has not been enough to quench such effects [
30]; this might be due to the molecule orientation or unbound parts of it.
In the Methylene Blue SERS spectra, the peaks that result from the most enhanced signals are at 1386 and 1617 cm−1 (PhGHep AuNPs) and at 1393 and 1619 cm−1 (RGHep AuNPs). As for Rose Bengal, such peaks are located at 1324, 1478 and 1603 cm−1 (PhGHep AuNPs) and at 1322, 1489 and 1605 cm−1 (RGHep AuNPs). Finally, for Neutral Red, although signal enhancement is not very intense, the strongest interactions are indicated by the peaks at 1336 (PhGHep AuNPs) and 1374 cm−1 (RGHep AuNPs). According to the assignation in the case of MB, the aromatic rings interact the most with the metal surface, being the same for Rose Bengal. Also, the chloride ions belonging to the dye structure, promote some adhesion to the metallic surface by the displacement of ions they induce in addition to the fact that they might also induce some aggregation on the colloid-dye system, which contributes to the generation of the Rose Bengal SERS signal. Lastly, for Neutral Red, the most acute peak corresponds to the C-N stretching vibration and considering that it possesses a NH2 group attached to an aromatic ring and is positively charged, it can be concluded that is the channel through which an interaction, though weak, is established.
When a molecule binds to a metal surface, it can be either physisorbed or chemisorbed. In the case of physisorption, the spectra of physisorbed molecules and free molecules are similar. However, when the molecules are chemisorbed on the metal surface, the position and relative intensities of the SERS bands are dramatically changed due to the overlapping of the molecular and metal orbitals that leads to the formation of a new metal-molecule SERS complex [
31]. Here, the probe molecules have been chemisorbed in relation to the 1 mM solution prepared from each dye.
Reactive grade heparin shows better interaction with analytes than the pharmaceutical grade one. This could be due to the higher degree of purity from the first one, with respect to pharmaceutical grade heparin, which is less pure, probably due to the fact that it is administered to humans, so its sulfation level is lower. Also, the acid nature of pharmaceutical grade nanoparticles might promote less contact between the nanoparticles surface and the analytes as will be discussed later.
2.3. IR Spectroscopy Measurements
Figure 6 depicts IR spectra taken from both heparin types, and in general, they exhibit rather comparable features differing only in the sulfate groups content [
32,
33,
34], especially in the glucosamine (941 cm
−1) and iduronate (800 and 816 and 820 cm
−1) sections of the spectra, meaning that reactive-grade heparin is more sulfated and has a larger molecular weight. Also, it is possible that the lower sulfation degree in the pharmaceutical-grade heparin might be due to the fact that it is administered to humans, and sulfates, at high concentrations are harmful. Besides this, the reducing power in each heparin is different; the reducing power is found in the glucosamine monosaccharide of heparin and if reactive-grade heparin is bigger and more sulfated, it means that there are more monosaccharides present in its structure, thus promoting more reducing efficiency during the nanoparticle synthesis, yielding a more monodisperse distribution, which is the opposite for pharmaceutical-grade heparin nanoparticles.
Figure 6.
IR spectra of pharmaceutical and reactive-grade.
Figure 6.
IR spectra of pharmaceutical and reactive-grade.
Heparin is strongly acidic and negatively charged because of its content of covalently linked sulfate and carboxylic acid groups. In heparin sodium, the acidic protons of the sulfate units are partially replaced by sodium ions, and the reason why heparin is a negatively charged molecule.
2.4. Measurements of pH
Measurements of pH were taken in order to find out how the acidity or basicity of the as-prepared colloids, as well as the mix between them and the dyes, affects the SERS signal. The corresponding results are shown in
Table 4. The pharmaceutical-grade heparin functionalized gold colloids had a slightly acidic value that in general diminished when the colloids were mixed with the dye analytes, becoming more acidic. On the other hand, for the reactive-grade heparin functionalized gold colloids (unlike the PhGHep AuNPs) the behavior was completely different. The initial pH value (4.86) went up when the dye analytes were added, but still remained in the acidic domain. These results suggest that a very acid environment does not favor SERS signal enhancement, whereas a close to neutral environment benefits it. It can be said that this last condition promotes interactions between nanoparticles and analytes, that are predominantly electrostatic, and allows ion displacement in the case of oppositely charged probe molecules. Another possible explanation for better interactions is that at higher pH values a hydroxyl-rich environment is generated resulting in stronger electrostatic interactions, especially for the positively charged dye. On the contrary, when the media is quite acid, the weak interactions are predominant, most likely of the pi-type, due to the probe molecules being aromatic [
35], and result in a protonation state that promotes much less adsorption of the analytes [
36].
Table 4.
pH measurements for nanoparticle syntheses and their mixtures with dyes.
Table 4.
pH measurements for nanoparticle syntheses and their mixtures with dyes.
| Sample | pH |
|---|
| Pharmaceutical-grade nanoparticle synthesis | 6.78 |
| PG Hep NPs-Methylene Blue | 6.24 |
| PG Hep NPs-Neutral Red | 6.10 |
| PG Hep NPs-Rose Bengal | 5.94 |
| Reactive-grade nanoparticle synthesis | 4.86 |
| PG Hep NPs-Rose Bengal | 5.87 |
| PG Hep NP-Methylene Blue | 6.30 |
| PG Hep NPs-Neutral Red | 6.60 |
