Development of Novel SERS Probe for Selective Screening of Healthy Prostate from Malignant Prostate Cancer Cells Using Zn(II)

Abstract

Even in 21st century, prostate cancer remains the second leading cause of cancer death for men. Since normal prostate gland contains the most Zn(II) and there are huge differences in Zn(II) content between the healthy and malignant prostate cancer cells, mobile zinc can be used as a biomarker for prostate cancer prediction. Current article reports the design of novel and highly efficient surface enhanced Raman spectroscopy (SERS) probe using p-(imidazole)azo) benzenethiol attached gold nanoparticle as a Raman reporter, which has the capability to identify prostate cancer cells based on Zn(II) sensing. A facile synthesis, characterization and evaluation as Zn(II) sensing Raman probe has been reported. Reported data indicate that after binding with Zn(II), Raman reporter attached gold nanoparticle forms assembly structure, which allows selective detection of Zn(II) even at 100 ppt concentration. Theoretical full-wave finite-difference time-domain (FDTD) simulation has been used to understand huge enhancement of SERS signal. Reported SERS probe is highly promising for in-vivo sensing of cancer, where near IR light can be easily used to avoid tissue auto-fluorescence and to enhance tissue penetration depth. Reported data show that SERS probe can distinguish metastatic cancer cells from normal prostate cells very easily with sensitivity as low as 5 cancer cells/mL. Designed SERS probe has the capability to be used as chemical toolkit for determining mobile Zn(II) concentrations in the biological sample.

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Current article reports the design of novel and highly efficient surface enhanced Raman spectroscopy (SERS) probe using p-(imidazole)azo) benzenethiol attached gold nanoparticle as a Raman reporter, which has the capability to identify prostate cancer cells based on Zn(II) sensing.

1. Introduction

Early stage identification of cancer is very important to cure from the aggressive disease^1–4. Mobile zinc is highly valuable component for prostate^2–10. It is now well documented that due to the presence of zinc transporter protein ZIP1, prostate tissue contains more than 10 times higher zinc than other tissues^5–10. Since the male prostate contains more zinc than any other soft tissue and there is a dramatic reduction in the zinc content of prostate tissue when it became cancerous^5–10, the mobile zinc concentration in the prostate can be used as a marker for early diagnosis of prostate cancer^2–10. For in vivo biological Zn(II) sensing, near infrared (NIR) light in biological I window between 650–950 nm needs to be used to provide maximum penetration through tissue^5–10. Since last decade, several types of metal ion sensors have been developed using aptamers, plasmonic nanoparticles and electrochemical methods^11–20. To date, most of the biological Zn(II) sensing via fluorescence imaging is reported using visible light, although substantial background noise from tissue autofluorescence is high in visible light and the tissue penetration depth is limited^6–10. Driven by the need, this article reports for the first time the development of novel and highly efficient surface enhanced Raman spectroscopy (SERS) probe using p-(imidazole)azo) benzenethiol attached gold nanoparticle, which has the capability to identify prostate cancer cells based on Zn(II) sensing, as shown in Figure 1. Using 785 nm biological I window excitation light, reported result shows that SERS probe is able to distinguish prostate metastatic cells from normal prostate cells.

Figure 1.

A) Scheme showing the synthetic path we have followed for the development of p-(imidazole)azo) benzenethiol Raman reporter. B) FT-IR spectrum from Raman reporter verifying the existence of-NH, –SH, –N=N, –C=N, –C=C,-C-S, C-C groups. C) Scheme showing the working principle for SERS based Zn(II) sensing using p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle. D) Schematic representation shows the working principle for the separation of prostate cancer cells from prostate normal cells using Zn(II) sensing.

Raman spectroscopy is an inelastic light scattering technique which has the capability to be used as fingerprint identification^21–30. Since in Raman spectroscopy, scattering efficiency is extremely weak, surface-enhanced Raman spectroscopy (SERS) based on nanotechnology is becoming an excellent bio-analytical tool^21–30. In case of SERS, the Raman signals intensity can be enhanced by 10⁸–10¹⁴ orders of magnitude via electromagnetic fields generated by the “hot spots”, as a result, SERS can be used for ultrasensitive sensing applications^24–32. In addition, SERS provides several advantages over fluorescence-based assays^21–30 and these are as follows: 1) near IR light can be easily used to avoid tissue autofluorescence and to enhance significantly the tissue penetration depth; (2) very narrow spectral widths in Raman bands allow multiple labels detection under single NIR light source and (3) due to the non-resonance condition, photo-bleaching issues are minimum for SERS. Since SERS has capability for non-destructive in vivo measurements with relatively large penetration depth, scientists are developing Raman probes which can be used for diagnostic accuracy, speed and cost-effective in-vivo sensing of cancer^{20–23, 26, 29–31}.

For the design of highly efficient Zn(II) sensing SERS probe, at first we have developed p-(imidazole)azo) benzenethiol as an excellent Raman reporter, which has the thiol group in one side, which helps to bind it with gold nanoparticle as shown Figure 1A and Figure 2A. Since in human body, the coordinating zinc in proteins is dominated by ligation to nitrogen atoms of imidazole from histidine^{4–10,33–34}, the imidazole moiety on the other side of the Raman reporter has been used to bind with Zn(II) selectively. As shown in Figure 1A and Figure 2A, in the presence of Zn(II), gold nanoparticle forms assembly structure via the formation of co-ordination bond between Zn(II) and p-(imidazole)azo) benzenethiol Raman reporter. In the assembly structure, Raman reporter attached gold nanoparticle forms “hot spots”, which enhances the electromagnetic field coupling of plasmonic particles via light-matter interaction^23–32. Since the presence of Zn(II) enhances the SERS signal intensity tremendously, our reported experimental data show that the detection limit for Zn(II) can be as low as 10 parts per trillion (ppt). Since in the prostate cancer cell, the amount of zinc decreased by 83% as compared to normal prostate tissue, mobile zinc can be used an excellent candidate for biomarker for prostate cells^5–10. In this manuscript, we have demonstrated that p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle based Zn(II) sensing based SERS assay can be used for the identification of prostate metastatic cells and for the separation of prostate cancer cells from prostate normal cells, as shown in Figure 1B.

Figure 2.

A) Scheme showing p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle synthesis and its use for Zn(II) sensing. B) TEM image of freshly prepared Raman reporter attached gold nanoparticle. C) TEM image of freshly prepared Raman reporter attached gold nanoparticle in the presence of Zn(II). D) SERS spectra from p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle in the presence and absence of Zn(II). E) Extinction spectra of Raman reporter (5 × 10⁻⁵M) and Raman reporter attached gold nanoparticle (1.3 × 10⁻⁹ M gold nanoparticle) in the absence and the presence of Zn(II). F) Figure shows the histogram of size distribution for Raman reporter attached gold nanoparticle in the absence of Zn(II) measured by DLS. G) Figure shows the histogram of size distribution for Raman reporter attached gold nanoparticle assembly in the presence of 100 ppt Zn(II) measured by DLS. H) Figure shows the histogram of size distribution for Raman reporter attached gold nanoparticle assembly in the presence of 10 ppm Zn(II) measured by DLS. I) Energy-Dispersive X-ray (EDX) spectroscopy elemental mapping from gold nanoparticle assemble structure shows the presence of Zn(II) in the aggregated nanoparticles. J) Photographs indicating colorimetric changes due to the formation of assembly structure after binding with Zn(II). J1: only Raman reporter, J2: raman reporter attached gold nanoparticle in the absence of Zn(II), J3: raman reporter attached gold nanoparticle in the presence of Zn(II).

2. Results and Discussions

2.1. Design and characterization of p-(imidazole)azo) benzenethiol attached gold nanoparticle based SERS probe

For the design of highly efficient Zn(II) sensing SERS probe, in the first step we have developed the Raman reporter p-(imidazole)azo) benzenethiol. The Raman probe was developed by modifying a literature procedure, as shown in Figure 1A. The experimental details have been discussed in the experomental section. In brief, 125 mg (1.0 mmol) of 4-aminothiophenol (4-ATP) was dissolved in 25 ml of a 1:1 ethanol-water mixture by sonicating for 10 minutes. Next, 10 mL of 0.5 N hydrochloric acid was slowly added into the 4-ATP solution. The amine was diazotized by adding 10 ml of aqueous 100 mg (1.4 mmol) sodium nitrite at temperatures below 5°C with constant stirring. The reaction mixture was allowed to stand at below 5°C for 10 minutes, and the resulting diazonium chloride salt solution was added drop wise with vigorous stirring to 65 mg of imidazole (65 mg, 1.0 mmol) in 50 ml 2% sodium carbonate water maintained at a temperature below 5°C. We have used ¹H NMR (500 MHz, DMSO-d₆, TSP), infrared and absorption spectroscopy to characterize the Raman reporter. Details of NMR assignment has been discussed in the experimental section and NMR spectra has been reported as Figure S1 and S2 in the supporting information. As shown in Figure 1B, the FT-IR spectrum exhibits strong peak at ~3435 cm⁻¹ corresponds to the vibrational mode of the –N-H groups. Quite strong peaks were also observed at ~2555 cm⁻¹ for the –S-H stretch, ~1632 cm⁻¹ for the –C=C stretch, ~1590 cm⁻¹ for the –C-N stretch, ~1473 cm⁻¹ for the –N=N stretch and ~1080 cm⁻¹ for the –C-S stretch respectively. In the absorption spectra, as shown in Figure 2C, we have observed a strong peak around 380 nm, which is due to the azobenzene, mainly in trans form.

In the next step, we have performed the synthesis of gold nanoparticle using gold chloride, sodium borohydride and sodium citrate using our reported method^{24,28,31–32}. After that, we have attached p-(imidazole)azo) benzenethiol via thiol group as shown in Figure 1C. At the end, unbound Raman reporters were removed via centrifugation. In the next step, we have used high-resolution JEM-2100F transmission electron microscope (TEM), dynamic light scattering (DLS) and UV-Vis spectroscopy to characterize p-(imidazole)azo) benzenethiol attached gold nanoparticle.

As reported in Figure 2B, the particle size is about 25 nm for Raman reporter attached gold nanoparticle. DLS measurement data reported in Figure 2F also indicate that the average size is ~25 nm for Raman reporter attached gold nanoparticle. Figure 2E shows the extinction spectra for p-(imidazole)azo) benzenethiol attached gold nanoparticle in the absence of Zn(II), where we have used 1.3 × 10⁻⁹ M gold nanoparticle. Experimental data clearly show the plasmon peak around 530 nm and azobenzene peak around 380 nm in the absence of Zn(II). Figure 2D shows SERS spectra from the Raman reporter p-((imidazole)azo) benzenethiol. The strongest Raman bands seemed to consist of the NH₂ rock at ~1380 cm⁻¹, –C-N stretch at ~1590 cm⁻¹, –N=N stretch at ~1473 cm⁻¹, –C-S stretch at ~1080 cm⁻¹ and C-N bend at ~1211 cm⁻¹.

2.2 Selective sensing of Zn(II) using our developed SERS probe

Since in human body, the coordinating of zinc with proteins is dominated by nitrogen atoms of imidazole from histidine^33–35, we have used the imidazole moiety of the Raman reporter for the detection of Zn(II) via co-ordination complex, as shown in Figure 2A. As shown in Figure 2C, in the presence of Zn(II), gold nanoparticle forms assembly structure via the co-ordination bond between Zn(II) and p-(imidazole)azo) benzenethiol Raman reporter. Figure 2J shows the colorimetric change from purple to blue color due to the formation of assembly structure. Figure 2E shows the extinction spectra for p-(imidazole)azo) benzenethiol attached gold nanoparticle in the presence of Zn(II), which indicates a broad band between 530–680 nm and it is due to the assembly structure formation by gold nanoparticle in the presence of Zn(II). DLS measurement, as reported in Figure 2G and 2H, also indicates assembly structure formation for Raman reporter attached gold nanoparticle in the presence of Zn(II). Energy-Dispersive X-ray (EDX) spectroscopy elemental mapping reported in Figure 2I clearly shows the presence of Zn and Au in the assemble nanoparticle. The observed Al peak is due to the sample grid and N peak is due to the Raman reporter. Figure S3 shows how the absorption peaks at 520 nm for SERS probe in the absence of Zn(II) and 650 nm for assembly structure in the presence of Zn(II) vary with the increasing concentration of Zn(II). The binding constant K was calculated by fitting the change of UV- Vis absorbance with Zn(II) concentration, as reported in Figure S4. The observed binding constant was 9.9 × 10⁴ M ⁻¹.

Since after binding with Zn(II), Raman reporter attached gold nanoparticle resulted in assembly structure which formed high intensity “hot spots”, SERS intensity from Raman reporter enhanced significantly. As shown in Figure 2D, around two orders of magnitude of SERS intensity enhancement is mainly from the enhanced electromagnetic field in the hot site, which is produced by resonant excitation of the surface electrons in the metal nanostructures due to an excitation laser light source. To understand better about the huge enhancement of SERS signal from Raman reporter via gold nanoparticle assembly formation, we have performed full-wave finite-difference time-domain (FDTD) simulation^36–38, as reported in the Figure 3A. Details on the finite-difference time-domain (FDTD) simulation calculation have been reported by us and other groups before^31,36–37. In brief, for the calculations, we have used FDTD simulation software package. We have used frequency dependent dielectric function of gold to calculate the electric filed enhancement in “hot spot”^31,36–37. For the comparison of experimental outcome with theoretical data, we have used experimental excitation wavelength for excitation of gold nano-assembly in our theoretical calculation^31,37. Polarized light at a wavelength of 785 nm was used along the y-axis, for FDTD simulation For FDTD simulation, we have used the amplitude of electric field as 10V/m and the count number as 0.99^31,37. For the nano-assembly structure, we have used the bent structure, similar to the observed experimental structure as shown in Figure 2C. For our calculation, the entire simulation is done with 0.001 nm mesh resolution with 4000 fs time^31,37. Figure 3A indicates that the electric field enhancement intensity can be enhanced by more than 20 times for gold nano-assembly containing four particles with respect to a monomer. Since the SERS signal enhancement is directly proportional to |E|⁴, in theory, approximately 5 orders of magnitude of SERS signal enhancement is expected in the presence of Zn(II) due to the formation of assembly structure.

Figure 3.

A) Simulated electric field enhancement |E|² profiles in arbitrary units for monomer and nanoparticle assembly. B) SERS spectrum shows how the SERS signal varies with the concentration of Zn(II). C) SERS intensity at 1380 cm⁻¹ as a function of Zn(II) concentrations in the range of 0 to 1500 ppt. A linear relationship has been found with R = .98. Inserted figure shows how SERS intensity at 1380 cm⁻¹ varies as a function of Zn(II) concentrations up to 680 ppb. D) SERS spectrum show how the SERS signal from Raman probe varies in the presence of other metal ions in the concentration of 10 ppm. E) Extinction spectra of Raman reporter attached gold nanoparticle in the presence of different transition metal ion.

To evaluate the sensitivity of the SERS probe, different concentrations of Zn(II) from one stock solution were evaluated. Figure 3B shows how the SERS intensity from Raman reporter varies in the presence of different concentration Zn(II). Figure 3C shows that the SERS intensity at 1380 cm⁻¹ as a function of Zn(II) concentration. Experimental data clearly show a linear relationship with R= 0.98, for the concentration in the range of 0 to 1500 ppt. Inserted figure shows how SERS intensity at 1380 cm⁻¹ varies as a function of Zn(II) concentrations up to 680 ppb, which indicates that only at lower concentrations, the SERS response follows the linearity. At higher concentrations, the SERS intensity change is less significant, as shown in the inserted Figure in Figure 3C and it is due to the fact that bigger assembly structure is alredy formed when the concentration is around 1500 ppt. Thus, it will form a logarithmic trend in Raman intensity, when the concentration is more than 1500 ppt. As shown in Figures 3B and 3C, the sensitivity of our assay for Zn(II) can be as low as 100 ppt, which is about 1–2 orders of magnitude higher than reported method^7,9,18–19. To determine the sensitivity, we have used SERS response at 1380 cm⁻¹. The sensitivity has been defined as the concentration of the Zn(II) that is required for 3 times larger SERS intensity than that of in the absence of Zn(II), after optimization with the standard deviation of the noise level. Since several other heavy metal ions are also present in body fluid, we have performed the selectivity experiment in the presence of other heavy metal ions. For this experiment, we have used the concentration 20 times higher (10 ppm) than Zn(II) ion concentration (500 ppb).

Since selectivity is very important parameter before it can be used for biological application, we have performed the selectivity experiment by evaluating the response over other potentially competing metal ions such as Fe²⁺, Hg²⁺, Pb²⁺, Cu²⁺, Co²⁺, Ni²⁺, Ca²⁺, Cd²⁺. Figure 3D shows the SERS response from Raman reporter-modified gold nanoparticles in the presence of various heavy-metal ions. Reported experimental data clearly show that p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle based SERS assay is highly selective for Zn(II). It is well documented that Zn(II) form strong complexes with imidazole which are the subunits of Zn–enzymes^39–40. The stronger affinity of Zn(II) for imidazole is due to the fact that Zn(II) forms strong tetrahedral and trigonal bipyramid complex with imidazole as reported by several group^39–40. To understand whether the gold nanoparticle attached p-(imidazole)azo) benzenethiol Raman reporter has stronger affinity towards Zn(II), we have performed extinction spectra response from Raman reporter-modified gold nanoparticles in the presence of various metal ions. As reported in Figure 3E, extinction spectra show the plasmon peak around 530 nm and azobenzene peak around 380 nm in the presence of Fe²⁺, Hg²⁺, Pb²⁺, Cu²⁺, Co²⁺, Ni²⁺, Ca²⁺, Cd²⁺ metal ions. On the other hand, as reported in Figure 2E, in the presence of Zn(II), we have observed a new broad band between 530–680 nm due to the assembly structure formation, other than the 380 and 530 nm band. Our experiment clearly indicates that p-(imidazole)azo) benzenethiol Raman reporter has a stronger affinity for Zn(II), over other potentially competing metal ions. As a result, Zn(II) helps to form assembly structure for gold nanoparticle attached p-(imidazole)azo) benzenethiol which enhances the formation of “hot spot” significantly.

2.3. Screening metastatic cancer cells from normal prostate cells uisng Zn(II) based Raman reporter

Since the mobile zinc is an excellent candidate for biomarker for prostate cells, to understand whether p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle based Zn(II) sensing using SERS assay can be used for the identification of prostate metastatic cells, we have performed SERS experiment using normal (PNT2-C2) and metastatic (LNCaP) prostate cells. For this purpose, both cells were cultured according to the manufacture guidelines. After that, normal and metastatic cells were incubated with ZnCl₂ for 12 hours. In the next step, we have done the detachment of Zn(II) infected cells using cell dissociation buffer. Next, for the stabilization of Raman reporter attached gold nanoparticle in cell media, we have developed thiol-terminated polyethylene glycol (PEG) coated Raman reporter attached gold nanoparticle. After that we have tested the stability of PEG coated Raman reporter attached gold nanoparticle in biological cell media for one hour and we have noted very good stability. The SERS spectra, as reported in Figure 4C, clearly show that SERS intensity from Raman reporter attached gold nanoparticle remains unchanged in the absence of cells, although cell media is present. Our data indicate that the PEG coated Raman reporter attached gold nanoparticle is quite stable in biological cell media. Next, for SERS measurement, we have added PEG coated p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle on infected cell solution for 15 minutes. The excess PEG coated Raman reporter attached gold nanoparticle in solution, which were not bound with Zn(II) on the extracellular cell surface, were removed by centrifugation. After that we have used a continuous wavelength 785 nm DPPS laser based portable Raman probe for SERS measurement. For this purpose, we have used ~2 mW of power of 785 nm light with the spot size on the sample around 200 μm in diameter. To understand whether there is any cellular damage during the SERS measurement, we have also perform cellular viability measurement before and after the experiment, which indicate that cellular viability remain same during the SERS measurement.

Figure 4.

A) TEM shows that Raman reporter attached gold nanoparticle forms very high amount of assembly structure on prostate normal cells. B) TEM shows that Raman reporter attached gold nanoparticle forms very small amount of assembly structure on prostate cancer cells). C) SERS spectrum shows how the Raman signal from PEG coated Raman reporter attached gold nanoparticle varies in the presence of normal prostate normal cells and prostate metastatic cells. D) Variability of SERS spectra from PEG coated Raman reporter attached gold nanoparticle in the presence of prostate normal cells. We have made PEG coated Raman reporter attached gold nanoparticle in 4 different batches. And for each batch, we have performed several experiments with different prostate normal cells cultured in different batches. E) Variability of SERS spectra from PEG coated Raman reporter attached gold nanoparticle in the presence of prostate cancer cells. We have made Raman reporter attached gold nanoparticle in 4 different batches. And for each batch, we have performed several experiments with prostate cancer cells cultured in different batches. F) SERS spectrum shows how Raman signal fom PEG coated Raman reporter attached gold nanoparticle varies in the presence of different amounts of prostate metastatic cells. Reported data show that the detection limit can be as low as 5 cells/mL. G) SERS spectra and images from LNCaP prostate cancer cells and LNCaP prostate cancer cells with SERS probe in the absence of Zn(II). H) SERS spectra and images from LNCaP prostate cancer cells with SERS probe in the absence and presence of Zn(II).

It is well documented that zinc plays very important roles in human via the formation of tightly-bound divalent ions in metalloproteins^2–10. There are also dynamic pools of free, labile, chelatable zinc ions, known as mobile zinc in many tissues and organs including the prostate^2–10. As we have discussed before, the healthy prostate contains very high concentrations of mobile zinc, on the other hand the mobile Zn(II) levels decrease about one order of magnitude during the development of prostate cancer^2–10. Our developed p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle based SERS sensor has been used for the sensing of free or mobile Zn(II) ions to separate healthy cells from prostate metastatic cells. For this purpose, p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle were modified with thiol-terminated polyethylene glycol (PEG) for high stability in physiological solutions.

Since healthy prostate contains very high concentrations of mobile zinc, PEG coated p-(imidazole)azo) benzenethiol Raman reporter attached gold nanoparticle forms huge assembly structure in the extracellular surface of cells due to the binding of mobile Zn(II) with p-(imidazole)azo) benzenethiol, as shown in Scheme 1. The mobile Zn(II), present in the extracellular surface of healthy prostate cells, will form co-ordination bond with p-(imidazole)azo) benzenethiol attached gold nanoparticle. Due to the high binding affinity between Zn(II) the imidazole-azo moiety, the intracellular mobile Zn(II) may also release from inside the healthy prostate cells and form co-ordination bond with p-(imidazole)azo) benzenethiol attached gold nanoparticle on the extracellular surface. As a result, the observed strong assembly formation in the extracellular surface of healthy prostate cells, as reported in Figure 4A, can be dure to the extracellular, as well as intracellular mobile Zn(II). High-resolution TEM picture as reported in Figures 4B, clearly show that the amount of assembly formation is much lower in case prostate metastatic cell due to the lack of high concentration of mobile Zn(II).

The SERS spectra, as reported in Figure 4C, show that the Raman intensity from PEG coated Raman reporter attached gold nanoparticle increases 4 times in the presence of prostate metastatic cell and the increment is around 30 times in the presence of prostate normal cell. Our data clearly show that PEG coated Raman reporter attached gold nanoparticle based Zn(II) sensing SERS assay can be used for the differentiation of prostate metastatic cell from prostate normal cell. Figure 4G shows the SERS spectra and images from LNCaP prostate cancer cells and LNCaP prostate cancer cells with SERS probe in the absence of Zn(II). We have used HORIBA’s Raman microscope to measure the SERS spectrum and image from LNCaP prostate cancer cells. Figure 4H shows the SERS spectra and images from LNCaP prostate cancer cells with SERS probe in the presence and absence of Zn(II). Reported data clearly show that SERS probe based Zn(II) sensing assay can be used for the differentiation of cancer cell with and without Zn(II). To understand the variability of the Raman reporter response, we have monitored the variability of the SERS signal from PEG coated Raman reporter attached gold nanoparticle made in different batches. We have also varied the prostate cells cultured in different batches. Experimental data reported in Figure 4D shows the relative standard deviation of the SERS signal intensity to be about 6.2 %, in the presence of prostate normal cells. For this purpose, we have made PEG Coated Raman reporter attached gold nanoparticle in 4 different batches and for each batch we have performed several different experiments by changing the prostate normal cells grows in different batches. Similarly, Figure 4E shows the relative standard deviation of the SERS signal intensity to be about 5.4 % in the presence of prostate cancer cells. In this case, we have made PEG coated Raman reporter attached gold nanoparticle in 4 different batches and for each batch we have performed several different experiments by changing the prostate cancer cells cultured in different batches.

Similarly, Figure 4E shows the relative standard deviation of the SERS signal intensity to be about 5.4 %, in the presence of prostate cancer cells. In this case, we have made PEG coated Raman reporter attached gold nanoparticle in 4 different batches and for each batch, we have performed several different experiments by changing the prostate cancer cells cultured in different batches. Prostate metastatic cell concentration dependent SERS study, as reported in Figure 4D, shows that Zn(II) sensing SERS based assay can be used for the detection of prostate metastatic cell even in the concentration of 5 cells/mL.

3. Conclusion

In conclusion, in this manuscript we have reported a highly efficient SERS probe for identifying prostate cancer cells based on Zn(II) sensing, using 785 nm biological I window excitation light. Experimental data demonstrated that SERS intensity from Raman reporter enhanced by more than two orders of magnitude in the presence of Zn(II) due to the formation of assembly structure. Theoretical data using FDTD simulations indicate that four huge enhancement of SERS intensity in the presence of Zn(II) is mainly due to huge electric field enhancement in “hot spot” formed by the assembly structure. Reported data show that SERS probe can be used for the selective detection of Zn(II) even at 100 ppt concentration. Our experimental result shows that Zn(II) sensing SERS probe is able to distinguish prostate metastatic cells from normal prostate cells. We have also shown that Zn(II) sensing SERS based assay can be used for the detection of prostate metastatic cell even at the concentration of 5 cells/mL. Although our developed SERS probe is able to distinguish prostate metastatic cells from normal prostate cells, it cannot be used to determined exact Zn(II) concentration in clinical prostate sample.

4. Experimental

Hydrogen tetra chloroaurate (HAuCl₄), sodium borohydride, sodium citrate, normal (PNT2-C2) prostate cells were purchased from Sigma-Aldrich (St. Louis, MO, USA). Metastatic (LNCaP) prostate cancer cells and growth media were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA).

4.1. Synthesis of p-((imidazole)azo) benzenethiol

The Raman reporter p-((imidazole)azo) benzenethiol was prepared by modifying a literature procedure, as shown in Figure 1A. In brief, 125 mg (1.0 mmol) of 4-aminothiophenol (4-ATP) was dissolved in 25 ml of a 1:1 ethanol-water mixture by sonicating for 10 minutes. Next, 10 mL of 0.5 N hydrochloric acid was slowly added into the 4-ATP solution. The amine was diazotized by adding 10 ml of aqueous 100 mg (1.4 mmol) sodium nitrite at temperatures below 5°C with constant stirring. The reaction mixture was allowed to stand at below 5°C for 10 minutes, and the resulting diazonium chloride salt solution was added drop wise with vigorous stirring to 65 mg of imidazole (65 mg, 1.0 mmol) in 50 ml 2% sodium carbonate water maintained at a temperature below 5°C. The reaction mixture was refrigerated for 12 hours. Following refrigeration, the mixture was acidified with 0.1 N hydrochloric acid until neutral (pH = 7). The solid yellowish-red product was separated by filtration, purified by crystallization from hot methanol, and dried under vacuum for future use. Yield: 200 mg, 98%, MP: 97°C. NMR data is reported in supporting information. ¹H NMR (500 MHz, DMSO-d₆, TSP): δ 12.7 (s, 1H, imidazole NH), 7.89 (d, J = 8.6 Hz, 2H, ArH), 7.77 (d, J = 8.7 Hz, 2H, ArH), 7.52 (s, 1H, ArSH), 7.36 (s, 2H, imidazole CH), ¹³C NMR (125 MHz, DMSO-d₆), 154.98 (imidazole-C), 151.55 (imidazole-C), 139.45 (Ar-C), 130.04 (Ar-C), 127.92 (Ar-C), 123.79 (Ar-C), Anal. Calcd for C₉H₈N₄S: C, 52.92; H, 3.95; N, 27.43. Found: C, 52.91; H, 3.97; N, 27.45, ESI-MS (+ve): m/z 205.03.

We have also used infrared and absorption spectroscopy to characterize the Raman reporter. As shown in Figure 1B, the FT-IR spectrum exhibits strong peak at ~3435 cm⁻¹ corresponds to the vibrational mode of the –N-H groups. Quite strong peaks were also observed at ~2555 cm⁻¹ for the –S-H stretch, ~1632 cm⁻¹ for the –C=C stretch, ~1590 cm⁻¹ for the –C-N stretch, ~1473 cm⁻¹ for the –N=N stretch and ~1080 cm⁻¹ for the –C-S stretch respectively. In the absorption spectra, as shown in Figure 1C, we have observed a strong peak around 380 nm, which is due to the azobenzene, mainly in trans form.

4.2. Synthesis of spherical gold nanoparticle

Gold nanoparticles of 25 nm in size were synthesized by using HAuCl₄, 3H₂O, sodium boro-hydride (NaBH₄) and ascorbic acid as we have reported before^{24,28,31–32}. In brief, 0.01 wt.% solution chlorauric acid was heated to boiling and then trisodium citrate dihydrate solution was quickly added. The solution changed color within several minutes to red. JEM-2100F transmission electron microscope (TEM) and UV-visible absorption spectrum were used to characterize the nanoparticles as shown in Figure 2.

4.3. Synthesis of Raman reporter p-((imidazole)azo) benzenethiol attached gold nanoparticle

We have attached p-(imidazole)azo) benzenethiol via thiol group as shown in Figure 2A. In the next step, we have used high-resolution JEM-2100F transmission electron microscope (TEM) to characterize p-(imidazole)azo) benzenethiol attached gold nanoparticle. As reported in Figure 2B, the particle size is about 25 nm for Raman reporter attached gold nanoparticle. Figure 2E shows the extinction spectra for p-(imidazole)azo) benzenethiol attached gold nanoparticle, which clearly shows a plasmon peak around 530 nm and azobenzene peak around 380 nm.

4.4. Synthesis of Raman Polyethylene glycol (PEG) -coated p-((imidazole)azo) benzenethiol attached gold nanoparticle

Next, for the stabilization of Raman reporter attached gold nanoparticle in cell media, we have developed thiol-terminated polyethylene glycol (PEG) coated Raman reporter attached gold nanoparticle. We have attached thiol modified PEG with gold nanoparticle via Au-S bond.

4.5 SERS Spectra Detection

For the SERS experiment, we have used a portable SERS probe, as we have reported before^{24,28,31–32}. For this purpose, we have used a continuous wavelength 785 nm DPPS laser as an excitation light source. InPhotonics 785 nm Raman fiber optic probe has been used for excitation and data collection. SERS data was collected using a miniaturized QE65000 spectrometer. A 10-second acquisition time and 5-scan averaging were used for the Raman data collection, using Ocean Optics data acquisition and Spectra Suite spectroscopy software.

4.6. FT-IR measurements

For the FTIR measurement, we have used Thermo Nicolet Nexus 870 FTIR spectrometer.

4.7. UV-Vis absorption measurements

For UV-Vis measurements, a Cary 300 spectrophotometer controlled by the new Cary Win UV software was used.

4.8. Cell culture

PNT2-C2 and LNCaP cells were cultured in a 5% CO₂ incubator at 37°C using RPMI-1640 medium supplemented with 10% premium fetal bovine serum (FBS) and antibiotics in 75-cm² tissue culture flasks according to the ATCC procedure.

4.9. FDTD simulation

For full-wave finite-difference time-domain (FDTD) simulation calculation, we have used FDTD simulation software package from Lumerical Solutions, as we and others have reported before^31,36–37. In this simulation, we have solved the macroscopic Maxwell’s equations in discretized space and discretized time. By doing this, we were able to follow the response of a nano-assembly to any applied EM field. Polarized light at a wavelength of 785 nm was used along the y-axis, for FDTD simulation. Since for the SERS measurement we have used ~60 V/m electric field, to compare with experimental result for FDTD simulation, we have used the amplitude of electric field as 10V/m and the count number as 0.9. For the nano-assembly structure, we have used the bent structure, similar to the observed experimental structure. For our calculation, the entire simulation is done with 0.001 nm mesh resolution with 4000 fs time.