**6. Bio-application**

*Zinc Oxide Based Nano Materials and Devices*

LSPR area.

**5. Cell viability test**

carried out as follows.

600 nm. The near-field distribution of the electric fields was calculated for 785 nm incident light and parallel plate boundary conditions with symmetry of the electric field. Even if the head width is increased, there is almost no change in the full scale, and the electric field is distributed in the vertical direction of the incident light as shown in **Figure 4**. The cross-sectional width of incident light is 2 μm, and the density of the nanorods in the region is constant. This suggests that the difference in SERS enhancement by Au thickness is due to concentration of sample at

Raman measurements of nanometer-sized biomarkers secreted from living cells require confirmation of the suitability of cells for SERS substrates. The toxic endogenous properties of gold nanoparticles have previously been reported [29], and ZnO nanorods are reported to be toxic to NIH 3 T3 fibroblasts [30]. Therefore, it is necessary to confirm whether the sensing chip is suitable for cell application through the evaluation of cytotoxicity, and cell culture and cell viability tests were

Breast cancer cell line of MDA-MB-231 was purchased from the Korean Cell Line Bank (Seoul, Korea). The breast cancer cells MDA-MB-231 were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM; Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) and a 1% penicillin–streptomycin solution (Life Technologies, Inc.) in a humidified 5% CO2 incubator at 37°C. Cell viability analyses were based on MTT (Sigma, USA) assays. Cells were plated at a density of 3 × 105 cells/well and incubated for 24 hours. After preparing the Au-ZnO substrate, the cell was treated with 5 mg/ml MTT for 30 min and then dissolved using DMSO. The absorbance was measured at 540 nm with an ELISA microplate reader (Multiskan EX, Thermo Scientific, USA). **Figure 5** shows the results of MTT assay of the death of the breast cancer cell line MDA-MB-231 according to SERS substrate condition. The MDA-MB-231 is commonly used in biotechnology applications such

**104**

**Figure 5.**

*Cell viability of MDA-MB-231 on each substrate by MTT assay.*

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a refractory disease that afflicts the vague pelvic pain when the urine enters the bladder and makes frequent urination [31]. There are various treatments based on oral agents [32–34], but they are still unsatisfactory with frequent recurrence of symptoms and Hunner's lesions [35]. Parallel with the development of therapeutic technology, early diagnosis of IC/BPS related to quality of life and detection of disease before development to chronic type can minimize patient's pain and increase treatment effect. Therefore, it is necessary to confirm the possibility of early detection of nanometer biomarkers from urine obtained from interstitial cystitis animal model using SERS substrate. IC/BPS animal models and comparative groups for these experiments were derived using 10-week-old female Sprague Dawley rats. The rats were instilled with 0.2 M HCl for 10 min using a 26-gauge angiocatheter in the bladders of four rats, followed by neutralization and saline wash. Other rats in the comparison group were used as vehicle instead of HCl injection. The voiding pattern was analyzed to confirm the reproducibility of animal modeling as in the previous paper [36]. Rat urine was collected in a 50 mL tube using a metabolic cage. The voiding pattern measured at a week after HCl injection was examined, and the collected urine was used as a sample for Raman measurement. Twenty-four hours of natural voiding patterns in the metabolic cage were recorded and analyzed using AcqKnowledge 3.8.1 software and an MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) at a sampling rate of 50 Hz. The volume change of the obtained raw data of urine was estimated by 1 mL unit as shown in **Figure 6a**. Irregular frequency of urinary dysfunction caused by bladder inflammation is observed in HCl-treated rats and is consistent with previous animal model studies [35, 36]. Steps and terraces in the graph are the excretion urine and the duration between voiding, respectively. The total amount of control and IC/BPS animal models for approximately 10 hours is 11 and 13 mL, respectively. However, compared with the amount, the frequency is 3 and 6 times. When the unit of step is 0.5 mL, the frequency is 4 and 11 times, and the difference in the voiding frequency is clearly revealed.

From the identified sample, a drop of 5 μL was applied to the SERS specimen, and the sample was allowed to spread for 60 min. After confirming that the droplets were dry and diffused, they were loaded onto a Raman spectroscope system attached to a microscope (IX-73, Olympus, Japan) and measured. As shown in **Figure 6b**, the diffused region of the sample can be confirmed by an optical microscope, and the region where the sample is diffused as in (c) can be confirmed to have a nanometer-scale porosity. In this area, Raman spectra were collected using a customized spectrometer (FEX-INV, NOST, Korea) with a 785 nm diode laser as the excitation source. The 1 mW of excitation light was focused on the sample through a 40 ×/0.6 NA objective with spot size ~2.4 μm. The spectrum of each point was measured 8 times in the range of 550 to 1500 cm<sup>−</sup><sup>1</sup> with a spectral resolution of 1 cm<sup>−</sup><sup>1</sup> and an integration time of 40 s at room temperature. The Raman spectrum was calibrated by measuring a silicon sample before the Raman measurements. To evaluate the spectral differences between control and IC/BPS of rat urine, principal component analysis (PCA) was introduced. A statistical analysis method of PCA reduces the number of variables in multivariate systems, and all of spectral range was used as variables. All analyses were conducted using XLSTAT 2018 software.

**Figure 6.**

*(a) Measurement of voiding function in control group (blue line) and IC/BPS animal group (orange line) at 7 days after HCl treatment. (b) Optical microscope images of a Raman measurement region diffused from a sample droplet into a nanoporous area and (c) magnified SEM image.*

As shown in **Figure 7a**, brown bars are indicated on the peak, which is the main factor above the graph drawn as the average of total data. The main peaks for the control and IP/BPS samples were observed at 641, 683, 723, 873, 1002, 1030, and 1355 cm<sup>−</sup><sup>1</sup> , which corresponded to C-C twisting mode of tyrosine [37, 38], ring breathing of nucleic acids for G [38, 39] and A [39, 40], C-C stretch of hydroxyproline [37, 41], symmetric ring breathing mode [37–41] and C-H in-plane bending mode of phenylalanine [37, 41], and CH3CH2 wagging mode of collagen [37, 41], respectively.

The peak at 1002 cm<sup>−</sup><sup>1</sup> has a considerably large value compared to the rest of the data, which is notable in literature referring to other peaks, as they relate to other biologies. To analyze Raman peaks, PCA is utilized as shown in **Figure 7b**. Clear discrimination and reliable separation between control and IC/BPS groups were observed. By plotting PC2 and PC3, the groups show clear distinctions between IC/ BPS urine and the control samples (dotted line). Raman spectrum measurements and PCA analysis showed that it is possible to distinguish between normal and diseased groups using gold-coated ZnO nanorod substrates that can be applied to early disease diagnostic sensing chips.

#### **Figure 7.**

*(a) Averaged Raman spectra for IC/BPS (blue line) and control (green line) of rat's urine. Standard deviations are painted around the spectra. (b) Principal component analysis results for urine of IC/BPS and control sample.*

**107**

*Surface-Enhanced Raman Spectroscopy (SERS) Based on ZnO Nanorods for Biological…*

In summary, we compared the differences in surface-enhanced Raman effect using RhB by adjusting the length and diameter of ZnO nanorods and the volume of deposited gold in ZnO-based SERS substrates. Electron microscopy images showed clustering on top of nanorods during gold deposition and showed nanometer level porosity. As the volume of deposited gold increases, the Raman signal also improves, but as the growth conditions of the nanorods change, the signal intensity also changes. This is because the Raman enhancement factor is determined by the enhancement by the SERS properties of metal and the concentration of the sample. Through the finite element analysis in the two-dimensional plane, signal enhancement was similar for 80 and 125 nm of Au-grain head. It was found that the enhancement of the Raman signal was determined by the wider surface area of gold. It was confirmed that this signal enhancement is made in the vertical direction of the rod, so that only nanometer targets trapped in the porous space can obtain enhanced signals. In addition, the SERS chips based on ZnO nanorods were found to have no coffee ring effect in measuring liquid samples and were also suitable for cell application experiments. An IC/BPS animal model was constructed for the bio-application, the voiding pattern was observed for urinary disease status, and urine was collected from the IC/BPS. The obtained urine was diffused into a ZnObased SERS chip having nanopores, and Raman was measured in the corresponding region. Statistical analysis of Raman signals obtained from nanometric level area showed that IC/BPS and normal animals were distinguished. Therefore, we can confirm that ZnO nanorod-based SERS has sufficient potential for early disease

*DOI: http://dx.doi.org/10.5772/intechopen.84265*

diagnosis by efficiently detecting nano-sized biomarkers.

This work was supported by the Basic Science Research Program (2018R1D1A1B07048562) and MRC grant (2018R1A5A2020732) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT), by the Ministry of Trade, Industry and Energy (MOTIE) under the Industrial Technology Innovation Program (10080726, 20000843), and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and

**Acknowledgements**

**Conflict of interest**

Welfare, Republic of Korea (HI18C2391).

The authors declare no conflict of interest.

**7. Conclusion**

*Surface-Enhanced Raman Spectroscopy (SERS) Based on ZnO Nanorods for Biological… DOI: http://dx.doi.org/10.5772/intechopen.84265*
