Abstract: Breast cancer has become one of the major hidden dangers to women’s health. The cure rate of advanced breast cancer is less than 10%, but this number in early breast cancer is as high as 90%. So how to find breast cancer earlier becomes the key issue. We utilize surface-enhanced Raman scattering (SERS) [1] to detect the Circulating Tumor Cells (CTC) in the plasma with the enhancement of silver nanostars (AgNS) [2]. CTC can be separated by plasma under the action of specific chips, but it is necessary to add auxiliary materials to get its surface-enhanced Raman spectroscopy. Then we can use spectroscopy analysis to determine the presence of tumor cells. So, we offer a simpler, faster, and more effective method for early diagnosis of breast cancer, and this method has great potential to be applied to clinical diagnosis.
Introduction
Circulating Tumor Cells (CTC) are a general term for various types of tumor cells present in peripheral blood. Malignant tumors can be transferred to other organs of the patient’s body through blood transmission, which is the leading cause of death in cancer patients. In recent years, CTC-based tumor detection monitors the trend of CTC type and quantity changes by capturing and detecting trace amounts of CTC in peripheral blood, so as to monitor tumor dynamics in real time, evaluate treatment effects, and achieve real-time individual treatment.
Raman spectroscopy is a molecular vibrational spectroscopy whose spectral information can reflect the structural characteristics of a molecule. However, Raman is weak in scattering effect, resulting in a weak spectral signal. Therefore, the Raman spectroscopy now uses surface enhancement effects—Surface enhanced Raman spectroscopy (SERS). What’s more, SERS detection can achieve fast, easy and repeatable non-destructive testing of the object to be tested without complicated sample preparation, which makes SERS detection widely applicable to various subject areas.
The combination of SERS detection and microfluidic chip enables rapid detection of live CTCs, and this method enables high-throughput detection, allowing a large number of cell spectra to be obtained in a short period of time. Obviously, this can greatly improve the detection efficiency.
Materials & Methods
Microfluidic Chip
Our microfluidic chip uses 3D printing technology, using Fused Deposition Modeling (FDM) printer, the material is polylactic acid (PLA) (Figure 1). 3D printing has the advantages of low price, fast and convenient, high design degree, and can meet the general precision of chip manufacturing. It should be noted that our chip needs to be fitted with a silicone hose to be used.
Synthesis and Characterization of Silver Nanostars (AgNSs)
Silver nanostars (AgNSs) are obtained by reduction of silver nitrate with neutral hydroxylamine and citrate [3]. This method involves two steps: neutral hydroxylamine reduction and trisodium citrate reduction. Mix 2.5 ml of sodium hydroxide solution and an equal amount of neutral hydroxylamine solution with stirring (500 rpm), continue stirring and add 45 ml of AgNO3 solution dropwise, the dropping rate is 2 ml per minute. After the completion of the dropwise addition, continue stirring for 5 minutes and then add 0.5 ml the trisodium citrate solution. Finally, continue to stir for 15 minutes. We characterized silver nanostars by transmission electron microscopy (TEM) (Figure 2) and ultraviolet (UV) absorption spectroscopy (Figure 3).
Surface enhanced Raman spectroscopy (SERS)
We mixed the silver nanostars with the cell suspension through a microfluidic channel to attach nanoparticles to the cell surface, and obtained the Raman spectrum of the cells under the excitation of a 532 nm laser. Normal blood cells and breast cancer cells are then distinguished based on the differences between the spectra [4-6].
Results
It is well known that the cell membrane is negatively charged, and the modified nanoparticle is positively charged, so it can be well bound to the surface of the cell membrane to effectively enhance the Raman signal of the cell. We process the resulting spectral data, which includes outlier removal, baseline smoothing and normalization. After these processes, specific algorithms are used to distinguish and classify these spectra.
Discussion
Since different proteins are distributed at different positions on the surface of the cell membrane, even if the spectra of the same cells are different, it is necessary to distinguish them based on the proteins specifically expressed by the cancer cells.
Conclusion
In conclusion, we have developed a simple, rapid, cheap and highly sensitive method for detection of live cancer cells, and this method can distinguish between normal cells and breast cancer cells to some extent. Finally, it has a high probability of being used clinically for screening for early breast cancer.
References
[1] Gokhan Demirel; Hakan Usta; Mehmet Yilmaz; Merve Celik; Husnuye Ardıc Alidag; Fatih Buyukserin. Surface-Enhanced Raman Spectroscopy (SERS): An adventure from plasmonic metals to organic semiconductors as SERS Platform. Journal of Materials Chemistry C. 2018; 20: 5314-5335.
[2] Creighton, J. A.; Blatchford, C. G.; Albrecht, M. G. Plasma Resonance Enhancement of Raman-Scattering by Pyridine Adsorbed on Silver or Gold Sol Particles of Size Comparable to the Excitation Wavelength. J. Chem. Soc., Faraday Trans. 2 1979, 75, 790−798.
[3] Guerrero-Martínez, A.; Barbosa, S.; Pastoriza-Santos, I.; LizMarzan, L. M. Nanostars Shine Bright for You. Colloidal Synthesis, Properties and Applications of Branched Metallic Nanoparticles. Curr. Opin. Colloid Interface Sci. 2011, 16, 118−127.
[4] Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and of the transferrin receptor in rat reticulocytes recycling. J Cell Biol. 1983; 97: 329-39.
[5] Harding CV, Heuser JE, Stahl PD. Exosomes: Looking back three decades and into the future. J Cell Biol. 2012; 200: 367-71.
[6] Denzer K, Kleijmeer MJ, Heijnen HF, Stoorvogel W, Geuze HJ. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci. 2000; 113: 3365-74.
