Ultrasensitive and Multi-Functional Rapid Screening Plasmonic Ag-Based SERS Substrate
Authors: Lee Fang Jing , Yen Ta-Jen 
Published: May 2020

Motivation

The complex interpretation of the cause for some diseases and the lack of specificity in clinical manifestations lead to the difficulty in making early diagnosis and treatment. So far, several analytical methods, including radiation immunoassay (RIA), fluorescence immunoassay (FIA)[1] and enzyme-linked immunoassay (ELISA) [2], have been widely applied in the field of biomedical research. However, the above-mentioned techniques still suffer some disadvantages. For example, these immunoassay take at least 4~6 hours to complete the incubation and detection process. Also, attenuation of the fluorescence signal-to-noise ratio is low which may cause inaccurate detection easily. [3]As a consequence, it is necessary to develop a new and effective method to obtain the ‘3S’ goal, which are the sensitive, specific and speedy detection of biomarkers.

Introduction

With the mentioned motivations in mind, a plasmonic Ag-based SERS substrate is proposed to implement the ultrasensitive detection of biomarkers while cardiac troponin I (cTnI), which is the most reliable biomarker for cardiovascular disease (CVD), is chosen as the analyte in this work. The proposed substrate is mainly composed of silicon nanowires array decorated with silver nanoparticles. By exploiting this substrate, the appropriate length and interspace of silicon nanowires may offer a high surface-to-volume ratio for wider area of detection. On the other hand, the silver nanoparticles can contribute localized surface plasmon resonance (LSPR) for SERS enhancement as well. The superiority of this substrate includes (1) facile fabrication, (2) low detection limit, (3) wide linear range of concentration (4) time-saving and (5) label-free detection. Apart from this, the cost of this SERS substrate is very low while it can achieve multiple and repeated measurements by one sampling, hence shows great potential for practical application in clinical diagnosis.

Experimental Procedures

To begin with, the silicon nanowires scaffold was fabricated by metal-assisted chemical etching (MaCE) procedure. As shown in Fig. 1, after the first absorption of silver nanoparticles on a cleaned silicon substrate, it was then being immersed into a HF/H2O2 solution for etching. Next, the residual silver nanoparticles were removed by concentrated HNO3 solution and then immersed the prepared substrate into a KOH/IPA solution to enlarge the interspace between silicon nanowires. Finally, silver nanoparticles were deposited on the silicon nanowires by electroless metal deposition (EMD). After applying 5 mL of different concentrations of cTnI on each fabricated substrate by using pipette, a Confocal Micro-Raman Spectroscopy equipped with 633nm laser source and 17 mW of power was utilized to excite the SERS substrates, while the spectra were collected in the continuous mode with the integration time of 10 s.

Results and Conclusions

The morphology of silver nanoparticles deposited on silicon nanowires with different KOH etching time were characterized by scanning electron microscopy (SEM). As shown in Fig. 2, silicon nanowires are free-standing on the substrate with silver nanoparticles distribute uniformly on the tips, while the interspaces of the silicon nanowires are significantly enlarged after the KOH etching. To startup, a preliminary Raman measurement of 1.616 ppb cTnI was obtained and consistent to the referred Raman spectrum as shown in Fig. 3 [4]. By eliminating the overlapped signals from silicon on 520 cm-1 and 900 cm-1, the more significant characteristic peaks of cTnI which located at around 1300 cm-1 and 1600 cm-1 are mainly considered for further analysis. Also, taking into account the discrepancy in the state of focus and power for each measurement, every Raman spectrum was normalized by the intensity of the silicon peak at 520.6 cm-1. Fig. 4 shows the Raman results of substrates with different KOH etching time for three different concentrations of cTnI, while all of the results show that the one with 1 min KOH etching gives the most enhancement. Furthermore, Fig. 5 shows both the Raman spectra of blank substrate that without applying any analyte and that of cTnI with different concentrations from 0.012 ppb to 1.616 ppb. Herein, a clean background signal was successfully obtained for the blank substrate, demonstrating that the proposed substrate might be applicable for not only cTnI but also other chemical molecules such as food additives and drug. Importantly, the plot for the concentration-dependent SERS intensity of the peaks at 1300 cm-1 and 1600 cm-1 (Fig. 6) show that, both of them obey a good linear relationship, with R2=0.93368 and R2=0.91904 respectively. These experiment results demonstrate that the proposed strategy displays high detection sensitivity for cTnI up to 0.012 ppb in a wide and linear detection range from 0.012 ppb to 1.616 ppb, which exhibits the potential to perform quantitative analysis. Meanwhile, the specificity of the analyte is also verified by the unique Raman fingerprint of cTnI, which presents the superior ability to neglect the complex incubation process needed in many immunoassay methods.

References

[1] R.T. Ranasinghe, T. Brown, Ultrasensitive Fluorescence-Based Methods for Nucleic Acid Detection: Towards Amplification-Free Genetic Analysis, Chemical Communications, 47, 3717-3735 (2011); doi: 10.1039/C0CC04215C

[2] Y.C. Ko, N. Mukaida, A. Panyutich, N.N. Voitenok, K. Matsushima, T. Kawai, T. Kasahara, A Sensitive Enzyme-Linked Immunosorbent Assay for Human Interleukin-8, Journal of Immunological Methods, 149, 227-235 (1992); doi: 10.1016/0022-1759(92)90254-q

[3] M. Guo, J. Dong, W. Xie, L. Tao, W. Lu, Y. Wang, W. Qian., SERS Tags-Based Novel Monodispersed Hollow Gold Nanospheres for Highly Sensitive Immunoassay of CEA, Journal of Materials Science, 50, 3329-3336 (2015); doi: 10.1007/s10853-015-8825-3

[4] M. Saliminasab, A. Bahrampour, M. H. Zandi, Human Cardiac Troponin I Sensor Based On Silver Nanoparticle Doped Microsphere Resonator, Journal of Optics, 14, 1-7 (2012): doi: 10.1088/2040-8978/14/12/122301

Figure 1