Detection of breast cancer based on novel porous silicon Bragg reflector surface-enhanced Raman spectroscopy-active structure Download: 870次
Breast cancer (BRC) is one of the most important malignant tumors affecting women’s health. The number of BRC deaths worldwide is approximately 520,000 per year[1]. According to a report by the World Health Organization’s International Agency for Research on Cancer in 184 countries and regions, the number of women with BRC in China is only smaller than that of the United States, which accounts for 11.9% of the world’s total. Although people have an increasing knowledge of BRC and more diversified treatment methods in the past 20 years, the incidence and mortality still exhibit a substantial increase[2]. One of the reasons for this problem is that patients are diagnosed with BRC quite late[3], and the current diagnosis of BRC is determined by the patient’s clinical manifestations and medical history, physical examination, imaging examination, X-ray, magnetic resonance imaging, or ultrasound, histopathology, and cytopathology[4]. However, these diagnostic techniques have insurmountable disadvantages. High cost and information lag have a great impact on the early diagnosis of BRC, causing patients to miss the best treatment time, and operative biopsy greatly deepens the patients’ pain[5]. Therefore, it is of great significance to develop a low-cost technology that can diagnose BRC efficiently and reduce pain.
Many research groups have developed BRC serum diagnostic techniques based on surface-enhanced Raman scattering (SERS) and infrared spectroscopy. Raman spectroscopy is a detection method with a “fingerprint” recognition ability for the detection of each molecule or sample, which shows that the Raman spectroscopy detector itself has a better specificity[6]. For example, Shafer-Peltier et al. obtained human BRC tissue from in vitro samples and detected BRC tissue using SERS technology[7]. Many researchers, such as Vargas-Obieta, mixed the BRC serum with gold glue or silver glue and detected BRC serum using the SERS technique[8]. Zheng et al. used a microfluidic chip to detect BRC serum by SERS[9]. However, the sensitivity, stability, biocompatibility, and enhancement of the Raman peak in these detection methods are unsatisfactory.
The substrate is critical in the application of SERS, and researchers in various countries are still exploring new low-cost and high-enhancement substrates[10]. One type of porous substrates that have attracted much interest because of their impressive properties for sensor applications is a PSi-based device. The most attractive feature of PSi is its compatibility to be used with the biomolecules, and it is widely applied to biological and biomedical research[11]. The main reason for choosing a PSi nanomaterial is that PSi has rough surface characteristics. The PSi Bragg reflector can be prepared by controlling the corrosion current, which is simple to operate and has a low cost[12]. Yue et al. prepared a single-layer PSi substrate and used it to detect echinococcosis serum[13]. Zhang et al. developed a PSi optical microcavity biosensor for detecting DNA and the detection limit is 43.9 nmol/L[14]. Futamata et al. achieved SERS detection of rhodamine 6G (R6G) molecules on single-layer PSi substrates[15]. Wang et al. developed a PSi surface grating and applied it to SERS research[16]. For these advantages, PSi materials can be used as a good SERS substrate.
In this Letter, a composite silver nanoparticles (Ag NPs) PSi Bragg reflector SERS substrate is experimentally optimized. N-type highly doped silicon wafers[17] with a greater porosity and specific surface area cannot only be used for SERS detection of macromolecular organisms but can also greatly improve SERS signal intensity[18]. At the same time, the substrate offers a high detection sensitivity, which is of great significance for the diagnosis of BRC. Finally, and more importantly, this Letter uses the composite Ag NPs PSi Bragg reflector SERS substrate to detect the early BRC serum collected in the clinic and uses the principal component analysis (PCA) and linear discriminant analysis (LDA) (PCA-LDA) method to carry out statistical analysis and calculate results to show that the accuracy, specificity, and sensitivity of the diagnosis are 95%, 96.7%, and 93.3%, respectively. Analysis of the data better shows that the optimized SERS substrate has a high accuracy and practicability, and this detection technology can serve as a new noninvasive clinical tool for cancer detection and make a great contribution to clinical medicine.
All serum samples were acquired from the First Affiliated Hospital of Xinjiang Medical University. Approval of the ethics committee was obtained to study human blood serum samples. In our experiment, 30 samples of fresh blood from early BRC patient serum and 30 samples of fresh blood from normal subjects were obtained. 3 mL fresh blood was collected from each patient without any anticoagulant, and the samples were placed in a clean environment at 37°C for 1 h for condensing. Then, a high-speed centrifuge (4000 r/min) was used to centrifuge the blood at 4°C for 5 min, and the top level of clear liquid was extracted to obtain the serum. The serum was subpacked in a centrifuge tube and stored in a refrigerator at −80°C for experiments.
To better verify the sensitivity of the substrate, we diluted the early BRC serum with deionized water in terms of volume ratio and placed it on an oscillator for 20 min to mix it evenly. We took the early BRC serum out of the freezer and diluted it immediately after ablation to prevent inactivation, and pipette 2 μL of it onto the prepared substrates. (The preparation process of composite Ag NPs PSi Bragg reflector SERS substrate is shown in the Supplementary Materials.)
The serum Raman spectra were recorded using a confocal Raman microspectrometer (LabRAM HR Evolution Raman spectrometer, HORIBA Scientific, Ltd.) with an excitation wavelength of 532 nm and an laser (12.5 mW). The spectral data were acquired in 5 s with a 50× objective, and the numerical aperture (NA) was 0.5. Three spectra were recorded for each sample in different positions, and the mean spectrum was taken for further analysis.
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Fig. 1. SEM images of the newly prepared SERS substrates: (a) surface morphology; (b) cross-sectional morphology; (c) partial enlarged view of (b); (d) surface morphology of the composite Ag NPs PSi Bragg reflector SERS substrate.
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Fig. 2. SERS spectra of early BRC serum samples from 30 patients on monocrystalline silicon, composite Ag NPs single-layer PSi substrates, and the composite Ag NPs PSi Bragg reflector SERS substrate.
The composite Ag NPs PSi Bragg reflector SERS substrate can enhance the SERS effect of the analyte[19]. The periodic change of the refractive index of the PSi Bragg structure increases the interaction time between light and material, enhances the magnetization, and improves the SERS signal intensity[20]. Our research team carried out X-ray diffraction detection and theoretically simulated the electric field distribution on single-layer PSi and multi-layer PSi. Though the calculation simulation and detection results showed that the diffraction peaks of the multi-layer PSi became wider and wider, the field intensity of the PSi photonic crystals was stronger than that of single-layer PSi[17,21]. It was proved by experiments and calculation simulation that the enhanced electric field on the surface of photonic crystals is conducive to achieving a stronger localized surface plasmon resonance (LSPR), which leads to the enhancement of Raman optical signals.
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Fig. 3. SERS spectra of early BRC serum samples (diluted in different proportions) on the composite Ag NPs PSi Bragg reflector SERS substrate.
The serum SERS spectra of 30 normal persons and 30 early BRC patients were analyzed by principal component analysis combined with linear discriminant analysis (PCA-LDA). And we have made the difference graph of the average normalized spectrum between the serum of healthy people and the early BRC patients (see Fig. S4 in the Supplementary Materials). It can be intuitively seen that the average normalized spectrum of serum of healthy people and early BRC patients has some differences in some Raman characteristic peaks. This provides a theoretical basis for further classification through algorithms.
First, the original spectral signal was corrected by LabSpec6 software and then normalized between 0 and 1 to reduce the influence of the laser power fluctuation and the slight differences in experimental conditions. Then, PCA was used to reduce the dimension of the band of interest, and the characteristic spectrum that best reflected the difference between normal people and BRC patients was identified for subsequent discriminant analysis.
The first three principal components (PC1, PC2, and PC3) with the largest variance contribution rate after PCA were selected for analysis. In Figs.
Fig. 4. (a) Plot of PC1 versus PC2 for the normal group versus the BRC group. (b) Plot of PC1 versus PC3 for the normal group versus the BRC group. (c) Plot of PC2 versus PC3 for the normal group versus the BRC group. (d) A three-dimensional mapping of the PCA results for the normal group and BRC group with PC1, PC2, and PC3 as the three axes. (The black triangle represents the normal group; the red triangle represents the BRC group.)
Generally, the accuracy of discriminant analysis increases with the number of principal components after dimensionality reduction[22]. The number of principal components increases (from PC1 to PC10); the cumulative variance contribution rate and the accuracy of LDA are shown in Fig.
Fig. 5. Cumulative variance contribution rate and LDA classification accuracy as a function of the number of principal components.
Considering the small sample size, to obtain more reliable diagnostic results, LDA uses the leave one out cross validation (LOOCV) method[22]. Table
Table 1. Confusion Matrix for the LDA Algorithm[22]
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In this Letter, the composite Ag NPs PSi Bragg reflector SERS substrate was successfully prepared by electrochemical etching and chemical reduction. The SERS response of the serum for 30 normal people and 30 early BRC patients was measured on the composite Ag NPs PSi Bragg reflector SERS substrate, and statistical analysis was carried out by PCA-LDA. The analysis results show that the diagnostic sensitivity is 93.3%, the specificity is 96.7%, and the total accuracy is 95%. The images show that the Raman signal intensity on the composite Ag NPs PSi Bragg reflector SERS substrate is more than three times higher than that on the single-layer PSi SERS substrate. The results of this exploratory study prove that the detection of early BRC serum based on a composite Ag NPs PSi Bragg reflector SERS substrate is with a stable, strong Raman signal, and an unmarked and noninvasive BRC diagnosis technology. In the future, this detection technology can serve as a noninvasive clinical tool for cancer detection, and its use can extend to food safety, jewelry identification, cultural relics identification, and other fields.
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Xiaorong Ma, Hong Cheng, Junwei Hou, Zhenhong Jia, Guohua Wu, Xiaoyi Lü, Hongyi Li, Xiangxiang Zheng, Chen Chen. Detection of breast cancer based on novel porous silicon Bragg reflector surface-enhanced Raman spectroscopy-active structure[J]. Chinese Optics Letters, 2020, 18(5): 051701.