English

• Simultaneous detection of different tumor markers is highly significant for disease evaluation and diagnosis because of the substantial complexity involved in cancer development [1-4]. Surface-enhanced Raman scattering (SERS)-based immunosensing methods have garnered increased interest in clinical diagnosis fields because to its exceptional benefits [5-11]. Currently, multiplex SERS immunosensing is usually achieved by labeling different Raman reporters onto antigen targeting antibodies, and then realizing the multi-SERS signal output [12-15]. However, the sensitivity of SERS immunosensing is limited by the weak intensity of common Raman labels [16]. Furthermore, the limited number of Raman labels and resolution of various Raman scattering signals hinder the throughput of this detection mode. Although the purpose of current multiplex SERS immunosensing has been achieved, the low detection sensitivity and the cumbersome analysis process are disadvantageous to the rapid and accurate screening of cancer markers.

  Over the last few decades, many researchers have demonstrated ways to improve SERS detection sensitivity through the design of noble metal nanoparticles with special structures to generate stronger enhancement of the electromagnetic field [17-21]. Au nanoparticles (AuNPs) are one of the commonly used SERS substrates due to their excellent local surface plasmon resonance properties, which can amplify the signal of Raman reporter molecules through local electromagnetic fields and charge transfer [22-26]. However, there is still a great challenge in the Raman signal amplification of AuNPs toward Raman-inactive reporter molecules. Nanozymes as artificial enzymes have attracted considerable attention due to a series of advantages [27-36]. Recently, nanozymes have been reported to catalyze some Raman-inactive organic dyes to Raman-active reporter molecules, and thus have become promising in SERS biosensing [37-41]. Additionally, it has been recently demonstrated that AuNPs possess multienzyme-mimicking activity [42-45]. Therefore, the combination of excellent SERS activity and the enzymatic properties of Au nanozymes provide an exceptional avenue for the development of sensitive SERS immunosensors.

  Photonic crystal microbeads as a periodically arranged material have seen use as coding carriers for multiplex detection because of their three-dimensional ordered spatial structure and the fact that their unique reflection spectra can be used to obtain a large amount of coding elements for meeting the requirements of high-throughput detection [46-49]. In this work, a versatile Au nanozyme Raman probe strategy was proposed to develop an ultrasensitive multiplex SERS immunosensor using encoded silica photonic crystal beads (SPCBs) (Scheme 1). A robust Au nanozyme with oxidase-like and peroxidase-like activity can doubly catalyze Raman-inactive 3,3′,5,5′-tetramethylbenzidine (TMB) into Raman-active oxidized TMB (ox-TMB) reporters to enhance the SERS signal twice. Moreover, the Au nanozyme probes as an extraordinary SERS substrate further enhances the Raman signals of the large number of ox-TMB Raman reporter molecules. SPCBs with different codes (reflection peaks of 502 nm and 600 nm, respectively) were used to encode tumor markers for detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). This research provides a promising Au nanozyme Raman probe strategy for amplifying SERS signals to realize the ultrasensitive multiplex SERS biosensing.

  Scheme 1

  

  Scheme 1.  Schematic diagram of Au nanozyme Raman probe strategy for multiplex SERS immunosensing.

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  The synthesized Au nanozyme was investigated via TEM as shown in Fig. S1A (Supporting information), exhibiting spherical morphology and good dispersion with an average size of about 15 nm (inset in Fig. S1A). Fig. S1B (Supporting information) presents the UV–vis spectrum of the synthesized Au nanozyme. A characteristic absorption peak at 530 nm indicates the successful preparation of Au nanozyme. The oxidase (OXD)-like and peroxidase (POD)-like properties of the Au nanozyme were evaluated by colorimetric method using the conversion of TMB at 652 nm. It can be seen from Fig. 1A that TMB (curve a) and TMB + H₂O₂ (curve b) display no obvious absorbance peaks. Similarly, the solutions of TMB (inset a) and TMB + H₂O₂ (inset b) are colorless. However, Au nanozyme + TMB displays an intense absorbance peak (curve c), and the solution of Au nanozyme + TMB turned light blue (inset c). This color can be ascribed to the oxidation of TMB catalyzed by Au nanozyme with OXD-like activity. As expected, Au nanozyme + TMB + H₂O₂ displays an even more intensive absorbance peak (curve d), and the solution of Au nanozyme + TMB + H₂O₂ corresponds to the blue change in the photograph (inset d), which can be attributed to the further oxidation of TMB catalyzed by Au nanozyme with POD-like activity. The electron paramagnetic resonance (EPR) spectroscopy was further verified by the enzyme-like activity of Au nanozyme by measuring the generation of ^•OH, unpaired electron and ^•O₂^- radicals. Compared with the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-H₂O₂ reaction, the DMPO-^•OH signal from DMPO-AuNPs-H₂O₂ shows an evident four-line spectrum of a 1:2:2:1 intensity, verifying the generation of ^•OH radicals that can oxidize TMB to produce blue ox-TMB (Fig. 1C). Furthermore, ^•O₂^- generation was also confirmed by EPR spectroscopy, showing specific six-line patterns of ^•O₂^- with DMPO as the trapping reagent. Significantly, more ^•O₂^- is obtained with enhanced EPR peaks in the presence of Au nanozyme, which matches the OXD-like properties of Au nanozyme by catalyzing cascade reactions (Fig. 1D).

  Figure 1

  

  Figure 1.  (A) UV–vis spectra and colorimetric photographs (insets) for characterizing dual enzyme-like activity of Au nanozyme: (a) TMB, (b) TMB + H₂O₂, (c) Au nanozyme + TMB, (d) Au nanozyme + TMB + H₂O₂. (B) Raman spectra of (a) TMB, (b) Au nanozyme + TMB, (c) Au nanozyme + TMB + H₂O₂. EPR spectroscopy of the (C) ^•OH and (D) ^•O₂^- spin adduct generated by Au nanozyme.

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  As shown in Fig. S2 (Supporting information), in the presence of AuNPs, the assignments of the Raman-enhanced vibrational peaks of 4-MBA (10^–5 mol/L) molecule at 1587 cm^-1 was significant enhanced, revealing that the Au nanozyme substrate has good SERS activity [50]. Fig. 1B shows the Raman spectra of TMB (curve a), Au nanozyme + TMB (curve b) and Au nanozyme + TMB + H₂O₂ (curve c). As shown in Fig. 1B, the Raman signal generated by the single TMB is very weak, indicating no Raman activity of the TMB molecule. While intense Raman signals could be observed from the Raman spectrum of Au nanozyme + TMB. This result indicates that a portion of Raman-inactive TMB was oxidized to Raman-active ox-TMB in the presence of O₂ owing to OXD-like activity of the Au nanozyme. In addition, a more significant Raman signal was observed from the Au nanozyme + TMB + H₂O₂. This is because the Au nanozyme with POD-like activity can further catalyze the oxidation of TMB to generation more ox-TMB. Moreover, AuNPs as an excellent SERS substrate can enhance the Raman signal of the ox-TMB reporter molecules [51].

  The feasibility of Au nanozyme tags boosting Raman signal enhancement strategy was investigated for the detection of tumor markers for the encoded-SPCBs immunosensor. The preparation of Au nanozyme tags (as Raman signal probes) was first characterized using UV–vis absorption spectroscopy (utilizing AFP as a preliminary target). As depicted in Fig. 2A, the single Au nanozyme only has an absorption peak at 532 nm (curve a), and the secondary antibody shows a characteristic protein absorption peak at 276 nm (curve b). Meanwhile, the Au nanozyme Raman probe shows two absorption peaks at 276 nm and 532 nm (curve c), indicating the successful formation of the secondary antibody linked Au nanozyme tag (Au-Ab₂) signal probes. X-ray photoelectron spectroscopy (XPS) was used to further characterize SPCBs and antibody-SPCBs. As shown in Fig. 2B, SPCBs have two typical peaks of Si 2p (103.04 eV) and O 1s (532.72 eV). SPCBs modified with antibodies showed the characteristic peaks of N 1s (400.09 eV) and a more intensive C 1s (284.8 eV) peak than SPCBs. And the high-resolution scans for N 1s of SPCBs-Ab₁ was shown in Fig. 2C. These data both indicate that the antibodies have been modified onto the surface of Raman photonic crystal microsphere substrate. As demonstrated in Scheme 1, Au nanozyme Raman probes were used to carry out the Raman signal enhancement for ultrasensitive multiplex SERS immunosensing. As shown in Fig. 2D, the Raman signal of the encoded-SPCBs Ⅱ immunosensor is very weak in the absence of AFP antigen (curve a), which is because the Au-Ab₂ signal probe cannot be captured by SPCBs-Ab₁ therefore the Raman-inactive TMB cannot be oxidized to Raman-active ox-TMB by the Au nanozyme tags. These results also demonstrate that the encoded-SPCBs immunosensor has no obvious non-specific absorption. Furthermore, the results indicate that the encoded-SPCBs Ⅱ immunosensor can recognize AFP to bind Au-Ab₂ signal probe via the sandwich immunoassay. The corresponding OXD-like activity of Au nanozyme tags of sandwich immunocomplex on encoded-SPCBs can catalyze the oxidation of TMB to Raman-active ox-TMB in the presence of O₂, which resulted in an enhanced Raman signal of ox-TMB reporter molecules (curve b). In the presence of AFP antigen and H₂O₂, the constructed immunosensor also showed a significant signal enhancement (curve c). This can be attributed to the POD-like activity of Au nanozyme tags which can further catalyze the oxidation of TMB to produce great number of Raman-active ox-TMB molecules. The Au nanozyme tags appear as an extraordinary SERS substrate greatly enhancing the detection signals of Raman reporter molecules. It demonstrates that the Au nanozyme Raman probes boosting Raman signal enhancement strategy can be applied for SERS immunosensing of tumor markers.

  Figure 2

  

  Figure 2.  (A) UV–vis spectra of (a) Au nanozyme, (b) Ab₂, (c) Au nanozyme-Ab₂. (B) XPS spectra of SPCBs and SPCBs-Ab₁. (C) High-resolution scans for N 1s of SPCBs-Ab₁. (D) Raman spectra of (a) SPCBs-Ab₁ + Au-Ab₂ + TMB, (b) SPCBs-Ab₁ + AFP + Au-Ab₂ + TMB, (c) SPCBs-Ab₁ + AFP + Au-Ab₂ + TMB + H₂O₂.

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  Encoded-SPCBs in this research were designed for multiplex SERS detection of tumor markers (CEA and AFP). The synthesized SPCBs were characterized using scanning electron microscope (SEM) and polarizing microscope (POM). Fig. S3 (Supporting information) shows the SEM image of SPCBs, where a smooth edge and spherically shaped morphology with a size of approximately 280 µm were observed. Insets in Figs. 3A and B show the amplified SEM images of SPCBs Ⅰ and SPCBs Ⅱ which were prepared via reaction at 45 and 35 ℃, respectively. The SPCBs were composed of SiO₂ nanoparticles with sizes of about 230 nm (inset a) and 260 nm (inset b), which arranged into an orderly hexagonal close-packed structure. Under polarizing microscope, SPCBs Ⅰ showed a green color with a reflection peak at 502 nm (Fig. 3A) while SPCBs Ⅱ exhibited a red color with a reflection peak position at 600 nm (Fig. 3B). Therefore, the encoded-SPCBs can be utilized to encode CEA and AFP antibodies, respectively. CEA or AFP antibody-modified encoded-SPCBs can specifically react with their target antigen within the mixture of CEA and AFP, then bind the Au-Ab₂ signal probe to form a sandwich immune structure for signal amplification. As shown in Figs. 3C and D, the OXD-like and POD-like Au nanozyme tags can catalyze TMB to Raman-active ox-TMB to produce significantly enhanced SERS signals. The mixed antigen of CEA and AFP can be accurately differentiated for multiple detection according to the reflection peak wavelength and the color of microscope image SPCBs.

  Figure 3

  

  Figure 3.  POM reflectance spectra of SPCBs (A) SPCBs Ⅰ: Prepared at 45 ℃, and the corresponding SEM image (inset a), (B) SPCBs Ⅱ: Prepared at 35 ℃, and the corresponding SEM image (inset b). Raman spectra of (C) encoded-SPCBs Ⅰ for CEA detection, (D) encoded-SPCBs Ⅱ for AFP detection.

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  The concentrations of Au nanozyme tags and H₂O₂ impact the enzyme-like activity and then greatly impact the SERS performance. Therefore, these parameters were optimized by changing the concentration of Au nanozyme tags and H₂O₂ during the detection process of tumor markers. Fig. S4 (Supporting information) shows that the SERS signal continuously enhances with increases in the concentration of Au nanozyme tags. The highest SERS signal was observed when the concentration of Au nanozyme tags was 2 mmol/L and when the concentration of H₂O₂ was 20 mmol/L as shown in Fig. S5 (Supporting information). Therefore, 2 mmol/L Au nanozyme tags and 20 mmol/L H₂O₂ were used in the following experiments. Subsequently, the activation and incubation time of Raman-active ox-TMB were also optimized at a concentration of 10 ng/mL CEA. Accordingly, the activation time of 20 min was found to possess the highest SERS signal (Fig. S6 in supporting information). In addition, incubation times from 5 min to 30 min was investigated and the incubation time of 30 min possessed the highest SERS signal (Fig. S7 in supporting information). As such, the activation time of 20 min and incubation time of 30 min were selected as the optimum for further experiments.

  Under these optimized analysis conditions, the intensities of Raman signal were monitored with increasing concentration of CEA or AFP (Figs. 4A and C). The calibration curves of the fabricated SERS immunosensor for multiplex detection of CEA and AFP are shown in Figs. 4B and D. Looking at Fig. 4A, it was clear that the Raman peak intensities at 1605 cm^-1 increased linearly with the increase of both tumor marker concentrations ranging from 0.001 ng/mL to 100 ng/mL (CEA) and 0.01–1000 ng/mL (AFP) with linear correlation coefficients of 0.9854 and 0.9871, respectively. The resulting detection limits were calculated to be 0.66 pg/mL (CEA) and 9.5 pg/mL (AFP). The comparison of the multiplex SERS immunoassay and previous immunoassay was listed in Table S1 (Supporting information). This demonstrates that the Au nanozyme Raman probes boosting Raman signal enhancement strategy has excellent assay properties for multiplex SERS immunosensing.

  Figure 4

  

  Figure 4.  Raman spectra of encoded-SPCBs at different concentrations of (A) CEA and (C) AFP and calibration curves for SERS immunosensing of (B) CEA and (D) AFP.

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  Anti-interference is an important basis for measuring the feasibility of the proposed multiplex SERS immunosensor. To test this, another antigen was used as an interfering substance when detecting a target antigen with a known concentration, and the anti-interference was judged by examining whether the Raman signal value changes. Fig. 5A illustrates that despite the continued increase in interfering AFP concentration, the Raman signal for CEA detection remained largely unaffected. Under the same circumstances, Fig. 5B illustrates that the Raman signal for AFP detection remained largely unaffected despite the continued increase in interfering CEA concentration. When the concentration of interfering antigens was changed within the range of 10–50 ng/mL, it was found that the SERS signal remained stable for detection of 10 ng/mL CEA and AFP antigens with less than 6.9% and 5% intensity variability, respectively. As shown in Fig. 5C, Raman signals were recorded with the respective injection of CEA (10 ng/mL), AFP (10 ng/mL), IgG (10 ng/mL), IgM (10 ng/mL) and PBS (0.01 mol/L), which explored the anti-interference property of the fabricated immunosensor. It is obvious that the addition of CEA leads to a more conspicuous Raman signal than that of other antigens, which is consistent with the anticipation. Under the same circumstances, Fig. 5D illustrates that the Raman signal for AFP leads to a more conspicuous Raman signal than that of other antigens. The resultant results suggest that fabricated immunosensor has high specificity for determination of target analytes. The experimental results show that the designed multiplex SERS immunosensing method can realize the simultaneous determination of multiple tumor markers without impacts from cross-reaction.

  Figure 5

  

  Figure 5.  (A) Raman spectra and intensity changes of 10 ng/mL CEA with different concentrations of incorporated interfering substance (AFP). (B) Raman spectra and its intensity changes of 10 ng/mL AFP with different concentrations of interfering substance (CEA). (C) Raman response of 10 ng/mL CEA, 10 ng/mL AFP, 10 ng/mL IgG, 10 ng/mL IgM and blank solution for studying the selectivity of the fabricated SERS immunosensor. (D) Raman response of 10 ng/mL AFP, 10 ng/mL CEA, 10 ng/mL IgG, 10 ng/mL IgM and blank solution for studying the selectivity of the fabricated SERS immunosensor. (E, F) Stability of multiplex SERS immunosensor across different encoded-SPCBs.

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  To study the stability of the constructed SERS immunosensors, 10 different encoded-SPCBs were randomly selected for the detection of 10 ng/mL CEA, and measured Raman signals were compared. As illustrated in Fig. 5E, all 10 encoded-SPCBs displayed Raman response and SERS signal remained stable. The corresponding histogram shows that the Raman signals minimally fluctuated (Fig. 5F). Therefore, the encoded-SPCBs have good stability, which lays a good foundation for the accuracy and reproducibility of the multiplex SERS immunosensor.

  To evaluate the practicability of the Au nanozyme tags enhanced SERS immunosensor for clinical analysis, serum samples were collected from a local hospital and analyzed without any prior treatment. Ethical approval was obtained from the Ethics Committee of Northern Jiangsu People's Hospital. In addition, written informed consent was obtained from all the participants prior for this study. The serum samples were diluted appropriately to the detection range of the immunosensor using 0.01 mol/L PBS (pH 7.4) before the measurement. The results of CEA and AFP detection in serum are listed in Table 1. The actual concentrations obtained were basically consistent with the reference values from the electrochemiluminescence immunoassay. Relative errors for the data obtained from clinical samples using the proposed SERS method were no more than 8.5%. These results clearly show that the encoded-SPCBs immunosensors based on Au nanozyme tags boosting Raman signal enhancement strategy can be applied to accurately monitor multiple antigens in human serum.

  Table 1

  

  Table 1.  Determination of CEA and AFP antigen (ng/mL) in human blood serum samples using the encoded-SPCBs (n = 5).

  DownLoad: CSV

  Biomarkers	Sample	1	2	3	4	5
  CEA (ng/mL)	Proposed method	0.776	1.31	1.91	7.23	108
  	Reference method	0.826	1.43	1.77	7.90	105
  	Relative error (%)	−6.0	−8.4	8.0	−8.5	2.9
  AFP (ng/mL)	Proposed method	1.09	1.382	2.88	4.41	76.01
  	Reference method	1.08	1.42	2.77	4.57	74.67
  	Relative error (%)	0.9	−3.3	4.0	−3.5	1.8

  In this work, a versatile Au nanozyme Raman probe strategy was proposed for developing an ultrasensitive multiplex SERS immunosensor employing encoded-SPCBs. The robust Au nanozyme shows high dual enzyme-mimicking activity and excellent SERS activity. Firstly, the OXD-like activity of the Au nanozyme probes can catalyze the oxidation of Raman-inactive TMB to Raman-active ox-TMB in the presence of O₂. Secondly, the POD-like activity of the Au nanozyme tags can catalyze Raman-inactive TMB to Raman-active ox-TMB in the presence of H₂O₂. The resulting large number of Raman-active ox-TMB reporter molecules results in significant Raman signal enhancement. Thirdly, Au nanozyme tags themselves act as an extraordinary SERS substrate and further enhance signal from Raman reporter molecules. The Raman signal enhancement plays a key role in improving the SERS detection sensitivity. The reflection peaks of encoded-SPCBs and the color of their microscope images were decoded to realize multiplexed SERS immunosensing of different tumor markers. The proposed method shows ultrahigh sensitivity, wide linear range, high selectivity, and stability, and has a high consistency with the test results of actual samples. The presented research showcases a universal and prospective nanozyme-induced signal amplification strategy to develop ultrasensitive multiplex SERS immunosensing.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

  CRediT authorship contribution statement

  Jiayin Li: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Linan Yang: Validation. Feng Shi: Supervision, Investigation. Yan Long: Investigation. Yuru Wang: Software. Daniel D. Stuart: Investigation. Hongbo Li: Writing – review & editing, Resources. Quan Cheng: Writing – review & editing, Supervision, Resources. Lingfeng Min: Writing – review & editing, Resources, Investigation. Zhanjun Yang: Writing – review & editing, Supervision, Investigation, Funding acquisition. Juan Li: Writing – review & editing, Supervision, Resources.

  Acknowledgments

  This work was financially supported by National Natural Science Foundation of China (Nos. 21475116, 21575125 and 22474124), the National Natural Science Foundation of Jiangsu Province (Nos. BK20221370, BK20211362), Key University Natural Science Foundation of Jiangsu-Province (No. 20KJA150004), the Project for Science and Technology of Yangzhou (No. YZ2022074), the Project for Yangzhou City and Yangzhou University corporation (No. YZ2023204), Cross cooperation project of Subei Peoples' Hospital of Jiangsu Province (No. SBJC220009), the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science (No. SKLACLS2405), and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX24_3728).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110883.

  
  
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  Scheme 1  Schematic diagram of Au nanozyme Raman probe strategy for multiplex SERS immunosensing.

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  Figure 1  (A) UV–vis spectra and colorimetric photographs (insets) for characterizing dual enzyme-like activity of Au nanozyme: (a) TMB, (b) TMB + H₂O₂, (c) Au nanozyme + TMB, (d) Au nanozyme + TMB + H₂O₂. (B) Raman spectra of (a) TMB, (b) Au nanozyme + TMB, (c) Au nanozyme + TMB + H₂O₂. EPR spectroscopy of the (C) ^•OH and (D) ^•O₂^- spin adduct generated by Au nanozyme.

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  Figure 2  (A) UV–vis spectra of (a) Au nanozyme, (b) Ab₂, (c) Au nanozyme-Ab₂. (B) XPS spectra of SPCBs and SPCBs-Ab₁. (C) High-resolution scans for N 1s of SPCBs-Ab₁. (D) Raman spectra of (a) SPCBs-Ab₁ + Au-Ab₂ + TMB, (b) SPCBs-Ab₁ + AFP + Au-Ab₂ + TMB, (c) SPCBs-Ab₁ + AFP + Au-Ab₂ + TMB + H₂O₂.

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  Figure 3  POM reflectance spectra of SPCBs (A) SPCBs Ⅰ: Prepared at 45 ℃, and the corresponding SEM image (inset a), (B) SPCBs Ⅱ: Prepared at 35 ℃, and the corresponding SEM image (inset b). Raman spectra of (C) encoded-SPCBs Ⅰ for CEA detection, (D) encoded-SPCBs Ⅱ for AFP detection.

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  Figure 4  Raman spectra of encoded-SPCBs at different concentrations of (A) CEA and (C) AFP and calibration curves for SERS immunosensing of (B) CEA and (D) AFP.

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  Figure 5  (A) Raman spectra and intensity changes of 10 ng/mL CEA with different concentrations of incorporated interfering substance (AFP). (B) Raman spectra and its intensity changes of 10 ng/mL AFP with different concentrations of interfering substance (CEA). (C) Raman response of 10 ng/mL CEA, 10 ng/mL AFP, 10 ng/mL IgG, 10 ng/mL IgM and blank solution for studying the selectivity of the fabricated SERS immunosensor. (D) Raman response of 10 ng/mL AFP, 10 ng/mL CEA, 10 ng/mL IgG, 10 ng/mL IgM and blank solution for studying the selectivity of the fabricated SERS immunosensor. (E, F) Stability of multiplex SERS immunosensor across different encoded-SPCBs.

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  Table 1.  Determination of CEA and AFP antigen (ng/mL) in human blood serum samples using the encoded-SPCBs (n = 5).

  Biomarkers	Sample	1	2	3	4	5
  CEA (ng/mL)	Proposed method	0.776	1.31	1.91	7.23	108
  	Reference method	0.826	1.43	1.77	7.90	105
  	Relative error (%)	−6.0	−8.4	8.0	−8.5	2.9
  AFP (ng/mL)	Proposed method	1.09	1.382	2.88	4.41	76.01
  	Reference method	1.08	1.42	2.77	4.57	74.67
  	Relative error (%)	0.9	−3.3	4.0	−3.5	1.8

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