Glucose Detection of 4-Mercaptophenylboronic Acid-Immobilized Gold-Silver Core-Shell Assembled Silica Nanostructure by Surface Enhanced Raman Scattering

The importance of glucose in many biological processes continues to garner increasing research interest in the design and development of efficient biotechnology for the sensitive and selective monitoring of glucose. Here we report on a surface-enhanced Raman scattering (SERS) detection of 4-mercaptophenyl boronic acid (4-MPBA)-immobilized gold-silver core-shell assembled silica nanostructure (SiO2@Au@Ag@4-MPBA) for quantitative, selective detection of glucose in physiologically relevant concentration. This work confirmed that 4-MPBA converted to 4-mercaptophenol (4-MPhOH) in the presence of H2O2. In addition, a calibration curve for H2O2 detection of 0.3 µg/mL was successfully detected in the range of 1.0 to 1000 µg/mL. Moreover, the SiO2@Au@Ag@4-MPBA for glucose detection was developed in the presence of glucose oxidase (GOx) at the optimized condition of 100 µg/mL GOx with 1-h incubation time using 20 µg/mL SiO2@Au@Ag@4-MPBA and measuring Raman signal at 67 µg/mL SiO2@Au@Ag. At the optimized condition, the calibration curve in the range of 0.5 to 8.0 mM was successfully developed with an LOD of 0.15 mM. Based on those strategies, the SERS detection of glucose can be achieved in the physiologically relevant concentration range and opened a great promise to develop a SERS-based biosensor for a variety of biomedicine applications.

However, a low affinity with metal surface and Raman scattering cross-section of polarizability limited the detection of glucose by SERS [17,22,28,29]. To achieve a high

Preparation of SiO 2 @Au@Ag
The SiO 2 @Au@Ag NPs were prepared in accordance with the steps outlined in [38]. The SiO 2 @Au NPs were prepared by incubating 10 mL of Au NP suspension with 2 mL of aminated silica NPs overnight. The colloids were centrifuged and washed thoroughly using EtOH. The NPs were then re-dispersed in 2.0 mL of complete EtOH to obtain 1 mg/mL SiO 2 @Au NPs in EtOH.
The SiO 2 @Au@Ag NPs were prepared in an aqueous medium via the reduction and deposition of Ag using ascorbic acid onto SiO 2 @Au NPs in PVP. Moreover, 200 µL of SiO 2 @Au (1000 µg/µL) was briefly dispersed in 9.8 mL of water that contained 10 mg of PVP, which was kept still for 30 min. Thereafter, 20 µL of silver nitrate (10 mM) was added to the suspension, followed by the addition of 40 µL of ascorbic acid (10 mM). The suspension was incubated for 15 min to completely reduce the Ag + ions to Ag 0 . By repeating the reduction steps, the final AgNO 3 concentration of 300 µM was controlled. The SiO 2 @Au@Ag NPs were obtained by the centrifugation of the suspension at 8500 rpm for 15 min, then the NPs were washed thoroughly using EtOH to remove any excess reagent. The SiO 2 @Au@Ag NPs were then re-dispersed in 1 mL of absolute EtOH to obtain a 200 µg/mL SiO 2 @Au@Ag NP suspension.

Behavior of SiO 2 @Au@Ag@4-MPBA in the Presence of Hydrogen Peroxide
100 µL of PBST containing 20 µg SiO 2 @Au@Ag@4-MPBA was added incubated with 100 µL of H 2 O 2 at various concentration for 1 h at 25 • C, followed by centrifugation for 15 min at 15,000 rpm to obtain the NP suspension. The prepared NPs were washed thoroughly using PBST to remove any excess reagent. The prepared NPs was then redispersed in 100 µL of PBST, to obtain a 200 µg/mL NPs suspension.

Glucose Detection of SiO 2 @Au@Ag@4-MPBA
One hundred microliters of PBST containing 20 µg SiO 2 @Au@Ag@4-MPBA was added, incubated with 100 µL PBST that contained various concentrations of glucose, and 100 µL of GOx for 1 h at 25 • C, followed by centrifugation for 15 min at 15,000 rpm to obtain the NP suspension. The prepared NPs were washed thoroughly using PBST to remove any excess reagent. The prepared NPs was then re-dispersed in 100 µL of PBST, to obtain a 200 µg/mL NPs suspension.

SERS Measurement
To obtain the surface-enhanced Raman spectra, a micro-Raman system with a 10-mW 532-nm diode-pumped solid-state laser excitation source and an optical microscope (BX41, Olympus, Tokyo, Japan) was utilized. The SERS signals were collected with a backscattering geometry using an objective lens with a magnification of 10× (0.90 NA, Olympus, Tokyo, Japan). The selected sites were measured randomly, and all the SERS spectra were integrated over a period of 5 s. The size of the laser beam spot was approximately 2.1 µm, and the SERS spectrum was obtained within the wavenumber range of 300-1700 cm −1 .

Results and Discussion
In this study, SiO 2 @Au@Ag was first immobilized by 4-MPBA as a Raman reporter and labeling molecule. Then this suspension was added into glucose solution in the presence of the GOx enzyme. GOx enzyme catalyzed and converted glucose to gluconolactone and H 2 O 2 . The presence of H 2 O 2 converted 4-MPBA to 4-MPheOH on the surface of SiO 2 @Au@Au. The variation of SERS signal of 4-MPBA was observed and extrapolated to the concentration of glucose in solution ( Figure 1). To prepare SiO 2 @Au@Ag, silica NPs (~150 nm) were first functionalized by 3-aminopropyltriethoxysilane (APTS) to prepare aminated silica NPs. Simultaneously, colloidal Au NPs (3 nm) were synthesized by THPC and incubated with the aminated silica NPs by to prepare Au NPs seed embedded with SiO 2 (SiO 2 @Au seed NPs), according to the method reported by Pham et al. [37,38,[42][43][44]. Subsequently, the Ag NPs on the surface of SiO 2 @Au seed were grown by reducing a silver precursor (AgNO 3 ) in the presence of ascorbic acid (AA) and polyvinylpyrrolidone (PVP) [37]. The silver ions reduced by AA were selectively grown onto SiO 2 @Au seed to generate the SiO 2 @Au@Ag nanostructure. According to a previous study, 4-MPBA was used for specific binding of glucose with the boronic acid motif in 4-MPBA, leading to a significant increase in the absolute intensity of the SERS signal of 4-MPBA. So, we modified the surface of SiO 2 @Au@Ag by 4-MBPA to generate SiO 2 @Au@Au@4-MPBA as a specific ligand for glucose. Subsequently, glucose was added in the reaction in the presence of glucose oxidase (GOx). GO catalyzed and converted glucose to glucolactone and hydrogen peroxide (H 2 O 2 ) in the reaction. H 2 O 2 converts 4-MPBA to 4-mercaptophenol (4-MPhOH). and labeling molecule. Then this suspension was added into glucose solution in the presence of the GOx enzyme. GOx enzyme catalyzed and converted glucose to gluconolactone and H2O2. The presence of H2O2 converted 4-MPBA to 4-MPheOH on the surface of SiO2@Au@Au. The variation of SERS signal of 4-MPBA was observed and extrapolated to the concentration of glucose in solution ( Figure 1). To prepare SiO2@Au@Ag, silica NPs (~150 nm) were first functionalized by 3-aminopropyltriethoxysilane (APTS) to prepare aminated silica NPs. Simultaneously, colloidal Au NPs (3 nm) were synthesized by THPC and incubated with the aminated silica NPs by to prepare Au NPs seed embedded with SiO2 (SiO2@Au seed NPs), according to the method reported by Pham et al. [37,38,[42][43][44]. Subsequently, the Ag NPs on the surface of SiO2@Au seed were grown by reducing a silver precursor (AgNO3) in the presence of ascorbic acid (AA) and polyvinylpyrrolidone (PVP) [37]. The silver ions reduced by AA were selectively grown onto SiO2@Au seed to generate the SiO2@Au@Ag nanostructure. According to a previous study, 4-MPBA was used for specific binding of glucose with the boronic acid motif in 4-MPBA, leading to a significant increase in the absolute intensity of the SERS signal of 4-MPBA. So, we modified the surface of SiO2@Au@Ag by 4-MBPA to generate SiO2@Au@Au@4-MPBA as a specific ligand for glucose. Subsequently, glucose was added in the reaction in the presence of glucose oxidase (GOx). GO catalyzed and converted glucose to glucolactone and hydrogen peroxide (H2O2) in the reaction. H2O2 converts 4-MPBA to 4-mercaptophenol (4-MPhOH). The characteristics of the SiO2@Au@Ag NPs are first shown in Figure S1. The transmission electron microscopy (TEM) images of the SiO2@Au and SiO2@Au@Ag NPs are shown in Figure S1a. Small-sized Au NPs (3 nm) were immobilized onto the surface of the SiO2 NPs when the colloidal Au NPs were incubated with the amine-functionalized silica NPs for 12 h at 25 °C (Figure S1a-i). After the reduction of AgNO3 in the presence of The characteristics of the SiO 2 @Au@Ag NPs are first shown in Figure S1. The transmission electron microscopy (TEM) images of the SiO 2 @Au and SiO 2 @Au@Ag NPs are shown in Figure S1a. Small-sized Au NPs (3 nm) were immobilized onto the surface of the SiO 2 NPs when the colloidal Au NPs were incubated with the amine-functionalized silica NPs for 12 h at 25 • C (Figure S1a-i). After the reduction of AgNO 3 in the presence of PVP, the surface of the SiO 2 @Au NPs contained many Ag NPs ( Figure S1a-ii). The UV-Vis spectra of SiO 2 @Au and SiO 2 @Au@Ag were investigated, as shown in Figure S1b. The suspension of the SiO 2 @Au NPs exhibited the maximum peak at~500-520 nm (Figure S1b-i). A broad band was observed from 320-700 nm, with the maximum peak at~450 nm, for the suspension of SiO 2 @Au@Ag NPs ( Figure S1b-ii). This indicated the generation of Ag shells, in addition to the creation of hot-spot structures, on the surfaces of the SiO 2 @Au NPs, thus yielding a continuous spectrum of resonant multi-modes [37,38,42,43].
pension of the SiO2@Au NPs exhibited the maximum peak at ~500-520 nm (Figure S1b-i). A broad band was observed from 320-700 nm, with the maximum peak at ~450 nm, for the suspension of SiO2@Au@Ag NPs ( Figure S1b-ii). This indicated the generation of Ag shells, in addition to the creation of hot-spot structures, on the surfaces of the SiO2@Au NPs, thus yielding a continuous spectrum of resonant multi-modes [37,38,42,43].

Adsorption of 4-MPBA on the Surface of SiO2@Au@Ag
It is believed that 4-MBPA can selectively interact with glucose in solution, so the SERS-active SiO2@Au@Ag were incubated with 4-MPBA solution for 1 h at 25 °C. Figure  2a showed the SERS bands of SiO2@Au@Ag@4-MPBA in ethanol solution. Compared to the SiO2@Au@Au without 4-MPBA (0 µM) and with 100 µM 4-MPBA, we found that the SiO2@Au@Ag in EtOH solution showed SERS bands at 432, 883, 1051, 1097, 1276, and 1455 cm −1 , assigned to the SERS bands of the EtOH solution. At 100 µM 4-MPBA, clear and new bands were observed at 417, 473, 1077, 1170, 1484, and 1583 cm −1 , indicating that 4-MPBA was successfully immobilized on the surface of SiO2@Au@Ag [18]. The detailed SERS bands of SiO2@Au@Ag@4-MPBA can be seen in Table S1. The SERS intensity of SiO2@Au@Ag@4-MBPA was also examined in the presence of various concentrations of 4-MPBA from 0.1 µM to 100 µM in EtOH solution. The SERS signal of 4-MBPA increased from 0.1 to 10 µM and achieved a saturation at 50 µM. In particular, the intensities of the Raman bands at 1583 cm −1 increased dramatically with 4-MPBA concentration (Figure 2b). Therefore, 50 µM 4-MBPA was chosen for further study. However, the SERS signal of EtOH was too strong compared to the SERS signal of SiO2@Au@Ag@4-MPBA, which can seriously affect the accuracy of analytical result. Therefore, we re-dispersed the SiO2@Au@Ag@4-MPBA in the PBST pH 7.0 ( Figure 3a). Indeed, the SERS bands of 4-MPBA in PBST was clearly observed, compared to those in EtOH. Figure 3a showed that the typical SERS bands of 4-MBPA were dominated by bands at 417 cm −1 , 473 cm −1 , 824 cm −1 , 998 cm −1 , 1021 cm −1 , 1077 cm −1 , 1170 cm −1 , 1484 cm −1 , and 1583 cm −1 . The SERS spectra at 998 cm −1 , 1021 cm −1 , and 1077 cm −1 , which are assigned to the C-C in-plane breathing, C-H in-plane breathing, and C-C in-plane breathing and C-S stretching, respectively ( Figure 3a, Table S1). The pair of intense bands at 1586/1573 cm −1 is ascribed to the original and OH --associated forms of C-C stretching. The SERS spectra However, the SERS signal of EtOH was too strong compared to the SERS signal of SiO 2 @Au@Ag@4-MPBA, which can seriously affect the accuracy of analytical result. Therefore, we re-dispersed the SiO 2 @Au@Ag@4-MPBA in the PBST pH 7.0 ( Figure 3a). Indeed, the SERS bands of 4-MPBA in PBST was clearly observed, compared to those in EtOH. Figure 3a showed that the typical SERS bands of 4-MBPA were dominated by bands at 417 cm −1 , 473 cm −1 , 824 cm −1 , 998 cm −1 , 1021 cm −1 , 1077 cm −1 , 1170 cm −1 , 1484 cm −1 , and 1583 cm −1 . The SERS spectra at 998 cm −1 , 1021 cm −1 , and 1077 cm −1 , which are assigned to the C-C in-plane breathing, C-H in-plane breathing, and C-C in-plane breathing and C-S stretching, respectively ( Figure 3a, Table S1). The pair of intense bands at 1586/1573 cm −1 is ascribed to the original and OH --associated forms of C-C stretching. The SERS spectra at 824 and 417 cm −1 were assigned as the C-C out-plane bending and the C-S stretching with weak intensities [18,34,45,46].

Behavior of SiO 2 @Au@Ag@4-MPBA in the Presence of Hydrogen Peroxide
First, the SiO 2 @Au@Ag@4-MPBA were used as a SERS substrate for glucose detection in the range of 1 to 10 mM ( Figure S2a). According to the previous report, the boronic acid group of 4-MPBA specifically binds to glucose, leading to a significant increase in the absolute intensity of the SERS signal of 4-MPBA, which was ascribed to the orientation change and the charge transfer effect [17,45]. However, the SERS signal of SiO 2 @Au@Ag@4-MPBA at pH 7.0 in our study did not show any significantly difference in the range of 1 to Nanomaterials 2021, 11, 948 6 of 13 10 mM glucose. Therefore, glucose oxidase enzyme was added to the glucose solution to convert glucose to gluconolactone and hydrogen peroxide to SiO 2 @Au@Ag@4-MPBA in our study. The behavior of SiO 2 @Au@Ag@4-MPBA in PBST pH 7.0 containing 1 mg/mL H 2 O 2 was investigated and is shown in Figure 3a.
First, the SiO2@Au@Ag@4-MPBA were used as a SERS substrate for glucose detection in the range of 1 to 10 mM ( Figure S2-a). According to the previous report, the boronic acid group of 4-MPBA specifically binds to glucose, leading to a significant increase in the absolute intensity of the SERS signal of 4-MPBA, which was ascribed to the orientation change and the charge transfer effect [17,45]. However, the SERS signal of SiO2@Au@Ag@4-MPBA at pH 7.0 in our study did not show any significantly difference in the range of 1 to 10 mM glucose. Therefore, glucose oxidase enzyme was added to the glucose solution to convert glucose to gluconolactone and hydrogen peroxide to SiO2@Au@Ag@4-MPBA in our study. The behavior of SiO2@Au@Ag@4-MPBA in PBST pH 7.0 containing 1 mg/mL H2O2 was investigated and is shown in Figure 3a.  (Figure 3a). Based on these results,  Figure S3). The dramatic increase in SERS ratio was observed in the range of 1 µg/mL to 1000 µg/mL of H 2 O 2 . The detection limit of H 2 O 2 based on the SERS ration of I 1583/1597 was calculated to be 0.31 µg/mL (3σ/k, where σ is the standard deviation of the blank and k is the slope of the calibration slope).
Next, the effect of experimental conditions for glucose detection on the SERS signal of SiO 2 @Au@Ag@4-MPBA in the presence of GOx were examined and optimized in Figure 4 and Figures S4-S7. concentration decreased from 200 µg/mL to 100 µg/mL and the SERS signals were almost unchanged after 100 µg/mL SiO2@Au@Ag@4-MPBA. Meanwhile, the SERS signals of SiO2@Au@Ag@4-MPBA in the presence of 5 mM glucose increased slightly when SiO2@Au@Ag@4-MPBA decreased to 67 µg/mL. After subtracting background signal, the SERS signal achieved the highest value at 67 µg/mL. For concentrations lower than 50 µg/mL, the SERS signal of the glucose-incubated SiO2@Au@Ag@4-MPBA suspension decreased sharply owing to the low diffraction of the suspension. After optimizing the detection conditions, the SERS spectra of the SiO2@Au@Ag@4-MPBA suspensions with various glucose concentrations were obtained. Figure 5 described the changes in SERS signal of SiO2@Au@Ag@4-MPBA after reacting with different concentrations of in glucose the range of 0.5 to 8.0 mM with and without 100 µg/mL GOx. The SERS signals of the nanomaterial suspensions at I390/417, I390/1077, I390/1583, I1170/417, I1170/1077, I1170/1583, I1597/417, I1597/1077, and I1597/1583 increased sharply when the glucose concentration was increased from 0.5 mM to 8.0 mM ( Figure S8). This implied that H2O2 was produced from GOx in the suspensions by the GOx, and that H2O2 was able to convert 4-MPBA to 4- To reduce the detection time of glucose, we attempted to combine the enzyme reaction and conversion of 4-MPBA to 4-MPheOH into one step, which is termed the "all-in-one" technique. We also conducted the glucose detection in two separate steps: The enzyme reaction followed by the conversion of 4-MPBA to 4-MPheOH, which is termed the "stepby-step" technique. For both techniques, the glucose concentration was fixed at 5 mM using 20 µg SiO 2 @Au@Ag@4-MPBA for 30 min while the GOx concentration was varied in the range of 10 −2 -10 3 µg/mL. The results showed that the SERS signal of the SiO 2 @Au@Ag@4-MPBA at 5 mM glucose and various concentration of GOx in both the "step-by-step" and "all-in-one" techniques were quite similar ( Figure S4). However, the SERS signal of SiO 2 @Au@Ag@4-MPBA in the "step-by-step" reached saturation earlier than that in the "all-in-one". As a result, its dynamic linear range was narrower than that of "all-in-one". Figure 4a showed that in general the SERS signal at I 390/1077 , and I 390/1583 increased with the GOx concentration in the range of 0.1 to 100 µg/mL and achieved the saturation at 100 µg/mL GOx, while the SERS ratio at I 390/417 increased with the GOx concentration in the range of 1 to 1000 µg/mL. Thus, 100 µg/mL GOx was utilized for further study. The incubation time of glucose detection by the SiO 2 @Au@Ag@4-MPBA was also performed in Figure 4b and Figure S5. The SERS ratio at I 390/1077 was saturated at 30 min, and another SERS ratio of the SiO 2 @Au@Ag@4-MPBA increased with incubation time until 1h. The gradual increase in the SERS signal of SiO 2 @Au@Ag@4-MPBA in the presence of GOx indicated that the enzyme reaction and conversion of 4-MPBA to 4-MPheOH took place simultaneously for 1h.
In addition, the effect of SiO 2 @Au@Ag@4-MPBA core-shell amount was observed in the range of 10 to 50 µg. The results can be seen in Figure 4c and Figure S6. It is well known that SERS signal depends on the Raman reporter of nanomaterials [39]. Therefore, when a greater amount of SiO 2 @Au@Ag@4-MPBA was added, more 4-MPBA were available on the surface of SiO 2 @Au@Ag, generating numerous detection sites for glucose. However, the SERS signals of the SiO 2 @Au@Ag@4-MPBA at various core-shell quantities were insignificantly different in our study, indicating that quantities of 4-MPBA molecules of SiO 2 @Au@Ag at 10 µg were enough to react with all H 2 O 2 generated by 5 mM glucose in the enzyme reaction. However, to ensure that the SiO 2 @Au@Ag@4-MPBA is sufficient to convert H 2 O 2 generated by higher glucose concentration in diabetes, we decided to use 20 µg SiO 2 @Au@Ag@4-MPBA for further study.
For Raman measurement, the concentration of SERS substrate is one of the most important factors affecting the SERS signal [39,41,44]. Figure 4d and Figure S7 showed the effect of SiO 2 @Au@Ag@4-MPBA concentration after glucose incubation in the presence of GOx on the SERS signal of 4-MPBA. In the absence of glucose, the SERS signal of SiO 2 @Au@Ag@4-MPBA suspension significantly decreased when SiO 2 @Au@Ag@4-MPBA concentration decreased from 200 µg/mL to 100 µg/mL and the SERS signals were almost unchanged after 100 µg/mL SiO 2 @Au@Ag@4-MPBA. Meanwhile, the SERS signals of SiO 2 @Au@Ag@4-MPBA in the presence of 5 mM glucose increased slightly when SiO 2 @Au@Ag@4-MPBA decreased to 67 µg/mL. After subtracting background signal, the SERS signal achieved the highest value at 67 µg/mL. For concentrations lower than 50 µg/mL, the SERS signal of the glucose-incubated SiO 2 @Au@Ag@4-MPBA suspension decreased sharply owing to the low diffraction of the suspension.
After optimizing the detection conditions, the SERS spectra of the SiO 2 @Au@Ag@4-MPBA suspensions with various glucose concentrations were obtained. Figure 5 described the changes in SERS signal of SiO 2 @Au@Ag@4-MPBA after reacting with different concentrations of in glucose the range of 0.5 to 8.0 mM with and without 100 µg/mL GOx. The SERS signals of the nanomaterial suspensions at I 390/417 , I 390/1077 , I 390/1583 , I 1170/417, I 1170/1077, I1170/1583 , I 1597/417 , I 1597/1077 , and I 1597/1583 increased sharply when the glucose concentration was increased from 0.5 mM to 8.0 mM ( Figure S8). This implied that H 2 O 2 was produced from GOx in the suspensions by the GOx, and that H 2 O 2 was able to convert 4-MPBA to 4-MPheOH on the surface of SiO 2 @Au@Ag. When the concentration of glucose increased higher than 6.0 mM, the SERS peak reached saturation. This result was due to complete conversion of 4-MPBA to 4-MPheOH on the surface of SiO 2 @Au@Ag@4-MPBA. Meanwhile, the SERS signal of SiO 2 @Au@Ag@4-MPBA in glucose without GOx remained unchanged, indicating that the increase of glucose in the solution did not significantly affect the SERS of 4-MPBA on the surface of SiO 2 @Au@Ag as mentioned in Figure 2. Therefore, we concluded that the SERS signal of SiO 2 @Au@Ag@4-MPBA in the glucose solution in the presence of GOx was the result of the combination of the GOx reaction and the conversion of 4-MPBA to 4-MPheOH on the surface of SiO 2 @Au@Ag.
A linear curve-fitting procedure was utilized for calibration. A significant relationship between the SERS signal ratio and the glucose concentration was found in the experimental data points ranging from 1.0 to 8.0 mM (calibration curve: y = 0.159 x + 0.707, where x is glucose concentration, y is the SERS ration, and R 2 = 0.99). The theoretical LOD was 0.15 mM, estimated by the 3sblank criterion. Our result demonstrates that our material can be utilized for glucose detection for diagnosis.
higher than 6.0 mM, the SERS peak reached saturation. This result was due to complete conversion of 4-MPBA to 4-MPheOH on the surface of SiO2@Au@Ag@4-MPBA. Meanwhile, the SERS signal of SiO2@Au@Ag@4-MPBA in glucose without GOx remained unchanged, indicating that the increase of glucose in the solution did not significantly affect the SERS of 4-MPBA on the surface of SiO2@Au@Ag as mentioned in Figure 2. Therefore, we concluded that the SERS signal of SiO2@Au@Ag@4-MPBA in the glucose solution in the presence of GOx was the result of the combination of the GOx reaction and the conversion of 4-MPBA to 4-MPheOH on the surface of SiO2@Au@Ag. A linear curve-fitting procedure was utilized for calibration. A significant relationship between the SERS signal ratio and the glucose concentration was found in the experimental data points ranging from 1.0 to 8.0 mM (calibration curve: y = 0.159 x + 0.707, where x is glucose concentration, y is the SERS ration, and R 2 = 0.99). The theoretical LOD was 0.15 mM, estimated by the 3sblank criterion. Our result demonstrates that our material can be utilized for glucose detection for diagnosis.
The interference behavior is an important factor for glucose detection since easily oxidizable species, such as ascorbic acid (AA), uric acid (UA), and bovine serum albumin (BSA), at various concentrations and fructose co-exist with glucose in blood samples [48][49][50]. Figure 5b depicted the evaluation of the selectivity of the SiO2@Au@Ag@4-MPBA as a SERS substrate for glucose detection at I390/417 in the presence of interfering species including 50 µM AA, 40 µM UA, 0.5% BSA, 1% BSA, 2% BSA, and 5 µM fructose were evaluated. Additionally, the SERS ration response of the SiO2@Au@Ag@4-MPBA in 5 mM glucose was examined as a reference, and the response SERS ratios at I 390/417 , I 390/1077 , I 390/1583 , I 1170/417 , I 1170/1077 , I 1170/1583 , I 1597/417 , I 1597/1077 , and I 1597/1583 in 5 mM glucose in the presence of interferences were simultaneously observed in Figure S9. In the presence of 50 µM AA, the SERS ratio of SiO2@Au@Ag@4-MPBA at I390/417 decreased 2% to 98%, while in the presence of 40 µM UA, it decreased to 95% compared to the SERS ratio of 5 mM glucose. The presence of 0.5% BSA caused an insignificant decrease in the SERS ratio at I390/417 to 96%. However the SERS ratio at I390/417 in the presence of 1% BSA or 2% BSA showed a significant decrease to 86 and 82%, respectively, because of the adsorption of BSA on the surface of SiO2@Au@Ag@4-MPBA [51]. The SERS ratio at I 390/417 of 5 mM glucose in the presence of 5 µM fructose decreased slightly to 98%. It meant that the detection of glucose by The interference behavior is an important factor for glucose detection since easily oxidizable species, such as ascorbic acid (AA), uric acid (UA), and bovine serum albumin (BSA), at various concentrations and fructose co-exist with glucose in blood samples [48][49][50]. Figure 5b depicted the evaluation of the selectivity of the SiO 2 @Au@Ag@4-MPBA as a SERS substrate for glucose detection at I 390/417 in the presence of interfering species including 50 µM AA, 40 µM UA, 0.5% BSA, 1% BSA, 2% BSA, and 5 µM fructose were evaluated. Additionally, the SERS ration response of the SiO 2 @Au@Ag@4-MPBA in 5 mM glucose was examined as a reference, and the response SERS ratios at I 390/417 , I 390/1077 , I 390/1583 , I 1170/417 , I 1170/1077 , I 1170/1583 , I 1597/417 , I 1597/1077 , and I 1597/1583 in 5 mM glucose in the presence of interferences were simultaneously observed in Figure S9. In the presence of 50 µM AA, the SERS ratio of SiO 2 @Au@Ag@4-MPBA at I 390/417 decreased 2% to 98%, while in the presence of 40 µM UA, it decreased to 95% compared to the SERS ratio of 5 mM glucose. The presence of 0.5% BSA caused an insignificant decrease in the SERS ratio at I 390/417 to 96%. However the SERS ratio at I 390/417 in the presence of 1% BSA or 2% BSA showed a significant decrease to 86 and 82%, respectively, because of the adsorption of BSA on the surface of SiO 2 @Au@Ag@4-MPBA [51]. The SERS ratio at I 390/417 of 5 mM glucose in the presence of 5 µM fructose decreased slightly to 98%. It meant that the detection of glucose by SiO 2 @Au@Ag@4-MPBA was highly selective. Thereby, the combination of GOx and SiO 2 @Au@Ag@4-MPBA exhibited excellent selectivity for glucose detection in the presence of interfering species, 50 µM AA, 40 µM UA, 0.5% BSA, and 5 µM fructose with negligible interference to the SERS signal of glucose.
The long-term storage of the SiO 2 @Au@Ag@4-MPBA was examined in Figure S10. First, 200 µ/mL SiO 2 @Au@Ag@4-MPBA was synthesized, re-dispersed in EtOH, and stored at 4 • C for one week. The SERS signal of 4-MPBA was measured every day and the SERS signal at 1583 cm −1 was monitored. As showed in Figure S10, the SERS signal was not decreased until seven days, indicating that the surface of SiO 2 @Au@Ag was not oxidized during storage in EtOH at 4 • C. However, the SERS signal slightly increased after four days, which might be from the partly aggregation of SiO 2 @Au@Ag.

Conclusions
We have developed a new SERS-based boronated nanoprobe of the SiO 2 @Au@Ag@4-MPBA for quantitative, selective detection of glucose in neutral condition. This work confirmed that 4-MPBA was converted to 4-MPhOH in the presence of H 2 O 2 . Moreover, it provided a new calibration curve to evaluate H 2 O 2 species in the range of 1.0 to 1000 µg/mL with LOD as low as 0.3 µg/mL. Moreover, the SiO 2 @Au@Ag@4-MPBA for glucose detection in the presence of GOx were optimized at 100 µg/mL GOx, 100 µg/mL GOx with 1-h incubation time using 20 µg/mL SiO 2 @Au@Ag@4-MPBA and measuring Raman signal at 67 µg/mL SiO 2 @Au@Ag. At the optimized condition, the calibration curve for selective glucose detection in the range of 0.5 to 8.0 mM was successfully developed with an LOD of 0.15 mM. The combination of GOx and our nanostructure also illustrated that our SERS probe can be coupled with other enzymes to greatly expand its applicability to biologically active targets.