The Strategy for the Sensing of β-Glu
The principle of the designed sensing platform for β-Glu detection was shown in Scheme 1. As illustrated in Scheme 1 (A), the BSA-Cu3(PO4)2·3H2O nanohybrids possessing peroxidase-like activity were facilely obtained through the spontaneously self-assembly reaction in phosphate-buffered saline (PBS) an room temperature. In the presence of hydrogen peroxide (H2O2), BSA-Cu3(PO4)2·3H2O can catalyze the oxidation of Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) to resorufin (7-hydroxy-3H-phenoxazin-3-one), which is accompanied with the fluorescence enhancement and the corresponding color change from colorless to dark magenta [Scheme 1 (B)] . While β-Glu and cyanogenic glycoside amygdalin (Amy) were added, the catalytic oxidation process was blocked by the enzymatic hydrolysate cyanide ions (CN-), which can greatly restrain the catalytic activity of BSA-Cu3(PO4)2·3H2O nanozyme [Scheme 1 (C)] . Meanwhile, the fluorescence decreased and the color changed from dark magenta to pink. Therefore, the changing of fluorescence intensity and hue parameters produced the corresponding signals to accurately detect β-Glu. Owing to the convenient, cost-effective and environmental friendly, it is expected that the present fluorescent and visual sensing system can be utilized to the β-Glu assay in real samples.
Peroxidase-like Activities of BSA-Cu3(PO4)2·3H2O
We investigated the peroxidase-like catalytic properties of BSA-Cu3(PO4)2·3H2O nanohybrids via acting BSA-Cu3(PO4)2·3H2O on Amplex Red solution (fluorogenic and chromogenic substrate) and H2O2 to initiates a redox reaction. As displayed in Fig. 1, the control experiments suggested that there are no marked fluorescence emission peaks between 565 nm and 800 nm with the single Amplex Red solution (curve a) and Amplex Red solution containing NFs (curve b) or H2O2 (curve c), separately. While NFs, Amplex Red and H2O2 were mixed, the mixed solution exhibited a remarkable fluorescence enhancement from the very low fluorescence background level that was almost flat (curve d). The fluorescence of the NF-Amplex Red-H2O2 system is significantly much higher than that of Amplex Red (a), NF-Amplex Red system (b) and H2O2-Amplex Red system (c), revealing that the BSA-Cu3(PO4)2·3H2O NFs possessed excellent peroxidase-mimicking catalytic activity. The fluorescence is derived from the oxidation product of Amplex Red that is known as resorufin [37,38]. The maximum fluorescence emission wavelength of the reaction mixture is around 584 nm at the excitation wavelength of 550 nm. Meanwhile, as displayed in the inset of Fig. 1, it can be observed inset that the colorless Amplex Red was rapidly oxidized to a typical dark magenta product, which can be easily discernible by the naked eyes.
We tested the fluorescence emission spectra of the reaction in which different concentrations of H2O2 were catalyzed by series of certain amount of BSA-Cu3(PO4)2·3H2O in order to further investigate the catalytic performance of the NFs. As depicted in Fig. 2 (A), the fluorescence emission intensity steadily increased as the increasing concentrations of H2O2 from 0.05 to 200 mmol·L-1. And a good linearity was founded between the fluorescence emission intensity and the H2O2 concentration [Fig. 2 (B) inset]. The above results confirmed the satisfactory peroxidase-like catalytic performance of BSA-Cu3(PO4)2·3H2O nanohybrids. Moreover, the incubating time was studied since it takes a crucial role during the enzymatic catalytic process. It can be seen from Fig. 3 that the fluorescence emission intensity enhanced as the increasing incubating time within a certain range, and reached a plateau at about 90 min that indicating the catalytic reaction was finished within 90 min.
As we all known, the catalytic performance of enzyme is closely associated with the reaction conditions. In this work, we first studied the influence of pH on the enzyme catalytic properties of the as-prepared NFs. As shown in Fig. 4, when the pH changed from 4.0 to 9.0, both the nanoflower catalytic activity and the HRP catalytic activity are higher in alkaline medium than in acidic medium using Amplex Red as substrate. But overall the NFs had better catalytic capacity when compared with HRP in the pH range of 4.0 to 9.0. The effect of temperature on the enzyme catalytic properties of the obtained NFs was subsequently tested from 4 ℃ to 50 ℃. From Fig. S1, it can be found that the NFs possess stronger ability of resistance to high temperature. Furthermore, we carried out a control experiment to contrast the stability of the NFs and HRP. The results in Fig. S2 suggested that the NFs catalytic activity basically unchanged in one month, but the HRP almost no longer exhibited catalytic activity only within 75 h, which is accordance with the previous work . The above experimental results indicated that the as-prepared nanozyme have advantage over the nature enzyme HRP and hold potential application in practice.
Optimization of the Sensing Platform
In order to acquire better performance for sensing β-Glu, we optimized the experimental parameters including incubation time, pH values, temperature and salt concentration. As displayed in Fig. 5 (a), the fluorescence emission intensity was found gradually decreased with the increasing of reaction time and reached a plateau at around 55 min [Fig. 5]. Therefore, 55 min was chosen for the further study. As exhibited in Fig. 5 (b), it can be observed that the fluorescence readout declined from pH 4.0 to pH 8.0 and increased from pH 8.0 to pH 10.0, the minimum value was appeared at pH 8.0. So 8.0 was chosen as the optimal pH value for β-Glu sensing platform. As shown in Fig. 5 (c), the fluorescence readout decreased from pH 4 ℃ to 25 ℃ and increased from 25 ℃ to 45 ℃, the minimum value was found at 25 ℃. Thus, the optimal reaction temperature for β-Glu sensing platform is 25 ℃. The salt concentration showed no obvious effect on the sensing platform, and we chose 50 mmol·L-1 NaCl for the subsequent experiments in this work.
Fluorescence Sensing of β-Glu
Under the optimum experimental conditions, further experiments were performed for β-Glu assay. Fig. 6 (a) recorded the fluorescence emission spectra of NFs-H2O2-Amplex Red-Amy system recorded from 565 nm to 800 nm at an excitation wavelength of 550 nm when addition of different concentrations of β-Glu activities from 0 to 1500 U·L-1.
We can noticeably found that the fluorescence emission intensity at 585 nm can be regulated by the β-Glu concentrations and decreased step by step until a platform, and the color of the mixed solution gradually becomes shallow from dark magenta to light magenta at the same time [Fig. 6 (c)]. Additionally, it can be calculated that from Fig. 1 that the fluorescence emission intensity at 584 nm decreased to 33.8% of the original fluorescence intensity of NFs-Amplex Red-H2O2 system. A quality linear range for β-Glu activity was established between fluorescence intensity ratio (I/I0) and β-Glu activity concentration form 0.5 U·L-1 to 1500 U·L-1 (0.5, 10, 20, 50, 100, 300, 500, 700, 900, 1100, 1300 and 1500 U·L-1). The linear fitting equation is I/I0 = 0.992-0.0002 [β-Glu] (U·L-1) with a correlation coefficient (R2) of 0.997. The limit of detection (LOD) defined by the 3σ equation (signal-to-noise ratio of 3) was calculated to be 0.33 U·L-1, which is comparable to LODs of the previous published approaches that are summarized in Table S1, suggesting that the present sensing method has satisfactory sensitivity. In addition, the sensing strategy displayed excellent detection range in comparison with the existing β-Glu assay methods. It should be further noted that the influence of single Amy or β-Glu had a negligible effect on the NFs-H2O2-Amplex Red system (Fig. 7).
Under the optimized reaction conditions, the selectivity and interference of the protein-inorganic hybrid nanoflower-based sensing platform were assessed by carrying out the experiment of various common coexisting substances that consist of several potential interfering enzymes (ACP and GOX), biological materials (L-cysteine, glucose and protamine) and inorganic ion (K+, Na+, Mg2+ Ca2+, NO3- and Cl-1). The concentration of coexisting ACP is 1.0 μU·mL-1. The concentration of coexisting GOX is 1.0 μg·mL-1. The concentration of coexisting L-cysteine, glucose and protamine is 1.0 μmol·L-1, respectively. The concentration of coexisting K+, Na+, Mg2+ Ca2+, NO3- and Cl-1 is 1.0 μmol·L-1, respectively. A relative error of ±5.0% is considered to be acceptable. As exhibited in Fig. 8, the protein-inorganic hybrid NF-based sensing system showed excellent specifcity for β-Glu determination, and the presence of these coexisting substances didn’t interfere the proposed sensing system for the β-Glu activity assay.
Sensing of β-Glu in Real Samples
To assess the reliability of the proposed sensing system, we applied it to the measurement of β-Glu in real samples of human serum samples, water samples and soil samples. To obtain spiked real samples, a series of three different fixed concentrations of β-Glu were added into the human serum samples, water samples and soil samples. Then, a recovery study was carried out on these real samples β-Glu assay and the results were given in Table S2. It can be seen that the average β-Glu recoveries for the human serum samples, water and soil samples were in the range from 96.2-104%. The relative standard deviations (RSDs, n=3) are lower than 5.0%. The above results reveal the proposed sensing platform held potential application for β-Glu activity assay.