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Abstract
The fluorescence detection platform has broad application in many fields. In this paper, we report a simple and efficient fluorescence detection platform based on the synergistic effects of Ag nanoparticles (Ag NPs) and polymethylmethacrylate (PMMA). Ag NPs were introduced to realize the plasmon enhancement fluorescence and a thin PMMA layer was used to adjust the distance between Ag NPs and riboflavin. The thin PMMA layer not only enhances the fluorescence by enhancing adhesion of substrate, but also optimizes the plasmon enhancement fluorescence effect by serving as the spacer. The fluorescence enhancement factor based on this platform shows a trend of increasing with the decrease of the concentration of riboflavin, and the detection of riboflavin is realized based on this feature, the lowest detectable concentration is as low as 0.27 µM. In addition to the detection based on plasmon enhancement fluorescence, the detection of riboflavin at low concentrations can also be realized by the shift and broadening of the fluorescence peak due to the Ag NPs. The combination of the two ways of plasmon enhancement fluorescence and shift of the fluorescence spectra is used for the detection of riboflavin. These results show that the platform has great potential applications in the field of detection and sensing.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Riboflavin is an indispensable part of our body, making a significant contribution to our metabolism. Riboflavin content is used to judge our health. When the riboflavin in the body does not reach the content we need, it would cause a series of inflammation in our body. Riboflavin is used to judge whether food is expired because of its excellent photosensitive properties. Riboflavin is also used in the treatment of inflammation, angina pectoris and other diseases because of its pharmacological properties [1–4]. In the face of multi-field applications, detection of riboflavin is essential, such as mass spectrometry, electrochemical detection, fluorescence detection, etc. [5–12]. Mass spectrometry can be used to detect very low content of riboflavin, but it is rarely reported in recent years because of the need for a large number of expensive chromatographic reagents and instruments [6]. Electrochemical detection can achieve the purpose of detecting riboflavin by selecting appropriate electrode materials to interact with riboflavin. However, electrochemical detection is easily affected by other electroactive compounds. The selection of appropriate electrode materials determines the performance of anti-interference ability of electrochemical detection [7–10]. Among the fluorescence detection methods, riboflavin has natural fluorescence response due to its own structure. Therefore, compared with above methods, the fluorescence detection method has the advantages of simplicity, rapidity and high sensitivity [11,12].
Currently, the main fluorescence detection methods for riboflavin include direct detection method, fluorescence resonance energy transfer method (FRET), and plasmon enhanced fluorescence method (PEF) [5,11–19]. It is difficult to detect riboflavin directly because the fluorescence intensity of riboflavin itself is weak, therefore, adsorbents or chromatographic agents are proposed to assist the detection [13]. Compared with the direct fluorescence detection method, the FRET method, which enhances the receptor signal by sacrificing donors, can be used to improve the sensitivity and selectivity of riboflavin fluorescence detection because of its simple operation and fast response, and can provide acc urate results [11,12,14–16]. Different from the fluorescence enhancement mechanism of direct detection and FRET, PEF method depends on the local surface plasmon resonance (LSPR) effect of plasmonic nanostructures. The LSPR effect of plasmonic structures can lead to enhancement of the excitation and modification of the emission quantum yield of fluorophores, which can further improve the fluorescence detection performance [20]. When the LSPR band matches the excitation/emission band well, the fluorescence of luminescent molecules will be greatly improved. For example, J. Zhang et al. prepared a recyclable up conversion fluorescence detection paper by combining up conversion nanorods and plasmon structure, which is used to detect human body fluids with different riboflavin concentrations and pH values, with a minimum detection of 0.01 ppm [18]. M. Subr et al. deposited silver nano islands on the surface of polytetrafluoroethylene (MS PTEF) by magnetron sputtering for surface enhanced fluorescence (SEF) of riboflavin, and can obtain 4-fold maximum fluorescence enhancement [19]. In addition, the fluorescence enhancement of fluorophores can also be adjusted by the distance between fluorophores and noble metal nanoparticles. The appropriate distance between nanoparticles and luminescent molecules is also the key to regulate fluorescence. When the distance is too close, the nonradiative attenuation is dominant, causing fluorescence quenching. When the distance is too far, the nanoparticles and fluorophores hardly interact with each other and have little effect on fluorescence. Only when the distance is appropriate, the fluorescence enhancement phenomenon will occur [21–29]. For example, H. He et al. prepared a molecularly imprinted polymer (MIP), which can obtain excellent riboflavin molecular fluorescence by regulating the distance between riboflavin and nanoparticles, and was used for the specific detection of riboflavin content in human urine [17]. In addition, the detection platform is also widely used in the field of riboflavin detection based on surface-enhanced raman spectroscopy (SERS), for example, A.I. Radu et al. reported the results of simultaneous detection of two B vitamins (riboflavin, vitamin B2 and cyanocobalamin, vitamin B12) by SERS, and the limit of detection (LOD) can be as low as 0.1 µM [30].
In this work, we designed a simple and efficient plasmonic platform for sensitive fluorescence detection of riboflavin based on LSPR effect. The platform is composed by drop-casted Ag NPs and spin-coated PMMA as spacer layer. The platform provided the strongest fluorescence enhancement of 14-fold for the directly detectable lowest concentration of 13.25 µM. Ag NPs were selected mainly because the well spectral overlap of LSPR band with both the excitation and emission band of riboflavin. Even after the PMMA layer is coated on the Ag NPs surface, the resonance band of the Ag NPs is slightly shifted due to the change of the refractive index around the Ag NPs. However, this slight shift will not have a great effect on the luminescence regulation due to the wide emission band of riboflavin [29], and it still has a good spectral overlap with the excitation and emission spectra of riboflavin. The results observed that the PMMA not only adjust the distance between riboflavin and Ag NPs, it also can improve the adhesion of the platform surface which induced that the fluorescence performance of Ri@PVA is further enhanced. In addition, PVA is selected because it is not only easy to process and has high transmittance, but also has good structural stability, biodegradable, biocompatibility, and film formation. Therefore, riboflavin was doped in PVA to prepare nanocomposite films [31–34]. We also simulated the distribution of local field of Ag NPs in the presence of PMMA layer by using FDTD simulation, the results are consistent with the experimental results. Finally, the platform is used for fluorescence detection of Ri@PVA in a wide range. And for the lower concentration, the samples can be detected by two ways of plasmon enhanced fluorescence and fluorescence spectra shift respectively. The combination of the two ways is novel for the fluorescence detection of riboflavin, and the platform can also be applied to the detection of other fluorophores and has a good application prospect.
2. Materials and methods
2.1 Materials
Silver nitrate (AgNO3, 99.80%), sodium borohydride (NaBH4, 98%) and hexadecyltrimethylammonium bromide (CTAB, 98%) were purchased from hwrk chemical. Trisodium citrate dihydrate (C6H5Na3O7·2H2O) was purchased from tianjin zhiyuan reagent. Ascorbic acid (AA, 99%) was purchased from inno-chem. Sodium hydroxide (NaOH, ≥96%), trichlormethane (CHCl3, ≥98%) and absolute ethanol (CH3CH2OH) were purchased from yuandong-chem. Polyvinyl alcohol (PVA, ≥99%) was purchased from kermel. Polymethylmethacrylate (PMMA) was purchased from acros organics. Riboflavin (C17H20N4O6, ≥98%) was purchased from sigma-aldrich. All materials were not further purified prior to use.
2.2 Synthesis of Ag nanoparticles
Ag nanoparticles (Ag NPs) were prepared by seed growth method. The seed solution was prepared by adding 0.6 ml of ice sodium borohydride solution (0.01 M) into a mixed solution of 0.5 ml of silver nitrate solution (0.01 M) and 19.5 ml of trisodium citrate dihydrate solution (0.5 mM). After vigorous stirring for 2 min, the color of seed solution changed from yellow to dark green. The growth solution was prepared by mixing 50 ml of CTAB solution (0.08 M) with 1.25 ml of silver nitrate solution (0.01 M) and standing at room temperature for 15 min. Then, 2.5 ml of AA solution (0.1 M) was added to the growth solution and stirred until colorless, then 1 ml of seed solution was added and react for 30 s, 0.5 ml of sodium hydroxide solution (1 M) was added for full reaction for 5 min, the solution changed from colorless to dark green. The prepared product was centrifuged at 6500 rpm for 20 min, the supernatant was removed, and the nanoparticles were dispersed in ultrapure water to obtain Ag NPs colloidal solution.
2.3 Synthesis of enhanced fluorescence platform
The silicon wafer of 1 cm × 2 cm was cleaned by multiple steps. Firstly, the silicon wafer was washed under the ultrapure water, then immersed in ultrapure water and ethanol for ultrasonic cleaning, and finally dried with nitrogen. PMMA solution was prepared by dissolving PMMA in chloroform at different mass ratios of 1:100, 1:200, 1:300, 1:500 and 1:600, corresponding to spacer layers with thickness of about 87 nm, 53 nm, 22 nm, 7 nm and 3 nm, respectively. 5 ml of PVA solution with a concentration of 2% and 5 ml of riboflavin solution with concentration of 0.54 µM, 5.32 µM, 13.25 µM, 26.5 µM, 53 µM, 80 µM, 106 µM, 160 µM were fully stirred for 1 h to prepare eight Ri@PVA sample solutions with riboflavin concentration of 0.27 µM, 2.66 µM, 6.63 µM, 13.25 µM, 26.5 µM, 40 µM, 53 µM, 80 µM respectively.
Figure 1 shows the preparation progress of the platform. Firstly, the Ag NPs substrate was obtained by drop-casting, 10 µl of Ag NPs colloidal solution was dropped onto the silicon wafer and evaporated water in a drying oven at 30 °C. Secondly, the spacer layer between the luminescent layer and Ag NPs was prepared by spin coating PMMA solution at 3000 rpm for 60 s, and dried on a 100 °C heating table for 30 min. Finally, the luminescent layer was prepared by spin coating Ri@PVA solution at 3000 rpm for 60 s.
2.4 Finite-difference time domain simulations
The electromagnetic field intensity and its distribution was simulated by FDTD method. The electric field distribution of nanoparticles in the presence of PMMA layer was simulated. In the simulation, the nanoparticles were selected according to the experimental data. The thickness and refractive index of PMMA spacer were selected as 7 nm and 1.49, respectively. The mesh order was set as 0.5 nm. The total field-scattered field source of light in the wavelength range 350 ∼ 550 nm was used as the incident light.
2.5 Characterization
The film thickness was measured by ellipsometer (J.A. wolam). The surface morphology of the sample was characterized by atomic force microscope (AFM). The morphology of nanoparticles was observed by transmission electron microscope (TEM). The resonance characteristics of nanoparticles were tested by U-3310 UV-visible spectrophotometer produced by the high tech company Hitachi . The fluorescence characteristics of the samples were recorded by FLS920 fluorescence spectrometer produced by an Edinburgh company in the UK.
3. Results and discussion
The molecular structure of riboflavin is shown in Fig. 2(a). The molecule has the characteristics of rigid plane structure due to the conjugated double bonds, which induces the natural fluorescence characteristics of riboflavin [5]. Figure 2(b) shows the excitation (blue line) and emission (red line) spectrum of riboflavin. As shown, the riboflavin has two excitation bands which peaks located at 370 nm and 445 nm respectively. Under excitation of 445 nm, the riboflavin has a green emission band located at 530 nm.
Fig. 2. (a) Molecular structure diagram of riboflavin. (b) Excitation spectrum of riboflavin (blue line), emission spectrum of riboflavin (red line) under the excitation at 445 nm.
To characterize the morphologies of Ag NPs, TEM measurement is carried out and the TEM image is show in Fig. 3(a). The Ag NPs are mainly composed of a small amount of rod nanoparticles and a large number of spherical nanoparticles. The size distribution (length and width) of rod nanoparticles is shown in Fig. 3(b)–(c), the average length of rod nanoparticles is 61.97 ± 12.05 nm and the average diameter is 21.57 ± 3.43 nm. The size distribution of spherical nanoparticles is shown in Fig. 3(d), and the average diameter is 31.08 ± 5.66 nm. Figure 3(e) shows the plasmon absorption bands of Ag NPs, which has two bands which peaks located at 535 nm, 424 nm respectively. The resonance band located at 535 nm represents the longitudinal LSPR of rod nanoparticles. The resonance band located at 424 nm represents the LSPR of spherical nanoparticles and the transverse LSPR of rod nanoparticles. The weak shoulder absorption located at 355 nm represents the LSPR of other small Ag NPs [35]. It is well known that fluorescence enhancement largely depends on the degree of spectral overlap between the excitation or emission band of emitters and the LSPR band of nanoparticles [24,36,37]. The two plasmon absorption bands of Ag NPs overlap well with the excitation and emission spectra of riboflavin, which can have a beneficial effect on the fluorescence enhancement of riboflavin.
Fig. 3. (a) TEM image of Ag NPs. (b) Length distribution statistics of rod nanoparticles. (c) The diameter distribution statistics of rod nanoparticles. (d) Size distribution statistics of spherical nanoparticles. (e) Absorption spectra of Ag NPs. (f) AFM image of the Ag NPs distribution near the ring. (g) AFM image of the Ag NPs distribution inside the ring after Ag NPs deposition. The inset is the position selected during measurement.
In order to analyze the Ag NPs distribution after drop-casting, the AFM image of the Ag NPs distribution are shown in Fig. 3(f)–(g), it can be observed that the distribution of Ag NPs is non-uniform, the Ag NPs solution leaves a circular ring with the evaporation, and the inset shows the position selected during measurement. This can be attributed to the coffee ring effect in the deposition process. The deposited nanoparticles are distributed in pattern of a coffee ring, that is, the droplets flowed from the central region to the edge during evaporation, the annular region formed after drying makes the Ag NPs relatively dense at the deposition edge [38,39]. Figure 3(f) shows the AFM image of the Ag NPs distribution near the ring. As shown, the Ag NPs distribution is relatively dense near the ring. Figure 3 (g) shows an AFM image inside the ring and relatively away from the ring. Compared with the nanoparticles near the ring, the Ag NPs inside the ring is less and the distribution is relatively sparse. In the experiments, in order to obtain the maximizing fluorescence intensity, the size and position of the light spot was carefully adjusted and the maximized fluorescence spectra were obtained when the light spot completely irradiated on the range with the densely arranged nanoparticles. The enhancement of fluorescence can be set as the average enhancement due to the relatively large spot size of the excited light.
To demonstrate the influence of the platform on the fluorescence, the fluorescence spectra are demonstrated based on platform of Ag NPs/PMMA with different thicknesses of PMMA space layer. And we also demonstrate the fluorescence based on the pure PMMA layer with different thickness as substrate to further determine the effect of the PMMA layer on the fluorescence, the results are shown in Fig. 4. As shown in Fig. 4(a), compared with pure Ri@PVA film, the enhanced fluorescence is obtained based on different thicknesses of pure PMMA substrates. The fluorescence intensity first increases with increasing of the thickness of the PMMA layer, reaches the maximum at 53 nm thick, and then decreases with the further increasing of the thickness. When the Ag NPs are introduced, as shown in Fig. 4(b), the fluorescence intensity increases first and then decreases with the increase of PMMA layer thickness due to the synergistic effect of Ag NPs and PMMA layer.
Fig. 4. (a) The fluorescence spectra of Ri@PVA based on pure PMMA substrates with different thicknesses. (b) The fluorescence spectra of Ri@PVA based on Ag NPs/PMMA platforms with different thickness of PMMA spacer. (c) Enhancement factor of pure PMMA substrate and Ag NPs/PMMA substrate with different thicknesses of PMMA layer (0 nm, 3 nm, 7 nm, 22 nm, 53 nm, 87 nm).
To analyze the effect of Ag NPs/PMMA substrate with different thickness of PMMA layer on the fluorescence enhancement, the enhancement factor was calculated using EF = F1/F0 by integrating the fluorescence intensity, where F1 and F0 are the fluorescence intensities of the samples with and without substrate, respectively [40]. The dependence of the fluorescence enhancement factor of Ri@PVA with pure PMMA and Ag NPs/PMMA substrates on the thickness of the PMMA layer is shown in Fig. 4(c). For pure PMMA substrates, the fluorescence enhancement factor shows a trend of first increasing and then gradually decreasing with the increase of the thickness of the PMMA substrate. When the thickness is 3 nm, 7 nm, and 22 nm, the fluorescence is enhanced by about ∼2.77-fold, ∼3.23-fold, and ∼10.01-fold respectively, reached a maximum of ∼18.17-fold when the thickness is increased to 53 nm, and then decreased to ∼5.67-fold when the thickness is further increased to 87 nm. For the platform Ag NPs/PMMA, the pure Ag NPs substrate without the PMMA layer enhances the fluorescence of Ri@PVA by about ∼2.24-fold. When PMMA with different thicknesses is introduced as a spacer layer, the fluorescence intensity of Ri@PVA is further enhanced. When the thickness is 3 nm and 7 nm, the fluorescence enhancement factors are almost equal, which are enhanced by ∼6.05-fold and ∼6.03-fold, respectively. When the thickness is increased to 22 nm, the fluorescence is enhanced by ∼11.49-fold. Similar with the pure PMMA substrate, the fluorescence enhancement factor reaches a maximum of ∼17.66-fold when the thickness is 53 nm. Then, the fluorescence enhancement factor decreases, reaching ∼5.68-fold at 87 nm with the further increasing of thickness. Compared with the pure PMMA substrate, the fluorescence enhancement factor of the Ag NPs/PMMA substrate is first larger than that of the pure PMMA substrate, especially for the thinner thickness of PMMA, such as 3 nm and 7 nm, which attributed to the synergistic effect of Ag NPs and PMMA layers [40]. When the thickness of PMMA is further increased to 53 nm and 87 nm, the enhancement factor of Ag NPs/PMMA substrate almost equal to that based on the pure PMMA substrate. The results show that Ag NPs can realize the regulation of luminescence when the thinner PMMA layer is introduced due to the plasmon enhanced fluorescence. And when the thickness of PMMA is further increased, the plasmon enhanced fluorescence is invalid due to the large distance between Ag NPs and Ri@PVA layer.
In order to analyze the mechanism of the enhanced fluorescence phenomenon based on pure PMMA layer, we measured the AFM topographic of the bare silicon surface, and the AFM topographic of the 7 nm thick PMMA layer selected for enhanced fluorescence. The results are shown in Fig. 5. Figure 5(a) corresponds to the AFM topographic of the bare silicon surface with an average roughness of about 0.188 nm, and Fig. 5(b) corresponds to the AFM topographic of the PMMA layer surface with an average roughness of about 0.312 nm. It is demonstrated that the average roughness of the surface of PMMA layer is greater than that of bare silicon. The height profile is demonstrated according to the AFM topography to evaluate the fluctuation of the film surface. Figure 5(c)-(d) shows height profile of the surface of bare silicon and PMMA layer respectively. The average height of PMMA layer surface is significantly greater than that of bare silicon surface. As we all know, fluorophores can produce stronger fluorescence on rough nanostructures, which undoubtedly improves the sensing performance of the fluorescence detection platform [41–43]. Therefore, we attributed the enhancement of the fluorescence caused by pure PMMA layer to the increase of surface adhesion of PMMA layer.
Fig. 5. (a) AFM topographic of bare silicon surface. and (b) AFM topographic of PMMA layer surface. (c) Height profile of bare silicon wafer. (d) Height profile of PMMA layer surface.
To further demonstrate the effect of the PMMA layer on the fluorescence, Fig. 6 shows the fluorescence spectra of Ri@PVA with different concentrations on a pure 7 nm PMMA layer, the corresponding concentrations of the samples are 80 µM, 53 µM, 40 µM and 26.5 µM. respectively. As shown in Fig. 6(a)–(d), the fluorescence intensity of Ri@PVA with different concentrations increases by 4.27-fold, 4.48-fold, 3.6-fold and 4-fold, respectively, due to the influence of PMMA layer. Compared with the samples on silicon wafer, PMMA substrate can realize the nearly 4-fold stable enhancement of the fluorescence at different concentrations. When the concentration of riboflavin continues to decrease, the fluorescence enhancement of nearly 4-fold can still be observed. The results further validate that the fluorescence enhancement of Ri@PVA layer on the surface of PMMA is due to the enhanced adhesion of PMMA surface compared with bare silicon wafer [43–46].
Fig. 6. Fluorescence spectra of Ri@PVA with different concentrations based on pure PMMA substrate, (a) 80 µM, (b) 53 µM, (c) 40 µM, (d) 26.5 µM.
Then we further investigated the effect of Ag NPs/PMMA substrate with the 7 nm PMMA as the space layer on the fluorescence of Ri@RVA. Figure 7(a)–(d) shows the fluorescence spectra of Ri@PVA with concentrations of 80 µM, 53 µM, 40 µM and 13.25 µM based on Ag NPs/PMMA substrate. When the maximum sample concentration is 80 µM, the fluorescence is enhanced nearly 5-fold under the effect of Ag NPs/PMMA substrate. When the sample concentration is reduced to 53 µM, the fluorescence is enhanced nearly 5.8-fold. When the sample concentration further decreases to 40 µM, the fluorescence increases to nearly 7.12-fold. For the lowest detectable concentration of Ri@PVA layer 13.25 µM, the enhancement factor reaches nearly 14-fold. The fluorescence is hardly detectable when the concentration is lower than 13.25 µM. Therefore, it’s hard to make sure the enhancement factor for the lower concentration Ri@PVA layer. Obviously, with the decreasing of concentrations, the enhancement factor of fluorescence of Ri@PVA based on Ag NPs/PMMA substrate gradually increases, this phenomenon is likely due to the significant contribution of Ag NPs [47], but this phenomenon will not appear when the PMMA layer is thick due to the invalidation of plasmon enhanced fluorescence at large distance. Therefore, this platform has great advantages for low concentration detection when the PMMA layer is thin. According to the results above, the fluorescence can realize nearly 4-fold enhancement based on the pure 7 nm PMMA layer. The platform of Ag NPs/PMMA can further improve the fluorescence performance of Ri@PVA layer with the decreasing of the concentrations of riboflavin. The fluorescence enhancement of Ri@PVA layer is attributed to the combined action of PMMA layer and Ag NPs. The PMMA layer not only enhances the fluorescence by enhancing adhesion of substrate, but also optimizes the plasmon enhanced fluorescence effect by serving as the spacer, thus further improving the detection of low concentration sample with the combination of the two characteristics [41]. This encourages the use of the platform for low concentration detection. So, the following we chose Ag NPs/PMMA with 7 nm PMMA layer as the substrate to detect the fluorescence of riboflavin.
Fig. 7. Fluorescence spectra of Ri@PVA with different concentrations based on Ag NPs/PMMA substrate, (a) 80 µM, (b) 53 µM, (c) 40 µM, (d) 13.25 µM.
To further verify the effect of Ag NPs/PMMA, we simulated the local electric field distribution of the platform Ag NPs/PMMA with the 7 nm PMMA as the space layer by using FDTD method. Figure 8(a)–(b) shows the local electric field distribution of rod nanoparticles/PMMA platform at 445 nm and 530 nm respectively. The local electric field intensity at 530 nm is greater than that at 445 nm, indicating that the rod nanoparticles can regulate the emission process better than the excitation process. Figure 8(c)–(d) shows the local electric field distribution of spherical nanoparticles/PMMA platform at 445 nm and 530 nm respectively. The local electric field of the platform at 445 nm is stronger than that at 530 nm, indicating that the spherical nanoparticles can regulate the excitation process better than the emission process. However, compared with the more stable regulation effect of spherical nanoparticles on excitation process and emission process, rod nanoparticles have better regulation effect. According to the simulation results, it is proved that the plasmonic enhancement is achieved by spectral matching [24]. When the rod nanoparticles and spherical nanoparticles are introduced into the PMMA layer, the surface of the platform has a great local electromagnetic field strength, which induced the enhancement of the electromagnetic field on the Ag NPs/PMMA platform. These results are consistent with the experimental results, reflecting the great potential in the field of riboflavin molecular detection.
Fig. 8. The electromagnetic field intensity distribution in the x-z plane of the platform at (a) 445 nm (rod nanoparticles), (b) 530 nm (rod nanoparticles), (c) 445 nm (spherical nanoparticles) and (d) 530 nm (spherical nanoparticles).
In order to investigate the performance of the prepared simple platform Ag NPs/PMMA for riboflavin detection, the detection platform Ag NPs/PMMA with 7 nm thick PMMA space layer is used for the detection of riboflavin with different concentrations. As shown in Fig. 9(a), the fluorescence spectra of samples with eight different concentrations were recorded based on the platform in the range from 0.27 µM to 80 µM. Obviously, the fluorescence intensity of the sample gradually decreases with the decreasing of concentration. The fluorescence spectra of samples with different concentrations were linearly calculated, as shown in Fig. 9(b). In the range of 0.27 µM to 80 µM, the fluorescence intensity increases with the increase of concentration and maintains a good linear relationship (R2= 0.984).
Fig. 9. (a) Fluorescence spectra of Ri@PVA with different concentrations base on Ag NPs/PMMA structure. (b) Based on Ag NPs/PMMA substrate, relationship of the fluorescence intensity and Ri@PVA concentration ranging from 0.27 µM to 80 µM. (c) Relation curve between spectral shift and Ri@PVA log concentration. The inset shows the normalized fluorescence spectra of Ag NPs and different concentrations of Ri@PVA.
In addition to the change in intensity, as shown in Fig. 9(a), the peak position of the fluorescence spectrum gradually blue shifts at the lower concentration. When the concentration decreased from 80 µM to 40 µM, the peak position is unchanged. The peak position shows a blue shift from 522 nm to 504 nm with a further decrease in the concentration of Ri@PVA from 26.5 µM to 0.27 µM. This is due to the scattering of nanoparticles. When nanoparticles are irradiated by incident light, the scattering spectrum would appear, especially when nanoparticles gather, the scattering would become more intense [48]. For the higher concentration of Ri@PVA, the fluorescence intensity is greater, the scattering of Ag NPs cannot be demonstrated. When the concentration of Ri@PVA is very low, the scattering of Ag NPs can be demonstrated. The scattering of Ag NPs makes the spectral blue shift for the Ri@PVA samples with low concentration [48], but has little effect on high concentration samples.
In order to further investigate the variation trend of blue shift with concentration, the curve between blue shift and log concentration at low concentration is calculated, as shown in Fig. 9(c). Obviously, there is a good linear relationship (R2= 0.998) between log concentration and the degree of blue shift at low concentration. When the concentration of Ri@PVA decreases to 26.5 µM, the peak began blue shifts and it is shifted by about ∼3 nm. For the concentrations of 13.25 µM, 6.63 µM, 2.66 µM and 0.27 µM, the peak blue shifts by about ∼5 nm, ∼8 nm, ∼10 nm and ∼18 nm respectively. To further analyze the effect of Ag NPs on low concentration samples, the spectrum of Ag NPs under 445 nm excitation was recorded. The inset in Fig. 9(c) shows the comparison between the fluorescence spectra of low concentration samples and the scattering spectrum of Ag NPs after normalization. As shown, the peak is blue shifted from 522 nm to 504 nm with the decreasing of the concentration. For the Ri@PVA with lower concentration based on Ag NPs/PMMA substrate, it is obvious that there is a difference in the spectral shape compared with the scattering spectrum of Ag NPs. The peak is shifted and the spectrum is broad at the long waveband. The results show that low concentration samples can still be detected by the spectral shape and the blue shift in this case, in addition to the detection based on the fluorescence intensity enhancement of this platform. The detection based on the shift of spectrum to detect samples also has been applicated in many monitoring platforms [49–51].
4. Conclusion
In this study, we introduced Ag NPs as the substrate to significantly modulate the fluorescence of riboflavin by adjusting the thickness of PMMA, and the platform was successfully used in the sensitive detection of riboflavin. The results show that the two plasmon absorption bands of Ag NPs overlap well with the excitation and emission spectra of riboflavin respectively, which can realize the well regulation of fluorescence. The PMMA spacer layer can not only regulate the distance between Riboflavin molecules and Ag NPs, but also improve the adhesion of the detection platform surface to a certain extent, and the detection performance is further improved. When the 7 nm PMMA spacer is selected, the enhancement factor shows a trend of increasing with the decrease of the concentration of riboflavin. In addition to the detection based on fluorescence intensity, the detection of riboflavin molecules can also be realized by the peak shift and broadening of the fluorescence peak due to the scattering of the Ag NPs at low concentration, the lowest detectable concentration based on the platform is as low as 0.27 µM. Our findings verify the feasibility of the platform. The combination of plasmon enhanced fluorescence and shift of the fluorescence spectra is novel for the fluorescence detection. This simple fluorescence detection platform would have broad application prospects in fluorescence detection and sensing.
Funding
Natural Science Foundation of Shandong Province (ZR2019MF068); Science and Technology Plan of Youth Innovation Team for Universities of Shandong Province (2019KJJ019); Introduction and Cultivation Plan of Youth Innovation Talents for Universities of Shandong Province; The Special Construction Project Fund of Shandong Province Taishan Scholars.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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