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Abstract
An all-optical fiber multi-layer surface plasmon resonance (SPR) biosensor based on a sandwich structure of polydopamine-MoSe2@Au nanoparticles-polydopamine (PDA-MoSe2@AuNPs-PDA) was designed for the detection of specific immunoreactions. By optimizing the multi-layer structure and the ratio of MoSe2: AuNPs, a sensitivity of 5117.59 nm/RIU has been obtained, which is more than double that of the only Au-filmed optical fiber SPR sensor. A large surface area was produced by integrating the MoSe2 primitive unit cell and the AuNPs into a hybrid plasmonic nanostructure of MoSe2@AuNPs, leading to optical fiber SPR signal amplification. The nanostructure of MoSe2@AuNPs was surrounded by the PDA layer to guarantee the efficient immobilization of the protein molecules on the optical fiber by strong covalent bond. This biosensor achieved a detection limit of 54.05 ng/mL for detecting the goat-anti-rabbit IgG, which demonstrated enhancements of 12.1%, 23.3% and 184.6% in comparison with three reported SPR biosensors decorated with PDA-AuNPs-PDA, PDA and Cysteamine-MoSe2@AuNPs-Cysteamine nanostructure, respectively. This biosensor achieved favorable selectivity and outstanding sensitivity compared with the reported SPR immuno-sensors, which will provide a miniaturized, rapid-response and label-free optical fiber bio-sensing platform for clinical diagnosis in the future.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
As an effective technique, surface plasmon resonance (SPR) has been applied for specific immunoassay and target bio-molecules detection. At present, SPR biosensors mainly have two types of structures: prism and optical fiber forms [1]. The optical fiber SPR biosensors have the advantages of miniaturization, low reagent consumption and mechanical flexibility [2], and also have been employed in compelling applications, such as the catheter-embedded biosensor using fiber grating [3] and the concanavalin-A functionalized optical fiber sensor for D-glucose detection [4]. The effective methods for enhancing the sensitivity of the optical fiber SPR biosensor [5] mainly included the use of multi-layer and nanoparticles structures.
As an important brunch of two-dimensional (2D) transition metal dichalcogenides (TMDCs), molybdenum selenide (MoSe2) has recently attracted strong interests [6]. The WS2 [7], MoS2 [8] and even MoSe2 [9] materials were used to enhance the sensitivity of the sensor. Compared with other TMDCs materials, MoSe2 has a larger layer spacing with a smaller energy band gap, leading to better sensing performance and higher optical absorption efficiency [10]. The simple gold film SPR sensor with low absorption of bio-molecules posed limitations on the sensitivity enhancement. As a good solution, the MoSe2 dielectric layer in the multi-layer structure can be used to enlarge the loading surface area [11]. Therefore, the reported dielectric layer of the multi-layer structure based SPR biosensor can be divided into the 2D-material layer and the oxide layer, including Cu/Ag/Au-indium tin oxide (ITO) multi-layer [12] with enhancement on the sensitivity and the TiO2/Graphene/MoSe2/WS2 multi-layer [13] with increment in the electric intensity. However, the introduction of the dielectric layer only increased the depth of evanescent field to a certain extent for enhancing sensitivity. To further amplify the SPR signal, the multi-layer structure with high-sensitivity nanoparticles should be introduced to enhance the coupling effect.
The local surface plasmas existing in AuNPs or discontinuous nanostructures can be excited by the incident light to produce the local surface plasmon resonance (LSPR) phenomenon [14]. The combination between the analyte and the nanoparticles will cause the changes in the thickness and the refractive index (RI) of the bio-molecular layer, which can be detected by the LSPR method based on the high-sensitivity nanostructures [15]. Therefore, materials coated with the AuNPs structure such as Graphene oxide/AuNPs for taking photothermal therapy [16] and detecting IgG [17] had been reported to enhance the coupling effect by the LSPR technology. Black phosphorus (BP) nanosheets was also coated with AuNPs to simultaneously enhance the hyperthermia in cancer therapy [18]. More recently, a MoS2 nano-flowers coupled with AuNPs were used to modify the gold plasmonic interface [19], which made a comprehensive use of the localized SPR and propagating SPR, as well as the regularity of the nano-flowered MoS2. Instead of directly adding the AuNPs on the 2D materials as mentioned above, integrating MoSe2 and Au nanoparticles within the hybrid nanostructure of the MoSe2@AuNPs functionalized multi-layer optical fiber SPR probe will lead to SPR signal amplification, which will show a further improvement in the bio-sensing sensitivity compared to the bare probe without any decoration.
In this work, we proposed an all-optical fiber multi-layer SPR biosensor, with a sandwich structure of polydopamine-MoSe2@Au nanoparticles-polydopamine (PDA-MoSe2@AuNPs-PDA). The sensing materials of MoSe2 was employed as the dielectric layer in the multi-layer structure. The sandwich structure of PDA-MoSe2@AuNPs-PDA was composed of the hybrid nanostructure of MoSe2@AuNPs and the covalent binding of PDA layers. This biosensor was fabricated for the detection of goat-anti-rabbit IgG. Experiments were also carried out using different types of fabricated SPR biosensors. The sensitivity and selectivity of these biosensors were also evaluated.
2. Materials and methods
2.1 Materials, instruments and sensing platform
The plastic clad silica (PCS) multi-mode optical fiber with a core diameter of 600 μm and a numerical aperture of 0.37 was purchased from Scitlion Technology. The MoSe2 isopropanol nano-dispersions (0.5 mg/mL) was from HaoNie Technology and the HAuCl4·4H2O (0.01%) was purchased from Beijing Chemical Works. The cysteamine, dopamine hydrochloride, Tris buffer (100 mM, PH 8.9), Tween 20 and the bovine serum albumin (BSA) were from Solarbio Science & Technology. rabbit IgG and goat-anti-rabbit IgG etc. were all procured from BOSTER Biological Technology. All other chemicals were of analytical reagent grade.
An all-optical fiber multi-layer SPR biosensor with PDA-MoSe2@AuNPs-PDA sandwich structure required following components: a tungsten halogen light source, an optical fiber SPR biosensor with a glass tube, a spectrophotometer (HR 4000 from Ocean Optics Co.) with a range of 200-1100 nm and a resolution of 20 pm, a thermostat steam bath vibrator and a computer. The experimental setup for the SPR bio-sensing system and the physical picture of sensor were illustrated in Fig. 1(A). The SPR phenomenon relies on the fact that evanescent wave generated by the total internal reflection of the light wave propagating along the fiber core to excite the surface plasmon wave at the interface between the metal layer and the external layer. Our model of SPR sensing platform contained the metal layer, the MoSe2 dielectric layer, the PDA film and the MoSe2@AuNPs nano-composite. The schematic diagram of the designed SPR sensor was shown in Fig. 1(B). The sandwich structure of PDA-MoSe2@AuNPs-PDA was composed of the hybrid nanostructure of MoSe2@AuNPs and the covalent binding of PDA layers.
Fig. 1. (A) The experimental setup of optical fiber SPR sensing system and the physical picture of sensor. (B) The schematic diagram of the optical fiber SPR sensing platform.
2.2 Construction of the SPR sensing platform with metal-MoSe2 multi-layer
Our previous work [20] illustrated the SPR sensing platform developed using a multi-mode optical fiber with a core diameter of 600 μm. We removed its coating and cladding for a section of 10 mm by a sharp blade. The compact layer of 5 nm Cr film and 50 nm Au film were successively deposited on the sensing region using the optical fiber rotating magnetron sputtering coater. Cr film could not excite SPR phenomenon, but as a very stable metal material, it could be used as a protective layer to coat on the fiber to prevent the gold film from falling off from the optical fiber. Thick Cr layer would affect the couple effect of SPR and reduced the transmission of optical signal, so a thin Cr film of 5 nm was adopted. The thickness of layer was calibrated by the crystal oscillator [21] and the parameters of the coater included the pressure of sputtering environment (2 × 104 Pa), the sputtering angle (80°) and the intensity of current (100 mA). A 100 μL MoSe2 isopropanol-based dispersion (0.5 mg/mL) dripped down and assembled on the metal layer via the electrostatic interaction. After that, a procedure of annealing at 50°C for 5 hours was required to maintain the adhesion strength between the MoSe2 and the gold film.
2.3 Synthesis of the MoSe2@AuNPs nano-composite
The MoSe2@AuNPs nano-composite including MoSe2 nanosheets and the gold nanoparticles was chemically synthesized by the hydrothermal method [22]. 1 mL AuNPs solution was immediately mixed with MoSe2 isopropanol-based dispersion (4 mL, 0.05 mg/mL) under vigorous stirring. The sodium citrate (1%) was used as the reductant accompanied by Tween 20 (0.05%) as the regulators, to ensure the generation of AuNPs on the surface of MoSe2 nanosheets in the reduction process. The above mixture was boiled for ∼5 minutes using a constant-temperature heating table. The product of MoSe2@AuNPs nano-composite was then cooled down to the room temperature and stored at 4°C for further use.
2.4 Functionalization of the PDA-based SPR biosensor
In order to apply our designed sensing platform for bio-sensing, an effective functional method was adopted using PDA film based on the sandwich structure. The functionalization procedure of the optical fiber SPR biosensor for IgG immunoassay included three steps. Firstly, the MoSe2-metal layer coated probe was immersed in the freshly prepared dopamine solution (2 mg/mL) in Tris-HCl buffer (10 mM, pH 8.5) and was shaken continually in a shaker for 15 minutes. Dopamine could be deposited on the MoSe2 layer and the discontinuous nanostructures of various materials using direct auto-oxidation and self-polymerization as the PDA layer in weak alkaline media [23]. No additional cross-linkers were required to improve the stability. Secondly, the PDA filmed optical fiber was immersed in the MoSe2@AuNPs nano-composite for 6 hours to produce the large functional surface. Finally, the sandwich structure of PDA-MoSe2@AuNPs-PDA was completed by coating the same PDA film. The superficial catechol groups played an important role for the reduction of Au3+ and the stabilization of AuNPs [24]. Moreover, PDA equipped with catechol groups was oxidized to quinine and interacted with the amino groups in the 1 mg/mL rabbit IgG through chemical bonds. The functionalized PDA-MoSe2@AuNPs-PDA-rabbit IgG optical fiber SPR biosensor was successfully produced for detecting the goat-anti-rabbit IgG. The whole schematic diagram of the immune modification process was shown in Fig. 2.
3. Results and discussion
3.1 Characterization of the sensor
The images of the gold filmed and MoSe2 coated optical fiber from the Field-emission scanning electron microscopy (FESEM) with a magnification of 50000x were shown in Fig. 3(A) and Fig. 3(B). The observed nanosheets structure verified the successful coating of MoSe2 layer and led to the increase of the reaction surface area. It can be found in the Fig. 3(C) that the surface topography of AuNPs is uniform. Obviously, plenty of MoSe2@AuNPs nano-structure with a homogeneous and dense distribution on the optical fiber can be observed in Fig. 3(D). AuNPs are formed at the edges of MoSe2 nanosheets, due to the high density of energetic defects near the edge of MoSe2 nanosheets. The thickness of the hybrid nano-structure was shown in Fig. 3(E). The composition of Mo, Se and Au elements on MoSe2@AuNPs nano-composite was analyzed using the Energy Dispersive X-Ray (EDX), as shown in Fig. 3(F). The above results showed that each step in the fabrication of the PDA-MoSe2@AuNPs-PDA multi-layer optical fiber SPR biosensor was successfully achieved in accordance with our design.
Fig. 3. Figures of the multi-layer SPR biosensor with PDA-MoSe2@AuNPs-PDA. (A) SEM images of gold filmed and (B) MoSe2 layer coated optical fiber. (C) The sample of AuNPs with size of 40 nm. (D) The synthetic MoSe2@AuNPs nano-composite. (E) The thickness of nano-structure on the optical fiber. (F) The energy dispersive X-ray (EDX) patterns of MoSe2@AuNPs.
3.2 Numerical simulations of the sensing platform
The optimization of the sensing performance and the resonant wavelength of AuNPs highly depended on the size, the shape, and the refractive indices of the surrounding media, which formed the bases of the LSPR bio-sensing [25]. We simulated the extinction spectra with different sizes of AuNPs by Mie theory and the multi-layer transfer matrix method [26]. The spectral profiles were exhibited in Fig. 4(A). The extinction efficiency represented the sum of the absorption and the scattering efficiencies, its maximum corresponded to the strongest coupling effect of LSPR, observed at the location of 548.67 nm. The peak value significantly increased and the corresponding resonant wavelength showed a red shift phenomenon with the size of AuNPs increasing from 10 nm to 50 nm. In our work, AuNPs with a size of 40 nm exhibited the superiority for designing the hybrid nanostructure. The size of AuNPs larger than 50 nm degraded the performance of the mixture colloids and produced the precipitate.
Fig. 4. Simulation results of (A) the extinction spectra with sizes of AuNPs increasing from 10 nm to 50 nm. (B) The optimization results of MoSe2 layer and the different ration of MoSe2: AuNPs.
An effective dielectric model is also employed to simulate the MoSe2@AuNPs nano-composite, which can be expressed as [27]: ${n_{eff}} = {f_{MoS{e_2}}} \times {n_{MoS{e_2}}} + {f_{AuNPs}} \times {n_{AuNPs}}$, where ${n_{eff}}$, ${n_{MoS{e_2}}}$,${n_{AuNPs}}$ represent the effective RIs of the MoSe2@AuNPs nano-composite, the MoSe2 and the AuNPs, respectively. ${f_{MoS{e_2}}}$, ${f_{AuNPs}}$ are the volume fractions of the MoSe2 and the AuNPs, which can be obtained from the EDX analysis. After optimizing the thickness of MoSe2 layer and the volume fraction ratio of MoSe2: AuNPs, calculating the effective dielectric function of each membrane, numerical simulations of transmission spectra were also carried out by the multi-layer media matrix method. The detection performance of sensor can be evaluated via the RI sensitivity defined as $S = {{\Delta \lambda } / {\Delta n}}$, where $\Delta n$ represents the interval of RI and $\Delta \lambda$ is the corresponding wavelength shift.
It can be seen from the simulation results in Fig. 4(B) that the sensitivity climbs first and then decreases with the thickness of MoSe2 layer from 0 to 100 nm, the doping ration of MoSe2 : AuNPs with 20% : 80%, 40% : 60%, 50% : 50%, 60% : 40%, 80% : 20%. The MoSe2 of 50 nm with the ratio of MoSe2: AuNPs of 4:1 demonstrated good performance. The existence of MoSe2 with a large real part of dielectric function absorbs strong energy from the incident light and dramatically enhances the sensitivity. However, the over-increase of the MoSe2 layer reduces the overlap between the analyte layer and the evanescent field, the SPR phenomenon becomes weak and the sensitivity decreases. Therefore, a stronger SPR coupling effect and signal cannot be obtained with the increasing of MoSe2 materials.
As seen from the Fig. 5, the distinct red shift phenomenon of the resonant wavelength was accompanied by a broadening and shallowing effect of the transmission spectrum. Moreover, the wavelength response sensitivity corresponding to the change of RI was improved by 79% after adding optimized multi-layer structure and MoSe2@AuNPs nano-composite. These were attributed to the change of the effective dielectric function with the wavelength and evanescent electric field distribution of multi-layer structure [28], which were in accordance with the experiment in our previous work.
3.3 Measurement of refractive index sensitivity
The only gold layer optical fiber and multi-layer structure with the use of MoSe2@AuNPs nanostructure optical fiber were successively immersed in the solution with an RI range of 1.333-1.358 (Equipped with Abbe Refractometer). The measurement spectra were plotted in Figs. 6(A) and 6(B). The resonant wavelength of the proposed sensor showed a red shift of 128.80 nm, which was two times higher than that of the gold filmed sensor (with a shift of 50.65 nm). Furthermore, an obvious broadening of spectrum was observed in the MoSe2@AuNPs nanostructure, which was in accordance with our simulation results in Fig. 5.
Fig. 6. Measurement transmission spectra of the solution with RIs in the range of 1.333-1.358 using (A) only gold layer sensor and (B) the multi-layer with PDA-MoSe2@AuNPs nano-composite sensor, respectively. (C) Linear fitting results and (D) the corresponding error of the optical fiber SPR sensors.
The figure of merit (FOM) defined as $FOM = {S / {FWHM}}$ is also employed to evaluate the performance of sensor. The FWHM was calculated by Matlab with the process of scanning spectrum data, extracting the baseline and achieving the half peak value. As the fitting results in Fig. 6(C) and the error in Fig. 6(D), our proposed sensor obtained a sensitivity of 5117.59 nm/RIU and a FOM value of 68.8, whose sensitivity was 152.7% higher than the control group. The FOM value of the sensor after adding MoSe2@AuNPs was 84.7% higher than the gold film sensor (FOM value of 37.24). It can be explained by the LSPR phenomenon enhanced by MoSe2@AuNPs nanostructure. Interestingly, the experimental results showed a better performance than the simulation results. PDA layer greatly enhances the coupling of energy in the experiments, while it cannot be simulated due to the limitations of the transfer matrix method in simulation. To verify the effect of PDA, we tested the refractive index experiment of PDA-Au layer SPR sensor and obtained a RI sensitivity of 3726.73 nm/RIU, which was 84% higher than the only gold film sensor. In such case, the wavelength is very sensitive to the change of RI in a small range around the AuNPs. Meanwhile, the MoSe2 with a large real part of dielectric function will absorb the strong energy from the incident light and can dramatically increase the effective RI to improve the sensitivity.
3.4 Specific binding of the PDA-MoSe2@AuNPs-PDA SPR biosensor
Figure 7(A) showed the experimental data from different fabrications of the MoSe2-metal layer based optical fiber SPR biosensors decorated with the PDA-MoSe2@AuNPs-PDA, the PDA, the PDA-AuNPs-PDA and the cysteamine-MoSe2@AuNPs-cysteamine (CS-MoSe2@AuNPs-CS) structure, respectively, for the detection of IgG immunoassay. They were all functionalized with rabbit IgG and exposed to goat-anti-rabbit IgG solutions (concentrations of 0.005, 0.01, 0.015, 0.02, 0.025, 0.05, 0.075, 0.1 mg/mL, respectively). A concept of limit of detection (LOD), defined as $LOD = {{{\delta \lambda } / S}_b}$, is applied to evaluate the sensing properties of the biosensor, where $\delta \lambda $ represents the resolution of the spectrophotometer (0.02 nm in this work), ${S_b}$ is the sensitivity of bio-sensing corresponding to the slope of fitting curve.
Fig. 7. (A) The binding curves of PDA-MoSe2@AuNPs-PDA SPR biosensor. (B) The linear relationship between the wavelength shift and the concentrations of IgG solution.
Four types of biosensors exhibited the best response to goat-anti-rabbit IgG with the concentration range of 5-25 μg/mL in the linear region. The linear fitting curves were shown in Fig. 7(B) with LOD results of 54.05, 60.61, 66.67 and 153.85 ng/mL, respectively. Our proposed PDA-MoSe2@AuNPs-PDA biosensor demonstrated enhancements of 12.1%, 23.3% and 184.6% in sensitivity compared to three other biosensors, respectively. The minimum shift of resonant wavelength was 8.39 nm at 5 μg/mL and the maximum was 20.51 nm at 100 μg/mL. These experiments demonstrated that the adulteration of the MoSe2 materials into AuNPs can further improve the performance of optical fiber SPR biosensor. The results can be explained from two expects: on the one hand, the large surface area and high loading capacity of MoSe2 layer are beneficial to carry a large amount of biomolecules and can thus improve the immobilization efficiency of SPR biosensor. On the other hand, the response of biosensor is improved by the electronic coupling between the localized plasmon of AuNPs and the surface plasmon wave associated with the MoSe2-metal layer. This will lead to the amplification of the SPR signals originating from the dielectric function changes of the substrate upon binding of the antigen and antibody.
As for the binding strategy, the abundant amino groups in Cysteamine (2 mM) can combine protein molecules by electrostatic adsorption and generate the self-assembled layer. But this type of physical absorption through Vander Waals force is reversible and the effect is far behind the covalent bond [29]. Therefore, the LOD of PDA-MoSe2@AuNPs-PDA SPR biosensor was 1.8 times lower than that of the cysteamine-immobilized biosensor. It should be ascribed to the PDA layer, since its rich catechol groups are easily oxidized into quinines [30], which can covalently bind protein molecules through the Michael addition and Schiff based reaction in the alkaline environment. Moreover, the smart sandwich structure can further increase the binding efficiency and can also guarantee the durability and the compactness of the multi-layer film. To provide a comprehensive assessment of the PDA-MoSe2@AuNPs-PDA optical fiber SPR biosensor, Table 1 listed the performance of other reported SPR biosensors for detecting IgG solution, reflecting the favorable sensing performance of our proposed biosensor.
Table 1. The LOD value comparison of our method with other reported SPR biosensors.
3.5 Selectivity and regeneration of the PDA-MoSe2@AuNPs-PDA SPR biosensor
To evaluate the selectivity of the PDA-MoSe2@AuNPs-PDA based optical fiber SPR biosensor, the test solution BSA and human IgG etc. with a concentration of 50 μg/mL were also applied. The shift of resonant wavelength was plotted in Fig. 8(A). The goat-anti-rabbit IgG exhibited a maximum shift of 16.96 nm and demonstrated the outstanding selectivity of our proposed biosensor. Most disease surveillance biomolecules such as carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) are essentially proteins, so our proposed SPR biosensor with the PDA-MoSe2@AuNPs-PDA structure provides a label free, real-time and miniaturized optical fiber sensing platform of disease diagnosis.
Fig. 8. (A) Wavelength shift measured for test solutions (BSA, human IgG, horse IgG, rabbit-anti-mouse IgG and goat-anti-rabbit IgG) of 50 μg/mL. (B) The wavelength shifts of IgG immobilized SPR biosensor with regeneration times from 0 to 3.
The IgG immobilized PDA-MoSe2@AuNPs-PDA SPR biosensor was regenerated by being immersed in the 10 mM NaOH solution for 10 min. The stability of immobilized biosensor was assessed by storing it at 4 °C in 10 mM PBS (pH 7.4) and using it to detect the 50 μg/mL goat-anti-rabbit IgG. The wavelength shift of PDA-MoSe2@AuNPs-PDA SPR biosensor was detected after 1-3 times of dissociation and regeneration. As seen from Fig. 8(B), there was tiny decrease in the specific binding signal and it kept good detection ability after regeneration. The wavelength shift was 14.5 nm at the first regeneration, which was 2.2 nm less than the original functionalized SPR biosensor. These results indicated the good regeneration ability and stability of our designed PDA-MoSe2@AuNPs-PDA SPR biosensor.
4. Conclusion
In summary, an immune-sensor based on a sandwich structure of PDA-MoSe2@AuNPs-PDA has been developed for detecting goat-anti-rabbit IgG. By optimizing the multi-layer MoSe2@AuNPs structure on the optical fiber, a double enhancement on the RI sensitivity has been achieved. A detection limit of 54.05 ng/mL was obtained, which was 1.8 times lower than that of conventional SPR biosensors. Our results illustrated that the uniform MoSe2@AuNPs nano-composite exhibited a significant enhancement effect on the SPR signals, and the PDA layer based sandwich structure can capture abundant protein molecules via persistent covalent bond. Therefore, our proposed biosensor provided a promising miniaturized platform for label-free detection and clinical diagnostics.
Funding
National Natural Science Foundation of China (61735011, 61775161, 61922061); Tianjin Science Fund for Distinguished Young Scholars (19JCJQJC61400); Ministry of Industry and Information Technology of the People's Republic of China (2013YQ030915).
Disclosures
The authors declare no conflicts of interest.
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