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Abstract
In this paper, a localized surface-plasmon resonance (LSPR) biosensor, which uses a U-shaped multi-mode fiber (U-MMF), is introduced and investigated. It is modified with a complex of three-dimensional (3D) gold nanoparticles and multilayer graphene as spacer: n*(Au/G)@U-MMF, where n denotes the layer number of gold nanoparticles. The gold nanoparticles were synthesized by reducing chloroauric acid. Graphene films were formed using a liquid/chemical method. The number of gold-nanoparticle layers was found to be critical for the performance of the sensor. Moreover, using the finite-difference time domain, 3D nanostructures, with a wide range of gold-nanoparticle layers, were explored. The sensor showed the sensitivity of 1251.44 nm/RIU, as well as high stability and repeatability; for the measurement-process of time- and concentration-dependent DNA hybridization kinetics with detection concentrations, ranging from 0.1nM to 100 nM, the sensor displayed excellent performance, which points towards a vast potential in the field of medical diagnostics.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Recently, localized surface plasmon resonance (LSPR) was proposed as one of the most feasible techniques to sense a wide range of physical, chemical, and biological parameters [1–4]. LSPR results from the resonant collective oscillation of the conduction electrons, which takes place in a subwavelength metallic nanostructure [5–7]. The frequently employed metals include gold and silver [8,9]. The LSPR exhibits very high sensitivity with respect to target detection for a highly sensitive localized electromagnetic field around the metal surface to the environmental refractive index (RI) [10].
Because optical-fibers display a range of advantages (e.g., long-distance sensing, low cost, lightweight, ease of use, as well as small size) [11–15], LSPR sensors, which are based on optic fibers have attracted increasing attention by researchers worldwide. The shape of the sensing probe allows the optical fiber LSPR sensors to be U-shaped [16], D-shaped [17], and Tapered [18], among other forms. Among the mentioned sensors, the U-shaped sensing probe exhibits various benefits (e.g., higher sensitivity, easier fabrication, and point-of-care approach) [8,19,20].
It has been previously reported that the performance of LSPR sensors can be controlled by the properties of the capping material, surface charge, and interparticle interaction, which are more efficient than the size of the particle [21]. Accordingly, it is important to investigate the capping material, the surface charge, and the interparticle interaction of the LSPR sensors. Hitherto, much effort was spent to boost the development of LSPR sensors. In 2013, by depositing graphene oxide over a monolayer of gold nanoparticles, chemically attached to a functionalized fused silica substrate, Michela Cittadini et al. prepared an LSPR gas sensor for selective detection of multiple gases [22]. In 2017, based on a graphene- and silver-nanoparticle hybrid structure, Jiang et al. proposed a novel U-bend optical fiber LSPR sensor, which has the capability to detect glucose and ethanol [23]. In 2019, using a graphene/ITO nanorod metamaterial hybrid structure, Yang et al. developed a U-bent optical fiber LSPR sensor that enables biomolecule detection [24]. In 2019, to detect a DNA-polymerase reaction, Johanna Roether et al. presented the application of nano-plasmonic LSPR technology that is coupled with microfluidics [25]. In 2019, Hang Song et al. developed a triangular silver nanoparticle U-bent fiber sensor with a sensitivity of 1116.8 nm/RIU [26].
Nevertheless, a considerable amount of limitations remains. For instance, sensors are easily affected by oxidation, which can likely lead to reduced stability and sensitivity. In addition, due to the sophisticated manufacturing processes, the sensor costs are high. The sensing effect primarily results from electromagnetic enhancement in the nanogap region between the metal nanoparticles, and the plasma oscillation is confined to a two-dimensional plane [5–7]. In contrast, bulk plasma resonance based on the three-dimensional structure has become an effective method to improve detection performance. In 2016, Kandammathe et al. developed a miniaturized plasmonic biosensor platform based on a hyperbolic metamaterial composed of three-dimensional multilayer metal [27]. In 2017, Zhen Li et al. utilized different layers of silver nanoparticles for surface-enhanced Raman spectroscopy analysis [28]. And in 2018, they combined multilayer silver nanoparticle structure with graphene oxide to achieve further performance improvements [29].
In the present study, an n*(Au/G)@U-MMF LSPR sensor is proposed, which uses a U-MMF, gold nanoparticles, and graphene. Firstly, graphene has a 2D honeycomb lattice of carbon with an sp2 structure and exhibits high biomolecular affinity and a large surface area (∼2630 m2 g−1), which facilitates the immobilization of molecules [30]. Secondly, graphene that is deposited on gold nanoparticles is capable of efficient charge transfer and enhancing the electric field strength at the nanointerface [31–35]. Third, graphene exhibits robust chemical inertness, which increases both the stability and the lifespan of the sensor. Moreover, graphene films act as spacers within the 3D structure. This facilitates vertical charge-density oscillations, converts the plasma oscillation mode from plane to 3D structure, and eventually produces a robust LSPR effect. In addition, using the 3D structure, a larger specific surface area can be created to accommodate more biomolecules, which maximizes the biological capacity of the structure. Accordingly, the hybrid structure is present to be a structure of hyperbolic super materials (HMMs) [36–38]. In this study, the LSPR spectrum of an n*(Au/G)@U-MMF(n = 1-5) sensor was investigated. The number of gold nanoparticle layers turns out to be critical for the performance of the sensor. 3*(Au/G)@U-MMF is the optimal structure, which shows strong anti-oxidation properties, a simple fabrication process, high sensitivity (1251.44 nm/RIU), as well as high stability, repeatability, and linearity. The proposed LSPR sensor was successfully used to measure time- and concentration-dependent DNA hybridization kinetics, with a detection limit as low as 0.1 nM. This indicates that a wide range of potential applications is possible, in areas like genetic screening and detection, disease biomarkers, transcriptional profiling, and single-nucleotides.
2. Materials and methods
2.1 Materials
Chloroauric Acid, Sodium hydroxide, Seignette salt, and D(+)-Glucose monohydrate were purchased from Sinopharm Chemical Reagent Co., Ltd(Shanghai, China). The 50% hydrazine hydrate solution (N2H4), Poly(allylamine hydrochloride) (PAH) were purchased from China Pharmaceutical Co., Ltd. The graphene-oxide dispersion (diameter: 50–150 nm), with a concentration of 0.5 g·L−1, was provided by Xianfeng Nano Technology Co., Ltd. The DNA, see Table 1, was purchased from Sangon Biotech Inc. (Shanghai, China). PBASE and N-dimethylformamide (DMF) were offered by Sigma-Aldrich (Shanghai, China). Phosphate-buffered saline (PBS, P5368-10PAK), at pH 7.4, was offered by Sigma-Aldrich (Shanghai, China). Probe aptamer solution, mis_DNA solution, and t_DNA solutions, with different concentrations, were prepared and diluted in PBS buffer solution for future use.
Table 1. The DNA used in this study.
2.2 Preparation of U-MMF
The synthesis process of n*(Au/G)@U-MMF is shown in Fig. 1. The figure suggests that a 30 cm long MMF (core/outer diameter = 62.5/125 µm) was taken to prepare the U-MMF. From the middle section, 2cm cladding was removed, using acetone and subsequent washing with deionized water. The treated fiber was first inserted into a capillary glass-tube (1 cm diameter) and then fixed on the iron stand. Subsequently, it was kept 1cm from the outer flame and heated. When all conditions were set, the inner diameter (1.5 mm) of the U-MMF remained constant, and the shape could be easily (and repeatedly) controlled.
2.3 Preparation of n*(Au/G)@U-MMF
Gold nanoparticles were synthesized by drawing upon the reducibility of glucose [39,40]. 2.5ml of 0.4% sodium hydroxide solution, 0.25ml of 0.25% Seignette salt solution and 0.45ml of 2% glucose solution were added to the gold chloride solution (24mM,2ml), respectively. While the sensitive area of the U-MMF was immersed in the reaction solution, gold nanoparticles were directly deposited on the surface of the U-shaped sensitive area. To synthesize consistent multiple gold-nanoparticle layers, based on the fact that the transmittance of U-MMF decreases with the increasing LSPR effect caused by the deposition of gold nanoparticles, We monitored the synthesis process in real-time based on transmission spectra. The deposition process was ended when the transmittance at the resonance peak reached 85%. Subsequently, the U-MMF was taken out and dried with nitrogen. Multilayer graphene was capped on the surface of the U-MMF by immersion in poly(allylamine hydrochloride) (PAH) solution (1 g·L−1) for 1h, and a subsequent reaction with both graphene-oxide dispersion (0.2 g·L−1; diameter: 50–150 nm) for 5h and hydrazine (N2H4, 50%) for 1h [41,42]. Then, the U-MMF was washed with deionized (DI) water and dried with nitrogen. By repeating the synthesis of the gold nanoparticle layer and graphene, the n*(Au/G)@U-MMF(n = 1-5) was obtained. In order to verify the repeatability of the sensors, the sensors of each structure are manufactured with a batch of 5.
2.4 DNA Detection
Firstly, the fabricated U-MMF was inserted into a 5 mL PBASE solution for 12 h, which, thereby, can act as a linker. The pyrene group of the PBASE solution could then undergo π–π stacking with graphene, and the succinimide portion could conjugate with probe aptamer modified by –NH2. Then the unmodified PBASE was removed by washing with DMF and deionized water three times. Subsequently, the probe aptamer with -NH2 was fixed on the U-MMF by inserting the U-MMF into the solution of 5 mL probe aptamer (1 µM) for 4 h. Then U-MMF was washed with PBS solution and deionized water respectively to remove the unreacted probe aptamer. Subsequently, the modified U-MMF could be used as an LSPR sensor for t_DNA detection based on DNA hybridization. The performance of the proposed sensors was evaluated with t_DNA solutions of various concentrations and mis_DNA solutions. The sensor was cleaned with a PBS solution before each detection. To monitor the DNA dissociation dynamics, the detected solution was substituted by a pure 0.01× PBS buffer to remove target DNAs. The dissociation was ended via the fast 60s immersion of 10 mM aqueous sodium hydroxide solution and rinsing with PBS solution. The entire detection process was monitored in real-time using the transmittance spectrum. [43,44]
2.5 Experimental setup
The experimental setup for the measurement of the U-MMF LSPR sensor is shown in Fig. 2. A white-light source (tungsten lamp, Ocean Optics HL-2000), with emission wavelengths of 360–2000 nm, was used as an excitation light source. The LSPR shifts of the sensor were obtained with a fiber optic spectrometer (PG2000, Ideaoptics Instruments). Using an electron microscope (SEM, Zeiss Gemini Ultra-55), the surface morphology of all 3D nanostructures was characterized. The multilayer graphene was studied using transmission electron microscopy (TEM JEM-3200FS, Tokyo, Japan).
3. Results and discussion
The U-MMF sensor with different gold-nanoparticle layers is shown in Fig. 3(a). The shape of the sensors indicates high uniformity and repeatability, while the inner diameter of the U-MMF and the length of the coating were nearly 1.5mm and 1 cm, respectively. The gradually darker colors suggest an increase in the thickness of the gold-nanoparticles layers. The SEM image of bare U-MMF, in Fig. 3(b), indicates a smooth surface of the U-MMF, which can facilitate the deposition of gold nanoparticles during the fabrication process. The morphology of gold nanoparticles, for one to five layers, is illustrated in Figs. 3(c)-(g). The diameter distribution of the gold nanoparticles was 10∼30 nm, with average diameters of about 20 nm. The figures indicate that gold nanoparticles produce significant differences in brightness. Due to the difference in the vertical distance of the gold-nanoparticle layers and the coverage of the multilayer graphene films, the gold nanoparticles on the top layer show higher brightness than on the bottom layer. Moreover, as the number of layers increased, multilayer graphene films were also clearly observed based on surface wrinkles and local blur. In other words, multilayer gold nanoparticles and graphene films on the U-MMF were obtained.
Fig. 3. (a) Photo of the U-MMF probe with different gold-nanoparticle layers. (b) SEM image of bare U-MMF. (c-g) SEM images of n*(Au/G)@U-MMF(n = 1-5). (h) TEM diagram of gold nanoparticles covered by multilayer graphene. (i) Raman spectra of graphene oxide and chemically-synthesized graphene.
TEM was performed to further characterize the structure of multilayer graphene, which was deposited on the surface of gold nanoparticles. Figure 3(h) suggests that the graphene capping on the surface of gold nanoparticles produced a uniform film, with ∼2.8 nm in thickness [42]. Raman spectra of graphene oxide and chemically-synthesized graphene are shown in Fig. 3(i).
Compared with the characteristic peaks of graphene oxide, the peak at 2905 cm−1 verified that graphene films were deposited on the gold nanoparticles [41]. The graphene films not only enhanced the performance of the LSPR sensor via effective charge-transfer but also led to the construction of a 3D nanostructure, which increases vertical charge-density oscillations.
To optimize the number of gold nanoparticle layers, the normalized transmittance spectra of the n*(Au/G)@U-MMF (n = 1-5) were collected and compared in the ethanol solutions with RI ranging from 1.340 to 1.352, respectively. As shown in Figs. 4(a)–(e), based on the red dashed line, the redshift and LSPR dip became increasingly pronounced as n increased. When n reached 3, the performance was optimal, with the most distinguishable redshift and LSPR dip, which decreased with n increasing to 4 and 5, due to the high thickness of the 3D overall nanostructure [45]. Figure 4(e) shows that the redshift is almost invisible, and the LSPR dip varies slightly with increasing RI. Moreover, compared to the n*(Au/G)@U-MMF structure, two types of U-MMF sensors were fabricated additionally and employed for ethanol detection. One type was where gold nanoparticles were deposited three times on the surface of the sensing area without graphene(3*Au@U-MMF). The normalized transmission spectra of 3*Au@U-MMF is shown in Fig. 4(f). The other type was gold nanoparticles were deposited three times for one layer of graphene film on the top ((3*Au)/G@U-MMF). The corresponding normalized transmission spectra are presented in Fig. 4(g). They indicate that the redshift of 3*Au@U-MMF was below (3*Au) /G@U-MMF, while both of them were inferior to 3*(Au/G)@U-MMF.
Fig. 4. (a-e) Normalized transmittance spectra of the n*(Au/G)@U-MMF (n = 1-5) in the ethanol solution, with RI ranging from 1.340 to 1.352, respectively. (f) Normalized transmittance spectra of the 3*Au@U-MMF in the ethanol solution with RI ranging from 1.340 to 1.352. (g) Normalized transmittance spectra of the (3*Au)/G@U-MMF in the ethanol solution with RI ranging from 1.340 to 1.352. (h) The redshift of the resonance wavelength of n*(Au/G)@U-MMF (n = 1-5),3*Au@U-MMF, and (3*Au)/G@U-MMF for RI from 1.340 to 1.352, respectively. (i) The FMHM of the n*(Au/G)@U-MMF (n = 1-5) in the ethanol solution with RI at 1.3445.
To reduce random errors of the RI, the weighted centroid algorithm was used to ensure the dip center wavelength, which can increase the measurement-accuracy effectively [23]. To compare the sensitivity of the sensors with the different structures mentioned, the wavelength shift, for RI from 1.340 to 1.352, is shown in Fig. 4(h). According to the histogram, the trend of the redshift variation, with different structures, was evaluated. The redshift of the resonance wavelength rose from 3.14nm to 20.13nm, with the number of gold nanoparticle layers increasing from 1 to 3. When the number of layers reached 4 and 5, the redshift continued to fall to 3.128 nm, which was almost invisible. The redshift of (3*Au)/G@U-MMF was significantly lower than for 3*(Au/G)@U-MMF but higher than the corresponding values for 3*Au@U-MMF and 1*(Au/G)@U-MMF. The comparison conclusively shows: (1) graphene can indeed effectively enhance the sensitivity of the sensor;(2) gold nanoparticles are critical to the sensing mechanism; (3) The 3D nanostructure, which was constructed by graphene films as spacers, is capable of effectively enhancing the LSPR effect. Furthermore, with the magnitude of the redshift, the sensitivity of the corresponding structure varies. We found that 3*(Au/G)@U-MMF was performing best, and the sensitivity could 1251.44 nm/RIU. The full-width-at-half-maximum (FWHM) acts as a valid parameter to measure sensor performance [46,47]. On the whole, a smaller FWHM of the resonance curve shows a better anti-interference capability and sensitivity of the sensor. In Fig. 4(i), the FWHM of n*(Au/G)@U-MMF (n = 1-5) was collected in the ethanol solution with RI at 1.3445. As shown in Fig. 4(i), the value for the FWHM continuously rose with n increasing from 1 to 3, which indicates the enhancement of sensor performance due to the multilayer structure. When n reached 4 and more, as the thickness of the overall nanostructure was out of range, the LSPR effect was down-regulated significantly, with the sensing noise increasing dramatically and FWHM becoming larger.
For a more in-depth assessment of the U-MMF sensor performance, with a wide range of gold-nanoparticle layer structures, the transmittance of n*(Au/G)@U-MMF (n = 1-5) at resonance wavelength was used to plot the LSPR response to the RI of the ethanol solution. As shown in Fig. 5(a), the coefficients of determination (R2) of the fit calibration curve for n*(Au/G)@U-MMF (n = 1-5) were 0.97246, 0.99169, 0.99837, 0.98534, and 0.7688, respectively. These numbers show that the transmittance at the resonant wavelength of 3*(Au/G)@U-MMF shows a better linear response to ethanol solutions with different values of RI. Furthermore, Fig. 5(b) illustrates the linear relationship between the redshift of n*(Au/G)@U-MMF (n = 1-5) and the corresponding RI for different ethanol concentrations. The values of R2 for the five variations above were 0.90002, 0.99022, 0.99577, 0.94943, and 0.77865, respectively. This suggests that 3*(Au/G)@U-MMF still performs best. In Fig. 5(c), the histogram is used to evaluate the linear-response capability of n*(Au/G)@U-MMF (n = 1-5). It is also suggested that 3*(Au/G)@U-MMF is the optimum configuration studied in this paper. Combined with the sensitivity discussion, it can be concluded that the 3*(Au/G)@U-MMF sensor has the highest sensitivity and the optimal linear response capability among the U-MMF sensors with all the other mentioned structures.
Fig. 5. (a) Transmittance at the resonance wavelength of the n*(Au/G)@U-MMF (n = 1-5) as a function of the corresponding RI. (b) The redshift of n*(Au/G)@U-MMF (n = 1-5) as a function of RI, respectively. (c) Histogram of the R2 values of the curves in transmission and redshift, respectively.
Besides the sensitivity and linear-response capability, stability is another critical element for the evaluation of sensing performance. To investigate the reproducibility of 3*(Au/G)@U-MMF, ten cycles of the normalized transmittance at a RI of 1.352 were studied under identical conditions. As shown in Fig. 6(a), the spectra stayed nearly constant, and the excellent reproducibility of the sensor could be confirmed after ten cycles. The response-speed serves as another critical indicator to assess sensor performance. In Fig. 6(b), the typical response-recovery characteristic of 3*(Au/G)@U-MMF in ethanol solution is plotted, with an RI of 1.352 at 532 nm. It is suggested that once immersed in the ethanol solution, the absorbance increases to 65%, immediately, in 0.3s. Such a fast response could be ascribed to the high surface-to-volume ratio, which was introduced by graphene and the 3D nanostructure. To investigate the repeatability of 3*(Au/G)@U-MMF in more detail, the ethanol solution at the RI of 1.352 was detected for seven cycles, using the same 3*(Au/G)@U-MMF sensor. As shown in Fig. 6(c), with the progress of multiple cycles, the absorbance shows only a small fluctuation, which suggests that the 3*(Au/G)@U-MMF sensor has excellent repeatability in multiple measurements.
Fig. 6. (a) Normalized transmittance spectra at the RI of 1.352 during ten cycles, based on 3*(Au/G)@U-MMF. (b) Typical response-recovery characteristic curves of 3*(Au/G)@U-MMF in ethanol solution with an RI of 1.352 at 532 nm. (c) Dynamic absorbance response of 3*(Au/G)@U-MMF for seven cycles in ethanol solution, with an RI of 1.352 at 532 nm.
LSPR is attributed to the resonant collective oscillation of the conduction electrons within a subwavelength metallic nanostructure. The enhanced local electromagnetic fields near the surface of the nanoparticle account for the intense signals observed in all surface-enhanced spectra [5–7]. Accordingly, the electric field distribution of the 3D structure of n*(Au/G)(n = 1-5), which was simulated using the FDTD method, has important implications. In order to eliminate the influence of various characteristics (propagation mode, propagation angle, wavelength, etc.) of U-MMF and intuitively observe the effect of the number of layers on the electric field, we constructed the simulation model through the extraction and abstraction of the experimental device [29,48–51]. The evanescent wave was directly used to activate LSPR to observe the effect of the number of layers on the electric field. Considering the conditions of evanescent wave generation and the range of refractive index, we selected TM polarized light with a 72-degree angle of incidence as the excitation source. In this case, the resonance wavelength of the localized surface plasmon was near 632 nm. The deviation of the wavelength may be caused by the difference between the ideal and actual conditions and the difference between the abstract simulation and the real experiment. We chose 632 nm as the wavelength of the light source. The simulation set-up of the structure n*(Au/G)(n = 1-5) can be seen in Fig. 7(a). As shown in Figs. 7(b)-(f), prominent electric fields occur in the nanogap region, which are produced by the thin graphene film between the gold nanoparticle layers. The excitation region of the electric fields was primarily located in the first three layers. The intensity of the electric fields within four layers and above was significantly weaker than for the first three layers. Figure 7(g) shows the electric-field enhancement variation (E/E0), which results from changes in the number of gold-nanoparticle layers. When the number of layers gradually rose from 1 to 3, the degree of electric field enhancement was increased significantly. Once the number exceeded three, the degree of electric-field enhancement would gradually decrease due to the limitation of the evanescent wave conduction distance and the thickness of the functional structure [5–7,45]. This is consistent with the experimental results.
Fig. 7. (a) Simulation set-up of the the structure n*(Au/G)(n = 1-5). (b-f) Electric-field distribution for the structure n*(Au/G)(n = 1-5), with the TM polarized light excitation at 632 nm wavelength. (g) Electric-field enhancement (E/E0) for the 3D structure with a wide range of gold nanoparticle layers.
To explore the suitability of the 3*(Au/G)@U-MMF sensor for practical use, the sensor was used to measure time- and concentration-dependent DNA hybridization kinetics. This is of high importance for genetic screening and detection, disease biomarkers, transcriptional profiling, and single-nucleotide variant discovery. As shown in Figs. 8(a)-(b), PBASE served as a link between graphene and probe aptamer, with its pyrene group facilitating π–π stacking with graphene at one end, and its succinimide portion conjugating with the probe aptamer modified by –NH2 at the other end [24]. Due to the complementarity of the base sequence between t_DNA and probe aptamer, the affinity between them was strong shown in Fig. 8(c). On the other hand, there was no complementarity between the base sequences of mis_DNA and probe aptamer, hence, they could not form a stable structure, as shown in Fig. 8(d).
Fig. 8. (a) Schematic showing the adding of the PBASE on the graphene surface. (b) Attaching of the probe aptamer. (c) After adding t_DNA onto the 3*(Au/G)@U-MMF sensor. (d) Adding mis_ DNA onto the 3*(Au/G)@U-MMF sensor.
To analyze the sensitivity and specificity of the 3*(Au/G)@U-MMF sensor, the normalized transmission-spectra were collected throughout the modification and detection processes. As shown in Fig. 9(a), with the resonance wavelength of the sensor in PBS solution at pH 7.4 as a control point, the entire PBASE modification process led to a redshift of 2.3 nm, which is probably due to the p-doping effect of the charge-transfer between the pyrene group and graphene. Subsequently, the modification process of probe aptamer caused a redshift of 11.15 nm, as interpreted by the electron-rich feature of the DNA. The normalized transmission spectra of the t_DNA solutions, with respective concentrations of 0.1nM to 100nM, were collected, see Fig. 9(b). It is suggested that the resonance wavelength displayed a very distinct redshift difference after sufficient complementary to equilibrium. To a certain extent, the complementation between t_DNA, at a higher concentration with probe aptamer, could cause localized RI to vary more noticeably, which can lead to a stronger redshift [44]. The redshift data during the process of modification and measurement was integrated into the histogram in Fig. 9(c). Using the resonance wavelength in the PBS solution at pH 7.4 as a reference point, the 3*(Au/G)@U-MMF reveals clear redshift-changes during the respective steps of modification and measurement. In Fig. 9(d), the resonance wavelength with probe aptamer and that after adding mis_DNA were 533.83nm and 532.88nm, respectively. The almost invariable resonance wavelength during the two stages suggests a non-bonding reaction between the probe aptamer and mis_DNA strands. The localized RI of the sensing area remained unchanged for the no-hybridization. We also detected mis_DNA samples with vary concentration, the same results were obtained. For a more in-depth evaluation of the sensor performance for DNA detection, the responses of the 3*(Au/G)@U-MMF sensor to a series of concentrations of t_DNA were recorded in real-time. 10 nM was taken as an example, see Fig. 9(e), where the process of DNA hybridization can be observed. After the t_DNA and probe aptamer had been sufficiently complementary to equilibrium, the process of dissociation was performed. A pure 0.01x phosphate-buffered saline (PBS) buffer was employed to dissociate and remove target DNAs with the transmission spectrum shown in Fig. 9(f). The resonant wavelength slowly decreased until it returned to the position before hybridization. The dissociation was ended via the fast 60s immersion of 10mM aqueous sodium hydroxide solution and rinsing with PBS solution. The successful recovery of the resonance wavelength at the end of the dissociation suggests a complete regeneration. Furthermore, the redshift data for hybridization and dissociation are included in Fig. 9(g). Using the resonance wavelength before hybridization as a control, the time-varying curve of the shift of the resonant wavelength during the whole detection process was plotted. It reveals a nonlinear variation. After immersion in the t_ DNA solution, the redshift increases initially but reaches a plateau at ∼70 min, which suggests that the t_DNA and probe aptamer are sufficiently complementary to equilibrium. After 70 min, the redshift was gradually reduced to the initial point, which indicates effective dissociation after DNA hybridization.
Fig. 9. (a) Normalized transmission-spectra of 3*(Au/G)@U-MMF during modification. (b) Normalized transmission-spectra of 3*(Au/G)@U-MMF in t_DNA solutions with respective concentrations of 0.1nM to 100nM after sufficiently complementary to equilibrium. (c) Histogram of the redshift of the resonance wavelength, at respective steps, during modification and binding. (d) Normalized transmission-spectra before and after adding mis_DNA. (e) Normalized transmission spectra of 3*(Au/G)@U-MMF in t_DNA solutions of 10nM during the binding process. (f) Normalized transmission-spectra of 3*(Au/G)@U-MMF in t_DNA solutions at 10nM during dissociation. (g) Real-time redshift for the binding- and dissociation-process of t_ DNA at 10nM.(h) Real-time wavelength shift for mismatched DNA and complementary target DNA.
To verify the prominent resolution for the DNA solution of the sensor at different concentrations, The changes in redshift, during DNA hybridization at different concentrations(0.1nM-100nM), were integrated as shown in Fig. 9(h). Using the Langmuir model for LSPR experiments:
4. Conclusion
In the present study, a novel LSPR biosensor using U-MMF, which was modified with the complex of 3D gold nanoparticles and multilayer graphene as spacers (n*(Au/G)@U-MMF), was investigated. We found that the number of gold nanoparticle layers is critical for the performance of the sensor. The configuration with 3*(Au/G)@U-MMF was found to be optimal in this paper. The sensor shows high sensitivity (1251.44 nm/RIU), linearity, stability, and repeatability, based on the merits of strong anti-oxidation and simple processing. In terms of applications, the sensor showed excellent performance for the measurement of time- and concentration-dependent DNA hybridization kinetics with detection concentrations from 0.1nM to 100 nM. This study suggests that 3D bulk resonance is a practical option to enhance the performance of LSPR sensors. Furthermore, a deeper understanding of combining 2D materials and LSPR sensors has been accomplished.
Funding
Natural Science Foundation of Shandong Province (ZR2019MF025, 2017GGX20120); National Natural Science Foundation of China (11674199, 61401258); Foundation of Shandong Provincial Key Laboratory of Biophysics (SD2019BP003); China Postdoctoral Science Foundation (2019M662423).
Disclosures
The authors declare no conflicts of interest.
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