Raman spectroscopy is widely applied in wood science because of its features of being nondestructive, rapidity, and high resolution. However, Raman scattering is weak, and the Raman signal is easily disturbed by autofluorescence arising from endogenous fluorescent molecules in biological tissue. In this work, a sensitive lignin detection platform was fabricated by a composite with a polyaniline (PANI) nanofiber and toluidine blue (TB) under the excitation of visible light. In this platform, TB acts as a specific marker for lignin, and a PANI nanofiber was used as a reinforcing reagent to improve the Raman intensity of TB. When wood slice is impregnated with TB/PANI, the lignin in wood can be precisely labeled with the TB, and the Raman intensity of TB had a threefold increase at 532 nm excitation. This TB/PANI detection platform is expected to make a significant contribution in qualitative and quantitative analysis of lignin to avoid autofluorescence in various lignin-based biosciences.
Lignin is a major component of the cell wall in wood; the content of lignin has a significant effect on wood performance; for example, lignin in plant fibers is generally regarded as undesirable by the pulp and paper industry, but the high concentration of lignin in the middle lamella between cells in wood is regarded as a positive benefit; thus, the determination of lignin quantity is very important in wood science [
Raman spectroscopic imaging has been increasingly used in the field of wood science for its ability to characterize various chemical compositions in wood cell walls with minimal sample preparation and provide spatial and spectral information about a sample simultaneously [
However, the sensitivity of Raman spectroscopy is somewhat poor as only a small number of incident laser photos are elastically scattered. To overcome this problem, many special Raman signal-enhancing techniques have been studied extensively [
Herein, we have prepared a PANI nanofiber and combined TB together for the sensitive detection of lignin in wood slice. In this platform, TB was chosen as a sensitive and specific biomarker for lignin, and the PANI nanofiber can improve the Raman intensity of TB. First, TB/PANI was assembled on wood slice by the impregnation method. Second, in the presence of target lignin, it would be labeled with TB and a powerful Raman signal was obtained by the approach between PANI and TB at the excitation wavelengths of 532 nm. This TB/PANIA platform had high specificity and sensitivity and can quantitatively detect lignin to avoid autofluorescence stimulated by the visible range light. The strategy of this detection system is shown in Scheme
Schematic illustration of lignin in wood detected by the TB/PANI platform.
All of the reagents including aniline, TB, p-toluene sulfonic acid (PTS), ammonium persulfate (APS), ethanol, and cyclohexane are of analytical grade. They were used in experiments without further purification. Deionized water was used throughout the experiment to prepare the solutions.
PANI was synthesized by using 0.5 g aniline, 0.01 g PTS, and 0.5 g APS in cyclohexane/water solution at room temperature. After magnetic stirring for 24 h, the mixture was washed with ethanol several times to remove the reaction byproducts; the dark green-colored PANI solution with a solid content of 2% was collected for further experiments. The TB/PANI was prepared by adding PANI to 2 mmol/L TB aqueous solution treated ultrasonically for 15 min. Experiments were performed with TB : PANI volume ratios of 1 : 1, 2 : 1, and 3 : 1, to understand the influence of PANI content on the Raman intensity of TB.
Wood slices were taken from an 8-year-old normal wood of
FTIR spectra of samples were collected with a BRUKER TensorII Fourier transform infrared spectrometer (America). The samples were analyzed using a diamond ATR accessory. The phase structure and purity of the samples were examined by X-ray diffraction (XRD) using a XRD-3 (PUXI, China) with a diffractometer with Cu K
Raman characterization of PANI, TB, and TP/PANI.
The TB/PANI was prepared by the ultrasonic-assisted impregnation method; ultrasonic vibrations in a liquid create pressure waves, resulting in the formation, growth, and implosion of millions of microscopic bubbles [
The UV-vis absorption spectra of PANI show photoadsorption at the wavelengths of 265 and 385 nm. While PANI is mixed with TB, the UV-vis adsorption is 281 and 625 nm (Figure
UV-vis spectra of TB, PANI, and TB/PANI (a), XRD images of PANI and TB/PANI (b), and SEM images of PANI (c) and TB/PANI (d).
XRD patterns of wood slice and wood slice impregnated with TB (2 mM), PANI, and TB/PANI (volume ratio of 2 : 1) are presented in Figure
XRD images of wood slice and TB/PANI/wood slice (a) and FTIR images of wood slice, PANI-wood slice, TB-wood slice, and TB/PANI-wood slice (b).
The morphology of the wood slice, TB-wood slice, and PANI-wood slice was observed using SEM (Figure
SEM images of wood slice, TB-wood slice, and PANI-wood slice.
When the wood slice was impregnated with TB/PANI (ratio of 1 : 1, 2 : 1, and 3 : 1) under ultrasonic dispersion for 30 min, the wood slice structure commendably remained. Aggregation disappears in the samples at different volume ratios of TB to PANI (Figure
SEM image of wood slice treated with TB/PANI at the volume ratio of 1 : 1, 2 : 1, and 3 : 1 after ultrasonic dispersion 30 min (a) and 60 min (b).
The fabrication process of the detection platform was characterized by using a Raman spectrometer; when the concentration of TB was 1.0, 2.0, and 3.0 mM, the difference of Raman intensity is not great, which ranges from 3000 to 4000; thus, 2.0 mM TB was used in this detection platform. The Raman peak of 1627 cm−1 and 1594 cm−1 belongs to TB and PANI, when the TB was mixed with PANI (volume ratio of 2 : 1), the peak shape of TB/PANI had the same trends with the TB, and the peak intensity of 1627 cm−1 was increased from 3000 to 8000 (Figure
Figure
Raman characterization of wood slice (a), PANI-wood slice (b), and TB-wood slice (c).
To study the analytical ability and real sample detection potential of the proposed TB/PANI platform, Raman characterization of wood slice treated with TB/PANI at various volume ratios was investigated. The Raman images showed the strongest peak of 1627 cm-1 in the cell corner, which were representative of lignin. The Raman intensity was 8000 at 1627 cm−1 when wood slice was treated with TB/PANI at a volume ratio of 1 : 1 (Figure
Raman characterization of wood slice with the TB/PANI volume ratio of 1 : 1 (a), 2 : 1 (b), and 3 : 1 (c).
To further determine whether the TB can act as a sensitive and specific biomarker for lignin, a filter paper and degreasing cotton which has no lignin were chosen as control samples. When the filter paper was treated with TB, no characteristic peaks of the TB (1627 cm-1) appeared (Figure
Raman characterization of the filter paper (a) and degreasing cotton (b).
The techniques for enhancing the Raman signals are mainly based on resonant Raman scattering (RRS) or surface-enhanced Raman scattering (SERS). For RRS, the resonance state arises when the excitation wavelength was resonant with a molecular transition. As for SERS, the two widely accepted mechanisms are the electromagnetic mechanism (EM) and chemical mechanism (CM). Briefly, the EM field is enhanced by the excitation of localized surface plasmon resonance when light interacts with metals [
To testify this hypothesis, Raman characterization of wood slice treated with TB and nonconductive polyglycol diglycidyl ether resin (DER736) is shown in Figure
Raman characterization of wood slice with the TB/DER736 volume ratio of 1 : 1 (a), 2 : 1 (b), and 3 : 1 (c).
These observations suggest that the CM dominates over the surface-enhanced effect in this study, charge transfer can occur between PANI and TB, and the similarity of the chemical structure between PANI and TB may be another factor contributing to the Raman enhancement.
In this study, a TB/PANI platform was prepared for the sensitive detection of lignin in wood slice. The PANI nanofiber was a potent enhancer to improve the Raman intensity of TB which acts as a marker of lignin in wood slice. At the 532 nm excitation, Raman intensity of TB could be improved more than three times to avoid autofluorescence. This lignin-recognizing platform could be utilized to proffer researchers a versatile, nondestructive, noninvasive, high-throughput analytical tool. In view of these advantages, we anticipate that this highly sensitive and selective method has potential to be applied to the lignin-based material area.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Hui Zhang, Liang Zhou, and Shengquan Liu conceived and designed the experiments. Hui Zhang and Jing Li performed the experiments. Liang Zhou and Jing Li analyzed the data. Hui Zhang and Jing Li wrote the paper. Liang Zhou and Shengquan Liu have given their approval for the final version of the manuscript. Hui Zhang and Jing Li contributed equally to this work.
The work was supported by the National Key R&D Program of China (2017YFD0600201), Postdoctoral Science Foundation of Anhui Province (2019B316), and Science and Technology Project of Education Department of Anhui Province (KJ2019A0200).