1. Introduction

In the past decades, surface-enhanced Raman scattering (SERS) spectroscopy with unique fingerprint identification at a single molecule level has been well developed as a fascinating optical technique for ultrasensitive determination of diverse chemical/biological molecules in many special applications [1–5]. It is generally accepted that the excellent SERS should be attributed to two mechanisms: (1) the dominant contribution of electromagnetic mechanism (EM) is originated from the strong and concentrated local electromagnetic field generated on the rough surfaces of plasmonic metallic (gold (Au), silver (Ag) or copper (Cu)) nanostructures [1,4,6]; (2) the minor effect of chemical mechanism (CM) is derived from the effective charge transfer (CT) between semiconductor-based nanosubstrates and absorbed probe molecules [3,7,8]. Usually, the ultrasensitive SERS detection is highly related to the corresponding distance between SERS substrates and probe molecules, since the longer interval implies the lower SERS activity. Even for plasmonic metallic SERS substrate with a relative long-range effect of EM enhancement, the corresponding Raman signals will dramatically decrease with an increase of the physical distance. So, it is highly desirable to shorten the distance between probe molecules and SERS nanosubstrates, ensuring the formation of strong interaction during incident laser excitation. Initially, the conventional SERS rigid substrates have been frequently prepared by the cleaned silicon plates or glass sheets grafted with SERS active nanomaterials (NMs) or functional nanoarrays [9–13]. Then, the colloidal probe molecules can be directly dropped on the rigid SERS substrates, which are very close to the “hotspots” of SERS active NMs. However, the main drawback is that the rigid SERS substrates are not suitable for SERS monitoring of probe molecules absorbed on non-smooth surfaces or curved structures owing to their mechanical stiff and brittle features.

Alternatively, the flexible and stretchable SERS substrates should be an advisable choice for the SERS detection on irregular surfaces, which have been developed and received increasing attention in many real-world scenarios [14–20]. For instance, a polydimethylsiloxane (PDMS) film decorated with wrinkled graphene (WG)/Au NPs has been used as the hybrid SERS substrate with high stretchability can operate normally under as high as 50% tensile strain without performance degradation [16]. Based on PDMS/Au nanostars flexible substrates, the SERS detection of thiabendazole pesticide on curved apple skin has been also illustrated in the previous work [14]. Besides the PDMS films, some other flexible supports were also adopted in the construction of stretchable SERS substrates, such as three-dimensional (3D) silica nanowires (SiO₂, NWs) [15], polyvinyl alcohol (PVA) [18,21], pyramid polymethyl methacrylate (PMMA) [20], the filter paper [19,22], etc. Obviously, these flexible SERS substrates with the aid of diverse soft backbone materials have shown remarkably multiple advantages over routine rigid SERS substrates: (1) they are conformed to many irregular surfaces and can be wrapped around curved surfaces for SERS detection; (2) they can be easily cut into some unconventional shapes for SERS detection in specific scenarios; (3) the stretchable and flexible substrates are suitable for optimizing SERS hotspots during tension or compression process. Moreover, compared to traditional artificial polymer-based flexible SERS sensors, the fascinating hygroscopic and biocompatible natural soft supports have the ability to provide a unique opportunity for further promoting SERS performance in biomolecular diagnostics. Especially, the unique hygroscopic capacity is particularly beneficial to concentrate the probe molecule solution and then enhance Raman signals, which is an effective approach for boosting SERS activity. Among these biocompatible soft supports, the silk fibroin fibers (SFF) served as a kind of widely used ancient natural material that can be directly derived from bombyx mori silkworms, which is composed of sericin and fibroin that are the major components of repetitive crystalline heavy chains (forming discrete β-sheet crystallites) and nanocrystalline light chains (forming amorphous domains) [23–25]. Nowadays, the SFF is widely explored as functional material in a broad range of applications from tissue engineering and drug release to microdevice realms due to its excellent biocompatibility, hygroscopic capacity, chemical stability in vitro and in vivo and tunable fabrication into various forms [26–28]. Therefore, the hygroscopic SFF-grafted with plasmonic NMs should be expected to provide an excellent SERS performance, which will be more suitable for ultrasensitive monitoring of biomolecules in widespread applications.

Herein, we report an appealing flexible hygroscopic SERS biocompatible sensor based on the SFF soft supports decorated with urchin-like Au/Ag nanoalloys (NAs). The hybrid SFF-Au/Ag NAs with a stronger absorption capacity (500∼1100 nm) and excellent hygroscopicity provide a much higher near-infrared (NIR)-SERS activity than that of bare urchin-like Au/Ag NAs. The main distinctive advantages of the established flexible hygroscopic SERS sensor are highlighted in the term of remarkably high NIR-SERS activity, excellent spatial uniformity and pronounced long-term durability. As for biomolecular detection, the NIR-SERS active sensor is applied to the ultrasensitive monitoring of folic acid (FA) molecules in the human sweat. It is well known that human sweat contains at least 60 different chemical constituents at varying concentrations. Among them, although the content of FA molecules is only ∼ 10 nM in human sweat [29], it is an indispensable constituent in the human body [30]. For humans, FA has been considered an important nutrient, playing an important role in cell division, growth and reproduction. The lack of FA can easily lead to metabolic disorders, loss of appetite, macrocytic anemia and leukopenia as well as the increased risk of cancer. Especially for pregnant women, insufficient FA intake will cause fetal growth retardation and some congenital diseases such as cleft lip and palate, brain abnormalities, heart defects [30]. Based on the interesting SFF-Au/Ag NAs in this work, the limit of detection (LOD) of FA molecules can be achieved at nanomole (nM, 1.04 × 10⁻⁹ M) level, giving rise to the ultralow analyses of biological molecules in the human body. These findings will be of great significance to establish a high performance flexible NIR-SERS biocompatible sensor for ultrasensitive assessment of other specific biomolecules in personal health management.

2. Experimental details

2.1 Materials

Silver nitrate (AgNO₃, 99.85%), chloroauric acid (HAuCl₄·4H₂O), folic acid (FA), dibasic sodium phosphate (Na₂HPO₄•12H₂O), sodium dihydrogen phosphate (NaH₂PO₄•2H₂O) and anhydrous phosphoric acid (H₃PO₄) were obtained from Sinopharm Chemical Reagent Co., Ltd. Ascorbic acid (AA, 99%), lithium bromide (LiBr) was purchased from Macklin. Rhodamine 6G (R6G) was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. Polyvinylpyrrolidone (PVP, Mw=40000) was purchased from TCI (Shanghai). Sodium carbonate (Na₂CO₃) was purchased from Sigma-Aldrich. The pH of the phosphate buffer (PB) solution was adjusted with Na₂HPO₄•12H₂O, NaH₂PO₄•2H₂O and H₃PO₄ and measured with a digital pH/ion meter. All reagents were analytical grade and directly used as supplied without further treatment. Deionized water used in the fabrication and measurement was prepared by the Millipore purification system (18.2 MΩ cm). Native B. mori silk cocoons were purchased from a local sericulturist in China.

2.2 Preparation of Silk Fibroin Solution

A silk fibroin solution was prepared according to methods previously [25]. First of all, we added 0.02 M Na₂CO₃ to 1L water and dissolved it completely. We added the chopped cocoon pieces when the water was boiling and kept them for 30 min to remove silk fibroin. Then, we rinsed the silk protein with deionized water while gently stirring on the stirring plate for 20 minutes. Secondly, the silk was dried overnight in a fume hood. When dried completely, the silk bundle was dissolved in a 9.3 M LiBr solution and incubated at 60 °C for 4 h. The silk fibroin solution was obtained after dialysis in DI water for 3 days.

2.3 Synthesis of urchin-like Au/Ag nanoalloys

The urchin-like Au/Ag nanoalloys (NAs) were prepared by a seed-mediated growth method, which is very similar to previous works [31–33]. In short, a well-polished Au metal used as the target was placed on the bottom of a rotating glass dish (∼400 rpm) filled with 2 cm depth of liquid solution (0.2 M AgNO₃, 0.08 M PVP and 20 mL DI water). First, Au/Ag nanoparticles (NPs) are obtained by laser irradiation Au metal with a Q-switched Nd-YAG laser beam, where the working wavelength of the laser beam is 1064 nm, the pulse duration is ∼10 ns, the energy is ∼250 mJ and the repetition frequency is 10 Hz. At the moment the pulsed laser arrives at the surface of the Au target, the Au element will boil and vaporize rapidly, resulting in the formation of explosive Au thermal plasma. The superheating Au plasma with highly non-equilibrium features can play a critical role in the subsequent nucleation process [34,35]. The strong interaction between the Au thermal plasma and the surrounding Ag ions makes the bimetallic Au/Ag nanocrystals nucleate during the rapid condensation stage of the plasma and the nucleation abruptly stops due to the exhaustion of the plasma vapor. To further increase the Ag content in the Au/Ag nanocomposite, we changed the laser to 532 nm laser irradiation. Finally, urchin-like Au/Ag NAs can be obtained by quickly mixing HAuCl₄ (5 ml, 2.5 mM), AA (1 ml, 0.08 M) and as-prepared Au/Ag NPs (2 mL, 0.1 M). Later, as-synthesized urchin-like Au/Ag NAs were washed and centrifuged (7000 rpm, 10 minutes) at least twice.

2.4 Preparation silk fibroin fibers (SFF) with Au/Ag NAs

Silk fibroin solution with a concentration of 6% (0.2 mL) and Au/Ag NAs solution at a concentration of 0.15 wt% (0.1 mL) were uniformly mixed, then coated on the silicon plate and dried for 12 hours. The SFF-Au/Ag NAs film and silicon plate detach automatically after drying.

2.5 Characterization of materials

The obtained precipitates were dropped on a copper mesh and dried in the fume hood for observation via transmission electron microscopy (JEOL-JEM-2100F). The morphologies of the samples were recorded through field emission scanning electron microscopy (FIB, Helios G4 UC). The contact angle of SFF-Au/Ag NAs film was tested by DSA 100. In addition, absorption spectra of the colloidal solutions were recorded by a UV-vis NIR spectrophotometer (UV-1800, Shimadzu). All the SERS signals were collected by a confocal microprobe Raman spectrometer (Renishaw Raman spectroscopy).

2.6 Surface-enhanced Raman scattering (SERS) substrate preparation

SFF-Au/Ag NAs film has been used as SERS substrate material. As for the preparation of R6G probe molecule solutions, 0.04 g R6G powder was dissolved in 10 mL deionized water (DI water) to form 0.01 M R6G solution, which will be separately diluted with DI water to prepare R6G solutions with different concentrations of 10⁻⁶–10⁻¹⁵ M. The solution of FA with different concentration were synthesized by the same method. All the SERS signals were collected by a confocal microprobe Raman spectrometer (Renishaw Raman spectroscopy) with a 50 × objective. The spot size of the Raman laser on the sample is ∼1µm. Excitation was provided using Renishaw high power diode Laser 785 nm CW laser with 300 mW output and 0.75 mW at the sample.

3. Results and discussion

In this work, the as-prepared urchin-like Au/Ag NAs were uniformly mixed with the as-prepared SFF solution, resulting in the formation of hygroscopic SFF-Au/Ag NAs. The absorption spectra of the obtained SFF-Au/Ag NAs, bare Au/Ag NAs and original SFF supports are illustrated in Fig. 1(a). The absorption spectrum of the original SFF supports reveals the weak absorbance capacity of these soft materials in the visible-NIR region. After grating with bimetallic NMs, the hybrid SFF-Au/Ag NAs have a stronger absorbance capacity in the visible-NIR region (500∼1100 nm) than that of bare urchin-like Au/Ag NAs. Meanwhile, the LSPR peak of the hybrid SFF-Au/Ag NAs or bare Au/Ag NAs is located at the same position of ∼790 nm. The 785 nm laser source that could be more sensitive to the resonance position of plasmonic NAs was selected in SERS tests, which is essential for getting better SERS activity. The morphologies of hybrid SFF-Au/Ag NAs and original SFF supports can be observed by SEM images, as shown in Figs. 1(b) and (c). It can be seen that the flexible SFF supports are covered by plentiful aggregated Au/Ag NAs with rugged surface structures. Moreover, urchin-like Au/Ag NAs with multi-tentacles can be visualized by TEM image in Fig. 1(d), and SFF flexible supports are wrapped around Au/Ag NAs. The enlarged TEM image (Fig. 1(e)) further confirms the construction of urchin-like nanostructure in this experiment. Moreover, the bimetallic nature of the nanoproducts is validated by elemental mapping images of the individual Au/Ag NAs. The relative ratio of Au to Ag in this bimetallic structure is calculated at about 96:4, implying the formation of Au/Ag NAs.

 

Fig. 1. (a) Absorption spectra of original SFF supports, bare Au/Ag NAs and hybrid SFF-Au/Ag NAs, respectively. (c) and (d) are SEM images of the hybrid SFF-Au/Ag NAs and original SFF supports, respectively. (d) and (e) are TEM and HRTEM images of SFF-Au/Ag NAs, respectively. The right images in (e) show the elemental mapping images of the individual Au/Ag NAs.

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Then, the SERS activity of the hybrid SFF-Au/Ag NAs is evaluated in Fig. 2. Initially, the interference of Raman signals originated from the pristine SFF can be ignored in the SERS measurements, since the Raman spectrum of this substrate (zero analyte) is too weak (black line in Fig. 2(a)). After loading probe R6G molecules on this original SFF sample, there is no obvious Raman peak that can be detected by 785 nm laser excitation (red line in Fig. 2(a)), supporting the negligible contribution of SFF-induced SERS enhancement. Subsequently, the 785 nm laser-excited NIR-SERS spectra of 10⁻¹³ M R6G molecules adsorbed on flexible SFF-Au/Ag NAs and bare urchin-like Au/Ag NAs were tested in this work. The dominating characteristic bands of R6G molecules at 614, 774, 1185, 1312, 1364, 1508 and 1650 cm⁻¹ are all clearly detected in SERS spectra, providing much enriched “molecular fingerprint” information. The positions and relative amplitudes of these spectra agree well with the previous results [14–16,36]. In detail, the two bands at approximately ∼614 cm⁻¹ and ∼774 cm⁻¹ are assigned to the C–C–C ring in-plane vibration mode and the C–H out-of-plane bend mode, respectively. The other bands at about ∼1185 cm⁻¹ and ∼1312 cm⁻¹ are separately related to the C–H in-plane bend modes and the N–H in-plane bend modes. The residual bands at approximately 1364, 1508 and 1650 cm⁻¹ are derived from the C–C stretching modes. As shown in Fig. 2(a), it can be seen clearly that the hybrid SFF-Au/Ag NAs provide a remarkably higher NIR-SERS activity than that of bare Au/Ag NAs. Moreover, the comparison results of different SERS peak intensities separately originated from SFF-Au/Ag NAs and bare Au/Ag NAs are summarized in Fig. 2(b). The quantitative comparisons in Fig. 2(b) further confirm the improved SERS signals can be detected on the obtained SFF-Au/Ag NAs, which are already ∼1.5 times higher than that of Au/Ag NAs. Considering the similar geometric nanostructures of plasmonic NAs used in two samples, the enhanced SERS activity of SFF-Au/Ag NAs should be related to the hygroscopic and flexible SFF supports, which will be revealed in the subsequent section.

 

Fig. 2. (a) SERS spectra of 10⁻¹³ M R6G molecules absorbed on different three substrates (SFF-Au/Ag NAs, bare urchin-like Au/Ag NAs and original SFF) and SFF without analyte. (b) The comparison results of different SERS peaks separately originated from the obtained flexible SFF-Au/Ag NAs and bare urchin-like Au/Ag NAs.

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To further evaluate the hygroscopic capacity of flexible SFF-Au/Ag NAs, the continuous monitoring of a drop of R6G/distilled water mixed solution was absorbed on this substrate. As illustrated in Fig. 3(a), it can be found that the corresponding contact angle (CA) of this liquid drop on SFF-Au/Ag NAs film dramatically changes from 95.4° at 0 min to 15.2° at 30 min. In contrast, as for the rigid cleaned silicon plate, the CA value of this liquid drop is nearly constant even up to 30 min, which slightly changes from 69.4° at the initial stage to 62.1° at 30 min, as shown in Fig. 3(b). Moreover, the real-time dependent SERS tests of this liquid drop adsorbed on the hygroscopic SFF-Au/Ag NAs were also performed in this work. The corresponding SERS spectra of R6G molecules versus the delay times with respect to the initial stage are shown in Fig. 3(c). Obviously, the SERS peak intensities of R6G molecules at a delay time of ∼120 min are much higher than that of shorter delay time regions (0-120 min). The variation of SERS peak intensity of R6G molecules at 1508 cm⁻¹ versus different delay times is shown in Fig. 3(d). It reveals that the SERS peak intensity significantly increases with an increase of delay time in the region of 0-120 min. The comparative results suggest that the SERS signal intensity collected at a sufficient molecular permeation time of 120 minutes is approximate ∼ 7.7 times higher than that of the measurement at 10 minutes. And after the sufficient molecular penetration time (>120 min), the stable SERS peak intensity can still be maintained for 7 days. In fact, the residual ∼95.7% SERS signal intensities can be retained after keeping them in normal condition for 7 days, implying the excellent long-term stability of SFF-Au/Ag NAs-based SERS sensors. Therefore, the excellent hygroscopic capacity of hybrid SFF-Au/Ag NAs can be revealed in this work, fascinating the probe molecule solution absorbed on the flexible substrate penetrates the underground area instead of flowing out from the surface. In this way, the hygroscopic SFF-Au/Ag NAs have the ability to concentrate the probe molecule solution, giving rise to an obvious higher SERS signal than that of bare Au/Ag NAs.

 

Fig. 3. (a) and (b) are the contact angle of hygroscopic SFF-Au/Ag NAs and clean silicon plate with probe molecular solution at 0 min, 10 min and 30 min, respectively. (c) Time-dependent NIR-SERS signals of R6G molecules absorbed on SFF-Au/Ag NAs at different delay times. (d) The evolution of the Raman peak intensity of R6G molecules at 1508 cm⁻¹ at different delay times.

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On the other hand, in order to verify NIR-SERS spatial uniformity of flexible SFF-Au/Ag NAs, twenty-five groups of Raman spectra of 10⁻⁹ M R6G molecules recorded from 25 random different points were performed in this work. Then, the variations of these peak intensities of R6G molecules at 614, 1185, 1312 and 1508 cm⁻¹ are illustrated in Fig. 4(b), supporting that the NIR-SERS signals can be well repeated on the obtained flexible substrate. In addition, as for the spatial variations of Raman peaks of R6G molecules at 614, 1185, 1312 and 1508 cm⁻¹, the mapping images of NIR-SERS peak intensities versus 25 points are also illustrated in Fig. 4(a). The corresponding relative standard deviation (RSD) values of NIR-SERS peak intensities at 614, 1185, 1312 and 1508 cm⁻¹ are calculated at about 2.41%, 1.35%, 2.76% and 1.49%, respectively, confirming the homogeneous distribution of SERS spectra on SFF-Au/Ag NAs substrate. Meanwhile, in order to further evaluate the corresponding NIR-SERS sensitivity, the Raman spectra of R6G molecules with different concentrations of 10⁻⁷−10⁻¹⁵ M absorbed on the substrate are shown in Fig. 4(c). It can be found that the NIR-SERS peak intensities gradually decrease as the molecular concentration decreases from 10⁻⁷ M to 10⁻¹⁵ M. Meanwhile, the dominant characteristic bands of R6G molecules such as 614, 1185, 1312 and 1508 cm⁻¹ can be clearly distinguishable even the concentration decreases to as low as 10⁻¹⁵ M. Moreover, the variations of the NIR-SERS peaks intensities of R6G molecules at 614, 1185, 1312 and 1508 cm⁻¹ molecules versus the logarithmic scale of R6G molecular concentrations (10⁻⁷−10⁻¹⁵ M) are separately depicted in Fig. 4(d). It reveals that the four groups of linear relationships can be obtained by plotting the different NIR-SERS peak intensities versus the logarithmical scale of molecular concentrations, giving rise to the precise SERS assessment of ultra-trace molecules in diverse real-world scenarios. For instance, the peak of 1312 cm⁻¹ changing as a function of molecular concentrations from 10⁻⁷ to 10⁻¹⁵ M shows the value of R² reaching 0.96, indicating the establishment of excellent linearity. Based on the results in Figs. 4(c) and (d), therefore, the corresponding limit of detection (LOD) of R6G molecules can be achieved at ∼1.18 × 10⁻¹⁵ M (fM), approaching the requirement for ultra-trace SERS detection. Based on the established hygroscopic SFF-Au/Ag NAs, the obtained ultralow LOD of this NIR-SERS sensor in this work is already better than that of many previous flexible SERS substrates, such as Ag nanolayer/rose petals (10⁻⁹ M) [37], stretchable freestanding WG/Au NPs hybrid platform (10⁻⁹ M) [16], flexible PVA/Ag NPs (10⁻⁵ M) [18], flexible PMMA/Ag NPs (10⁻¹³ M) [20], rGO/Ag NPs (10⁻⁹ M) [38], etc. As for plasmonic Au or Ag nanostructures, it has been well known that the excellent SERS performance with enhanced activity and ultralow LOD is highly related to the pronounced EM enhancement, which is related to the formation of plentiful hotspots on rough surfaces. Compared with relatively smooth surfaces of spherical Au or Ag NPs that were frequently used in previous flexible substrates, the urchin-like Au/Ag NAs with enormous nano-antennas immersed into hygroscopic SFF supports can offer much more SERS accessible hotspots [32,39], which can be easily accessible to probe molecules. In this way, the urchin-like Au/Ag NAs hotspots presented in the flexible SFF supports greatly reduce the distance between SERS hotspots and probe molecules, which is suitable for dramatically enhancing Raman signals of probe molecules in close proximity.

 

Fig. 4. (a) The related mapping diagrams of these SERS peak intensities, respectively. (b) The variations of Raman peak intensities of R6G molecules (10⁻⁹ M) at 614, 1185, 1312 and 1508 cm⁻¹ versus 25 different points on the flexible SFF-Au/Ag NAs. (c) Based on the obtained SFF-Au/Ag NAs, the SERS spectra of R6G solution at different concentrations (10⁻⁷−10⁻¹⁵ M). (d) The relationships between the SERS peak intensities at 614, 1158, 1312 and 1508 cm⁻¹ and the logarithmic concentrations of R6G molecules.

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Besides the above NIR-SERS detection of traditional R6G dye molecules, then the ultrasensitive monitoring of FA molecules absorbed on the as-prepared SFF-Au/Ag NAs was also investigated in this paper. Based on the NIR-SERS active sensor in this work, Raman signals of FA molecules with different concentrations (10⁻²−10⁻⁹ M) in distilled water solutions are shown in Fig. 5(a). It can be seen that the plentiful characteristic bands of FA molecules are clearly identified at 683, 856, 921, 1194, 1251, 1357 and 1608 cm⁻¹ [40–44], offering abundant “molecular fingerprint” information. On the other hand, the NIR-SERS peak intensities of FA molecules gradually decrease with a decrease of molecular concentration (10⁻²−10⁻⁹ M). Additionally, Fig. 5(b) shows that the variations of three groups of Raman peak intensities of FA molecules at 683, 1194 and 1608 cm⁻¹ versus the logarithmical scale of the corresponding FA concentrations (10⁻²−10⁻⁹ M). The established linear responses ranging over a wide range of FA concentrations in Fig. 5(b) will be also favorable for the exact quantitative assessments of FA molecules in diverse applications. Then, the LOD value of FA molecules in the presence of SFF-Au/Ag NAs is achieved at nanomole (nM) level, which is also comparable with some interesting previous works based on Ag NPs/metal-organic framework hybrids [41], Au nanogratings [42] or GO/Ag NPs hybrids [43]. In order to further evaluate the long-term durability of flexible SFF-Au/Ag NAs, the corresponding NIR-SERS signals of FA molecules absorbed on the same SERS substrate were separately recorded within 7 days. In detail, the variation of Raman peak intensity of FA molecules at 1608 cm⁻¹ versus different delay days with respect to the initial stage is shown in Fig. 5(d). It reveals that the residual 82.4% SERS signal intensity can be retained after keeping them for 7 days, supporting that the substrate possesses excellent long-term durability toward SERS monitoring of FA molecules. Therefore, the established NIR-SERS biocompatible sensor has a great potential for rational diagnosis of FA level in the human body.

 

Fig. 5. (a) The NIR-SERS spectra of FA molecules with different concentrations (The scale bar is 10000 counts) absorbed on the obtained SFF-Au/Ag NAs. (b) The plots of the Raman peak intensities of FA at 683, 1194, 1251 and 1608 cm⁻¹ versus the logarithm concentrations of FA molecules. (c) NIR-SERS spectra of 10⁻⁵ M FA molecules absorbed on the same sample versus different delay days. (d) The variation of Raman peak of FA molecules at 1608 cm⁻¹ versus different delay days.

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Finally, the obtained SFF-Au/Ag NAs-based NIR-SERS sensor was also practically applied to the ultrasensitive monitoring of FA level in the human sweat. Before NIR-SERS assessment of FA molecules in real-world scenarios, the effect of pH variation on these tests should be evaluated in this work, since the exact pH value of a normal human body will be slightly changed in the range of 6.0-8.0 at different physiological states. In this way, the Raman spectra of 10⁻⁵ M FA molecules immersed into three groups of solutions with different pH values of 6.0, 7.0 and 8.0 are separately shown in Fig. 6(a). It can be seen that the effect of pH changes (6.0∼8.0) can be rationally ignored during the NIR-SERS test of FA molecules in the presence of SFF-Au/Ag NAs, since the Raman signals can be well reproduced in Fig. 6(a). Moreover, Fig. 6(b) shows the variations of SERS peak intensities of FA molecules at 1608, 1194 and 683 cm⁻¹ versus different three pH values. The maximal RSD value in Fig. 6(b) is less than 8.9%, implying that the receivable SERS tests of FA molecules in different pH solutions can be established in this work. Then, we recruited five volunteers and separately collected 10 µL sweat from each of them for further NIR-SERS detection of FA molecules in the presence of SFF-Au/Ag NAs. Then, the NIR-SERS signals of different five human sweats are shown in Fig. 6(c). Considering that the real sample of human sweat indeed contains many different chemical/biomolecular constituents, the Raman spectral lines of FA molecules in the range of 800∼1800cm⁻¹ inevitably become more complex in Fig. 6(c). Fortunately, the relatively isolated dominant characteristic band of FA molecules at 683 cm⁻¹ in Fig. 6(c) can be well maintained in the human sweats, which is consistent with that of pure FA in Fig. 6(a). In this work, the Raman signal of FA molecules at 683cm⁻¹ was selected to evaluate the FA level in the human sweat. According to the established linear relationship of Raman peak intensity of FA molecules at 683 cm⁻¹ (Fig. 5(b)), the quantitative calculations of FA concentration in different human sweat are illustrated in Table 1. The experimental results in this work illustrate that the slight FA fluctuation in different human sweats can be monitored by this NIR-SERS active sensor. On the other hand, it can be concluded that the concentration region of FA molecules in human sweat is located at 9∼13 nM (Table 1), which is also similar to the previous report [29]. Taken together, the established flexible hygroscopic NIR-SERS active sensor holds significant potential for ultrasensitive assessment of other biomolecules in many specific applications.

 

Fig. 6. (a) NIR-SERS spectra of 10⁻⁵ M FA molecules under different pH solutions (pH: 6.0, 7.0 and 8.0). (b) The corresponding Raman peak intensities of FA molecules at 683, 1194 and 1608 cm⁻¹ under different three pH conditions. (c) The NIR-SERS spectra of FA molecules in the different human sweats of five volunteers. (d) The NIR-SERS peak intensities of FA molecules at 683 cm⁻¹ derived from different sweats of five volunteers.

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Table 1. The parameters corresponding to FA of actual volunteers’ sweat

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4. Conclusions

In conclusion, an interesting flexible hygroscopic SFF-Au/Ag NAs-based SERS biocompatible sensor has been established for ultrasensitive monitoring of FA level in the human sweat. The hybrid SFF-Au/Ag NAs with a stronger absorption capacity in the range of 500-1100 nm and excellent hygroscopicity provide a remarkable higher 785 nm laser-excited NIR-SERS activity than that of bare urchin-like Au/Ag NAs. The corresponding LOD of R6G molecules can be achieved at ∼1.18 × 10⁻¹⁵ M (fmol/L) in this work, which is already better than many previous flexible SERS substrates. Furthermore, the as-prepared SFF-Au/Ag NAs-based SERS sensor has been practically applied to the ultralow detection of FA molecules with LOD as low as 10⁻⁹ M. Based on the established linear relationships between dominant characteristic bands of FA molecules and the corresponding wide concentrations (10⁻²–10⁻⁹ M), the slight FA fluctuation can be monitored in different human sweats. Thus, this novel NIR-SERS active sensor is highly attractive for real-time human health monitoring in the near future.