Highly uniform self-assembled monolayers of silver nanospheres for the sensitive and quantitative detection of glutathione by SERS

Article abstract of DOI:10.1039/d1dt01474a

The homeostasis and imbalance of glutathione (GSH), an important antioxidant in organisms, are one of the key signals that reflect the health of organisms. In this paper, a novel SERS sensing platform based on Ag film@Si that self-assembled using silver nanospheres was proposed, which was used for the highly sensitive and selective detection of GSH. With the aid of an oil/water/oil three-phase system, the nano-silver film was self-assembled and finally deposited on silicon wafers. The heterobifunctional crosslinking agentN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which contains pyridine rings and disulfide bonds, was involved in the exchange reaction between the sulfhydryl groups and disulfide bonds. With the addition of GSH, the breakage of disulfide bonds was promoted, thereby enhancing the SERS signal of SPDP. GSH can be detected sensitively by detecting the changes in the SPDP signal. The detection limit of GSH is 10 nM, and the method is still highly stable when the external environment is serum or other more complex environments.

Full text of DOI:10.1039/d1dt01474a

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Highly uniform self-assembled monolayers of
silver nanospheres for the sensitive and
quantitative detection of glutathione by SERS†
Cite this: Dalton Trans., 2021, 50,
10436
a
Cailing Jiang,a Feixiang Huang,*b Yi Chen *a and Li Jiang
The homeostasis and imbalance of glutathione (GSH), an important antioxidant in organisms, are one of
the key signals that reflect the health of organisms. In this paper, a novel SERS sensing platform based on
Ag film@Si that self-assembled using silver nanospheres was proposed, which was used for the highly
sensitive and selective detection of GSH. With the aid of an oil/water/oil three-phase system, the nano-
silver film was self-assembled and finally deposited on silicon wafers. The heterobifunctional crosslinking
agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which contains pyridine rings and disulfide
bonds, was involved in the exchange reaction between the sulfhydryl groups and disulfide bonds. With
the addition of GSH, the breakage of disulfide bonds was promoted, thereby enhancing the SERS signal of
SPDP. GSH can be detected sensitively by detecting the changes in the SPDP signal. The detection limit
of GSH is 10 nM, and the method is still highly stable when the external environment is serum or other
more complex environments.
Received 6th May 2021,
Accepted 23rd June 2021
DOI: 10.1039/d1dt01474a
rsc.li/dalton
exogenous and endogenous compounds.2 At present, the
global COVID-19 epidemic situation is still grim. Alexey
Introduction
Glutathione is a low-molecular-weight thiol tripeptide, divided Polonikov et al. have shown that with aging, endogenous gluta-
into oxidized (GSSG) and reduced (GSH) forms.1–3 Most gluta- thione deficiency may be an important reason for the severe
thione in cells is in the reduced form (>95%), and the normal symptoms and even death of COVID-19 patients.2,10 Therefore,
concentration ranges from 0.5 to 10 mM.2 As an important detecting the changes in the serum glutathione level is of great
antioxidant in vivo, it participates in many metabolic processes significance for early diagnosis of the disease.
and its homeostasis and imbalance have attracted widespread
The active group of glutathione is sulfhydryl (–SH), and the
attention.4 Reactive oxygen species are products of normal standard method for detecting the sulfhydryl group is with the
metabolism in the body. During the period of metabolism and aid of the sulfhydryl reagent DTNB, which yields 2-nitro-5-thio-
immune response, the level of glutathione will increase as the benzoate (TNB2−) through thiol–disulfide bond exchange with
level of reactive oxygen species increases. The dynamic balance GSH. The absorbance was measured at 412 nm to indirectly
between the two supports the adjustment of the redox system realize the quantitative determination of GSH.11 In addition,
and signal transmission.4–6 Compared to the normal breast varieties of methods for detecting glutathione have been pro-
tissue, the level of glutathione in breast tumors is significantly posed in recent years, like fluorometry,12–15 high performance
higher.7 But some diseases also seem to be worsened by lower liquid chromatography (HPLC),16,17 electrochemical analysis18,19
glutathione levels.4 Mitochondria, for example, are important or colorimetry.20–22 However, there are disadvantages such as
sources of reactive oxygen species, and damage to mitochon- high cost and complicated experiment procedures. It is still
drial function is usually related to glutathione depletion.4,8,9 worthwhile to develop low-cost, fast, and efficient methods.
In addition, glutathione levels are also significantly reduced in
With the unique optical properties of metal nanoparticles,
esophagus, stomach and liver tumors.7 Another function of combined with the molecular fingerprint information provided
glutathione that cannot be ignored is the detoxification of by Raman spectroscopy, the surface-enhanced Raman spec-
troscopy (SERS) technology has attracted widespread attention
in the field of biomolecular detection.11,23,24 SERS utilizes the
aCollege of Optical and Electronic Technology, China Jiliang University, Hangzhou
isoplasmon effect of metals25 (gold, silver, copper, etc.) to
310018, P. R. China. E-mail: chenyi@cjlu.edu.cn
bDepartment of TCM Gynecology, Hangzhou Women’s Hospital, Hangzhou 310018,
adsorb specific Raman signal molecules on or near their sur-
faces to enhance the standard Raman intensity of the mole-
P. R. China. E-mail: fxhuang2009@163.com
cules, and the enhancement effect is more significant in the
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1dt01474a
hot spots formed by the adjacent metal nanojunction or its
10436 | Dalton Trans., 2021, 50, 10436–10445
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edges.26–28 As an ultra-sensitive vibration spectrum technology, paper realized the quantitative and selective detection of gluta-
the two main advantages of SERS are that the signal is very thione. It has the advantages of being quick and clear, simple
strong and can realize the detection of a single molecule, fol- to prepare and environmentally friendly, which has provided a
lowed by great specificity.29 On the whole, the application of basis for practical application.
SERS can be divided into two categories: labeled and label-free
detection.30 Kuligowski et al. developed a label-free method for
detecting glutathione in whole blood using AgNPs as the SERS
Experimental section
substrate. The relationship between the GSH concentration
Chemicals and materials
and SERS signal was established by combining SERS and
Silicon (111) wafer (boron-doped) was bought from Kaihua
Shunchen Electronic Technology Co., Ltd. Ascorbic acid (AA), tri-
sodium citrate, citric acid and polyvinylpyrrolidone (PVP) were
purchased from Yuanye Biological Technology Co., Ltd
(Shanghai, China). Sodium hydroxide (NaOH), dichloromethane
(CH2Cl2) and n-hexane were purchased from Macklin
Biochemical Technology Co., Ltd (Shanghai, China). Silver
nitrate (AgNO3) was purchased from Xiya Reagent. DMSO,
chromatography, the sensitivity reached the limit when the
concentration of glutathione was 13 μM.31 In order to improve
the measurement accuracy, the research team combined
isotope labeling (IS) and SERS to realize the direct quantifi-
cation of GSH.24 Compared to label-free detection, the use of
isotope labeling for labeled detection improved the selectivity
and repeatability of SERS detection, whereas the cost was rela-
tively high and label processing was relatively complex. It is
well known that GSH, as a part of the cysteine residue, has a
small Raman cross section and a low Raman response.
Detecting glutathione without labeling is very difficult.
Therefore, more researchers have turned to labeled detection.
Wang et al. reported a SERS probe of 4-MP functionalized gold
flowers. When detecting ClO−, the SERS spectrum would
change and then GSH was added and the SERS spectrum
would recover the SERS spectrum of the original substrate, so
as to realize the simultaneous determination of ClO− and
GSH.32 This method realized the high-precision ratio detection
N-succinimidyl-3-(2-pyridyldithio)propionate
(SPDP),
L-homocysteine were purchased from Aladdin Biochemical
Technology Co., Ltd (Shanghai, China). Glutathione, glutamic
acid, D-glucose anhydrous, L-cysteine, L-histidine, L-lysine, and
L-glycine were purchased from Bomeibio (Hefei, China).
Phosphate buffered saline was purchased from TransGen
Biotech. All reagents were used without further purification.
High-purity water (18.2 MΩ cm) was used in all the experiments.
Synthesis and self-assembly of Ag film@Si
of the two, but the seed growth method was relatively strict. Silver nanoparticles were synthesized through the reduction
Based on the thiol–disulfide bond exchange reaction, Wei et al. method referring to the literature,36 where AgNO3 was typically
designed a PDEA-modified AgNPs@Si substrate to achieve the reduced by ascorbic acid. Concretely, 40 mL of aqueous solu-
specific detection of glutathione.11 However, there is still room tion containing ascorbic acid (0.6 mM) and trisodium citrate
for improvement in the synthesis of complex reagents.
(3 mM) was prepared and adjusted to different pH values (6.0,
The convenience and versatility of the self-assembled SERS 7.0, 8.0, 9.0, 10.0, or 10.5) by adding 0.2 M citrate acid or 0.1 M
substrate have attracted many researchers to devote themselves NaOH solution. 0.1 M AgNO3 solution (0.4 mL) was injected
to its research and development,33,34 especially the high port- into the mixed solution under a stirring speed of 900 rpm in a
ability of the liquid–liquid interface self-assembled SERS sub- 30 °C oil bath. The reaction solutions were observed to change
strate. Silver nanoparticles of various shapes including nano- from colorless to yellow and then brown (at pH 10.5) or blue
spheres, nanowires, nanorods, nanoflowers and so on are turbid (at pH 6.0, 7.0, 8.0, 9.0 and 10.0). After 15 min, no
widely used in the SERS sensor platform. In this paper, a new further change occurred, indicating that the reaction was com-
type of Ag film@Si SERS substrate was designed and plete. Then, it was heated at 80 °C for 2 hours to promote
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was used intraparticle maturation. A condenser was used to prevent the
as the Raman reporter to achieve the highly consistent and evaporation of the solvent. After being heated over and down
highly sensitive detection of GSH. Considering the perfect geo- to room temperature, the silver nanoparticles were transferred
metric symmetry of spherical nanoparticles35 and the local to a 50 mL centrifuge tube and stored (4 °C) for future assem-
electromagnetic field formed by the seamless contact between bly applications.
the edges of adjacent particles, spherical silver nanoparticles
Ag NP monolayer films were prepared according to the
can generate rich hot spots, enhance the SERS signal of target method reported by Lin et al.37 Firstly, 6 mL of nanoparticle
detection objects and improve the sensitivity. The self- solution was mixed with 9 mL of ultrapure water. After cen-
assembled spherical silver nanoparticles form a dense silver trifugation at 7500 rpm for 20 min, the precipitates were col-
film on the silicon wafer to form an SERS platform for solid lected and dispersed into 1 mL of PVP ethanol solution
phase detection, which improves the uniformity and repeat- (1 wt%, 5.5 W). Then, it was shaken vigorously and allowed to
ability, with low cost, simple synthesis, and portability. SPDP stay overnight. After centrifugation, the PVP-coated Ag NPs
is a heterobifunctional crosslinking agent with a pyridine di- were obtained and redispersed in 1 mL of ethanol. To assem-
sulfide group. The addition of glutathione promoted the ble the PVP-coated Ag NPs, 400 μL of PVP stabilized nano-
breaking of the disulfide bond, thereby enhancing the SERS particle solution and 800 μL of dichloromethane were mixed
signal of SPDP. The SERS sensor platform constructed in this with 2 mL of water in a 5 mL centrifuge tube. After shaking
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thoroughly and allowing to stand for a while, the centrifuge 1 μM and 2 μM. 0.1 mM SPDP was prepared by weighing SPDP
tube was slightly tilted and then 800 μL of n-hexane was added solids and dissolving them in 10 mL of DMSO. In order to
slowly. A silver nanofilm was then formed at the interface make the silver film fully contact the analyte, the silver film
between n-hexane and water. Then, n-hexane was slowly was soaked in a centrifuge tube containing 50 μL of SPDP for
extracted and the nanofilm was transferred onto the hydro- 2 hours and then taken out for further measurement. 0.1 mM
philic silicon wafers.
SPDP (50 μL) was incubated with different concentrations of
GSH (50 μL) for 2 hours at room temperature. Silver films were
soaked in the mixture for 2 hours and then taken out for
further measurements. In order to avoid any accidental
phenomena in the experiment, the experiment was repeated
several times. The selectivity was tested by comparing the
SERS signals of GSH and other amino acids with similar
chemical structures including glutamic acid, D-glucose anhy-
drous, L-cysteine, L-histidine, L-lysine, L-glycine and
L-homocysteine.11 A solution of 1 μM of different amino acids
was prepared by weighing the corresponding solids and dissol-
ving them in 10 mL of PBS. Each amino acid solution was
mixed and incubated with SPDP for two hours, and then SERS
measurement was carried out as described above.
Instruments
The UV-vis absorption spectra of a 1 : 10 dilution of the silver
nanoparticles with high-purity water were recorded using a UV
spectrometer (TU-1901, PERSEE, Beijing). The morphological
features of the Ag NP monolayer film were detected by a field-
emission scanning electron microscope (SEM, Hitachi
SU-8010). The Raman spectra were recorded by a confocal
micro-Raman spectrometer (Horiba LabRAM Evolution 800) at
a wavelength of 532 nm using a solid state laser. A microplate
reader (FlexA-200, ALLSHENG, Hangzhou) was used to dynami-
cally measure the absorbance.
Surface enhanced Raman spectroscopy measurement
The standard stock solution of GSH (0.1 mM) was prepared by
dissolving 0.307 mg of GSH solids in 10 mL of PBS. Then, it
was gradually diluted to the required series of concentrations
with the same solvent and stored at 4 °C for further use. In
Results and discussion
Characterization and evaluation of the Ag film SERS platform
order to obtain the linear dynamic range and detection limit Ag film@Si is used to establish the SERS sensor platform by
of the detection method, a gradient concentration of GSH was the self-assembly of the synthesized silver nanoparticles onto
prepared, including 10 nM, 25 nM, 50 nM, 100 nM, 500 nM, silicon wafers. Fig. 1A is an SEM image of colloidal silver nano-
Fig. 1 (A) The SEM image of silver nanoparticles randomly dispersed on a silicon wafer. (B) The ultraviolet–visible absorption spectrum of colloidal
silver nanoparticles. (C) The SEM image of the self-assembled Ag film@Si. (D) The histogram of particle size distribution of the monolayer silver film.
10438 | Dalton Trans., 2021, 50, 10436–10445
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particles, the shape of the silver nanoparticles was spherical distribution of the synthesized silver nanoparticles is uniform.
and the diameter distribution was relatively uniform, about In order to evaluate the SERS performance of the Ag film@Si
30–50 nm. The UV-vis absorption spectra of the colloidal silver substrate, the SERS signals of SPDP were detected at an exci-
nanoparticles are shown in Fig. 1B, which indicate that the tation wavelength of 532 nm. Fig. 2A shows the Raman signal
AgNPs have an absorption peak roughly located at 402 nm and of solid SPDP (Fig. 2A(a)) and SERS signal enhanced by Ag
the half-peak width is 62 nm, which indirectly reflects the film@Si (Fig. 2A(b)). The band at 529 cm−1 is attributed to the
slight difference in particle size among the nanoparticles. The disulfide bond vibrations39 and the main vibrations at 985,
results are consistent with other studies38 on the synthesis of 1046, 1085 and 1116 cm−1 are all from the pyridine ring.11,38
silver nanoparticles, indicating that silver nanoparticles were Since SPDP contains disulfide bonds, it will interact with the
successfully synthesized.
silver surface.40–42 The SPDP solution was tested under the
The process of the self-assembly of silver nanoparticles same conditions, as shown in Fig. 2A(b), the main vibration at
refers to the three-phase oil/water/oil system proposed by Lin 1000, 1049, 1084 and 1121 cm−1 are from the pyridine
et al.,37 which overcomes the shortcomings of uneven sub- ring.11,38 Moreover, the Ag film@Si greatly enhanced the SERS
strates due to the irregular aggregation of silver nanoparticles. signal of SPDP, which reflects the excellent enhancement per-
The specific description is shown in Scheme 1. The PVP- formance of the self-assembled SERS sensor platform. The
coated Ag NPs, dichloromethane and water were successively uniformity of the substrate was evaluated by a thermodynamic
added to
a 5 mL centrifuge tube. After being shaken chart and relative standard deviations of the mapping area.
thoroughly and left standing, a tight accumulation layer was The size of the mapping area was 16 μm × 16 μm, the scanning
formed at the oil/water interface composed of dichloro- step was 1 μm. Fig. 2 shows the content of the mapping spec-
methane/water. The centrifuge tube was properly inclined and trum of the SPDP molecules at a concentration of 0.1 mM, the
another layer of oil-phase n-hexane was added. Since water is intensity heat map (Fig. 2B) and 3D waterfall diagram (Fig. 2C)
more dense than n-hexane, the Ag NPs spontaneously rise to indicate the high uniformity of the SERS signals. In order to
the water/n-hexane interface to form a bright yellow metal film quantitatively assess the uniformity of the SERS signal, the
with the help of oil–water interfacial tension. After n-hexane relative standard deviation (RSD) of the intensity at 1551 cm−1
was sucked out, the Ag film was loaded onto the silicon wafer. in the mapping area was calculated which was 8.41% (Fig. 2D),
From the SEM image of the Ag film@Si, as shown in Fig. 1C, indicating the uniformity of the Ag film@Si SERS sensor plat-
the distribution of Ag film is uniform. The particle size statisti- form in a wide range. Simultaneously, in order to explore the
cal distribution of the Ag film is shown in Fig. 1D. The average repeatability of the SERS platform, the SERS signals of SPDP
diameter of the Ag NPs is 41.91 7.1 nm and the particle size on five different batches of Ag film@Si were compared. For
Scheme 1 (A) Schematic diagram of the Ag film@Si SERS platform detecting GSH. (B) The chemical structure diagram of the reaction process of
SPDP and GSH.
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Fig. 2 (A) (a) The Raman spectrum of SPDP powder, (b) the SERS spectrum of 0.1 mM SPDP on Ag film@Si. (B) The intensity heat map of the SERS
spectrum of 0.1 mM SPDP; the size of mapping area was 16 μm × 16 μm. (C) The SERS spectrum of SPDP scanned by multiple points on Ag film@Si.
(D) The 3D histogram of SERS intensity corresponding to the mapping area.
details, see the ESI (Fig. S1†). The RSD value of 9.64% at SPDP is increased from 10−7 M to 10−3 M, the SERS signals are
1000 cm−1 is calculated quantitatively, indicating that the continuously enhanced and they reach saturation when the
SERS platform had good repeatability.
concentration was higher than 10−4 M. Therefore, it was deter-
mined to use 0.1 mM SPDP for subsequent experiments.
Among them, the SERS spectrum of the solvent DMSO is
SERS detection of glutathione
N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) is a hetero- shown in Fig. 3A(a), with its characteristic Raman peak at
bifunctional crosslinking agent with amine and sulfhydryl reac- 677 cm−1, which is attributed to the C–S stretch vibration.46
tivity, which consists of N-hydroxysuccinimide ester (NHS) and Fig. 3B(b) shows the SERS spectra of 0.1 mM SPDP tested
pyridine disulfide groups and disulfide bonds in its spacer arm. under the same conditions after one month interval. There is
Fig. S2† shows the process of the chemical reaction with the aid little difference in the SERS intensity before and after the com-
of the chemical structure. Fig. S2A† indicates that when the di- parison, indicating the high stability of the substrate. Fig. 4A
sulfide group of SPDP reacts with thiol, the disulfide bond will compares the SERS intensity of SPDP with or without gluta-
be broken. The chemical structure in Scheme 1 shows the reac- thione. After SPDP was incubated with GSH for two hours, the
tion process, and a detailed description is also given in the reduced glutathione with active sulfhydryl groups breaks the
ESI.† Reduced glutathione is one of the reduced thiols, and the disulfide bonds of SPDP. Pyridine-2-mercapto was replaced
exchange reaction of the thiol disulfide bond can promote the and combined with the silver nanoparticles through thiol–
breaking of the disulfide bond (Scheme 1B). The pyridine di- silver bonds,42 thus greatly enhancing the SERS signal of
sulfide group of SPDP and the sulfhydryl group of glutathione SPDP. In order to explore the uniformity of the SERS platform
undergo the exchange reaction of the sulfhydryl disulfide bond at this time, we set a range of 16 μm × 16 μm for point-by-
to generate the NHS ester with the sulfhydryl group and pyri- point scanning to record the mapping spectrum in the pres-
dine-2-mercapto or pyridine-2-thione,43,44 which will be bound ence of 1 μM GSH. Fig. 4B and C, respectively, show the inten-
with silver through an Ag–S bond.45
sity heat map and 3D waterfall map of the mapping area; the
Through the research of Kuligowski et al., we have learned RSD of the SERS intensity at 1000 cm−1 in this area is found to
that the Raman scattering cross section of glutathione is very be 7.65% through quantitative calculation, which comprehen-
small,24,31 which makes it difficult to carry out label-free detec- sively showed that the existence of GSH did not affect the uni-
tion and has low sensitivity. Therefore, here SPDP is used as formity of the substrate and further reflected the high uni-
the Raman reporter molecule. Scheme 1 shows the procedure formity of the substrate.
of the indirect detection of glutathione with the aid of this
In order to evaluate the sensitivity of the Ag film@Si SERS
molecule. As shown in Fig. 3A, when the concentration of platform, a series of prepared concentrations of GSH and
10440 | Dalton Trans., 2021, 50, 10436–10445
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Fig. 3 (A) The SERS spectra of different concentrations of SPDP on Ag film@Si, from a to f are 0 M, 10−7 M, 10−6 M, 10−5 M, 10−4 M, and 10−3 M. (B)
(a) The SERS spectrum of 0.1 mM SPDP on Ag film@Si and (b) the SERS spectrum of (a) after one month of storage.
Fig. 4 (A) (a) The SERS spectrum of 0.1 mM SPDP on Ag film@Si and (b) the SERS spectrum of SPDP on the surface of Ag film@Si after mixing and
incubating with 1 μM GSH. (B) The heat map of the SERS intensity of SPDP after mixing and incubation with 1 μM GSH; the size of the mapping area
was 16 μm × 16 μm. (C) The SERS spectrum of multi-point scanning in the mapping area. (D) The 3D histogram of the SERS intensity corresponding
to the mapping area.
SPDP were mixed and incubated for two hours, and the tive Raman intensity tended to saturate. As can be seen from
measured SERS spectra are shown in Fig. 5A. It is not difficult the vignette of Fig. 5B, when the concentration of GSH is in
to find that when the concentration of GSH gradually the range of 10–500 nM, the relative intensity of Raman peak
increased (Fig. 5A(b–i)), the SERS intensity of SPDP is also at 1000 cm−1 is linearly related to GSH concentration, with an
increasing (that is, positive correlation) and higher than that R2 value of 0.9918. At the same time, Fig. 5C(a) and (b) show
of SPDP without GSH (Fig. 5A(a)). Here, the SERS intensity of the SERS spectrum of the newly prepared Ag film@Si and the
the characteristic peak located at 1000 cm−1 is counted for substrate after one month of storage for the detection of 1 μM
further analysis. The relative Raman intensity (ISPDP
/
GSH, respectively. It can be seen that the SERS intensity values
+
GSH
I
SPDP) is obtained through quantitative calculation, and the had little difference, which showed the high stability of the
correlation between it and the concentration of GSH is shown SERS platform. In addition, the interference of adding GSH on
in Fig. 5B. The results show that the relative Raman intensity the repeatability of the SERS platform was also explored, and
at 1000 cm−1 increases with the increase in GSH concen- the SERS signals on the surface of Ag film@Si of five different
tration. When the concentration is higher than 1 μM, the rela- batches of silver films were compared. For details, see the ESI
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Fig. 5 (A) The SERS spectra of SPDP after incubation with different concentrations of GSH, from a to i are 0 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100
nM, 250 nM, 500 nM, 1000 nM, and 2000 nM. (B) Count of the relative Raman intensity change curve of SPDP at 1000 cm−1 after incubation with
different concentrations of GSH. (C) The linear plot of the relative Raman intensity at 1000 cm−1 in the GSH concentration range of 10 nM to 500
nM. (D) (a) The SERS spectrum of SPDP after incubation with 1 μM GSH and (b) the SERS spectrum of (a) after standing for one month.
(Fig. S1†). An RSD value of 9.64% at 1000 cm−1 was calculated test conditions remain the same as before. Fig. 6A(b–h) shows
quantitatively, indicating that the SERS platform had good the SERS signals of SPDP after adding seven different amino
repeatability.
acids, respectively. The original SERS signals of SPDP are
In order to further evaluate the SERS detection performance shown in Fig. 6A(i). Fig. 6A(a) shows the SERS spectrum when
of Ag film@Si for GSH, the above test results were compared 1 μM GSH was added. Compared to the SERS spectra of other
with other research results, as shown in ESI Table S1.† The amino acids, the intensity is significantly greater. In order to
results show that the cost analysis of liquid phase detection is quantitatively evaluate the interference degree of other amino
relatively high, the time is longer and the sensitivity is low. The acids on the SERS platform, the SERS intensity value with the
solid phase detection method used in this paper is simple and characteristic peak located at 1000 cm−1 was statistically ana-
quick to prepare, easy to transport and carry, and has high sen- lyzed. The relative intensity obtained is shown in the histo-
sitivity. The SERS detection method based on Ag film@Si is a gram of Fig. 6B. With the addition of GSH, the relative Raman
reliable, rapid and sensitive method for glutathione evaluation.
intensity of SPDP expanded by 3.48 times, which is higher
than the relative Raman intensity of SPDP caused by other
similar amino acids. Therefore, the SERS platform can realize
Selectivity and practical application of Ag film@Si
Serum is the gelatinous liquid of plasma after fibrinogen has selective detection of GSH.
been removed, which provides essential substances for cell
In order to explore the possibility of practical applications
growth, such as amino acids, vitamins, and lipids. To explore of this method, real serum samples were selected as the exter-
the selectivity of the indirect detection of GSH by the above nal environment for GSH. Normal human serum was pre-
SERS platform, the response of other metabolites with similar treated first and then 0.1 mM SPDP and 1 μM GSH were pre-
chemical structures, such as glutamic acid, glycine, histidine, pared with serum as the solvent. Then the two were mixed and
lysine, glucose, homocysteine and cysteine, on the SERS plat- incubated for two hours, and SERS detection was carried out
form was considered. The abovementioned various amino according to the above method. Fig. 7A(a) shows the SERS
acids (1 μM) were mixed and incubated with SPDP, and SERS spectrum of SPDP in serum, Fig. 7A(b) shows the SERS spec-
determination was carried out according to the same steps. All trum of 0.1 mM SPDP in serum containing 1 μM GSH, Fig. 7A
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Fig. 6 (A) Similar amino acid (from a to h are GSH, Cys, Hcy, Lys, His, Gly, glu, and glucose) SERS spectra of SPDP after incubation and (i) the SERS
spectra of SPDP. (B) The relative Raman intensity histogram of SPDP at 1000 cm−1 after incubation with similar amino acids.
Fig. 7 (A) (a) The SERS spectrum of 0.1 mM SPDP in serum, (b) the SERS spectrum of 0.1 mM SPDP in serum containing 1 μM GSH, (c) the SERS
spectrum of 0.1 mM SPDP diluted with PBS, and (d) the SERS spectrum of 1 μM GSH dissolved in PBS and 0.1 mM SPDP mixed and incubated. (B)
The histogram of the relative Raman intensity of SPDP at 1000 cm−1
.
(c) shows the SERS spectrum of 0.1 mM SPDP diluted with
PBS, and Fig. 7A(d) shows the SERS spectrum of 1 μM GSH dis-
Conclusions
solved in PBS and 0.1 mM SPDP after mixed incubation. It is In this paper, a novel SERS sensing platform based on simply
obvious that there is a little noise in the spectrum of SPDP in synthesized and self-assembled Ag film@Si is proposed for the
the serum, but the spectral intensity has not changed much, detection of GSH with high sensitivity and selectivity. With the
and the characteristic Raman peak position has not changed. aid of the oil/water/oil three-phase system, silver nanofilms
In order to quantitatively evaluate the interference degree of were self-assembled and finally deposited on a silicon wafer.
the external environment, the relative Raman intensity of the The heterobifunctional crosslinking agent SPDP, which con-
statistical characteristic peak at 1000 cm−1 is plotted in a histo- tains pyridine rings and disulfide bonds, was involved in the
gram as shown in Fig. 7B. The relative Raman intensity in exchange reaction between the sulfhydryl groups and disulfide
serum at 1000 cm−1 is 3.25, which is slightly lower than that in bonds. With the addition of GSH, the breakage of disulfide
PBS. Thus, it can be seen that the SERS sensing platform bonds was promoted, thereby enhancing the SERS signal of
designed by this scheme has the possibility of practical appli- SPDP. By detecting the change in the SERS signal of SPDP,
cations, which provides a basis for the quantitative detection GSH can be detected sensitively. The minimum detection sen-
of glutathione. In addition, the results were compared with sitivity of GSH is 10 nM, and a good linear relationship of GSH
those of DTNB colorimetry, as detailed in ESI and Fig. S3.† is found in the range of 10–500 nM. Simultaneously, Ag
After 90 min of reaction, the absorbance value of the serum film@Si also shows good uniformity and the relative standard
containing 1 μM GSH exceeds the maximum value of the stan- deviation of the SERS intensity is about 7.65%. When other
dard value. At this time, the DTNB colorimetric method may similar amino acid interferences were explored, the SERS plat-
have failed. The SERS detection method based on Ag film@Si form could realize the selective detection of GSH. Considering
can avoid the possible data deviation caused by enzyme activity the clinical detection of GSH levels when the external environ-
damage and has high sensitivity. Therefore, when the external ment is serum or other more complex environments, the
environment becomes more complicated, the SERS platform DTNB colorimetry reached its limit at 1 μM GSH. Moreover,
can provide a detection possibility for actual detection.
this method is susceptible to enzyme activity damage and has
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poor stability. Therefore, the method proposed in this paper 17 L. Wei, Y. Song, P. Liu and X. Kang, Mikrochim. Acta, 2018,
has the prospect of highly sensitive and quantitative detection
of GSH.
185, 321–328.
18 Y. Wang, L. Jiang, L. Chu, W. Liu, S. Wu, Y. Wu, X. He
and K. Wang, Biosens. Bioelectron., 2017, 87, 459–
465.
Conflicts of interest
19 Z. Wang, P. Han, X. Mao, Y. Yin and Y. Cao, Sens. Actuators,
B, 2017, 238, 325–330.
20 Y. Xianyu, Y. Xie, N. Wang, Z. Wang and X. Jiang, Small,
2015, 11, 5510–5514.
There are no conflicts to declare.
21 V. Kumar, D. Bano, D. K. Singh, S. Mohan, V. K. Singh and
S. H. Hasan, ACS Sustainable Chem. Eng., 2018, 6, 7662–
7675.
22 Z. Chen, L. Chen, L. Lin, Y. Wu and F. Fu, ACS Sens., 2018,
3, 2145–2151.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China under Grant No. 51972292 and the
Fundamental Research Funds for the Provincial Universities of
Zhejiang under Grant No. 2020YW09.
23 X. Ji, W. Wang, X. Li, Y. Chen and C. Ding, Talanta, 2016,
150, 666–670.
24 Á. Sánchez-Illana, F. Mayr, D. Cuesta-García, J. D. Piñeiro-
Ramos, A. Cantarero, M. D. L. Guardia, M. Vento, B. Lendl,
G. Quintás and J. Kuligowski, Anal. Chem., 2018, 90, 9093–
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