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Article
A SERS-Fluorescence Dual-Mode pH Sensing
Method Based on Janus Microparticles
Shuai Yue, Xiaoting Sun, Ning Wang, Yaning Wang, Yue
Wang, Zhangrun Xu, Ming-Li Chen, and Jian-Hua Wang
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13321 • Publication Date (Web): 24 Oct 2017
Downloaded from http://pubs.acs.org on October 26, 2017
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ACS Applied Materials & Interfaces
A SERS-Fluorescence Dual-Mode pH Sensing
Method Based on Janus Microparticles
Shuai Yue, Xiaoting Sun, Ning Wang, Yaning Wang, Yue Wang, Zhangrun Xu*,
Mingli Chen, Jianhua Wang
Research Center for Analytical Sciences, Northeastern University, Shenyang 110819,
P.R. China.
ABSTRACT: A surface enhanced Raman scattering (SERS)-fluorescence dual-mode
pH sensing method based on Janus microgels was developed, which combined the
advantages of high specificity offered by SERS and fast imaging afforded by
fluorescence. Dual mode probes, pH dependent 4-mercaptobenzoicacid and carbon
dots, were respectively encapsulated in the independent hemispheres of Janus
microparticles fabricated via a centrifugal microfluidic chip. Based on the obvious
volumetric change of hydrogels in different pH, the Janus microparticles were
successfully applied for sensitive and reliable pH measurement from 1.0 to 8.0, and
the two hemispheres showed no obvious interference. The proposed method
addressed the limitation that sole use of the SERS-based pH sensing usually failed in
strong acidic media. The gastric juice pH and extracellular pH change were measured
respectively in vitro using the Janus microparticles, which confirmed the validity of
microgels for pH sensing. The microparticles exhibited good stability, reversibility,
biocompatibility and ideal semipermeability for avoiding protein contamination, and
they have the potential to be implantable sensors to continuously monitor pH in vivo.
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KEYWORDS: Dual-mode pH sensing, SERS, Fluorescence, Janus microparticles,
Gastric juice pH, Extracellular pH
█ INTRODUCTION
The pH sensors have been widely used because accurate pH sensing is always
important in various fields of scientific research and technology, such as disease
diagnosis,1 environmental analysis,2 chemical process control3 and so on.
Electrochemical and optical pH determinations are the most common methods for
precise pH measurement. Although electrochemical pH sensing is a reliable tool for
many analytical tasks, it suffers disadvantages in big size, rigid design, mechanical
fragility and is especially not suitable for small volume sample measurements.4
Optical pH sensor that is miniaturized to micro/nano size can provide an alternative
solution for the situation where electrode is inadaptable. Over the past decades,
optical pH sensors based on SERS and fluorescence have attracted great interests due
to their high sensitivity, good biocompatibility, pH reversibility, small size, low cost,
mass production, continuous measurements and in vivo monitoring. A SERS pH
sensor is usually realised by labeling the pH-sensitive Raman active molecules, such
as
2-aminothiophenol
(2ATP),5
4-mercaptobenzoicacid
(4MBA)6
and
p-aminothiophenol (pATP)7 on Ag and Au nanoparticles and measuring their Raman
signal transformation in different pH environments. However, it is worth noting that
the traditional SERS pH sensing usually fails in acidic medium where the pH is below
approximately 5.5,8 which is caused by the probe aggregation. Besides, high ionic
strength medium9 and complex biological system10 also lead to the unreliable SERS
detection. Moreover, compared with fluorescence imaging, SERS imaging is much
slower.11 The fluorescence pH sensors, usually using quantum dots,12 carbon dots,13
silicon nanoparticles,14 fluorescent proteins15 and organic molecules16 as probes, have
been applied to measure pH, however they are always hampered by photo-instability
and vulnerability in the harsh chemical environment.
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The embedding of functional nanoparticles within stimuli-responsive hydrogels has
attracted wide interest, as it provides a new generation of sensors. These sensors are
equipped with a synergistic enhancement of each component performance: the
improvement of hydrogel mechanical strength and reduction of nanoparticle
aggregation.17 After interaction with target analytes, the stimuli-responsive hydrogels
undergo an obvious volumetric change, which have shown superior functions in
diverse sensing fields, including fluorescence,18-19 SPR20 and photonic21 hydrogel
sensors. Enormous efforts have been made toward encapsulating fluorescent
nanoparticles into responsive hydrogels, and various highly sensitive, highly selective
and continuous fluorescence sensing methods based on responsive hydrogels were
proposed, such as glucose sensing,18 pH sensing,19 temperature sensing,22 lactate
sensing23 and so on. In recent years, smart hydrogels encapsulating AuNPs or AgNPs
have gained considerable attention in SERS,24-25 which combined responsive behavior
and optical property, offering the reversible plasmon coupling and hot spot effect. As
we know, SERS possesses high photostability and molecular recognition ability over
fluorescence, while fluorescence imaging is much faster than SERS. Thus
SERS-fluorescence dual-mode method26-28 is promising in quick imaging and reliable
target sensing. Embedding SERS and fluorescence nanoparticles into different
compartments of hydrogels perhaps opens a new prospect for developing dual-mode
sensing methods.
This study aims to address the limitation of traditional SERS pH sensing which is
unreliable below pH 5.5 and provide a dual-mode pH sensing method. Here, we
fabricated a Janus microgel via a centrifugal microfluidic device29-30 for
SERS-fluorescence dual-mode pH sensing. To combine their individual advantages,
the labeled AuNPs and carbon dots (CDs) were separately encapsulated into the two
hemispheres of Janus microgels. On the basis of the tunable plasmon coupling and the
ratio of carboxyl/aromatic Raman bands intensity, SERS-based pH sensing approach
was constructed. Fluorescent microgels proved to be capable of pH detection based on
pH responsiveness of both CDs and microgels. We investigated pH sensing
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performances of SERS and fluorescence single-phase microgels, and then applied the
Janus microgels to measure pH. We also systematically investigated their
reversibility, photostability, biocompatibility and size selectivity. The Janus microgels
were successfully applied to detect gastric juice pH and monitor extracellular pH.
█ MATERIALS AND METHODS
Materials. Sodium alginate (low viscosity) and FITC (wt 389) were purchased
from Sigma-Aldrich Corporation. Calcium chloride (96%), trisodium citrate
dihydrate, chloroauric acid (HAuCl4·4H2O), dimethyl sulphoxide (DMSO),
phosphoric acid (85%), boric acid, acetic acid (98%), and sodium hydroxide (96%)
were purchased from Sinopharm Chemical Reagent Corporation, Shanghai, China.
Carboxymethyl cellulose (CMC, M.W. 90000) were purchased from Aladdin
Industrial Corporation, Shanghai, China. 4-Mercaptobenzoic acid (4MBA) was
purchased from Shanghai Macklin Biochemical Co., Ltd. Polysorbate 20 was
purchased from Alfa Aesar Corporation, Tianjin, China. Bovine serum albumin
(BSA) was purchased from Beijing Ordered Star Biological Technology Co., LTD.
FITC-tagged BSA (wt 68,000) was purchased from Beijing NobleRyder Technology
Co., Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
purchased from KeyGEN BioTECH. The Britton-Robinson (BR) buffer solutions
with pH values from 1.0 to 8.0 were prepared by mixing 0.04 M phosphoric acid, 0.04
M boric acid and 0.04 M acetic acid, and then adjusting pH by using 0.2 M NaOH.
All the above chemical reagents were used as received without further purification.
Deionized water was used throughout the experiments.
Microfluidic Synthesis of Nanoparticle-embedded Monophasic and Janus
Hydrogel Particles. AuNPs were synthesized using the classical Frens method.31 100
mL of 0.01% (w/v) HAuCl4 aqueous solution was stirred and heated to boiling, and 1
mL of 1% (w/v) sodium citrate solution was quickly added to HAuCl4 boiling
solution. The refluxing lasted for 15 min, and then the solution was cooled to room
temperature and reserved for use. Before the microparticle fabrication, 200 µL of 0.05
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mM 4MBA and 200 µL of 2% (w/v) BSA aqueous solutions were added into 10 mL
of as-prepared colloidal gold under stirring to obtain the stable labeled AuNPs
(AuNPs-(4MBA)-BSA),10 which were concentrated to 1 mL subsequently. 0.04g
sodium alginate and 0.03g CMC were together dissolved into the 1 mL concentrated
colloidal gold. Afterwards, the mixture was injected into eight radial channels on the
microfluidic chip, and 10 wt% CaCl2 solution with 0.5 wt% polysorbate 20, serving as
crosslinking agent, was introduced into circumjacent chambers opposite the channel
outlets. The centrifugal platform was offered by a spin coater on which the chip was
mounted and fixed by the vacuum chuck. The chip was then centrifuged at 4900 rpm
for 90s. The microgels containing labeled AuNPs were formed quickly in CaCl2
solutions. Thus the SERS pH sensing microgels were produced. The resultant
microparticles can be used as SERS pH sensors directly after washing away CaCl2
with water for three times. The fluorescence microparticles were produced via the
same method, except that the labeled AuNPs were replaced by CDs. For Janus
microgel fabrication, the two aforementioned solutions with labeled AuNPs and with
CDs were respectively injected into a pair of adjacent channels and then centrifuged
as above. Figure 1A shows the schematic diagrams of the chips for the fabrication of
single-phase and Janus microparticles. And the pH-induced swell and shrink of
single-phase and Janus microparticles encapsulating labeled AuNPs and CDs were
shown in Figure 1B.
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Figure 1. (A) Schematic illustrations of fabrication of single-phase microparticles (upper left) and
Janus hydrogel microparticles (lower left) using the centrifugal microfluidic chips. (B) pH-induced
swell and shrink of single-phase hydrogel microparticles encapsulating AuNPs-(4MBA)-BSA (upper
right), single-phase hydrogel microparticles encapsulating carbon dots (middle right), and Janus
hydrogel microparticles encapsulating AuNPs-(4MBA)-BSA and carbon dots respectively in duals
semispheres (lower right).
pH Sensing Based on Microparticles. To investigate their pH sensing
performances, the microparticles were incubated with BR buffer with various pH
values (from 1.0 to 8.0) for 15 min before measurement. The SERS measurements
were conducted using a confocal Raman microscope (XploRA ONE, Horiba Jobin
Yvon, France) with an excitation wavelength of 638 nm, a 10 × objective and a high
resolution grating (1200 cm-1). The signals were acquired for 10 s. The fluorescence
imaging was conducted by using a confocal laser-scanning fluorescence microscope
(CLSM, FV1200, Olympus, Japan) and then analyzed by Image J software (National
Institute of Mental Health, USA). A stereomicroscope (Stemi 2000-C, Zeiss,
Germany) was used to capture the bright field images of the microparticles.
Gastric Juice pH Sensing and Extracellular pH Monitoring. A gastric juice
sample was collected from a volunteer patient from Center Hospital of Liaoning
Electric Power. The extracellular pH monitoring and cytotoxicity were studied based
on Michigan Cancer Foundation-7 cancer cells (MCF-7). When the cells spread to
about 70% of the bottom of the culture bottle, the culture medium was renewed.
Thereafter, 100 µL of the culture media were respectively taken out after 0 h, 12 h, 24
h and 36 h for the following pH analysis. The Janus microparticles were measured
after 15 min incubation in the culture media taken out.
█ RESULTS AND DISCUSSION
SERS pH Sensing Using pH Sensitive Hybrid Microparticles Encapsulating
AuNPs-(4MBA)-BSA. As one of the important natural polysaccharide hydrogels,
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alginate has many carboxyl groups acting as anionic pH-responsive moieties.
Carboxyl groups are protonated at low pH and deprotonated at high pH, which makes
the microgels capable of shrinking at low pH and swelling at high pH. The pure
alginate microparticles will gradually crack and dissolve at pH 8.0. In the present
work, another anionic pH responsive hydrogel CMC was added to enhance the
stability of the microgels in alkalescent medium. To demonstrate their pH
responsiveness, the monodisperse microgels were incubated in BR buffer solutions
(pH from 1.0 to 8.0) for 15 min, and images of the microgels were collected by a
stereomicroscope, as shown in Figure 2. We observed that the particle size increased
sharply with the increase of pH, which proved that they had obvious pH
responsiveness. Although the microgels seemed a little wrinkled at pH 8.0 due to the
swell, the pH detection was not influenced. Besides, color gradual variations with
different pH are due to the change in surface plasmon resonance absorption, which is
caused by the increasing spatial distance between gold nanoparticles.32 The distance
between metal nanoparticles is important for SERS detection sensitivity, because the
coupling of the surface electromagnetic fields between two or more nanoparticles is
the main source of SERS, thus hot spot effect will be enhanced or reduced with the
pH stimulation.
Figure 2. Optical microscopic images of single-phase hydrogel microparticles encapsulating
AuNPs-(4MBA)-BSA after 15 min incubation respectively in BR buffer solutions with pH ranging
from 1.0 to 8.0. Scale bars are 200 µm.
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The SERS spectra of 4MBA in different pH mediums are shown in Figure 3A. The
two prominent peaks at 1077 cm-1 and 1585 cm-1 are assigned to the breathing
vibration of aromatic ring. The other important peak at about 1395 cm-1 is attributed
to -COO- stretching vibration, which is pH sensitive. The calibration curve of SERS
pH sensing was obtained based on the peak intensity at 1077 cm-1 with pH ranging
from 1.0 to 8.0 (Figure 3B). SERS signals were enhanced with pH values decreasing
from 8.0 to 1.0, especially within the range of 3.0 to 1.0. Interstitial crevices between
metal nanoparticles are widely known as hot spots, which can provide extraordinary
enhancement for SERS signals. The pH-sensitive hydrogels can swell or shrink
according to pH change, thus altering interparticle distance and SERS enhancement.
The pKa of alginate and CMC are about 3.2, so -COO- and COOH can transform
reversibly at this critical point. When pH is below 3.2, hydrogen bond interactions
between COOH groups lead to a significant shrink, creating more hot spots and
enhancing the 4MBA Raman signals further. On the contrary, microparticles swell
significantly due to electrostatic repulsion between the -COO- groups as pH increases.
Except for the pH-responsive microgels, pH sensitive 4MBA probes in microgels
were also used for SERS pH detection. When spectra at different pH were normalized
to the peak at 1077 cm-1, we noticed the peaks at 1395 cm-1 increased gradually with
pH increase due to the deprotonation of COOH, which was in agreement with the
previous report,6 as shown in Figure 3C. So, another calibration curve of SERS pH
sensing was obtained based on the intensity ratio of the peaks at 1395 cm-1 and 1077
cm-1 with pH ranging from 1.0 to 8.0, as shown in Figure 3D. The ratio increased
significantly within the pH range from 3.0 to 8.0, which is not consistent with the pKa
5.9 of 4MBA. This is mainly because 4MBA molecules were surrounded with
hydrogel and BSA, and more effective protons were needed to give rise to the
transformation between -COOH and -COO-.6 And the transformation between
–COOH and -COO- in hydrogels at pH 3.2 also was exhibited in spectra. The two
SERS pH sensing calibration curves were established on the basis of different
mechanisms and showed complementary sensitive ranges, 1.0 to 3.0 and 3.0 to 8.0
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respectively. Thus, more accurate results can be obtained by choosing the appropriate
calibration curve for different pH measurements. Additionally, owing to the
-COOH/-COO- reversibility with pH, it is expected that the Raman peak intensity at
1077 cm-1 and intensity ratio of Raman peak at 1395 cm-1 and 1077 cm-1 would be
reversible. The expectation was proved by measuring the intensities and ratios under
pH 3.0 and 7.5 for 5 cycles, as displayed in Figure S1. The results indicated the pH
sensing microgels have promising potential for continuous pH monitoring. In
conclusion, the SERS microgel offers the following advantages in pH sensing. First, it
is stable and usable in strong acidic medium, overcoming the disadvantage of
traditional SERS pH sensors which often fail in the medium with pH below about 5.5.
Second, two calibration curves with complementary sensitive ranges were established
for more reliable measurements. Third, the microgels were reversible with pH change,
which is valuable in real-time pH monitoring.
Figure 3. (A) pH dependent SERS spectra of microparticles encapsulating AuNPs-(4MBA)-BSA in
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BR buffer solutions. (B) pH calibration curve based on Raman intensity of the peak at 1077 cm-1 with
pH from 1.0 to 8.0. (C) Parts of 4MBA SERS spectra were normalized to the peak at 1077 cm-1. (D)
pH calibration curve based on Raman intensity ratio of the peaks at 1395 cm-1 and 1077 cm-1.
Fluorescence pH Sensing Using pH Sensitive Hybrid Microparticles
Encapsulating CDs. Fluorescence pH sensing microgels encapsulating pH
responsive CDs were fabricated here. The pH sensing ability, fluorescence stability
and reversibility were investigated successively. As shown in Figure 4A, the CLSM
images visually showed the pH-induced change in size and fluorensence intensity of
the beads with the emission wavelength of 405 nm. When the pH increased from 1.0
to 8.0, the hybrid microparticles gradually swelled until stretching to the maximum
limit before cracking, whereas the corresponding fluorescence intensity inversely
decreased. Based on the fluorescence intensity, the calibration curve was depicted in
Figure 4B. Fluorescence intensity was the average optical density value of six random
microparticles, which were treated with BR buffer solutions with different pH and
analyzed by Image J software. Hydrogel and CDs in the hybrid microparticles are
both pH responsive, so both of them may cause fluorescence change of the
microparticles in different pH media. In this work, a pH-sensing CD was selected to
fabricate the microgels encapsulating CDs.33 The fluorescence intensity of the CDs
decreased linearly as the pH increased. The effect of the hydrogel on the fluorescence
intensity shoud be due to the microparticle size change induced by pH alteration.
Since CDs in different pH circumstances can also influence the fluorescence intensity,
we adopted a strategy of evaporation to change the size of the microgels instead of
changing pH. The CLSM images of the microgels were captured before and after
water loss, as shown in Figure 4C. After water evaporation, the microgel became
much smaller, and its fluorescence was more prominent, which demonstrated that the
microgel size played an important role in the fluorescence intensity. This was because
that local refractive index of surrounding hydrogels increased owing to the shrink,
which induced the enhancement of Rayleigh scattering.18 On the contrary, the
reduction of fluorescence intensity induced by the swell could be explained from two
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respects. One was that the reduction of the Rayleigh scattering caused by the swell
reduced local refractive index of surrounding hydrogels. The other reason was that
CDs could be quenched by the surface electron change.34 When microgels swelled, the
elastic tension, which was created by hydrogen bonds between -OH/-COOH on
polymer chain and -NH3/-COOH on CDs surface, changed the electron state on CD
surface, resulting in fluorescence quench.18 In short, the pH sensing sensitivity of
microgels benefited from the pH-sensing CDs and microgel size change, which
provided a synergistic effect to enhance the determination sensitivity owing to their
coincident impact tendency.
Figure 4. (A) CLSM images of microgels encapsulating pH responsive carbon dots after incubation for
15 min in BR buffer solution with pH 1.0-8.0 (scale bar, 200 µm). (B) Calibration curve of the
microgels fluorescence intensity against pH value. (C) Fluorescence intensities of the microgels before
and after water loss (scale bar, 100 µm).
We estimated the reversibility of microgel fluorescence upon 5 reversible cycles at
pH 3.0 and 7.5, as illustrated in Figure S2. A slight decline of fluorescence intensity
was observed after the first cycle, which may be attributed to the loss of a small
amount of tiny sized CDs at the swell stage. Fortunately, the fluorescence intensity
showed a good reversibility and kept about 85% of the original intensity after the
following cycles. The average diameter of CDs (5-20 nm) is 10 nm, and the dense
cross-linked alginate/CMC could confine most of the CDs in the microgels via
physical entanglements and hydrogen bonding force. We also investigated the
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fluorescence stability of the hybrid microgels under continuous excitation by 405 nm
light for 3 h, as shown in Figure S3. The fluorescence intensity of the hybrid
microgels remained basically unchanged, which demonstrated the excellent
photostability of the hybrid microgels. Such microgels with highly reproducible and
stable fluorescent signals have a great promise for continuous pH monitoring.
SERS-Fluorescence Dual-Mode pH Sensing Using Janus Microparticles. We
further constructed Janus microgels encapsulating labeled AuNPs and CDs for
SERS-fluorescence dual-mode pH sensing. To investigate the pH sensing
performance of the Janus microgels, the microgels were incubated with BR buffer
solutions (pH from 1.0 to 8.0) for 15 min. Figure 5A shows the typical Janus
microparticles with distinct interfaces. SERS spectra were acquired from random
points on the SERS hemispheres under different pH conditions, as shown in Figure
5B. The fluorescence hemispheres loaded with CDs emitted blue fluorescence, as
shown in Figure 5C. Although feeble fluorescence in the other hemispheres was
observed, it did not cause obvious interference between SERS and fluorescence
detection. The Raman excitation wavelength (638 nm) is far from fluorescence
excitation wavelength (405 nm) and emission wavelength (390-500 nm), ensuring that
individual detections would not interfere each other. In the SERS-fluorescence
dual-mode pH sensing method, the advantages of molecular recognition capability of
SERS and fast imaging of fluorescence were combined. The fluorescence signal acted
as a fast indicator and the SERS signal was applied to distinguish specific targets. In
addition, the SERS can still provide pH information even when the fluorescence is
influenced in harsh chemical environment. Thus the dual-mode pH sensing method is
of great importance to actualize the complementary advantages and acquire more
comprehensive and reliable data.
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Figure 5. (A) Optical images of Janus microgels in BR buffer solutions with pH from 1.0 to 8.0 (scale
bar, 200 µm). (B) SERS spectra of SERS hemispheres in BR buffer solutions with pH from pH 1.0 to
8.0. (C) CLSM images of Janus microgels in BR buffer solution with pH from 1.0 to 8.0 (scale bar, 200
µm).
The Janus microparticles with an injectable size have demonstrated good pH
detection performance with stability and reversibility, which hold a great promise for
in vivo continuous pH monitoring. Besides, the biocompatibility of Janus microgels
was investigated through the classical MTT assay. As expected, no obvious
cytotoxicity was observed in Figure S4, which was attributed to the nontoxic
components of the microgels. It is expected that more dual-mode sensing platforms
will be constructed based on the Janus microgels for applications.
Size Selectivity of Janus Microparticles. It was clearly demonstrated that proteins
could interfere SERS6,35 and fluorescence36 detection. Fortunately the Janus
microparticles can prevent the diffusion of big molecules within small meshes in the
microgels, thus protein interference will be avoided. To investigate size selectivity of
the Janus microgels, they were respectively incubated in FITC (wt 389) solution and
FITC-tagged BSA (wt 68,000) solution for 15 min prior to CLSM imaging. As
displayed in Figure 6A, FITC entered into the microparticle, while FITC-tagged BSA
did not, which demonstrated the BSA could not diffuse into the microgels. Albumin,
as the most abundant protein in blood, can bind strongly on the surfaces of AuNPs37
and CDs38, so it is used to investigate the protein interference in the dual-mode pH
sensing method. Afterwards, SERS signals of the Janus microparticles in 100 µL BR
buffers (pH 7.5) with and without 1 mM BSA were compared, so were the
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fluorescence signals. As shown in Figure 6B, SERS spectrum acquired from the
SERS hemispheres in the buffer with BSA was comparable to that without BSA. The
fluorescence intensity of the fluorescence hemispheres in the buffer with BSA was
identical with that without BSA, as illustrated in Figure 6C. These agreements were
attributed to the fact that dense cross-linked microgels kept BSA out and avoided the
interference from BSA nonspecific adsorption. The size selectivity made the pH
sensing microgel a reliable pH sensor even in the complex biological matrix with a
large quantity of proteins.
Figure 6. (A) CLSM images of Janus microgels in aqueous solutions of FITC-tagged BSA (wt 68,000)
(a) and FITC (wt 389) (b). (B) SERS spectra acquired from SERS hemispheres in pH 7.5 BR buffer
with 1 mM BSA (red) and pH 7.5 BR buffer without BSA (blue). (C) CLSM images of Janus
microgels in pH 7.5 BR buffer with 1 mM BSA (a) and pH 7.5 BR buffer without BSA (b). (Scale bar,
100 µm).
Gastric Juice pH Sensing and Extracellular pH Monitoring. After the
successful demonstration of SERS and fluorescence pH sensing performances based
on the hydrogel microparticles, we further used them to detect pH value of real
biological samples. Gastric juice pH value, as a physiological parameter for screening
atrophic gastritis and gastric tumor, is usually detected in clinical diagnosis.39
Chlorhydria often causes overgrowth of helicobacter pylori and other bacteria, which
always ultimately results in the development of gastric cancer.40 Gastric cancer
patients mostly exhibit achlorhydria with pH value above 4.0, whereas gastric juice
pH value of normal people maintains at pH 1.8-3.5. With the proposed method above,
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the gastric juice pH of a volunteer patient was measured using the Janus
microparticles. As displayed in Figure 7, the fluorescence image and SERS spectra of
Janus microparticles in the gastric juice were acquired for pH determination. For
comparison, the fluorescence image and SERS spectra of the Janus microparticles
before being treated with gastric juice samples were shown in Figure S5. The pH
values measured by SERS and fluorescence were 3.8±0.2 and 4.1±0.1 respectively.
The pH calibration curve based on Raman intensity ratio of the peak at 1395 cm-1 and
the peak at 1077 cm-1 was used for the SERS detection. In clinic, pH indicator paper
is usually used for screening test, and pH meter is used for precise test once the pH
exceeds the normal range. The gastric juice sample was also detected using a pH
meter (PB-10, Sartorius), and the pH value was 3.98. The results obtained by the two
methods were basically consistent. Compared with the method using the pH meter,
the dual-mode method is accurate, sensitive and reliable for estimating gastric juice
pH with a rather small amount of sample consumption (100 µL). To the best of our
knowledge, it is the first time that the pH value of strong acidic body fluid was
measured via a SERS method. The SERS-fluorescence dual-mode pH sensing method
would be a promising method for pH screening of minute samples.
Figure 7. Fluorescent image (A) and SERS spectra (B) of Janus microparticles after being treated with
gastric juice samples. (Scale bar, 100 µm).
In addition, we applied the Janus microparticles for monitoring extracellular pH
change of tumor cells (MCF-7). The extracellular pH (pHe) decreases as protons (H+)
constantly are excreted from tumor cells to maintain the intracellular pH (pHi) stable
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and avoid intracellular acidosis. And the acidic pHe leads to more serious invasion of
tumor cells, cell gene mutation, increased drug resistance as well as tumor migration,
proliferation and metastasis.41 Previous studies have demonstrated that the pHi of
solid tumors maintained within a range of 7.0-7.2 and the pHe is acidic (6.2-6.9),
whereas pHi and pHe of normal cells are basic (7.2-7.4).42-43 We monitored the pHe
change by using the SERS-fluorescence dual-mode method. The pH value of the fresh
cell culture medium was 7.4±0.7 measured by SERS and 7.3±0.1 by fluorescence,
which were close to the pH value of 7.60 measured via a pH meter. Figure 8 shows
pH change of the culture medium over time. The culture medium pH obviously
decreased with the increase of MCF-7 cell culture time, and dropped to 5.5-6.0 when
the cells were cultured for 36 h. This decreasing trend agrees with typical pHe
variation during cell growth, metabolism and proliferation.44-45 We expect the
SERS-fluorescence dual-mode pH sensing microgels could be applied for in-vivo
tumor pH monitoring.
Figure 8. The pH values were determined based on Raman intensity ratio of the peaks at 1395 cm-1 and
1077 cm-1 (a) and the fluorescence intensity (b), respectively.
█ CONCLUSIONS
We proposed a SERS-fluorescence dual-mode detection method for pH sensing and
imaging. The single phase and Janus microgels with desirable uniformity, which were
both fabricated utilizing the centrifugal microfluidic chips, contributed to the
improvement of sensing reproducibility. The dense cross-linked microparticles
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successfully encapsulated the labeled AuNPs and CDs without obvious leakage. The
dense networks obviated big molecule access, which effectively guaranteed that
SERS and fluorescence signals were free from interference of macromolecules. Since
the labeled AuNPs were stabilized in microgels, the microgels for SERS detection had
a highly sensitive response to pH change ranging from pH 1.0 to 8.0. The
conventional pH-sensitive SERS probes usually failed below pH 5.5, whereas the
resultant microgels can provide two complementary sensitive ranges, from 3.0 to 8.0
and from 1.0 to 3.0. To the best of our knowledge, no SERS pH sensors have such
wide acidity range. At the same time, the microgels encapsulating pH-dependent
carbon dots also exhibited good pH sensitivity, and thus the SERS-fluorescence
dual-mode pH detection method based on Janus microparticles was established. The
Janus microparticles encapsulating the probes into divided hemispheres showed no
obvious interference for dual-mode detection, and combined the advantages of
molecular recognition capability from SERS and fast imaging from fluorescence.
They were successfully applied for measuring gastric juice pH and monitoring
extracellular pH change. The Janus microparticles are expected to construct more
dual-mode or multitarget sensors for small molecule analysis and cell secretion
determination, which are now underway in our lab.
█ ASSOCIATED CONTENT
Supporting Information is to submit additional figures.
█ AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-24-83687659; *E-mail: xuzr@mail.neu.edu.cn
Notes
The authors declare no competing financial interest.
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█ ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China
(21675020 and 21375012) and the Fundamental Research Funds for the Central
Universities (N160506002).
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