SERSâFluorescence Dual-Mode pH-Sensing Method Based on Janus

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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 3



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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 4


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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, 6


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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 8


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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 10


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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 11



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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 13



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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, 14


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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 15



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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 16


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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: [email protected] Notes The authors declare no competing financial interest.

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SERSâFluorescence Dual-Mode pH-Sensing Method Based on Janus

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