Chemical Sensing on a Single SERS Particle - ACS Sensors (ACS

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Chemical Sensing on a Single SERS Particle Ying Ma,*,† Kittithat Promthaveepong,† and Nan Li*,‡ †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore ‡ Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapore S Supporting Information *

ABSTRACT: We report a new chemical sensing platform on a single surface-enhanced Raman scattering (SERS) particle. A cabbage-like Au microparticle (CLMP) with high SERS enhancement was applied as an ultrasensitive SERS substrate. A new Raman reporter bis[4,4′-[dithiodiphenyl azo-phenol] (DTDPAP) was synthesized to display multiple fingerprints and high reactivity toward sodium dithionite. The reaction of DTDPAP with sodium dithionite was in situ monitored by SERS on a single CLMP. The DTDPAP fingerprint change is dependent on the sodium dithionite concentration, providing a simple and sensitive method for sodium dithionite profiling. KEYWORDS: stimuli-responsive Raman reporter, azobenzene, SERS, single particle, sensor ince the first discovery in the 1970s, surface-enhanced Raman scattering (SERS) has been regarded as an ultrasensitive analytical technique with high sensitivity down to single molecule level via identifying the fingerprints of specific analytes.1 It has been widely used in the fields of environmental monitoring,2,3 diagnostics, and biological detection with rapidity, high sensitivity, and selectivity.4 One promising application of SERS technique is that it serves as a versatile tool for in situ monitoring of the catalytic reactions owing to its significant advantages: (1) directly monitoring the fingerprint vibrational change induced by the catalytic reactions; (2) high SERS enhancement leading to single molecular sensitivity; (3) avoiding interference by the solvents.5 It has been used to in situ monitor the reduction of 4nitrothiophenol (4-NTP) to 4-aminothiophenol (4-ATP) by NaBH45−11 and the cyclic redox of 4-aminothiophenol.12 However, research in this area is very limited, and most of the strategies focused on the improvement of SERS substrate to achieve intense signals rather than the diversity of catalytic reactions. One reason is that it is still a big challenge to prepare a SERS substrate with a simple method but possessing high SERS enhancement and good reproducibility. The other reason is the lack of commercial Raman reporters for specific catalytic reactions. Recently, we developed a simple strategy to spontaneously grow 3D cabbage-like Au microparticles (CLMPs) using N-(3amidino)-aniline (NAAN) as a reducing agent.13 The resulting CLMP has several advantages as a SERS substrate: (1) high SERS enhancement and reproducibility (enhancement factor of ∼108 and reproducibility of 8.7% for 100 measurements) owing to the densely packed Au microplates; (2) its larger size than the diameter of laser beam (10 μm Vs ∼ 1 μm), providing the intense SERS signal and allowing the in situ monitoring of chemical reaction on a single particle; (3) excellent stability in

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various solvents. Therefore, a single CLMP can serve as a SERS platform to monitor the chemical reactions. Stimuli-responsive Raman reporters (SRRRs) are Raman reporters with stimuli-responsive properties. Their Raman fingerprints change under the external stimulus, allowing the detection of their corresponding stimulus using SERS signal as a readout. A good SRRR should contain two essential parts: (1) responsive group toward some particular stimulus; (2) functional group with high affinity to SERS substrate (mostly thiol or disulfide groups). Some SERS sensors have been reported based on the SRRRs. For example, purpald-based Raman reporter showed good sensing ability toward formaldehyde.14 Raman reporter containing quinone moieties was able to monitor the intracellular redox potential changes.15 4Acetamidobenzenesulfonyl azide (4-AA) and p-aminobenzenethiol (ABT) were utilized for the detection of hydrogen sulfide (H2S)16 and nitric oxide (NO)17 in living cells, respectively. Since the first report of its trans−cis photoisomerization, the photoswitchable SERS properties of azobenzene have been mostly studied.18,19 One SERS sensor was also reported to detect nitrite based on the nitrite-triggered catalytic conversion of p-aminothiophenol to p,p′-dimercaptoazobenzene.20 In this research, we synthesized a sodium dithionite-responsive Raman reporter DTDPAP, which shows high reactivity toward sodium dithionite, an important reducing agent in the textile and pulp industry. The chemical reaction of DTDPAP and sodium dithionite can be in situ monitored on a single CLMP. The SERS spectra of CLMP exhibited the sodium dithionite concentration-dependent change, providing a sodium dithionite Received: October 5, 2016 Accepted: December 29, 2016 Published: December 29, 2016 A

DOI: 10.1021/acssensors.6b00617 ACS Sens. XXXX, XXX, XXX−XXX

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subsequently washed with DMF and water three times. The modified CLMPs were dissolved in 20 μL water and stored in the fridge for further use. Monitoring of the Chemical Reaction by SERS. Sodium dithionite solution (10 μL, 400 μM) was mixed with 90 μL DTDPAPmodified CLMP solution. After vigorous stirring for 10 min, the CLMPs were collected by centrifugation and washed with excessive water three times. Finally, 20 μL diluted CLMP solution was deposited on a silicon wafer. After drying in air, the dark field image of CLMP was taken. The SERS spectra of CLMPs were measured on individual CLMP. To in situ monitor the chemical reaction, 300 μL diluted DTDAPAmodified CLMP solution was injected into a round plate with a diameter of 2 cm. After gently removing the excessive water and drying in air, its optical microscope image was taken to ensure the isolated CLMP dispersion. Subsequently, 2 mL 20 μM sodium dithionite solution was gently injected into the plate containing CLMPs, and the corresponding SERS spectra on a single CLMP were measured at a time interval of 30 s. SERS Sensing of Sodium Dithionite. To detect sodium dithionite, 10 μL DTDPAP-modified CLMP solution was mixed with 90 μL sodium dithionite solutions with varied concentrations for 10 min. The resulting CLMP was collected by centrifugation and washed with water. After dissolving into 100 μL water and depositing on a silicon wafer, SERS spectra on an individual CLMP were measured. SERS spectra of 10 CLMPs were measured for standard deviation calculation. Selectivity of SERS Sensor. 200 μM Na2SO4, Na2SO3, Na2S, and NaHS were mixed with DTDPAP-modified CLMP solutions for 10 min and their corresponding SERS spectra on a single CLMP were measured.

sensor with high sensitivity and selectivity compared with the reported methods.

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EXPERIMENTAL SECTION

Materials. HAuCl4, 4,4′-dithiodianiline (DTDA), sodium nitrite, phenol, sodium dithionite, Na2SO4, Na2SO3, Na2S, and NaHS were purchased from Sigma. NAAN was synthesized according to ref 10. All other reagents were analytical grade and used as received. Ultrapure water (resistivity up to 18.2 MΩ cm) was used throughout the experiment. Characterization. Scanning electron microscopy (SEM) (JEOL JSM-6700F field emission SEM) was used to study morphologies of CLMP. UV−vis absorption spectra were measured by a Varian Cary60 spectrophotometer. SERS spectra were recorded by a XploRA PLUS Raman microscope (Horiba/JY, France) using a 785 nm laser excitation source. The incident laser power was kept at 1 mW and total accumulation times of 10 s were employed. Nuclear magnetic resonance (NMR) spectra were acquired on a 600 MHz NMR spectrometer (Premium Shielded Narrow Bore, Agilent Technologies, CA, USA) at room temperature. Synthesis of CLMP. Cabbage-like Au microparticles (CLMPs) were synthesized according to our previous report.13 Simply, 8 μL 10% HAuCl4 (2 mM) was dissolved in 1 mL of 1 mM HCl containing 0.8 mg PVP, and the mixture was cooled down to 4 °C. 4 mg NAAN (16 mM) in 1 mM HCl was subsequently injected. After vigorous stirring for 20 s, the mixture was left to stand at 4 °C for 24 h. The final solution was centrifuged at 5000 rpm for 5 min, and the pellet was excessively washed with N-methyl-2-pyrrolidone (NMP) and water to get CLMPs. Synthesis of Raman Reporter Bis[4,4′-[dithiodiphenyl azophenol] (DTDPAP). DTDPAP was synthesized via the azo coupling reaction,21 and the synthetic route is shown in Scheme 1. Simply, 4,4′-

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RESULTS AND DISCUSSION Synthesis of DTDPAP. The Raman reporter DTDPAP was synthesized via the typical azo coupling reaction. The resulting DTDPAP consists of two key functional groups: (1) the disulfide group has high affinity to CLMP via the formation of Au−S covalent bond, providing the chemical enhancement and improving the stability of SERS probe;22 (2) Azobenzene moiety is the stimuli-responsive functional group. It exhibits multiple fingerprints due to the presence of -NN- bond18 and high reactivity toward sodium dithionite, which can cleave the -NN- bond to the phenylamine group (Figure 1a),23 resulting in the disappearance of the azo moiety fingerprints.

Scheme 1. Synthetic Route of DTDPAP

dithiodianiline (DTDA) (2.3 mmol) was dissolved in 20 mL of 0.5 M HCl solution. After cooling to 4 °C by ice water, NaNO2 (5.1 mmol) in 2 mL cooled water was added dropwise under vigorous stirring. After 20 min stirring, phenol (5.5 mmol) in 10 mL of 1 M cooled NaOH solution was added dropwise. The mixture was stirred for 5 min, and the solution pH was adjusted to 10 using 1 M NaOH. The resulting solution was continually stirred at 4 °C for 30 min and subsequently at room temperature for 1 h. The precipitate was collected by centrifugation and washed with water three times. Yellowish DTDPAP was received after lyophilization (yield = 95%). 1 H NMR (DMSO-d6): 6.9 (d, 2H, Ph), 7.5 (m, 2H, Ph), 7.7 (2H, Ph) 7.8 (2H, Ph). MS (ESI) m/z: calcd for C24H18N4O2S2:458.1; [M + H]+ found: 459.2 (See Figure S1). Reactivity of DTDPAP with Sodium Dithionite. To study the reactivity of DTDPAP with sodium dithionite, 5 μM DTDPAP was dissolved in methanol/water mixture (1/1 v/v). Sodium dithionite solution (1 mM in water) was subsequently injected. After continually stirring for 10 min, the solution photos were taken, and their corresponding UV absorption spectra were measured. To study the response time, 1 mM sodium dithionite solution was added into 5 μM DTDPAP in methanol/water, and their corresponding UV absorption spectra were measured with a time interval of 0.5 min. To study the selectivity of this reaction, 5 μM DTDPAP solutions were mixed with 1 mM Na2SO4, Na2SO3, Na2S, NaHS for 10 min, their corresponding UV absorption spectra were measured. Preparation of SERS Nanoprobe. The DTDA- and DTDPAPmodified CLMPs were prepared via the self-assembly strategy. Simply, 10 μL CLMP water solution was injected into 0.5 mL 1 mM DTDA or DTDPAP dimethylformamide (DMF) solutions. After vigorously shaking for 4 h, CLMPs were collected by centrifugation and

Figure 1. (a) Schematic demonstration of DTDPAP reaction with sodium dithionite. UV absorption spectra of (b) 5 μM DTDPAP in MeOH/water (1/1, v/v) and (c) DTDPAP solution after incubation with 1 mM sodium dithionite. Inset b and c: the corresponding photos of DTDPAP solutions.

Monitoring of the Chemical Reaction by UV Absorption Spectra. As shown in Figure 1b, DTDPAP has a yellow color in MeOH/H2O solution, and its corresponding UV absorption spectrum shows an intense absorption peak at 368 nm owing to the π−π* absorption of trans azobenzene.24 The DTDPAP solution color turned to pale and finally B

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provides a possibility for in situ monitoring of the azobenzene cleavage reaction on a single CLMP. Figure 3a demonstrates

colorless after incubation with sodium dithionite solution, illustrating the cleavage of azobenzene moiety. This observation is consistent with the disappearance of UV absorption peak at 368 nm in Figure 1c. Time-dependent UV absorption spectra shows that the cleavage reaction almost finished in 2 min (Figure S2a). We also studied the reactivity of DTDPAP with other sulfur-carrying ions such as Na2SO4, Na2SO3, Na2S, and NaHS, which are the major interferents for sodium dithionite detection. The UV absorption spectra of DTDPAP solutions did not show obvious change, revealing the good selectivity of this reaction toward sodium dithionite (Figure S2b). Monitoring of the Chemical Reaction by SERS Spectra. Figure 2a shows the SEM image of individual

Figure 3. (a) Schematic demonstration of the in situ monitoring of the chemical reaction on a single CLMP. (b) Time-dependent SERS spectra change and (c) the corresponding peak intensity changes at (red) 1145 and (black) 1433 cm−1.

the in situ monitoring of DTDPAP reduction reaction by sodium dithionite. Isolated CLMP was deposited on a plastic plate by injecting a diluted DTDPAP-modified CLMP solution. After focusing on a single CLMP under a microscope, its SERS spectrum was recorded. The aqueous solution was gently removed without disturbing the CLMP, and 2 mL of 20 μM dithionite solution was subsequently added to the plate. After that, SERS spectra of the same CLMP were measured at a time interval of 30 s. Figure 3b shows the time-dependent SERS spectra change of a single DTDPAP-modified CLMP. As the reaction proceeded, the peak intensities of azobenzene moiety at 1145, 1399, and 1433 cm−1 gradually decrease. However, the peak intensities at 1076 and 1585 cm−1 corresponding to the DTDA domain do not exhibit apparent change, showing that the cleavage of azobenzene moiety does not affect the DTDA domain of DTDPAP. No change in visible peak intensities after 5 min reveals the completion of the reaction. Figure 3c demonstrates that the SERS intensities at 1145 and 1433 cm−1 have a dramatic decrease in the first 4 min, and almost reach a plateau after 5 min, indicating the completion of this reaction. SERS Detection of Dithionite. Sodium dithionite is one of the essential chemicals used in the textile industry as a reducing agent; it is also used as a bleaching agent in reductive bleaching processes in paper pulp.27 Moreover, it can lower the redox potential in physiology experiments.28 Several methods have been reported for the detection of sodium dithionite. For example, the titration method with methylene blue was a wellknown method, but it has poor selectivity over other sulfurcarrying compounds such as sulfate and sulfide.29 Although electrochemical methods by measuring its redox potential demonstrated better performance, the complicated preparation of the electrochemical electrode and lower sensitivity limited its application.30 Based on the fast SERS response of DTDPAP-modified CLMP to dithionite, we envision a sodium dithionite sensor using individual CLMP as a SERS sensing platform. To quantitively detect sodium dithionite, different concentrations of sodium dithionite solutions were incubated with DTDPAPmodified CLMP for 10 min, and their corresponding SERS spectra are shown in Figure 4a. As sodium dithionite concentrations increase, the SERS peak intensities at 1145 and 1433 cm−1 decrease, indicating the sodium dithionite concentration-dependent azobenzene cleavage. The SERS peak intensities at 1145 and 1433 cm−1 show linear decrease with dithionite concentrations from 0.5 to 20 μM (Figure 4b,c). The limit of detection (LOD) was calculated according to 3 σ/slope, and the LOD of 0.08 and 0.12 μM was acquired for the SERS intensities recorded at 1145 and 1433 cm−1, respectively. These LOD are much lower than the reported electrochemical and

Figure 2. (a) SEM and (b) dark-field microscope images of a single CLMP. (c) Schematic demonstration of DTDPAP-modified CLMP and its corresponding Raman fingerprints change in the presence of sodium dithionite. SERS spectra of (d) DTDA-, (e) DTDPAPmodified CLMP, and (f) DTDPAP-modified CLMP after incubation with sodium dithionite.

CLMP, which has a cabbage-like structure and the nanometersized gaps between microplates create high density of hotspots for SERS enhancement.22 Figure 2b shows the dark-field microscopic image of CLMP deposited on a silicon wafer. The single bright dot indicates the isolated CLMP, revealing that SERS spectrum on an individual CLMP was measured. As illustrated in Figure 2c, a monolayer of DTDPAP was modified on CLMP after mixing with DTDPAP in DMF. Sodium dithionite can reduce the DTDPAP and convert it to DTDA, leading to its Raman fingerprint change. Figure 2d−f shows the SERS spectra of DTDA-, DTDPAP-modified CLMP and DTDPAP-modified CLMP after incubation with sodium dithionite. DTDA-modified CLMP exhibited two characteristic SERS peaks at 1076 and 1585 cm−1 assigned to the mixed mode of the C−C stretching, the C−H in-plane bending vibration, and the parallel C−C vibration stretching vibration, respectively (Figure 2d). This is consistent with the Raman spectrum of DTDA powder (Figure s3a).25 After the azo coupling reaction, the resulting DTDPAP-modified CLMP displays the peaks at 1076 and 1585 cm−1 corresponding to DTDA domain. Moreover, three new peaks located at 1145, 1399, and 1433 cm−1 appeared (Figure 2e), which are ascribed to the “b2” bands of azobenzene. This spectrum is consistent with the Raman spectrum of pure DTDPAP power (Figure S3b), indicating the formation of DTDPAP monolayer film.26 Interestingly, the SERS peaks of azobenzene moiety disappeared after incubation of DTDPAP-modified CLMP with sodium dithionite solution (Figure 2f), revealing the cleavage of azobenzene by sodium dithionite. This result is consistent with the previously mentioned color and UV absorption spectra change of DTDPAP. In Situ Monitoring of the Reaction by Single-Particle SERS. The intense, reproducible SERS signal of CLMP C

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Figure 4. (a) SERS spectra of DTDPAP-modified CLMP in the presence of different concentration of sodium dithionite. The SERS intensity (b) at 1145 and (c) 1433 cm−1 vs sodium dithionite concentrations.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

titration methods, which have the LOD of 8 μM30 and 0.5 mM,29 respectively. We also studied the SERS response of other sulfur-carrying compounds, including Na2SO4, Na2SO3, Na2S, and NaHS. Compared with SERS response of sodium dithionite (20 μM), no apparent SERS signal changes were observed after incubation of DTDPAP-modified CLMP with 10× concentrations (200 μM) of Na2SO4, Na2SO3, Na2S, and NaHS solutions (Figure 5), revealing the good selectivity of this SERS

ORCID

Ying Ma: 0000-0003-2204-0663 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by research funding from the Singapore Millennium Foundation and the National Medical Research Council for funding support (NMRC/CIRG/1358/ 2013).

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REFERENCES

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Figure 5. SERS responses of different sulfur-containing ions. The concentrations are 20 μM for Na2S2O4, and 200 μM for other ions.

probe over other sulfur-carrying ions. Therefore, we envision that this SERS sensor can be practically applied for sodium dithionite detection owing to its fast response (5 min), low LOD (0.08 μM), and good selectivity over other sulfur-carrying ions.

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CONCLUSION In conclusion, we have developed a SERS sensing platform on a single particle. A CLMP was used as a sensitive SERS substrate, and a new azobenzene-carrying Raman reporter DTDPAP was synthesized as a SRRR. The SERS probe showed high reactivity with sodium dithionite, enabling a SERS sensor for sodium dithionite profiling with high sensitivity and good selectivity. This strategy may also provide a platform for the in situ monitoring of other chemical or even enzymatic reactions. Given the specific responsive Raman reporters, this approach can be applied for environmental and biological sensing.

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sodium dithionite, UV absorption spectra of DTDPAP in the presence of different concentrations of other surfurcarrying ions, Raman spectra of DTDA and DTDPAP (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00617. Mass spectrum of DTDPAP, time-dependent UV absorption spectra of DTDPAP in the presence of D

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