Synthesis and Characterization of a Disulfide Reporter Molecule for

Article pubs.acs.org/ac

Synthesis and Characterization of a Disulfide Reporter Molecule for Enhancing pH Measurements Based on Surface-Enhanced Raman Scattering Latevi Lawson*,†,§ and Thomas Huser†,‡ †

NSF Center for Biophotonics Science and Technology, University of California, Davis, Sacramento, California 95817, United States Department of Internal Medicine, University of California, Davis, Sacramento, California 95817, United States § Department of Chemistry, University of California, Davis, Davis, California 95616, United States ‡

S Supporting Information *

ABSTRACT: In this paper, we describe the synthesis and characterization of 2,5-dimercaptobenzoic acid as a novel pH-sensitive disulfide reporter molecule for surface-enhanced Raman scattering (SERS) capable of inducing the controlled aggregation of gold (Au) colloids in solution without the addition of salts. While weak acids have been shown to yield some pH sensitivity as reporter molecules for SERS measurements, the reproducibility and signal strength of nanoparticle probes based on such molecules can vary greatly. This limited reproducibility depends greatly on the salt-induced aggregation of the colloidal nanoprobes, which is required in order to obtain SERS signals strong enough to probe individual clusters. This complicates their use in live cell sensing applications. We show that our approach results in primarily bridged nanoparticles comprising a pH-sensitive nanoprobe that can quantify accurately pH values well below 5.5. The robustness and sensitivity of this system makes it a powerful tool for measuring pH values on the nanoscale under in vitro conditions.

S

(oMBA) and p-mercaptobenzoic acid (pMBA) have been used as successful pH reporters,1−4 as well as the pyridine derivative 4-mercaptopyridine (4-Mpy).5,6 Although these pH-sensing probes have shown great promise, the design of new pHsensing reporters optimized for SERS, i.e., molecular designs that optimize both signal intensity and signal consistency, is still an ongoing process. It is now well established that SERS enhancements are typically greatest at the junctions between nanostructures, locations on and between nanoparticles that produce strong SERS that have been termed “hot-spots”. These hot-spots are readily available on roughened metal surfaces but rather poorly controlled, and structures that are specifically designed to provide junctions in which reporter molecules can be placed have been explored. However, these junctions are not native to colloidal metal nanoparticles used for SERS measurements in cells, and therefore, aggregates or multimers are often induced to achieve optimal enhancement, but this process is, again, poorly controlled.7,8 Many SERS measurements rely on the spontaneous aggregation of nanoparticles in solution or induce aggregation via the addition of salt or by a reduction in pH. Recently, in an attempt to harness the full potential of SERS, there has been a large push toward using and designing molecules that increase multimer formation. These multimer inducers can serve as both reporters for imaging and/or as nanoparticle bridges.9 In 2005, Moskovits showed that 1,4-

ince its discovery over 30 years ago, surface-enhanced Raman scattering (SERS) has become a highly popular technique for analyzing chemical species and chemical reactions near and on surfaces. Different implementations of SERS allow one to follow these events with high spatial and temporal resolution by monitoring extended surfaces by narrowband Raman imaging, by point-spectroscopy, or by bulk-sampling of spectral changes. The SERS effect represents the combined enhancements in the Raman signature of molecules chemically adsorbed or bound to a metal surface that supports optically induced surface plasmons. Metals such as silver (Ag), gold (Au), platinum (Pt), copper (Cu), and many others have been utilized as enhancement scaffolds for reporter molecules. It has been shown that under special circumstances the enhancement produced by SERS can lead up to 1015 times greater signals than those obtained by normal Raman spectroscopy, allowing for chemically specific imaging and spectroscopy that can compete with the strong signals generated by fluorescent probes and the endogenous autofluorescence generated by biological samples. Recently, significant strides regarding in vitro pH sensing have been made utilizing SERS. The chemically specific nature of Raman spectroscopy and the robust signals generated by SERS have even allowed for reliable and reproducible pH measurements within individual cells. This has largely been achieved through the design of novel pHsensitive Raman-active nanoprobes, i.e., SERS probes designed with reporter molecules that undergo spectrally distinguishable changes as a function of pH. Typically, weak acids that are chemically bound to the surface of nanoparticles comprise such probes. Benzoic acid derivatives such as o-mercaptobenzoic acid © 2012 American Chemical Society

Received: December 6, 2011 Accepted: March 20, 2012 Published: March 20, 2012 3574

dx.doi.org/10.1021/ac203103s | Anal. Chem. 2012, 84, 3574−3580

Analytical Chemistry

Article

benzene dithiol could be used to influence silver (Ag) colloids to form clusters and produce very large SERS signals.10 Here, we introduce the synthesis and application of 2,5-dimercaptobenzoic acid (2,5-DMBA) as a dithiol molecule that is capable of influencing the controlled aggregation of Au colloidal nanoparticles while also functioning as an excellent pH reporter system. We also show that this pH-sensitive Raman-active probe has excellent properties to function as an intra- and extracellular pH sensor.

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MATERIALS AND METHODS Spontaneous Raman spectra of intermediates and the final product during the synthesis of 2,5-DMBA were obtained on an Olympus IX71 inverted optical microscope. A diode-pumped solid-state laser (785 nm, Crystalaser, Reno, NV) served as the excitation source, providing up to 32mW of laser power focused to a diffraction-limited spot after passing a narrow-band cleanup filter, neutral density filter, and a dichroic beam splitter. Raman spectra were obtained by coupling the Raman-scattered light into an Acton Spectra Pro 2300i imaging spectrometer utilizing a back-thinned Princeton instruments Pixis 100BR CCD camera as detector. Localized SERS measurements on substrate-immobilized nanoparticles were undertaken using an automated digital optical microscope (iMic, Till Photonics, Munich, Germany). The microscope was controlled with custom-written MATLAB software. An Argon/Krypton ion laser (Innova 70c spectrum, Coherent Inc., Santa Clara, CA) restricted to less than 1 mW output power provided the excitation source at 647 nm. Localized SERS spectra were collected on a Spectra Pro 2300i spectrometer (Acton Research) and a Pixis 100 CCD sensor (Princeton Instruments) attached to the iMic platform. Dynamic light scattering (DLS) of colloidal particle solutions was conducted with a Zetatrac system (Microtrac), and a Varian 50 bio (Cary) spectrometer was used for UV−vis measurements. In order to remove autofluorescence contributions from our spectra, we subtracted all broadband background from our spectral measurements after data collection using an automated background subtraction routine as first described by Lieber et al.11 This allowed us to standardize the probe’s pH sensitivity in a flow cell, which we could then use to compare with SERS measurements of the probe particles taken within living cells. The automated subtraction routine was applied to all SERS spectra using a fourth order polynomial with 100 iterations,11 followed by normalizing the spectra to the β(CH) vibrational mode at 1034 cm−1. The background subtraction was performed on the spectra from 500 up to 1800 cm−1. 2,5-Dimercaptobenzoic acid was synthesized in a three step process following a protocol similar to the one used by Corbett et al.12 as detailed below. Corresponding Raman spectra of the intermediates are shown in Figure 1. Methyl 2,5-bis(dimethylcarbamothioyloxy)benzoate: (O-linked-2,5). Anhydrous N,N-dimethylformamide (DMF, 20 mL) was used under a nitrogen atmosphere to dissolve methyl 2,5-dihydroxybenzoate (5.02372 g, 29.9 mmol). While stirring, the solution was cooled to 0 °C in an ice bath. 1,4Diazabicyclo[2.2.2]carbamyl (DABCO; 13.4 g, ∼4 eq) was added in six portions at 4 °C, followed by adding 20 mL of a 6.8 M solution of N,N-dimethylthiocarbamoyl chloride (∼4 eq) in DMF dropwise to the suspension over 5 min. The solution was allowed to warm to room temperature for approximately 24 h. The mixture was poured into 200 mL of water and stirred

Figure 1. The normal Raman spectra outlining the synthesis of 2,5dimercaptobenzoic acid. In reaction (i), the starting material methyl 2,5-dihydroxybenzoate (a), DABCO, and thiocarbamoyl chloride were suspended and stirred vigorously in DMF at 0 °C and then stirred at room temperature for 24 h. Reaction (ii) required heating a solution of thiocarbamate (b) in diphenyl ether at temperatures ranging between 230 and 240 °C for approximately 4 h (10 mL of diphenyl ether per gram of thiocarbamate). In reaction (iii), (c) was suspended in 175 M of argon purged KOH in diethyleneglycol and then heated to 105 °C for 30 min. After cooling to room temperature, argon purged water was added to the reaction mix followed by the rapid addition of 10% HCl to yield the final product (d). The O-linked intermediate products show Raman stretching vibrations roughly at 1720 and 2942 cm−1, indicating the presence of the ester and more sp3 C−H functionalities, respectively. The S-linked intermediate products show vibrations at approximately 1655, 1720, and 2940 cm−1, indicating the presence of the amide, ester, and sp3 C−H functional groups, respectively. The acid products have peaks at roughly 1625, 2558, and 3056 cm−1 delineating the carboxylic acid, thiol, and sp2 C−H functional groups, respectively.

for 20 min until a white precipitate had formed. The precipitate was collected via suction filtration and then washed extensively with H2O. The resulting crystals were placed under vacuum for 24 h (9.21 g, 90% yield). Methyl 2,5-bis(dimethylcarbamoylthio)benzoate: (Slinked-2,5). O-Linked-2,5 was suspended (6.00 g, 17.5 mmol) in 67 mL of diphenyl ether under a nitrogen atmosphere. The suspension was heated to 240 °C for approximately 5 h. The resulting solution was allowed to cool to roughly 35 °C and then poured into 160 mL of hexane. Using an ice bath, the solution was cooled to 5 °C and sat at this temperature until the maximum amount of badge crystals had formed (25 min). The crystals were collected via suction filtration and washed with copious amounts of warm hexane. The product (4.20 g, 70% yield) was placed under vacuum for 48 h. 2,5-Dimercaptobenzoic Acid (2,5-DMBA). A 1.75 M solution of KOH in diethylene glycol was purged with argon gas for 4 h. Under nitrogen gas, S-linked-2,5 (4.00 g, 11.68 mmol) was dissolved in the KOH solution and then heated to approximately 105 °C for about 45 min. The solution was allowed to cool to room temperature and then poured into 500 mL of H2O that had been purged with argon gas for 2 h. This was followed immediately by the addition of 50 mL of 12.1 normal HCl. The white precipitate was collected using suction filtration and washed extensively with water. The product was dried under vacuum for 48 h yielding dry product (1.60996 g, 74% yield). LC-MS (m/z): [M − H]− calculated for 3575



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Figure 2. Significant shifts are visible in both, the UV−vis (a) and DLS (b to c) profiles after the addition of 5 mL of a saturated solution of 2,5DMBA to 3 mL of Au nanoparticles. The UV−vis red-shift from 532 nm to 548 nm indicates that the average size of the nanoparticles has increased. In addition, the DLS histograms show the change of average particle size from 35.4 to 72.0 nm, indicating the formation of dimer-like particles.

DMBA) have peaks within the 3000 and 3100 cm−1 region of their Raman spectrum. sp2 carbon−hydrogen stretches of benzene are known to appear in this region. We observe this vibration at roughly 3060 cm −1 in every compound. Intermediate products O-linked-2,5 and S-linked-2,5 have Raman peaks at 2942.1 and 2936.9 cm−1, respectively, while the final product 2,5-DMBA is void of peaks in this region. These peaks are attributed to the sp3 carbon−hydrogen stretching vibration of the ester and amide groups and generally appear within 2700 and 3000 cm−1. The lack of these bands in 2,5-DMBA indicates the absence of ester and dimethylamide functional groups. The starting materials, O-linked-2,5, and Slinked-2,5 have vibrations at 1712.4, 1720.3, and 1725.5 cm−1, respectively. Vibrations here are due to the carbonyl stretch of an ester bound to an aromatic ring.14,15 As expected, the acid product does not show this ester vibration but has a vibration at 1627 cm−1 due to the carbonyl stretch of aromatic carboxylic acids.16 It is worth mentioning that S-linked-2,5 has an amide carbonyl stretch mode at 1651.8 cm−1. This stretching vibration is not found in O-linked-2,5 and thus confirms a successful rearmament as described by Newman17 in the production of Slinked-2,5. Unlike the intermediates, 2,5DMBA has two peaks

C14H8O4S4 and C7H5O2S2, 184.2 and 185.2; found, 184.2 and 185.1, respectively. 13C NMR (400 MHz, CD3OD): δ = 168.19, 135.50, 133.18, 132.09, 131.36, 127.79, 127.08. 1H NMR (400 MHz, CD3OD): δ = 7.93−7.92 (m, 1H, ArCH), 7.263−7.265 (m, 2H, 2 × ArCH). Raman υmax = 2583.0 rel cm−1 (m, S−H), 2541.8 rel cm−1 (m, S−H), 1627.5 rel cm−1 (m, 2 × CO), 1597.312 rel cm−1 (s, CC ring breathing stretch). Gold Nanoparticle Synthesis. Gold (Au) nanoparticles (NPs) were synthesized via citrate reduction.13 HAuCl4 (24 mg) was dissolved in 50 mL of deionized milli-Q water. The solution was then heated until it began to boil. A 1% (by mass) solution of sodium citrate was added rapidly but dropwise to the boiling solution. A total of 5 mL of sodium citrate was added to the boiling solution. The solution continued to boil for 25 min and then sat overnight. The UV−vis spectrum of the wine-colored solution showed a maximum absorption peak at 530 nm. DLS measurements determined the colloids in solution had an average diameter of 35.4 nm (see Figure 2).

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RESULTS AND DISCUSSION As can be seen from the bulk Raman spectra shown in Figure 1, the starting material, intermediates, and final product (2,53576



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at 2531 and 2572.2 cm−1. Lee et al. observed similar splitting patterns with o-mercaptobenzoic acid (2517 and 2529 cm−1).16 They suggested that the splitting is due to the ortho-thiol hydrogen-bonding with both the hydroxyl and the carbonyl oxygen of the carboxylic acid. The absence of a doublet at ∼2550 cm−1 in o-toluenethiol18 and in 4-mercaptobenzoic acid1,2 supports Lee’s contention. Also, despite having nonequivalent thiols, the Raman spectrum of toluene-3,4-dithiol19 does not show splitting but only exhibits a single peak with a very slight shoulder within the thiol stretching region. The mass spectrum for 2,5-DMBA reveals two distinctive fragmentations that are commiserate in height, with m/z ratios of 185.1 and 184.2. The fragmentation indicates the presence of the monomeric and dimeric forms of the molecule, respectively. Hydrogen bonding with carboxylic acid should stabilize the ortho-thiol (thus increases the pKa of this thiol); therefore, the dimeric species is most likely coupled via a disulfide bond at the meta position leaving the ortho-thiol free to interact inter- and intramolecularly. The final step in the synthesis of 2,5-DMBA required an acidic environment to crystallize the product. However, the Raman spectra of 2,5-DMBA has a S−S stretching band at 504 cm−1,20 as well as an ortho-thiol stretching band at ∼2550.0 cm−1, and thiol deformation peaks at 917.0 and 978.9 cm−1 (see Figure 1d and Table 1), indicating Table 1. Vibration Assignment for 2,5-DMBAa frequencies of 2,5-DMBA (cm−1) NRS (acidic) 504 855.3

NRS (basic)

SERS (pH 2.70)

446.6 468.3 847 875.4

917 978.9 1053 1098 sh 1124 1176 1256 1545 1582 1627 2531 2572 3054

SERS (pH 11.2)

875.4

1040 1100 1127

1034 1098 1118 sh

1036 1094 1127

1152 1259 1525 1565

1157 1266

1157 1266

1566

1564

3045

Figure 3. The reporter molecule 2,5-DMBA has similar Raman and SERS profiles with slight differences due to metal adhesion. (a) The normal Raman spectrum of 2,5-DMBA at a basic pH of ∼12.0. (b)The normal Raman spectrum of 2,5-DMBA at an acidic pH of ∼5.0. (a’)The SERS spectrum of 2,5-DMBA at a basic pH of 11.2. (b’)The SERS spectrum of 2,5-DMBA at an acidic pH of 2.72. (c) The SERS spectrum of 2,5-DMBA bound to Au nanoparticles at various pH ranging from basic to acidic. Each spectrum is the average of several spectra where the background was subtracted via a fourth order polynomial fit and normalized to the peak at ∼1034 cm−1. Upon changes in pH, we observed significant changes in the SERS peaks at ∼847, ∼875, ∼1128, and ∼1165 cm−1.

assignment ν(S−S) aromatic ν(S−S) aromatic β(CCC) + ν(C− COOH) γ(COO−) (S−H) def (S−H) def b β(CH) β(CCC) + ν(C−S) β(CCC) + v(C−S) + ν(C−COOH) a β(CH) β(CH) a ν(CC) b ν(CC) ν(CO) ν(S−H) ν(S−H) ν(CH)

2,5-DMBA to the solution of Au nanoparticles. A saturated solution of 2,5-DMBA (5 μL) in ethanol was pipetted into 3 mL of colloidal Au solution. Prior to the introduction of the reporter molecule, the Au solution had a single maximum absorption peak at 530 nm in its UV−vis spectrum. Absorption at this wavelength is indicative of individual nanoparticles averaging approximately 30 nm in diameter.21,22 DLS measurements support our UV−vis analysis; DLS shows that the average size of the particles in solution was ∼35 nm. Four days after the addition of 2,5-DMBA, the characterization by UV−vis spectroscopy indicated a shift in the plasmon resonance, yielding a new maximum absorption at 548 nm (see Figure 2). Red shifts of the plasmon peak are an indicator of aggregate formation.23 DLS measurements taken after the addition of the reporter molecule now resulted in an average particle size of 72 nm. Although we see an increase in particle diameter, it is not known whether this agglomeration is due to the physical linkage of Au colloids through the reporter’s ortho and meta thiol groups or by way of the combinatorial nature of the reporter, where disulfide bonds lead to the formation of a mega structure capable of caging the colloids. This was not further investigated in this work but will require additional attention at a later stage. It is, however, interesting to note that the particle

NRS = normal raman spectroscopy; v = stretching; β = in plane deformation; a = symmetric; b = anti-symmetric; sh = shoulder. a

the presence of both the thiol and disulfide groups. Solid 2,5DMBA is not soluble in acidic aqueous media but instead readily dissolves in alkaline solutions. The Raman spectra at a pH of ∼12.0 shows almost complete deletion of the S−H stretch at ∼2550.0 cm−1 and S−H deformations at 917.0 and 978.9 cm−1, while also showing the opposite trend for the S−S stretching bands at 446.6 and 468.3 cm−1 (see Figure 3). These trends suggest that alkaline solutions promote disulfide bond formation as we would expect. Other bands of interest are highlighted in both Figure 3 and Table 1 Our reporter molecule is not readily soluble in nonalkaline aqueous solutions, and thus, ethanol was used to introduce the 3577



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Figure 4. (a) The intensity ratio between the 1156 and 1128 cm−1 modes displays excellent pH sensitivity well into the acidic range. The intensities were obtained by first fitting the region within 1000 and 1200 cm−1 of each spectrum to the sum of four Lorentzians, then reconstructing each vibrational mode from values obtained from the fit, and finally retrieving the maximum value from the peak apex and plotting it against pH. The peak ratio obtained from nanoprobes in adenocarcinoma cells is shown indicated by a triangle. (b) SERS spectra of the probes as a function of pH. Each spectrum is the average of several SERS measurements for the nanoprobes at various pH taken in a flow cell. (c) Wide field image of of adenocarcinoma cells; the red circle indicates the location where measurements were taken. (d) Corresponding SERS spectrum taken from nanoprobes within the cell. (e) The contribution (red peaks) of each vibration to spectrum (d) after fitting to the sum of four Lorentzians. This is shown in comparison to the peak contributions from SERS calibration measurements obtained in a flow cell at pH 4.40.

The SERS spectra of our probe molecule show significant changes as a function of pH (see Figure 3c). The spectra in Figure 3c are the average of typically about 20 individual spectra taken at various pHs. The most spectrally distinguishable pH-sensitive bands are the ν(C−COOH) band at 847.0 cm−1, the δ(COO−) band at 875.4 cm−1, the β(CCC) + ν(C− S) band at 1098 cm−1, the β(CCC) + ν(C−S) + ν(C−COOH) band at 1128 cm−1, and the β(CH) band at 1156 cm−1. Although these bands are observable in the clean environment of a flow cell, signal fluctuations and background contributions render some of these peaks mute (see Figure S1, Supporting Information) or unreliable for intracellular applications; therefore, we choose to use the peaks within the 1000 and 1156 cm−1 for range pH quantification. These peaks display robust, reliable signal changes in vitro that are easily distinguishable from spectral noise. Conversely, the close proximity of these bands to one another makes it difficult to adequately determine each peak’s true spectral contribution. Quantifying the proton concentration based on spectral intensity requires an accurate way of measuring the peak apexes of these pH sensitive bands. Lorentzian functions are often used to fit Raman peaks because of their particular shape.24,25 Therefore, in order to extract more accurate intensities from all the pH-sensitive peaks, we fit the convoluted region between 1000 and 1156 cm−1 to the sum of four Lorentzians using eq 1:

size of the aggregates equilibrated at a diameter that is roughly double the diameter of the single particles, indicating the formation of mostly dimers and trimerized particles rather than higher order aggregates. Interestingly, SERS spectra from the 2,5-DMBA aggregates taken under acidic and basic conditions strongly resemble their normal Raman counterparts. Each SERS spectrum in Figure 3 is the average of typically about 20 individual spectra taken at pH 2.70 and pH 11.2. We verified that 2,5-DMBA is bound to the surface of the nanoparticles based on the lack of S−H stretching bands at 2531.005 and 2572.209 cm−1 and also the lack of S−H deformation bands at 917.0 and 978.9 cm−1 in the acidic SERS spectrum. Both, the normal Raman and SERS spectra obtained at basic pH indicate the presence of the δ(COO−) vibration at 875.4 cm−1 and have similar spectral profiles within 1000 and 1200 cm−1. The acidic SERS spectrum and its normal Raman analogue both show the ν(C−COOH) bands at 847.0 and 855.3 cm−1, respectively, while both also exhibit just three distinguishable signals within the 1000 to 1200 cm−1 region. As expected, the β(CCC) + ν(C−S) + ν(C−COOH) vibration is very intense and obscures the β(CCC) + ν(C−S) band in the acidic normal Raman spectrum. Oddly, the direct comparison of the SERS data as a function of pH shows that the β(CH) mode at 1156 cm−1 increases with increasing pH (Figure 3); this trend is also seen in the normal Raman measurements and in the SERS of oMBA. The fact that this increase in signal is seen in oMBA suggests that 2,5-DMBA may share a similar nanoparticle binding mechanism, where the orthosulphur (S) atom and the oxygen in the carboxylic acid group are both interacting with the metal surface.

4

f (x ) =

i

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⎞ I(i)Γ(i)2 ⎟+C 2 2 ⎝ (x(i) − x0) + Γ(i) ⎠ ⎛

∑⎜

(1)



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Here, I is the peak intensity, Γ is the full width at half-maximum (fwhm) of each peak, xo is the spectral position at which the desired peak is observed, and C is a constant. The data in this region were reconstructed using the results from the fit, and the peak intensities were extracted from the reconstructions (see Figure S2, Supporting Information). We chose to quantify the pH using the peaks at 1128 and at 1156 cm−1. Since these peaks showed a pH-sensitive profile, we decided to use their ratio as an indicator of pH. The ratios of these two peaks were plotted against pH and ultimately also used as a reference for live cell measurements (see Figure 4a). Each point along our curve is the average peak ratio of peaks taken from approximately 20 spectra, and therefore, their standard deviation is a reflection of the probes’ spectral consistency as a function of pH. As we compare ratios collected at each pH, it becomes clear that when the pH decreases our probes precision increases. Note that the line in Figure 4a is just a guide to the eye and not derived from a true fit. To determine the function of our probe molecules in a SERS construct under more challenging conditions, we also performed in vitro measurements with cells in culture. Approximately 17 000 adenocarcinoma cells were seeded in a glass bottom Petri dish using 2.7 mL of media (see Figure 4c). Our pH-sensitive nanoprobes were introduced to the cells by adding 300 μL of the nanoprobe solution to the Petri dish. The probes were incubated with the cells for 48 h; afterward, the media was exchanged. SERS measurements were collected on probes taken up by the cells by focusing on particle clusters inside the cells as indicated in Figure 4c. The data collected from the adenocarcinoma cells were analyzed similar as the SERS spectra obtained in the flow cell calibration measurements. We first compared the spectral profiles of the SERS reporters obtained in the flow cell to those of the spectra obtained from within the adenocarcinoma cells. In Figure 4a, the spectral profile of the nanoprobes in the flow cell for different pHs is shown, and in Figure 4d, the spectral profile of probes taken up by one of the carcinoma cells is shown. We noticed that the spectrum in Figure 4d closely resembled the spectra taken at pH 4.40 in Figure 4b. Observing the blue shift and intensity change of the β(CCC) + ν(C−S) + ν(C− COOH) band at 1128 cm−1 and also its intensity relative to the β(CH) at 1156 cm−1, it is obvious from the spectra that the probe is contained within an acidic environment. Endosomes in mammalian cells have acidic interiors with pH ranging from pH 4.70 to 6.0; late endosomes are at the lower end of this range. Although using different cell lines, other benzoic acid derived SERS probes have also shown acidic pH inside cells and concluded that the probes were incorporated into the cell via the endocytosis pathway. Therefore, we concluded that our probe has also been transported into the cell via endocytosis. The SERS spectra of taken up pH probes were fit to the sum of four Lorentzians (cf. eq 1), and the fitted contributions of each vibration within the 1000 to 1200 cm−1 regions were obtained; the results were compared to that taken for our probe at a known pH of 4.40 (Figure 4e). Similarly to before the ratio of the β(CH) at 1156 cm−1 to β(CCC) + ν(C−S) + ν(C− COOH), the band at 1128 cm−1 was calculated and compared to the calibration data obtained ex vitro. The ratio in vitro was calculated to be 1.34, corresponding to a pH of approximately 4.7, seen as a triangle in Figure 4a. This is consistent with the probe being incorporated into late endosomes and/or lysosomes. The incorporation of the probes into endosomes also protects them from intracellular thiolated molecules that

might compete for the gold nanoparticle surface against our reporter molecules. Also, the SERS spectra show no evidence for such a process taking place.

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CONCLUSIONS Although the commercially available pH-sensitive SERS reporter molecule pMBA works well for following pH dynamics,1 it required pH calibration on a particle by particle basis because of significant particle to particle variations. Furthermore, below a pH of approximately 5.5, this probe becomes rather unreliable3,4 because its pH sensitive range is more basic than for the probes shown here. The carboxylate v(COO−) vibration used to determine pH becomes irresolvable below this threshold pH. Here, we have shown that the SERS performance of the dithiol 2,5-DMBA on Au nanospheres has good pH resolution at and below a pH of 7, with good resolution maintained well below a pH of 5.5. To the best of our knowledge, no other SERS-active probe has been able to produce similarly good pH resolution across almost the entire acidic range on the simple, inexpensive nanoprobe system reported here. Similar reports required the use of more complex particle sytems, i.e., the use of colloidal scaffolds that are composed of more than one material, or nanoparticles with a hollowed core. Such scaffolds require multiple time-consuming steps for their synthesis. In conclusion, we have demonstrated the successful synthesis of 2,5-DMBA, a disulfide molecule that can both function as a reproducible pH-sensitive SERS reporter and induce the controlled aggregate formation in Au colloids. Saturation of the pH-sensitive peaks is achieved at a pH of approximately 8.4, and therefore, the operational range of the probe is well suited for pH measurements in biological samples. First data obtained by incubating our probes with MDA-MB231 adenocarcinoma breast cancer cells provided excellent indication that the reporter molecule can maintain robust pHsensitive signals even when utilized in cell cultures.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funding from the National Science Foundation. The Center for Biophotonics, an NSF Science and Technology Center, is managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. We would like to thank Dr. Astra Chang and Prof. Jan Nolta (UC Davis) for providing the adenocarcinoma cells utilized in this study.

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REFERENCES

(1) Talley, C. E.; Jusinski, L.; Hollars, C. W.; Lane, S. M.; Huser, T. Anal. Chem. 2004, 76 (23), 7064−7068. (2) Schwartzberg, A. M.; Oshiro, T. Y.; Zhang, J. Z.; Huser, T.; Talley, C. E. Anal. Chem. 2006, 78 (13), 4732−4736.

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