Objective In some research fields and practical applications, the accurate determination of pH is extremely important. For example, in biology, the change in intracellular pH plays an important role in cell growth, apoptosis, ion transport, homeostasis, and enzymolysis. There are three main methods for traditional detection of pH: pH indicator; pH test paper; glass pH electrode. The principles of the pH indicator and test paper are essentially the same, and the pH value is determined from color change after the reaction using the indicator and test paper. However, because of the color change, the pH value of the solution is determined according to a standard colorimetric card, which is easily affected by subjective consciousness. At the same time, the detection target range is small; for example, pH test paper cannot display the pH value of oil. Furthermore, the accuracy of the pH value is limited by the method used. The use of a glass pH electrode is the main method for accurately determining pH, but it is not suitable for long-term use owing to susceptibility to mechanical damage, electrical interference, long preparation time, complex calibration, and other reasons; moreover, the detection targets are limited. Therefore, it is necessary to find a simple, noncontact, and reliable method to detect pH. In this study, a pH optical probe with good sensitivity, selectivity, reversibility, and stability was constructed using SERS technology, Au NPs as reinforcement substrate, and R6G as a probe molecule. Our basic strategy and research results are expected to be helpful for the combination of pH detection with other fields and the tracking of pH changes in single molecules and tissue cells.

Methods In this study, Au NPs were used as the enhancement substrate and R6G was used as the probe molecule to output SERS signals. The pH value of the sample solution was rapidly and conveniently measured. First, Au NPs were prepared via chemical reduction, and the silicon wafers were modified using the soaking method to obtain four types of dispersed distributions; the particle size of the SERS-enhanced substrate was within the range of 30--100 nm. Second, the morphology, particle size, particle dispersion, and LSPR resonance frequency of Au NPs were characterized using scanning electron microscopy (SEM) and ultraviolet visible absorption spectroscopy. We explored the uniformity and reproducibility of the enhanced Raman signal and the influence of different R6G concentrations and Au NPs size on the SERS spectrum and finally select the appropriate molecular concentration of Au NPs and R6G probe for subsequent experiments. Subsequently, we measured the SERS spectrum changes of the probe molecules under different pH conditions and determined the linear relationship between the peak area ratio of SERS and the pH value of R6G at 1363 and 1314 cm ^-1 so as to design the pH sensor. In addition, we conducted experiments on recovery, stability, H ⁺ selectivity, and real sample pH value to explore the performance of the pH sensor.

Results and Discussions The 45 nm Au NPs substrate exhibited better enhancement effect, stability (Fig.2) and uniformity of the Raman signal (Fig.3). Because of the different inclination orientation of the deprotonated and protonated R6G molecules adsorbed on the enhanced base surface in closed- and open-loop forms, R6G exhibited different SERS activities at different pH levels (Fig.4). We found a linear relationship between the SERS spectral peak area ratio of R6G and pH value at 1363 and 1314 cm ^-1 to design the pH sensor. Experimental results show that the SERS signal of R6G can be stabilized for more than 2 h at room temperature. The sensor showed good recoverability when the pH value of the sample solution was changed back and forth between 7 and 3 (Fig.5). After the introduction of other interfering metal cations in the pH detection process, the probe showed better selectivity of H ⁺ (Fig.6). The detection of the actual sample pH revealed that the probe has good analytical performance and that it is easy to detect the pH value in acidic mediums (Table 1).

Conclusions We explored the method of pH sensing using R6G molecules adsorbed on Au NPs. In the process of pH increasing from small to large values, R6G molecules changed from protonation in the form of an open ring to deprotonation in the form of a closed loop, and the angle between the R6G molecules on Au NPs' surface increased gradually, resulting in the interference of stretching vibration of the C—N bond connected with the benzene ring and C—C bond of the aromatic ring. We found a linear relationship between pH and the C—C and C—N frequencies.

Results show that the probe has superior sensitivity, selectivity, reversibility, and stability. The SERS signal can be stabilized for more than 2 h at room temperature. Moreover, the probe has the ability to detect dynamic changes in pH, which can reflect the change in pH in actual acidic mediums. Compared with traditional pH-detection methods and other optical pH sensors based on the spectral response of synthetic probes, the pH sensor prepared in the present work has the advantages of high sensitivity, nondestructive detection, and good biological stability. The application range of the pH sensor is widened, the selected probe molecules are easy to obtain and economical, and the phenomena of photolysis and photobleaching do not occur easily in the detection process. The rhodamine group produces cationic stimulated SERS signals with strong fluorescence; this has good application prospects in SERS ultrasensitive single-molecule detection. Moreover, it is easy to be combined with other fields and has greater application prospects in tracking the pH changes in single molecules and tissue cells.