隨著科技的日新月異，pH值的檢測在許多領域當中已是不可或缺的一項指標，酸鹼值的測量方式有很多，目前大多數量測酸鹼值的方式為使用傳統玻璃薄膜電極的pH meter，優點為快速、簡單且便於攜帶，但是電極非常脆弱且準確度僅能到達小數點後一位；而螢光具有極高的靈敏度與螢光染劑結合檢測酸鹼值近年來陸續的被發表，因此本研究嘗試開發出螢光酸鹼感測器，使用不同的解離常數(pKa)酸鹼指示劑或修飾酸鹼指示劑的官能基，來增加可檢測範圍。
首先需要會隨pH改變，而螢光放光強度也會改變的酸鹼指示劑，我們先選用茜素紅(Alizarin Red S, ARS)作為此酸鹼感測器的主要酸鹼指示劑，根據文獻顯示，最酸的兩個pka分別為5.5及10.8，在Phosphate buffer pH 2-12中，利用485 nm作為激發波長，在pH酸性的範圍會出現570 nm以及鹼性的範圍會有670 nm的放光，利用這個特性推算出未知溶液的酸鹼值，並將ARS的羥基接上硼化合物改變pKa和UV吸收以及螢光的強度，也可改變在不同pH值的環境下，有不同的螢光強度回應，使可偵測的pH值範圍改變，以增加可以利用的範圍，並使用不同酸鹼指示劑螢光素( Fluorescein, Flu )來擴增我們的偵測範圍。
在緩衝溶液的配置中利用Debye-Hückel equation來分別校正在0.1 M的濃度情況下的Phosphate buffer (PB)、Acetate buffer (Ac)以及Sodium bicarbonate buffer (Sbc)的pKa，再利用Henderson-Hasselbalch equation計算出欲配置的pH值緩衝溶液所需要加入的酸鹼鹽克數。使用我們的方法配置出的buffer pH值可精準到小數點後第二或甚至第三位。
接著為了使我們的偵測訊號能夠有更好的R2，因此分別針對螢光儀參數 (slit、激發波長) 以及指示劑的濃度作改變，以優化出最佳的偵測條件，再利用優化好的條件決定出每種酸鹼指示劑可以偵測的範圍，總結出三種指示劑所能覆蓋的範圍，ARS為PB pH 5.00-5.90以及PB pH 10.00-11.35；Flu為PB pH 2-3 (HCl、NaOH調配)、Ac pH 4-5以及PB pH 5.00-6.35；ARS-PBA Complex為pH 3.50-5.00，而在精密度及準確度的分析也能夠超越傳統的pH meter，並且在螢光法和吸收法的精密度及準確度比較中，證明螢光法是具有更高的靈敏度。
最後，我們利用Forster cycle求出ARS的理論lowest excited singlet state pKa，並推得化合物如果帶比較多推電子基，其excited state的pKa通常都會比ground state的pKa低上許多，因為形成共軛鹼後的電子會提供到芳香環上，使結構變得更穩定，讓氫更容易解離，因此讓激發態的pKa變得更低，但是利用Forster cycle所求出的激發態pKa，只能幫助我們做基本的判斷，但得到的結果無法到很精準，因為由Van't Hoff equation的公式得知，如果測量的溫度條件無法固定，其解離常數便會受到影響，故無法得到準確的pKa*值但是可以知道其改變的趨勢。

With the development of technology, the detection of pH value has become an indispensable indicator in many fields. At present, most methods for measuring pH value are pH meter using glass thin film electrodes. It is fast, simple, convenient and easily to carry, but the electrode is very fragile and the accuracy only reach one decimal. Fluorescence has extremely high sensitivity and the combination of fluorescent acid-base sensor to detect pH value has been published in recent years. In this study, we tried to develop a fluorescent acid-base sensors which used different dissociation constant (pKa) acid-base sensor to increase the detectable range.
First, we need a acid-base sensor that changes with pH and the intensity of fluorescence emission. We choose Alizarin Red S (ARS) as the main compound of this sensor. According to the literature, there are two main pKa in ARS. One is 5.5; the other is 10.5. In phosphate buffer pH 2-12, using 485 nm be the excitation wavelength, the emission wavelength will be 570 nm in the acidic range and 670 nm in the alkaline range. The property can be calculated the pH value of the unknown solution. The hydroxyl group of ARS is connected to a boron compound to change the pKa, UV absorption and the fluorescence intensity. The pH range was changed to increase the available range and a different dye Fluorescein (Flu) was used to expand our detection range.
The pKa of Phosphate buffer (PB), Acetate buffer (Ac), and Sodium bicarbonate buffer (Sbc) at 0.1 M concentrations were corrected according to the Debye-Hückel equation. and the Henderson-Hasselbalch equation was used to prepare the required pH within the range of pKa±1 in these buffers. The buffer pH can be accurate to the second or the third decimal by our method.
In order to make our detection signal have a better R2, the parameters of the fluorometer (slit, excitation wavelength) and the concentration of dye were changed to optimize the best detection conditions. Then used the optimized conditions to determine the detectable range and summerized the range that the three dyes can cover. ARS is PB pH 5.00-5.90 and PB pH 10.00-11.35; Flu is PB pH 2-3, Ac pH 4-5 and PB pH 5.00-6.35; ARS-PBA Complex is pH 3.50-5.00. The analysis in precision and accuracy can also surpass the traditional pH meter. The comparison between the absorption and the fluorescence method also proves that the fluorescence method is a better detection method and has higher sensitivity than the absorption method.
Finally, we used the Forster cycle to find the theoretical lowest excited singlet state pKa of ARS and deduced that if the compound has more electron-donating groups, the excited state pKa is usually lower than the ground state pKa. Electrons of conjugation base will be donated to the aromatic ring that making the structure more stable and hydrogen ion easier to dissociate, so making the excited state pKa lower than the ground state pKa. The excited state pKa obtained by the Forster cycle that only can help us do the basic judgment. According to the Van't Hoff equation that if the measured temperature conditions can not be fixed, the pKa will be affected, so the accurate pKa* value cannot be obtained.

1. Brisset, J.-L.; Benstaali, B.; Moussa, D.; Fanmoe, J.; Njoyim-Tamungang, E., Acidity control of plasma-chemical oxidation: applications to dye removal, urban waste abatement and microbial inactivation. Plasma Sources Science and Technology 2011, 20 (3).
2. Shalaby, A. A.; Mohamed, A. A., Determination of acid dissociation constants of Alizarin Red S, Methyl Orange, Bromothymol Blue and Bromophenol Blue using a digital camera. RSC Advances 2020, 10 (19), 11311-11316.
3. Braslavsky, S. E., Glossary of terms used in photochemistry, 3rd edition (IUPAC Recommendations 2006). Pure and Applied Chemistry 2007, 79 (3), 293-465.
4. Drummen, G. P., Fluorescent probes and fluorescence (microscopy) techniques--illuminating biological and biomedical research. Molecules 2012, 17 (12), 14067-90.
5. Thomas, O.; Burgess, C., UV-Visible Spectrophotometry of Water and Wastewater (Second Edition). Amsterdam: Elsevier 2017.
6. Dong, Z.; Le, X.; Zhou, P.; Dong, C.; Ma, J., An “off–on–off” fluorescent probe for the sequential detection of Zn2+ and hydrogen sulfide in aqueous solution. New J. Chem. 2014, 38 (4), 1802-1808.
7. Galardon, E.; Tomas, A.; Roussel, P.; Artaud, I., New fluorescent zinc complexes: towards specific sensors for hydrogen sulfide in solution. Dalton Trans 2009, (42), 9126-30.
8. Kaushik, R.; Kumar, P.; Ghosh, A.; Gupta, N.; Kaur, D.; Arora, S.; Jose, D. A., Alizarin red S–zinc(ii) fluorescent ensemble for selective detection of hydrogen sulphide and assay with an H2S donor. RSC Advances 2015, 5 (97), 79309-79316.
9. Roger, T.; Raynaud, F.; Bouillaud, F.; Ransy, C.; Simonet, S.; Crespo, C.; Bourguignon, M. P.; Villeneuve, N.; Vilaine, J. P.; Artaud, I.; Galardon, E., New biologically active hydrogen sulfide donors. Chembiochem 2013, 14 (17), 2268-71.
10. Springsteen, G.; Wang, B., Alizarin Red S. as a general optical reporter for studying the binding of boronic acids with carbohydrates. Chem Commun (Camb) 2001, (17), 1608-9.
11. Baeyer, A., Ueber eine neue Klaese von Farbetoffen. Ber. Dtsch. Chem. Ges. 1871, 4, 555−558.
12. Tabor, C.; Pu, Y.; Wang, W.; Dorshow, R. B.; Alfano, R. R.; Kajzar, F.; Kaino, T.; Koike, Y., Picosecond polarization spectroscopy of fluorescein attached to different molecular volume polymer influenced by rotational motion. In Organic Photonic Materials and Devices XIV, 2012.
13. Anthoni, U.; Christophersen, C.; Nielsen, P. H.; Püschl, A.; Schaumburg, K., Structure of red and orange fluorescein. Structural Chemistry 1995, 6 (3), 161-165.
14. I, N. K.; Sawyer, W. H., Spectral Properties of the Prototropic Forms of Fluorescein
in Aqueous Solution J. Fluoresc. 1996, 6 ( 3), 147-157.
15. Lavis, L. D.; Rutkoski, T. J.; Raines, R. T., Tuning the pKa of Fluorescein to Optimize Binding Assays. Analytical Chemistry 2007, 79 (17), 6775-6782.
16. Amat-Guerri, F.; Costela, A.; Figuera, J. M.; Florido, F.; Sastre, R., Laser action from rhodamine 6G-doped poly (2-hydroxyethyl methacrylate) matrices with different crosslinking degrees. Chemical Physics Letters 1993, 209 (4), 352-356.
17. Dujols, V.; Ford, F.; Czarnik, A. W., A Long-Wavelength Fluorescent Chemodosimeter Selective for Cu(II) Ion in Water. Journal of the American Chemical Society 1997, 119 (31), 7386-7387.
18. Wu, M. M.; Llopis, J.; Adams, S.; McCaffery, J. M.; Kulomaa, M. S.; Machen, T. E.; Moore, H.-P. H.; Tsien, R. Y., Organelle pH studies using targeted avidin and fluorescein–biotin. Chemistry & Biology 2000, 7 (3), 197-209.
19. Wang, B.; Guan, X.; Hu, Y.; Su, Z., Synthesis and photophysical behavior of a water-soluble fluorescein-bearing polymer for Fe3+ ion sensing. Journal of Polymer Research 2008, 15 (6), 427-433.
20. Han, J.; Loudet, A.; Barhoumi, R.; Burghardt, R. C.; Burgess, K., A Ratiometric pH Reporter For Imaging Protein-dye Conjugates In Living Cells. Journal of the American Chemical Society 2009, 131 (5), 1642-1643.
21. Morris, M. C.; Depollier, J.; Mery, J.; Heitz, F.; Divita, G., A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nature Biotechnology 2001, 19 (12), 1173-1176.
22. Shi, W.; Li, X.; Ma, H., Fluorescent probes and nanoparticles for intracellular sensing of pH values. Methods Appl Fluoresc 2014, 2 (4), 042001.
23. Brooks, W. L. A.; Sumerlin, B. S., Synthesis and Applications of Boronic Acid-Containing Polymers: From Materials to Medicine. Chemical Reviews 2016, 116 (3), 1375-1397.
24. Sun, X.; James, T. D., Glucose Sensing in Supramolecular Chemistry. Chemical Reviews 2015, 115 (15), 8001-8037.
25. Liu, J.; Yang, K.; Shao, W.; Qu, Y.; Li, S.; Wu, Q.; Zhang, L.; Zhang, Y., Boronic Acid-Functionalized Particles with Flexible Three-Dimensional Polymer Branch for Highly Specific Recognition of Glycoproteins. ACS Applied Materials & Interfaces 2016, 8 (15), 9552-9556.
26. Wang, L.; Zhang, J.; Kim, B.; Peng, J.; Berry, S. N.; Ni, Y.; Su, D.; Lee, J.; Yuan, L.; Chang, Y.-T., Boronic Acid: A Bio-Inspired Strategy To Increase the Sensitivity and Selectivity of Fluorescent NADH Probe. Journal of the American Chemical Society 2016, 138 (33), 10394-10397.
27. Springsteen, G.; Wang, B., A detailed examination of boronic acid–diol complexation. Tetrahedron 2002, 58 (26), 5291-5300.
28. Dipak K. Palit, H. P., Tulsi Mukherjee" and Jai P. Mittal, Photodynamics of the S, State of some Hydroxy- and Am ino-su bstituted Naphthoqu inones and Anth raqu inones. J. CHEM. SOC. FARADAY TRANS. 1990, 86 (23), 3861-3869.
29. Brooks, W. L. A.; Deng, C. C.; Sumerlin, B. S., Structure-Reactivity Relationships in Boronic Acid-Diol Complexation. ACS Omega 2018, 3 (12), 17863-17870.
30. Swindlehurst, B.; Narayanaswamy, R., Optical Sensing of pH in Low Ionic Strength Waters. Optical Sensors 2004, 281-308.
31. Detailed discussion of pKa vs pKa0 of the buffer. http://www.reachdevices.com/Protein/pKa_explanation.html.
32. Steinegger, A.; Wolfbeis, O. S.; Borisov, S. M., Optical Sensing and Imaging of pH Values: Spectroscopies, Materials, and Applications. Chemical Reviews 2020, 120 (22), 12357-12489.
33. Xu, X. Y.; Yan, B., An efficient and sensitive fluorescent pH sensor based on amino functional metal-organic frameworks in aqueous environment. Dalton Trans 2016, 45 (16), 7078-84.
34. Safavi, A.; Abdollahi, H., Optical sensor for high pH values. Analytica Chimica Acta 1998, 367 (1), 167-173.
35. Werner, T.; Wolfbeis, O. S., Optical sensor for the pH 10–13 range using a new support material. Fresenius' Journal of Analytical Chemistry 1993, 346 (6), 564-568.
36. Posch, H. E. L., Marc J. P.; Wolfbeis, Otto S., Towards a gastric pH-sensor: an optrode for the pH 0- 7 range. Fresenius Z Anal Chem 1989, 334, 162-165.
37. Baldini, F.; Bechi, P.; Bracci, S.; Cosi, F.; Pucciani, F., In vivo optical-fibre pH sensor for gastro-oesophageal measurements. Sensors and Actuators B: Chemical 1995, 29 (1), 164-168.
38. Park, H.-R.; Mayer, B.; Wolschann, P.; Koehler, G., Excited-State Proton Transfer of 2-Naphthol Inclusion Complexes with Cyclodextrins. The Journal of Physical Chemistry 1994, 98 (24), 6158-6166.
39. Jamal, M. M. E.; Mousaoui, A. M.; Naoufal, D.; Tabbara, M. A.; Zant, A. A. E., Effect of Operating Parameters on Electrochemical Degradation of Alizarin Red S on Pt and BDD Electrodes. Portugaliae Electrochimica Acta 2014, 32, 233-242.
40. Murugesan, R.; Bajasekar, B.; Thanulingam, T. L.; Shunmugasundaram, A., Correlation of ground and excited state dissociation constants of trans-para- and ortho-substituted cinnamic acids. Chem. Sci. 1992, 104 (3), 431-436.
41. Gold, V., Ed., Advances in Physical Organic Chemistry. London: Academic Press, 1976.
42. Kubo, Y.; Ishida, T.; Kobayashi, A.; James, T., Fluorescent alizarin-phenylboronic acid ensembles: Design of self-organized molecular sensors for metal ions and anions. Journal of Materials Chemistry 2005, 15, 2889-2895.