Fluorescent pH Sensors for Broad-Range pH Measurement Based on

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Novel Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore Jing Qi, Da-Ying Liu, Xiaoyan Liu, Shiquan Guan, Fengli Shi, Hexi Chang, Huarui He, and Guangming Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00053 • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 24, 2015

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Analytical Chemistry

Novel Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore Jing Qi,† Daying Liu,† Xiaoyan Liu,† Shiquan Guan,‡ Fengli Shi,‡ Hexi Chang,‡ Huarui He,*‡ Guangming Yang*† † ‡

Department of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China Heowns Biochem Technologies LLC, 6 Lanyuan Road, Tianjin 300384, P.R. China

ABSTRACT: We constructed a series of novel optical sensors for determination of broad-range pH based on a single fluorophore and multi-ionophores with different pKa’s. These optical sensors use photo-induced electron transfer (PET) as the signal transduction and follow the design concept of “fluorophore-spacer-receptor (ionophore)” which employs 4-amino-1,8-naphthalimide as the single fluorophore, ethyl moiety as the spacer and a series of phenols and anilines as the receptors. Key to the successful development of this sensor system is that coupling the receptors with six different pKa values with a single fluorophore produces the correct optical properties. This rational design affords a series of optical pH sensors with unique fluorescence property and accurately tunable pH measurement ranging from 1 to 14 pH units. Because of covalent immobilization of the indicators, these sensors demonstrate excellent stability, adequate reversibility and satisfactory dynamic range up to full pH ranges (pH 1 – 14).

The pH sensors are found frequently in a wide range of applications required in various fields of science and technology such as chemical process control, medical diagnosis and environmental analysis, as well as industrial applications.1-7 There are mainly two kinds of pH sensors such as electrochemical sensor and lately fiber optical sensor.8,9 Although electrochemical pH sensors are well-established and can be used as reliable tools for a large number of analytical tasks, a number of disadvantages of the pH electrode including frequent calibration, the susceptibility to electrical interference and corrosion by alkaline solutions or fluoride ions limit its usefulness.10 In the last three decades, many efforts have been directed toward the development of optical pH sensors11, especially fiber optic pH sensors.12-20 Optical pH sensors are based on reversible changes in the indicator’s structures induced by pH and translated into changes in spectroscopic phenomena such as absorption,21 reflectance,22 luminescence.23 Among these techniques, the fluorescent pH sensor23-28 has gained considerable attention due to its particularly high sensitivity. For real-world applications, especially for on-line monitoring where the frequent calibration is not possible, the fluorophore plays a key role in the design of fluorescent sensors. Therefore, searching for fluorophores with good properties including excellent photostability, large Stokes shifts and high quantum yield for optical sensing is a challenge for the research efforts in analytical chemistry. Among those fluorophores reported in the literatures, aminophthalimide, which has been used as fluorescent brightening agents, intracellular biomarkers, DNA photocleavers, and electro-luminescent copolymers,29-33 is an excellent choice with superb stability and high quantum yield. A series of 1,8-naphthalimide ester analogues with good ‘‘offon’’ switching of fluorescence upon encountering the correct target have been applied in a number of areas including colora-

tion of polymers,34,35 fluorescent markers in biology,32 electroluminescent materials,36,37 and also photo-induced electron transfer (PET) sensors.38-40 The intra-molecular photo-induced electron transfer (PET) process of naphthalimide fluorophores has been studied in details.41-43 PET regularly occurs when certain photoactive materials interact with light, and the format of “fluorophorespacer-receptor (ionophore)” is the most commonly exploited approach for the design of fluorescent sensors and switches.44 The fluorophore is covalently linked to receptor by means of a non-π-electron-conjugating spacer group, e.g. alkyl group with one to four carbons. Typically, the receptor contains an oxygen or a nitrogen atom, the electrons of which can ligate the analyte ion. In the absence of a bound cation or proton, the HOMO (highest occupied molecular orbital) of the unbound receptor has a higher energy than the half-filled HOMO of the excited fluorophore. This energy difference drives rapid electron transfer from the receptor to the excited-state fluorophore, thus the fluorescence is quenched, or “switched off”. When the receptor is bound to a cation or proton, the energy level of the receptor is lower than that of the HOMO of the excited fluorophore. Therefore, the receptor is stabilized and the electron transfer is not energetically favored, thus, the fluorescence is “switched on” (as shown in Figure 1).44 Although many different types of fluorescent pH probes and sensors including fluorescein derivatives,24,45-47 BODIPY dyes,48-56 rhodamines,57-59 and naphthalimide derivatives60-62 have been reported, most of them didn’t respond to pH over a broad range or required use of different multi-fluorophores. To the best of our knowledge, few reports have been published concerning determination of pH change over a broad range in the presence of receptors with different pKa values and naphthalimide as the single fluorophore so far. Here, we report a

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series of novel fluorescent sensors for determination of broadrange pH based on PET using a series of ionophores with different pKa values (N,N-bis(2-methoxyethyl)aniline, N,Ndiethylaniline, 2,6-dichlorophenol, 2-chlorophenol, phenol, 2,6-dimethoxyphenol) as the receptors, 4-amino-1,8naphthalimide as the single fluorophore, and ethyl moiety as the linker (Figure 2). The benzoic acid moiety is used for covalent immobilization onto hydrophilic polymer support for reversible measurements.

Figure 1. Mechanism of Photoinduced Electron Transfer (PET) using phenols and anilines as ionophores, aminonaphthalimide as fluorophore.

Figure 2. Structures of ionophores and fluorophore used in this work.

EXPERIMENTAL SECTION Reagents. Solvents and reagents used in the synthesis of sensors were purchased form Heowns Biochem Technologies LLC (Tianjin, China). Column chromatographic purification was carried out on silica gel (200-300 mesh) obtained from

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Heowns Biochem Technologies LLC (Tianjin, China). Tris(hydroxymethyl)aminomethane (Tris), citric acid, concentrated hydrochloric acid, sodium hydroxide, sodium tetraborate decahydrate, sodium chloride, and phosphoric acid used in the preparation of pH buffer solution were of analytical grade reagents and purchased from Sigma-Aldrich (Shanghai, China). The amino-cellulose fibers used in the synthesis of fluorescent dyed particles were purchased from Heowns Biochem Technologies LLC (Tianjin, China). All solutions were prepared using deionized water. The hydrophilic hydrogel D4 and the polyester sheets (MELINEX), obtained from Heowns Biochem Technologies LLC (Tianjin, China). Instruments. Intermediate and target products were measured by 1H NMR and 13C NMR spectra recorded using a 300 MHz instrument (Varian) in DMSO-d6 or CDCl3 with TMS as a standard. Data were presented as follows: 1H NMR chemical shift (in ppm on the scale relative to δTMS = 0), multiplicity (s = singlet, d = doublet, t = triplet, and m = multiplet), coupling constant (J/Hz), and interpretation. 13C NMR chemical shift values were given in ppm on the scale (δTMS = 0). HRMS (EI) spectra were obtained on the 6520 Q-TOF LC/MS (Agilent). The sensor polycarbonate cassette was made by ultrasonic plastic welding machine BRANSON 900 series ( BRANSON, US ). Fluorescence spectra were measured on the Gangdong F280 fluorometer (Tianjin, China). Absorption measurements were performed on Gangdong UV−4501S spectrophotometer (Tianjin, China). pH values were measured with JENCO 6173 pH (Shanghai, China). Water was purified by using Molgene 1810 A Deionizer (Shanghai, China). Synthesis. Figure 3 showed the synthetic route of six pH dyed particles. The detailed procedures and characterization of the new compounds were described in the Supporting Information. Preparation of Buffer Solutions with Different pH. The buffer solutions covering wide range of pH were prepared as following: Tris(hydroxymethyl)aminomethane (Tris, 3.0250g), citric acid (5.2500g), sodium tetraborate decahydrate (9.5340g), and phosphoric acid (2.8800g) and sodium chloride (8.7660g) were dissolved in 1500 mL deionized water, diluted to 2L by adding more deionized water. The pH meter was calibrated at 24.2 °C with standard buffers of pH 6.86 and 9.18. In order to keep the ionic strength approximately constant, all buffers were prepared using the same stock solution. A series of 14 buffers with different pH values (1.10, 2.15, 3.11, 4.11, 5.12, 6.26, 7.35, 8.20, 9.11, 10.51, 11.14, 12.24, 13.28, 13.96) were prepared by adding appropriate amount of conc. HCl or 4.5M sodium hydroxide solution. Preparation of Sensor. Sieved (100-µm) indicator immobilized amino-cellulose fiber (0.2 g) was stirred 24 h into a D4 hydrogel dispersion (3.8 g) containing 10% solids in 90% w/w ethanol/water. The resulting dispersion was knife-coated onto a 150-µm polyester sheet such that the indicator layer dried to a thickness of 15 µm. A second hydrogel layer consisting of 3% (w/w) carbon black (Degussa Corporation) in the same hydrogel dispersion was then knife-coated and allowed to dry overnight. The sample layer is covered with the ion-permeable blackened hydrophilic polymer (carbon black layer) and the total thickness is about 20µm. This overcoat protects the sensing layer from ambient light and optical interferences by the


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Figure 3. Synthesis of the pH dyed particles.

sample.63 A sensor disk 25 mm in diameter was then prepared in large sheets that can be separated into individual sensor disc. One disc was fixed on the polycarbonate cartridge to be hydraulic molded. The polyester sheet containing sensor disc was placed into plastic flow-through pump which allowed the pH buffer solution to be introduced and measured. Measurement of the Fluorescence Quantum Yield ΦFlu and Molar Absorption Coefficient ε. In this work, the fluorescence quantum yield (ΦFlu) and molar absorption coefficient (ε) of compound 3a, 3b, 3c, 3d, 3e, and 3f were measured according to the published literature64,65. We selected the rhodamine 10165,66as the reference substance. RESULTS AND DISCUSSION Design of the Ionophores. Generally, a single pH indicator can only cover up to 3 pH units. In order to measure the full range of pH, at least five pH indicators with evenly distributed pKa are required. Phenols and anilines have been proven to be efficient ionophores in the preparation of PET-based pH sensor.50-55 Existence of hydroxyl and amino groups provide many possibilities for further modification of the ionophores. The phenolic hydroxyl group, its pKa value is about 10,46 is an attractive factor in organic synthesis. By attaching electronwithdrawing groups such as halogen (o-chloro and o,odichloro) or electron-donating groups such as alkyl or alkyloxy (o-methyl, p- methyl, o-methoxy or p- methoxy) to the benzene ring, one can modulate the electron density of the phenolic hydroxyl groups, causing the change of pKa’s accordingly. By introducing one chlorine atom at ortho position, the pKa of phenol can be reduced from 10 to 8.5,67 decreasing about 1.5 units. Similarly, an ionophore with pKa near 7 can be obtained by introducing two chlorine atoms at ortho positions (o,odichloro).51 Contrarily, phenols with higher pKa’s can be achieved by introducing electron-donating groups such as methyl or methoxy groups. For the easiness of organic synthesis,

o,o-dimethoxy phenol is chosen as the ionophore with a pKa higher than 10. A key starting material, syringaldehyde, is commercially available and its aldehyde group can be converted into ethyl amine by a few simple steps. Thus, 2,6dimethoxyphenol is selected as the ionophore for the pKa higher than 10. It is well known50-55 that fluorescence intensity of phenolbased PET sensor is “switched on” in a relatively acidic medium (pHpKa) while aniline-based PET sensor has the similar pH trend as that of phenol. The aniline-based PET sensor also “switched on” in a relatively acidic medium (pHpKa). Electrochemical measurements show that PET in several amino-modified dyes is thermodynamically favorable, underlining the importance of kinetic aspects to the process.52 Upon recognition of the analyte (protons or other metal ions), the oxidation potential of secondary amine increases less than that of tertiary amine after the protonation of the amino moieties. Therefore, the secondary amine doesn’t show significant changes in the emission properties as a function of pH.52 Thus, the tertiary amine is much more preferred over the secondary amine. The successive protonation of the tertiary amine moiety enhances the fluorescence intensity. The changes are of such magnitude that they can be considered as representing two different ‘‘states’’, where the fluorescence emission is ‘‘switched off’’ in a relatively alkaline solution and ‘‘switched on’’ in an acidic solution. Consequently, there is pH-response affiliated with the protonation of the tertiary amine, which is characterized by the corresponding pKa value, with a usual sensitive pH-range of pKa ±1.5. N,Ndiethylaniline, whose pKa is about 5.5,46 meets the requirement of covering pH range of 3.5 -7. For the ionophore with a pKa lower than 3, a similar approach was taken by introducing an electron-withdrawing group, chlorine, at the ortho position of



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N,N-diethylaniline. Unfortunately, the result obtained with this ionophore was not satisfactory due to too low quantum yield. Enlightened by the low basicity of N,N-bis(hydroxyethyl)aniline, methoxyethyl was introduced through nitrogen atom of the aniline, yielding N,N-bis(2-methoxyethyl)aniline, which is used as ionophore covering the most acidic range of pH (1 – 3). In summary, the selected ionophores are, in the sequence of increasing pKa, N,N-bis(2-methoxyethyl)aniline, N,Ndiethylaniline, o,o-dichloropheol, o-chlorophenol, phenol and o,o-dimethoxyphenol. The structures are shown in Figure 2, along with the selected fluorophore. Selection of the Fluorophore. Conjugation of a well established and efficient recognition site with a suitable signaling moiety is the most popular and rewarding approach, which has been widely used for the design of specific and sensitive fluorescence pH sensors. Single fluorophore bearing diverse pHsensitive groups is strongly preferred over multi-fluorophores for several reasons: (i) fixing excitation and emission spectra wavelengths in the practical application and instruments design; (ii) avoiding the mutual effects of different fluorophores and ensuring the stability and accuracy of measurement; (iii) strengthening the unity of data to facilitate calculation process. In principle, many well known fluorophores such as rhodamine, fluorescein, BODIPY and coumarine etc. could be chosen as the single fluorophore, but amino-1,8-naphthalimide derivatives are strongly preferred owing to their many advantages. We chose to use 4-amino-1,8-naphthalimide as the single fluorophore based on following reasons: it absorbs in the visible region (λ ~ 470 nm), emits in the green (λ ~ 550 nm), with Stokes shifts of ca. 80 nm, and possesses high fluorescence quantum yield and excellent fluorescence properties based on PET (Figure 1). Most importantly, it has been proven that this fluorophore can be covalently immobilized on to hydrophilic polymer support and widely used in many applications in the real world environments.43,51,68 Fluorescence Spectra. Firstly, a few concepts in this work are defined as follows: fluorescent indicator means those simple compound 3a, 3b, 3c, 3d, 3e, and 3f. When the indicator is immobilized onto hydrophilic polymer support (aminocellulose fiber in this work), it is called fluorescent dyed particle (compound 4a, 4b, 4c, 4d, 4e, and 4f) which is embedded into the hydrogel and cast into thin film to produce optical sensor ( 5a, 5b, 5c, 5d, 5e, and 5f) respectively.

solutions. The pH of those buffer solutions were set at the pH values 2 units lower than individual pKa: 4a, pH=1.10; 4b, pH=3.11; 4c, pH=5.12; 4d, pH=7.35; 4e, pH=9.11; 4f, pH=10.51. (left: excitation spectra, fixed emission at 550 nm, right: emission spectra, fixed excitation at 470 nm).

Figure 4 showed the excitation and emission spectra of six pH dyed particles (4a, 4b, 4c, 4d, 4e and 4f) suspended in the different buffer solutions. In order to obtain spectra with nearly full fluorescence intensity, the suspensions of six dyed particles were scanned in buffer solution with the pH values 2 units lower than individual pKa: 4a, pH=1.10; 4b, pH=3.11; 4c, pH=5.12; 4d, pH=7.35; 4e, pH=9.11; 4f, pH=10.51. As expected, while the pH value is changing, all the corresponding fluorescent dyed particles exhibit a characteristic excitation (absorption) band of 4-amino-1, 8-naphthalimide at ~470 nm with a weak green color emission at ~550 nm. Notably, PET process can be explained more clearly by using this real example shown in Figure 4. The excitation and emission maxima are nearly invariant even by changing various ionophores with different pKa. This is the characteristic of a PET process and is different from intramolecular charge transfer (ICT) in conjugated systems, in which the emission maxima of the fluorophore change upon binding of an analyte. The invariability of the spectra is unique to PET-based sensors and enables us to construct a new series of pH sensors by simply mixing different fluorescence dyed particles with different pKa’s. Dynamic Response of a Film Sensor with a Single Dyed Particle. Using film sensor 5d as an example, Figure 5 (a) showed the dynamic response of film sensor 5d (ochlorophenol as the ionophore) by varying pH from 6.26 to 12.24, with the excitation and emission wavelength set to 470 nm and 550 nm, respectively. As expected, the fluorescence intensity went down gradually with the increase in pH value of the sample increased. The increasing pH caused more deprotonation of the hydroxyl group of the phenol moiety, which is believed to be responsible for the PET quenching. A similar mechanism can be applied to the sensors using the aniline derivatives as ionophore (sensor 5a, 5b). A stable signal was obtained within about 1 min, which meets the requirements for most practical application for on-line monitoring. The slight drift of the signal at the beginning was caused probably by the improper setting of the instrument, in which the excitation light was too strong. This can be avoided just by turning down the brightness of the excitation light. Figure 5(b) showed the calibration curve of film sensor 5d. F denotes the measured fluorescence intensity at a given pH and Fi denotes the fitted fluorescence intensity using Boltzmann function equation shown as following: (Fmax-Fmin)/(Fi-Fmin)=1+10^[W(pKa-pH)]

Figure 4. Fluorescence excitation and emission spectra of fluorescent dyed particles (4a, 4b, 4c, 4d, 4e and 4f) in different buffer

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(1)

Where Fmax was the maximum fluorescence intensity, Fmin was the minimum fluorescence intensity, W was empirical parameters describing width of the calibration curve. From the graph, one can see that the experimental data can be fitted quite well using above-mentioned equation. Two calibration curves overlapped perfectly. All other five sensors were measured in a similar way (as shown in Figure S2-S6) and the results are summarized in Table S1.


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Table 1. Optical properties of fluorescence indicator 3a-3f.

Compound

3a

3b

3c

3d

3e

3f

log(ε(acid)/ M-1cm-1)

4.32 ±0.05

4.32 ±0.02

4.10 ±0.03

4.38 ±0.03

4.18 ±0.04

4.50 ±0.04

log(ε(base)/ M-1cm-1)

4.29 ±0.03

4.29 ±0.01

4.13 ±0.03

4.39 ±0.02

4.20 ±0.05

4.50 ±0.04

ΦFlu(acid)

0.61 ±0.03

0.61 ±0.05

0.23 ±0.02

0.39 ±0.04

0.34 ±0.01

0.10 ±0.01

ΦFlu(base)

0.035 ±0.010

0.082 ±0.004

0.008 ±0.001

0.010 ±0.002

0.010 ±0.005

0.035 ±0.008

To better compare the response among the six film sensors, the maximum fluorescence intensity (Fmax) was normalized from the original data by using Fmax = 1 in the Eq (1) as shown in Figure 6. Obviously, the six calibration curves cover entire range of pH from 1 – 14, and more importantly the fluorescence on-off trend are very consistent among each other. As discussed earlier, all six sensors follow the same tendency: lower pH of the sample leads to higher fluorescence intensity of the sensor. These tendencies facilitate the fact that a new series of pH sensors with different sensing range can be created by means of mixing proper amount of fluorescence dyed particles.

Figure 5. (a) Dynamic response of optical film sensor 5d in buffer solution and (b) The corresponding calibration curve. F denotes the measured fluorescence intensity and Fi denotes the fitted fluorescence intensity using Boltzmann function equation. The excitation: 470 nm, emission: 550 nm.

The results in Table S1 showed that the pKa values of all six sensors are distributed very evenly across the entire pH range. As mentioned before, the useful range of an individual film sensor can only cover up to about 3 pH units. The empirical parameters (W), which not only describe the width of the calibration, also imply the steepness of the sensor slope, lies between 0.55 and 0.68. In addition, fluorescence quantum yield ΦFlu and molar absorption coefficient ε for all six compounds (3a, 3b, 3c, 3d, 3e, 3f) were also measured, and were listed in Table 1. The profiles of fluorescence quantum yield ΦFlu vs pH for 3a, 3b, 3c, 3d, 3e and 3f are performed on excitation at 470 nm wavelength in DMF/buffer solution (1/9, v/v). Molar absorption coefficient ε is in units of M-1 cm-1. The results in Table 1 showed that aniline ionophores ( 3a, 3b) and phenol ionophores (3c, 3d, 3e) had very similar ε and ΦFlu except for 3f, which were different from the reported literature.69,70 We assumed that electron-richness of the ionophore 3f was responsible for the lower fluorescence quantum yield ΦFlu due to the electron-donating effect of two methoxy groups. Besides the invariability of the fluorescence spectra mentioned above, there are additional requirements for the preparation of a broad range pH sensor by means of simply mixing those fluorescence dyed particles with a well defined weight ratio. The low intensity of sensor 5f can be compensated by adding more dyed fluorescence particle.

Figure 6. The normalized calibration curves of all six sensors (5a, 5b, 5c, 5d, 5e, 5f). The excitation: 470 nm, emission: 550 nm.

Creation of a Series of pH Sensors with Different Sensing Ranges. Many practical applications require specific pH sensing range, e.g. pH 1 – 4 for fermentation of vinegar,4 pH 3 – 6 for fermentation of winery,6 pH 6 – 8 for fresh water aquaculture, pH 7 – 9 for sea water aquaculture7 and pH 9 - 11 for alkaline fermentation.5 Theoretically, many pH sensors with different sensing range could be constructed by using six fluorescence dyed particles with different pKa’s. For the purpose of demonstration, we chose seven typical combinations for some specific applications and the results were summarized in Figure 7 and Table 2. Sensor #1 uses 100% fluorescence dyed particle 4c to measure pH 6.0 – 8.5 for the possible application of fresh water aquaculture; Sensor #2 is composed of fluorescence dyed particle 4b and 4c in a 1:1 ratio and measures pH 5.0 – 8.5, which could be applied in winery fermentation and fresh



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change every 0.01 pH change (%/0.01pH), decreases gradually. For example, the slope of sensor #1, which contains only one dyed fluorescence particle, was 0.56%/0.01pH, while sensor #6, which consists of five dyed fluorescence particles, was only 0.16%/0.01pH. The slope has dropped about 40%. The extension of measuring range is at the expense of sensor slope. It strongly suggests that one needs to keep the slope value within a reasonable range, while trying to mix more dyed fluorescence particles to extend the pH sensing range. Ionic Strength and Photo-stability. To better describe the effect of cross-sensitivity to ionic strength (IS) and the photostability, we tested the sensor under different IS and continuous illumination over a longer period of time according to the reported literature19. Figure 7. Seven pH film sensors made with different amount of dyed particle in D4 hydrogel. Sensor #1, (Dye: 4c 100%); Sensor #2, (Dyes: 4b/4c = 50/50); Sensor #3, (Dyes: 4c/4d = 50/50); Sensor #4, (Dyes: 4c/4d:/4e = 31.3/31.3 /37.4); Sensor #5, (Dyes: 4b/4c/4d/4e = 23.5/23.5/23.5/29.5); Sensor #6, (Dyes: 4a/4b/ 4c/4d/4e = 30.8/15.8/15.8/15.8/21.8); Sensor #7, (Dyes: 4a/4b/4c/ 4d/4e/4f = 28/13/13/13/19/14).

water aquaculture; Sensor #3 is made of fluorescence dyed particle 4c and 4d in a 1:1 ratio and measures pH 6.0 – 10.5, which could be suitable for the application of fresh and sea water aquaculture; Sensor #4 uses a mixture containing 31.3% fluorescence dyed particle 4c and 31.3% of 4d and 37.4% of 4e, measures pH 6.0 – 12.0 for the possible application of fresh water, sea water aquaculture and alkaline fermentation; Sensor #5 employs a mixture composed of 23.5% fluorescence dyed particle 4b, 23.5% of 4c, 23.5% of 4d and 29.5% of 4e, measures pH 3.5 – 12.0, which is suitable for the application of winery fermentation, fresh/sea water aquaculture and alkaline fermentation; Sensor #6 and 7 involve five or more dyed particles, enable us to measure almost full range of the pH. The detailed composition can be found in Table 2. As described above, various sensors with specific measuring pH ranges could be obtained by different combination of dyed particles. Table 2. Summary of seven pH film sensors with different combination of dyed fluorescence particle 4a to 4f.

Sensor composition dyed particle

Weight (%)

Useful range (pH)

Slope (%/0.01 pH)

4c

100

6.0-8.5

-0.566

4b/4c

50/50

5.0-8.5

-0.446

4c/4d

50/50

6.0-10.5

-0.373

4c/4d/4e

31.3/31.3/37.4

6.0-12.0

-0.286

4b/4c/4d/4e

23.5/23.5/23.5/29.5

3.5-12.0

-0.216

4a/4b/4c/4d/ 4e

30.8/15.8/15.8/15.8/21.8

1.0-12.0

-0.159

4a/4b/4c/4d/ 4e/4f

28/13/13/13/19/14

1.0-14.0

-0.142

As the measuring range of pH becomes wider and wider by mixing more kinds of dyed fluorescence particles into one sensor, one of the key parameter, the sensor slope (last column in Table 2), which is defined as percentage fluorescence signal

Figure 8. (a) Calibration curves of sensor #7 measured at different ionic strengths. (b) Photodegradation profiles of sensor #7 and the reference (immobilized carboxy-fluorescein) in a buffer solution (acidic: pH 3.11; basic: pH 9.11) when illuminated with the fluorometer (fixed excitation at 470 nm).

Purposely, the broadband sensor #7 was chosen as the example because it was composed of six dyed particles. If one dye had strong ionic strength dependence or poor photostability, then the fluorescence signal of broadband sensor #7 would drift. Figure 8a showed the calibration curves of sensor #7 under different ISNaCl. The ISNaCl changed from 50 to 400mM, the calibration curve of sensor #7 drifted slightly upwards. The photo-stability of this sensor was tested through the fluorescence due to the higher sensitivity (as shown in Figure 8b). As one can see, the sensor #7 was exposed to a


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continuous illumination for about 3 h in the acidic and basic solution, respectively. It showed that the fluorescence intensity of the sensor #7 decreased about 3%, while the intensity of the reference fluorescein sensor in a basic solution decreased more than 25%. All the above data indicated that all dye particles (or sensors) had an acceptable ionic strength dependence and photo-stability. The signal drift resulted from these effects can be corrected in the practical applications. Batch-to-Batch Reproducibility. We repeatedly immobilized compound 3c and 3d onto amino-cellulose fiber five times, respectively, and received ten different fluorescent dyed particles (4c1, 4c2, 4c3, 4c4, 4c5, 4d1, 4d2, 4d3, 4d4, 4d5). Five different film sensors #3 (#31, #32, #33, #34, #35) were produced by simply 1:1 mixing of fluorescent particle 4c1 and 4d1, 4c2 and 4d2, and so on. The results of five film sensors were summarized in Table 3 (the fluorescence intensity was normalized to the first batch 4c1+4d1). The results in Table 3 showed that all five-batch film sensors behaved similarly except the fluorescence intensity, which varied from 3% to 12%. These results suggested that individual calibration of each batch was required. For the application in real world, however, this issue can be minimized by scaling up the batch size, since calibration of an individual batch which is enough to manufacture up to million sensor discs will become much less burden in the real-world application. In fact, this strategy is been taken to manufacture the sensor disc in the real production line. Table 3. Summary of five different film sensors #3.

Figure 9. The dynamic response of the sensor #7 to broad range pH 1.10 - 13.28, by shuttling from pH 1.10 to various pH: 3.11, 5.12, 7.35, 9.11, 11.14 and 13.28.

CONCLUSION We have described a series of novel optical sensors suitable for measurement of pH up to full ranges (1 -14). This design of the optical sensors follows the fluorophore -spacer- receptor approach using intramolecular PET-based signal transduction. Key to the development of these sensors is the design of phenol- and aniline-containing hydrogen ionophores, coupled with single fluorophore, 4-amino-1,8-naphthalimide. A pH sensor with any sensing range can be constructed by simply mixing six fluorescence dyed particles with proper weight ratio. The sensors showed excellent stability, reversibility and fast response time and are being used for on-line pH monitoring in many real world applications.

Film sensor #3

Fmax/Fmin

pH useful range

Slope (%/0.01 pH)

#31(4c1+4d1)

1/0.040

6-10.5

-0.373

ASSOCIATED CONTENT

#32(4c2+4d2)

0.97/0.037

6-10.5

-0.378

Supporting Information

#33(4c3+4d3)

0.95/0.035

6-10.5

-0.367

#34(4c4+4d4)

1.07/0.043

6-10.5

-0.369

#35(4c5+4d5)

1.06/0.041

6-10.5

-0.370

Detailed synthetic procedures, NMR, HRMS, dynamic response of optical film sensors (5a, 5b, 5c, 5e, 5f) and the reversibility and stability of sensor #7. This material is available free of charge via the Internet at http://pubs.acs.org.

Reversibility, Response Time and Stability. Figure 9 showed the dynamic response of sensor #7 to full range of pH (1 – 14). The reversibility and stability were examined by shuttling pH from 1.10 to various pH’s: 3.11, 5.12, 7.35, 9.11, 11.14 and 13.28 and repeating this procedure three times. The sensor showed an excellent reversibility over the broad range of pH from 1 to 13. A stable signal was generally reached in about 60 seconds, and returned to its initial value in less than 50 seconds. The stability of the sensor was excellent as well. As one can see from the graph, the initial fluorescence intensity of pH 1.10 remained constant value near 1.0 normalized photon counts, even after thirty-six times of buffer shuttling from 1.10 to 13.28. Furthermore, fluorescence intensity changes upon switching from pH 1.10 to 13.96 also demonstrate the reversibility and stability of sensor #7 (Figure S7). In fact, all these discussed sensors are being used for continuously on-line monitoring pH in various fields including abovementioned four applications.

AUTHOR INFORMATION Corresponding Author

* Guangming, Yang: Tel, +86-22-23501230. Email, [email protected] * Huarui, He: Tel, +86-22-83717408. Email, [email protected] Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript.

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT The work was supported by National Natural Science Foundation of China (Nos. 20941004, 21071084, 21071085 and 90922032), MOE (IRT-0927), Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry and Tianjin Natural Science Foundation (No. 11JCYBJC03500). The authors acknowledge the helpful discussions and collaboration from co-workers within Heowns Biochem Technologies LLC.



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REFERENCES (1) Li, X. H.; Gao, X. H.; Shi, W.; Ma, H. M. Chem. Rev. 2014, 114, 590-695. (2) Tang, Y. L.; Liu, Y.; Cao, A. L. Anal. Chem. 2013, 85, 825830. (3) Peng, H. S.; Stolwijk, J. A.; Sun, L. N.; Wegener, J.; Wolfbeis, O. S. Angew. Chem. Int. Ed. 2010, 49, 4246-4249. (4) Wang, J.; Fung, D. Y. C. Crit. Rev. Microbiol. 1996, 22, 101138. (5) de Ory, I.; Romero, L. E.; Cantero, D. J. Food Eng. 2004, 63, 39-45. (6) Robinson, J. The Oxford Companion to Wine, 3rd ed.; Oxford University Press, 2006, 267-279. (7) Cahill, P. L.; Hurd, C. L.; Lokman, M. Aquaculture 2010, 306, 153-159. (8) Galster, H. pH Measurement: Fundamentals, Methods, Applications, and Instrumentation, VCH, New York. 1991. (9) Suah, F. B. M.; Ahmad, M.; Taib, M. N. Sens. Actuators, B 2003, 90, 182-188. (10) Shamsipur, M.; Abbasitabar, F.; Zare-Shahabadi, V.; Shahabadi.; Akhond, M. Anal. Lett. 2008, 41, 3113-3123. (11) Wencel, D.; Abel, T.; McDonagh, C. Anal. Chem. 2014, 86, 15-29. (12) Wang, X. D.; Wolfbeis, O. S. Anal. Chem. 2013, 85, 487-508. (13) Arregui, F.J.; Otano, M.; Fernandez-Valdivielso, C.; Matias, I. R. Sens. Actuators B 2002, 87, 289-295. (14) Jin, Z.; Su, Y.; Duan, Y. Sens. Actuators, B 2000, 71, 118-122. (15) Gotou, T.; Noda, M.; Tomiyama, T.; Sembokuya, H.; Kubouchi, M.; Tsuda, K. Sens. Actuators, B 2006, 119, 27-32. (16) DeGrandpre, M. D.; Baehr, M. M. Anal. Chem. 1999, 71, 1152-1159. (17) Wolfbeis, O. S. Anal. Chem. 2002, 74, 2663-2678. (18) Aigner, D.; Borisov, S. M.; Orriach Fernández, F. J.; Fernández Sánchez, J. F.; Saf, R.; Klimant, I. Talanta 2012, 99, 194 -201. (19) Aigner, D.; Borisov, S. M.; Petritsch, P.; Klimant, I. Chem. Commun. 2013, 49, 2139-2141. (20) Aigner, D.; Borisov, S. M.; Klimant, I. Anal. Bioanal. Chem. 2011, 400, 2475-2485. (21) Liu, Z.; Luo, F.; Chen, T. Anal. Chim. Acta 2004, 510, 189194. (22) Sotomayor, M.D.P.T.; Paoli, M.A.D.; Oliveira, W.A. Anal. Chim. Acta 1997, 353, 275-280. (23) Cajlakovic, M.; Lobnik, A.; Werner, T. Anal. Chim. Acta 2002, 455, 207-213. (24) Lobnik, A.; Oehme, I.; Murkovic, I.; Wolfbeis, O. S. Anal. Chim. Acta 1998, 367, 159-165. (25) Nivens, D. A.; Schiza, M. V.; Angel, S. M. Talanta 2002, 58, 543-550. (26) Ji, J.; Rosenzweig, Z. Anal. Chim. Acta 1999, 397, 93-102. (27) Michael, K. L.; Taylor, L. G.; Walt, D. R. Anal. Chem. 1999, 71, 2766-2773. (28) Aigner, D.; Ungerböck, B.; Mayr, T.; Saf, R.; Klimant, I.; Borisov, S. M. J. Mater. Chem. C 2013, 1, 5685–5693. (29) Niu, C. G.; Zeng, G.M.; Chen, L. X.; Shen, G.L.; Yu, R. Q. Analyst 2004, 129, 20-24. (30) Konstantinova, T.; Spirieva, A.; Petkova, T. Dyes Pigm. 2000, 45, 125-129. (31) Patrick, L. G. F.; Whiting, A. Dyes Pigm. 2002, 55, 123-132. (32) Stewart, W. W. J. Am. Chem. Soc. 1981, 103, 7615-7620. (33) Yao, W.; Qian, X. H.; Hu, Q. Y. Tetrahedron Lett. 2000, 41, 7711-7715. (34) Hu, Y.; Wang, B.; Su, Z. J. Appl. Polym. Sci. 2009, 111, 19311935. (35) Bojinov, V.; Grabchev, I. Dyes Pigm. 2003, 59, 277-283. (36) Zhu, W. H.; Minami, N.; Kazaoui, S.; Kim, Y. J. Mater. Chem. 2003, 13, 2196-2201.

Page 8 of 9

(37) Wang, Y.; Zhang, X. G.; Han, B.; Peng, J. B.; Hou, S. Y.; Huang, Y.; Sun, H. Q.; Xie, M. G.; Lu, Z. Y. Dyes Pigm. 2010, 86, 190-196. (38) Tian, H.; Gan, J. A.; Chen, K. J.; He, J.; Song, Q. L.; Hou, X. Y. J. Mater. Chem. 2002, 12, 1262-1267. (39) Grabchev, I.; Qian, X. H., Bojinov, V.; Xiao, Y.; Zhang, W. Polymer 2002, 43, 5731-5736. (40) Grabchev, I.; Chovelon, J-M.; Qian, X. H. New J. Chem. 2003, 27, 337-340. (41) Tian, H.; Xu, T.; Zhao, Y. B.; Chen, K. C. J. Chem. Soc., Perkin Trans. 2 1999, 545-549. (42) Le, T. P.; Rogers, J. E.; Kelly, L. A. J. Phys. Chem. A 2000, 104, 6778-6785. (43) He, H.; Mortellaro, M. A.; Leiner, M. J. P.; Young, S. T.; Fraatz, R. J.; Tusa, J. K. Anal. Chem. 2003, 75, 549-555. (44) de Silva, A. P.; Nimal Gunaratne, H. Q.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515-1566. (45) Lin, H. J.; Szmacinski, H.; Lakowicz, J. R. Anal. Biochem. 1999, 269, 162-167. (46) McNamara, K.P.; Nguyen, T.; Dumitrascu, G.; Ji, J.; Rosenzweig, N.; Rosenzweig, Z. Anal. Chem. 2001, 73, 3240-3246. (47) Liu, Y.H.; Dam, T.D.; Pantano, P. Anal. Chim. Acta 2000, 419, 215-225. (48) Baruah, M.; Qin, W.; Basarić, N.; De Borggraeve, W. M.; Boens, N. J. Org. Chem. 2005, 70, 4152-4157. (49) Boens, N.; Leen, V.; Dehaen, W. Chem. Soc. Rev. 2012, 41, 1130-1172. (50) Sun, K. M.; McLaughlin, C. K.; Lantero, D. R.; Manderville, R. A. J. Am. Chem. Soc. 2007, 129, 1894-1895. (51) He, H.; Mortellaro, M. A.; Young, S. US patent, 8,097,725 2006/0121623 A1. (52) Aigner, D.; Freunberger, S. A.; Wilkening, M.; Saf, R.; Borisov, S. M.; Klimant, I. Anal. Chem. 2014, 86, 9293-9300. (53) Lin, J.; Liu, D. Anal. Chim. Acta 2000, 408, 49-55. (54) Wolfbeis, O. S.; Daub, J.; Gereis, T.; Kollmannsberger, M.; Heinl, S.; Werner, T.; Huber, C.; Boila-Goekel, A.; Leiner, M. J. P., US patent, 6,001,999, 1999/085 812. (55) Gereis, T.; Huber, C.; Wolfbeis, O. S.; Daub, J. Chem. Commun. 1997, 1717-1718. (56) Werner, T.; Huber, C.; Heinl, S.; Kollmannsberger, M.; Daub, J.; Wolfbeis, O. S. Fresenius J. Anal. Chem. 1997, 359, 150-154. (57) Best, Q. A.; Xu, R. S.; McCarroll, M. E.; Wang, L. C.; Dyer, D. J. Org. Lett. 2010, 12, 3219-3221. (58) Praveen, L.; Saha, S.; Jewrajka, S. K.; Das, A. J. Mater. Chem. B, 2013, 1, 1150-1155. (59) Li, Z.; Wu, S. Q.; Han, J. H.; Han, S. F. Analyst 2011, 136, 3698-3706. (60) Georgiev, N. I.; Bojinov, V. B.; Nikolov, P. S. Dyes Pigm. 2011, 88, 350-357. (61) Cox, R. P.; Higginbotham, H. F.; Graystone, B. A.; Sandanayake, S.; Langford, S. J.; Bell, T. D. M. Chem. Phys. Lett. 2012, 521, 59-63. (62) Kriltz, A.; Löser, C.; Mohr, G. J.; Trupp, S. J. Sol-Gel Sci. Technol. 2012, 63, 23-29. (63) Leiner, M. J. P.; Hartmann, P. Sens. Actuators, B 1993, 11, 281-289. (64) Brouwer, A. M. Pure Appl. Chem. 2011, 83, 2213-2228. (65) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; 54. (66) Karstens T.; Kobe K. J. Phys. Chem. 1980, 84, 1871-1872. (67) Lide, D. R.; Haynes, W. M. CRC Handbook of Chemistry and Physics, 2010, 8-45. (68) Tusa, J. K.; He, H. J. Mater. Chem. B, 2005, 15, 2640-2647. (69) de Silva, A. P.; Rice, T. E. Chem. Commun. 1999, 163-164. (70) de Silva, A. P.; Nimal Gunaratne, H. Q.; Gunnlaugsson, T. Tetrahedron Lett. 1998, 39, 5077-5080.


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TOC Graph


Fluorescent pH Sensors for Broad-Range pH Measurement Based on

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