A fluorescent colorimetric pH sensor and the influences of matrices on sensing performances

Abstract

A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488 nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pK_a).

1. Introduction

Measuring pH is a widely applied principle, however, is important for environment, ecology, agriculture, industry, food, and biology. Optical pH sensors based on changes of emission intensity and lifetime have been intensively studied as they can be non-invasive or minimally invasive, disposable, easily miniaturized (down to sub-micrometer), and simple to process (as a coating or solid layer on optical fibers and certain surfaces) for investigation of biology, environmental analysis, medical diagnosis, and process control [1 – 4]. Some optical pH sensors have been integrated with other sensors for simultaneous monitoring of biological and environmental (multi)-parameters [5–9]. In order to fulfill the requirements for pH sensing in environment, agriculture, industry, and biology for extracellular pH values, optical pH probes needed to be physically incorporated or chemically immobilized with a proton permeable polymer matrix, such as collagen, polyurethane hydrogel, or poly(2-hydroxyethyl methacrylate) [5–9]. Leaching of the physically doped sensors from the matrices is a significant problem [5] which may result in signal instability, inaccuracy of the resultant measurements, decreased long-term applicability, and potential cytotoxicity for cells. To alleviate the leaching problem, optical sensors have been conjugated into suitable matrices through either chemical grafting of the probes onto matrices [9] or polymerization of the probes with the precursors/monomers of the matrices [6, 7, 8, 10]. We have been investigating thin film (membrane)-based sensors derived from polymerizable monomeric pH probes [10] and their dual sensors [7,8] for extracellular pH analysis. The pH probe was polymerized with other necessary monomers including a crosslinker to form a stable gel membrane (Figure 1). Thus the probes were chemically immobilized in the matrices formed by the polymerization of the monomers and crosslinkers. It is a generally accepted fact that if the chemical structures of the probes are different, their sensing performances are different even if they are in the same polymer matrix. However, little information is available on how the structures of the matrices for the same sensing probes in terms of matrix compositions, charge densities, and crosslinker concentrations and structures affect the sensing capabilities of the sensors.

Figure 1.

The pH probes and other monomers used for the sensor film preparation.

Consequently, scientists are currently focusing on the development and use of fluorescence - based colorimetric approach for sensing studies [11–17] either through fluorescence intensity ratios [11–16] or by fluorescence lifetime referencing method [17]. The materials suitable for colorimetric imaging, which can be taken by conventional digital color cameras, should possess at least two separated emissions either from the same fluorophore or from different fluorophores. The colorimetric imaging is believed to be able to (1) act as an alternative scientific instrument of spectrofluorophotometer for ratiometric imaging with one excitation wavelength and multiple (three wavelengths of red-green-blue (RGB)) readouts and (2) produce small and inexpensive analytical devices.

Herein, we will address a few universal aspects for pH sensor study. (1) To investigate the influences of the matrix compositions on the sensing performance. We chose to study two monomeric pH probes, SM1 [18] and SM2 [19] (Figure 1). SM1 is based on the mechanism of intramolecular charge transfer to manipulate the responses [20]. SM1 exhibits a strong green emission at basic conditions, while producing a weak emission at acidic conditions. SM2 is based on a photo-induced electron transfer (PET) mechanism [20]. SM2 shows strong emission at acidic conditions, while weak emission is produced at basic conditions. (2) To prepare ratiometric sensors with high sensitivity and a broad dynamic range for pH value measurement. We integrated SM1 and SM2 into the same matrix. The dual color pH sensor (PSM1,2) will possess two emission colors suitable for ratiometric measurements and will achieve high sensitivity because of their opposite pH responsive trends. (3) Because of the well separated emission spectra of the individual SM1 and SM2, the emission changes of the integrated dual color sensor can not only be measured using spectrofluorophotometer, but can also be visualized by eyes or cameras. Therefore, this dual color sensor is also an emission-based colorimetric sensor. This study divulges mechanics behind the development of advanced small and inexpensive analytical devices, which may broaden the application of these sensors in the future.

2. Experimental Section

2.1 Materials and reagents

All chemicals and solvents were of analytical grade and were used without further purifications. N,N’-dimethylformamide (DMF), trimethylsilylpropyl acrylate (TMSPA), 2-hydroxyethyl methacrylate (HEMA), acrylamide (AM), 2-(methacryloyloxy)ethyltrimethyl ammonium chloride (METAC), poly(ethylene glycol)-dimethacrylate (PEGDMA, M_n = 550), and azobisisobutyronitrile (AIBN), were commercially available from Sigma-Aldrich (St. Louis, MO) and were used without further purification. SM1 and SM2 were synthesized according to previous publications [18, 19]. Ethyloxylate trimethylolpropane triacrylate (SR454®) is a commercial product of Sartomer Company (Exton, PA). 2-(Methyacryloyloxy)ethylsulfonic acid sodium salt (MESA) was purchased from TCI America (Portland, OR). Doubly distilled water was used for the preparation of the buffer solutions. Britton-Robinson (B-R) buffers composed of acetic acid, boric acid, phosphoric acid, and sodium hydroxide were used for tuning pH values. For fluorescence measurements, quartz glass from University Wafer (South Boston, MA) was cut into squares of 1.31 cm × 1.31 cm using a dicing saw (Microautomation, Billerica, MA).

2.2 Polymerization – the preparation of PHEMA-co-PAM copolymer sensor thin films

Thin films were prepared according to our published protocols [7, 8, 10, 21]. The preparation of the membrane is given in Figure 1. More detailed schematic drawing of the film preparation is given in Supplementary Data (S-Figure 1). The components for the films, possessing different ratios of polyHEMA (PHEMA), polyAM (PAM), polyPEGDMA (PPEGDMA), polyS454 (PSR454), poly(METAC) (PMETAC), and/or poly(MESA) (PMESA) were given in Table 1 and Table 2. Film of F5 is described here as a typical example. 1 mg of the monomeric SM1, 800 mg of HEMA, 150 mg of AM, 50 mg of PEGDMA, and 10 mg of AIBN were dissolved in 1 mL DMF as a stock solution. 15 µL of the stock solution was added onto the surface of the TMSPA-modified quartz glass and covered with a clean but untreated cover slip to make a sandwich structure. Using TMSPA to modify the quartz glass was to enable the sensors and matrices to be chemically grafted onto a quartz substrate [7, 8, 10, 21]. The thickness was controlled using 25 µm Kapton tape (DuPont, Wilmington, DE). The sandwich set-up was placed into a vacuum oven, which was then evacuated and refilled with nitrogen three times. Polymerization was carried out under nitrogen at 80 °C for 1.5 hours in the oven. The quartz glasses with polymer membranes were removed from the oven, with Kapton tape and non-surface modified glass being removed from the polymerized membrane surface. The polymer membranes on the quartz glasses were washed three times using methanol to remove any remaining non-polymerized monomers and residual DMF. The films were dried and stored in the dark at room temperature.

Table 1.

Compositions of the films of PSM1 (F1 to F14), their sensitivity, and pK_a values.

Films	Polymer compositions and their weight ratios						Sensitivity (IpH=9/IpH=3)f)	pKa
	Main matrices		Crosslinkers		Charges
	PHEMA	PAM	PEGDMA	SR454	PMETAC (+)	PMESA (−)
F1 a)		95	5				57	6.58
F2 a)	95		5				35	6.24
F3 a)	15	80	5				59	6.41
F4 a)	50	45	5				60	6.36
F5 a)	80	15	5				102	6.67
F6 a)	80	15	5		5		53	5.62
F7 a)	80	15	5		10		28	5.10
F8 a)	80	15	5		20		25	4.66
F9 a)	80	15	5			10	102	6.86
F10 a)	80	15	5			20	108	6.89
F11 b)	80	15	10				107	6.63
F12 c)	80	15	20				68	6.03
F13 d)	80	15		5			31	6.31
F14 e)	80	15		10			12	5.53

^a)Each film contains a 5% PEGDMA crosslinker

^b)The film contains a 10% PEGDMA crosslinker

^c)The film contains a 20% PEGDMA crosslinker

^d)The film contains a 5% SR454 crosslinker

^e)The film contains a 10% SR454 crosslinker

^f)The sensitivity of the sensing films to pH values was calculated according to the ratios of fluorescence intensities (I at 520 nm) at pH = 9 and pH = 3 for comparison.

Table 2.

Compositions of the films of PSM2 (F15 to F29), their sensitivity, and pK_a values.

Films	Polymer compositions and their weight ratios						Sensitivity (IpH=3/IpH=9)i)	pKa
	Main matrices		Crosslinkers		Charges
	PHEMA	PAM	PEGDMA	SR454	PMETAC (+)	PMESA (−)
F15 a)	95	0	5				3.5	5.63
F16 a)	80	15	5				5.7	5.33
F17 a)	50	45	5				4.0	5.93
F18 a)	15	80	5				3.3	6.17
F19 a)	0	95	5				3.2	6.00
F20 a)	80	15	5			5	5.3	7.73
F21 a)	80	15	5			20	4.5	7.67
F22 a)	80	15	5		5		4.1	5.87
F23 a)	80	15	5		20		4.1	5.13
F24 b)	80	15	10				4.1	5.57
F25 c)	80	15	20				3.2	4.60
F26 d)	80	15	40				1.7	--- h)
F27 e)	80	15		5			2.7	6.61
F28 f)	80	15		20			2.1	4.81
F29 g)	80	15		40			1.3	--- h)

^a)Each film contains a 5% PEGDMA crosslinker

^b)The film contains a 10% PEGDMA crosslinker

^c)The film contains a 20% PEGDMA crosslinker

^d)The film contains a 40% PEGDMA crosslinker

^e)The film contains a 5% SR454 crosslinker

^f)The film contains a 20% SR454 crosslinker

^g)The film contains a 40% SR454 crosslinker

^h)no reliable pK_a can be calculated

ⁱ⁾The sensitivity of the sensing films to pH values was calculated according to the ratios of fluorescence intensities (I at 612 nm) at pH = 3 and pH = 9 for comparison.

2.3 Instruments and characterization

An oxygen plasma cleaner (Harrick Plasma, Ithaca, NY) was used for quartz glass surface activation. A Shimadzu UV-3600 UV-VIS-NIR spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) was used for absorbance measurements. A Shimadzu RF-5301 spectrofluorophotometer was used for fluorescence measurements. For easy measurement of the films in liquid solutions, quartz glass was cut with a dicing saw into squares of 1.31 cm × 1.31 cm, which can fit diagonally into a quartz fluorescence cuvette to enable the sensing membrane be positioned at an angle of 45° to the excitation light. The pH values were determined with a digital pH meter (Thermo Electron Corporation, Beverly, MA) calibrated at room temperature (23 ± 2 °C) with standard buffers of pH 10.01, 7.00, and 4.01.

For getting color images of the dual color pH sensor using a camera, a 488 nm laser under a Nikon Eclipse Ti Microscopy was used to excite the sensor film. A Nikon D3000 camera was connected to the microscope for taking pictures.

3. Results and discussions

3.1 pH responses of the sensing membranes derived from SM1 (PSM1)

3.1.1 Film preparation

PHEMA, PAM, and their composites were chosen as the matrices, since they are excellent ion permeable hydrogels [22–25]. PEGDMA and SR454 were used as the crosslinkers with different hydrophilicity and hydrophobicity. PMETAC was used a positive charge polymer and PMESA was used as a negative charge polymer. Fourteen films (Table 1) were prepared for the investigation of the influence of various weight ratios of PHEMA, PAM, crosslinkers, and charge densities on the sensing performances. F1 is a PAM sensing film without PHEMA components. F2 is a PHEMA sensing membrane without PAM segments. F3, F4, and F5 are three sensing films with different weight ratios of PHEMA to PAM. F6 to F10 are PHEMA-co-PAM derived films with tunable charge densities by the copolymerization of the neutral HEMA and AM with the charge-containing METAC or MESA. F5, F11, and F12 are the films using different weight percentages of PEGDMA in the PHEMA-co-PAM matrix. F13 and F14 are the films using SR454 as the crosslinkers.

3.1.2 Influences of matrix compositions on sensing performance

Figure 2 showed the typical UV-Vis absorption, emission spectra of F5 at various pH values, and its sensing mechanism. The F5 film showed stronger emissions at basic conditions and weaker emissions at acidic conditions. All these changes are due to the differences of the intramolecular charge transfers of the fluorescein moieties from a conjugated dianionic tautomer at basic conditions to a non-conjugated lactone tautomer at acidic conditions (Figure 2C) [26]. More detailed structures of the tautomers of the fluorescein moieties by pH were given in Supplementary Data (S-Figure 2). The dianionic tautomer is a conjugated moiety, which exhibited strong absorption and strong green emissions. While the lactone tautomer is a non-conjugated segment, which neither absorb the light around 488 nm significantly nor emit light strongly.

Figure 2.

pH responses of F5 film. A) changes of absorbance by pH; B) changes of fluorescence by pH at an excitation wavelength of 488 nm; C) Simplified structural changes of the fluorescein moieties at strong basic and acidic conditions. Detailed intermediates between the two states were given in supporting S-Figure 2; D) fluorescence intensity (at 520 nm) ratios of F5, from pH 3 to pH 9. I₀ is the fluorescence intensity at pH 3.

Fluorescence intensity change by ratio at 520 nm was plotted at Figure 2D. The sensor has a dynamic range from pH 5.5 to pH 7.5, indicating its suitability for biological application. The fluorescent intensity at pH 9 is 100 fold larger than that at pH 3, showing the high sensitivity of the pH sensor.

The fluorescence intensity changes can be described well by a sigmoidal function (Boltzmann fitting) as shown in equation 1 and plotted in Figure 2D.

$$\frac{I}{I_0}=\frac{m1-m2}{1+\text{exp}(\frac{PH-P{K}_a^{\text{'}}}{P})}+m2$$ (1)

I and I₀ are the fluorescence intensities measured at varying pH values and that at the lowest pH value (pH 3) used during the titration, respectively. m1, m2, pK_a^’, and p are empirical parameters describing the initial value (m1), the final value (m2), the point of inflection (pK_a^’), and the width (p) of the sigmoidal curve. The apparent pK_a value (pK_a^’) for F5 was calculated to be 6.67 for F5. The fitting was highly reliable with a correlation coefficient (R²) of 0.995.

Figure 3A showed the ratios of the fluorescence intensities at various pH values via the intensities at pH = 3 of the films F1 – F5. Exact ratios and pK_a values were given in Table 1. The sensor film of F5 with a ratio of HEMA to AM of 80:15 by weight exhibited the best sensitivity (e.g, the highest value of I_pH=9/I_pH=3) among the few compositions we studied. This result indicated that the chemical compositions of the PHEMA-co-PAM changed the H⁺ permeability. It is known that both the PHEMA and PAM have good swelling properties and ion permeability. However, their swelling and ion permeability could be further improved by their copolymerizations with various ratios. Suitable composite materials can increase the swelling degree (W_∞) and therefore increase the range of pore sizes and size distribution in the hydrogel film [27], which affects the pH responses of the sensors.

Figure 3.

A) Fluorescence intensity (at 520 nm) ratios of films of F1 – F5, from pH 3 to pH 9. I₀ is the fluorescence intensity at pH 3; B) Influences of charges on sensing performances. I₀ is the fluorescence intensity at pH 3; C) Influences of cross-linkers and their concentrations on sensing performances. I₀ is the fluorescence intensity at pH 3.

We found that charges of polymer matrices also affected the sensing activities of membranes (Figure 3B). Increasing the density of negative charge on the film by MESA did not change the sensitivity to pH. In contrast, increasing the density of positive charge by METAC decreased the response sensitivity and also shifted the pK_a values to low pH values. This should be attributed to that ion permeability and the water swelling properties of the various composite materials were affected by the charged polymers.

Crosslinker densities and species also affected the sensing performances (Figure 3C). Increasing the crosslinker densities decreased the sensitivity and shifted the pK_a to low values, which can be found by a comparison of F5, F11, and F12 or a comparison of F14 and F15. When there is more crosslinker present, the network becomes much tighter, which may result in the decrease of swelling ability of the hydrogel and therefore the difficulty of protonation and deprotonization of the pH sensor in the matrices. Using SR454 instead of PEGDMA decreased the sensitivity significantly. Most likely, the films with the hydrophobic and short crosslinker of SR454 possess much tighter networks and smaller swollen ratios than those with PEGDMA.

3.2 pH responses of the sensing membranes derived from SM2 (PSM2)

3.2.1 Membrane preparation and sensing mechanism

Table 2 lists the films of PSM2 (F15 – F29) with various compositions. Sixteen films were prepared for investigation of the influence of various weight ratios of PHEMA, PAM, crosslinkers, and charge densities on the sensing performances.

Figure 4 shows the typical UV-Vis absorption and emission spectra of F16 at various pH values. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. Because of the strong electron-donating and -withdrawing units conjugated within the sensing moiety, SM2, the fluorophore exhibits an absorption maximum around 500 nm and emits in the red spectral window. A quasi-isosbestic point from the absorbance spectra was observed at 528 nm, showing that the sensor response to pH is through a single acidification and basification mechanism. The emission maxima shifted from 640 nm at pH 9 to 610 nm at pH 3 with the emission intensity increases as the pH decreases. The emission intensity change follows a sigmoidal (Boltzmann fitting, equation 1). The fluorescence intensity changes and their curve fittings are shown in Figure 7C. The apparent pK_a value (pK_a^’) was 5.33 with a correlation coefficient of 0.995.

Figure 4.

pH responses of film of F16. A) changes of absorbance by pH; B) changes of fluorescence by pH at an excitation wavelength of 488 nm; C) Mechanism of the sensor of PSM2; D) fluorescence intensity (at 612 nm) ratios of F16, from pH 3 to pH 9. I₀ is the fluorescence intensity at pH 9.

Figure 7.

Color images of the colorimetric pH sensor PSM1,2 at different pH (A – G) from pH 9 to pH 3 and the ratios of the intensities at green and red channels at various pH (H).

Fluorescence intensity change of PSM2 was ascribed to photo-induced electron transfer (PET) in the pH sensor being suppressed by the protonation of the amino group. When a fluorophore is attached to an electron quencher (usually one or more nitrogen-containing functional groups which are non-conjugated to the fluorophore), PET occurs between them (Figure 4C) [6–10]. In the piperazinyl group of SM2, the nitrogen atom in NCH₂CH₂ is not directly connected to the TCF-conjugated fluorophore, of which the NCH₂CH₂ moiety is a strong electron donor. PET occurs from the lone electron pair of the amine group to the acceptor TCF-containing fluorophore, making the sensor weakly fluorescent. At lower pH, however, the protonation of the amino group diminishes the PET effect and, in turn, leads to restoration of the fluorescence originating from the fluorophore. Hence, a remarked increase in emission intensity was observed at low pH.

3.2.2 Influences of matrixes on sensing performance

Figure 5A showed the ratios of the fluorescence intensities at various pH values versus the intensities at pH = 9 of the films F15-F19. Exact ratios and pK_a values were given in Table 2. The sensor film of F16 with a ratio of HEMA to AM of 80:15 by weight exhibited the best sensitivity (e.g, the highest value of I_pH=3/I_pH=9). This phenomenon is consistent with that of PSM1, showing the influences of the chemical compositions of the PHEMA-co-PAM on the sensing behaviors.

Figure 5.

A) Influences of the ratios of HEMA and AM on sensing performances of PSM2; B) Influences of charges on sensing performances of PSM2; C) Influence of PEGDMA as the crosslinker on sensing performances of PSM2; D) Influence of SR454 as the crosslinker on the sensing performance of PSM2. For easy comparison, 5% of PEG was kept in D.

Figure 5B plots the charge influences on the sensing performances. Increasing the density of negative charge on the film by MESA did not significantly change the sensitivity to pH, however, it did significantly shift the pK_a from 5.33 of F16 to ~7.7 of F20 and F21. Increasing the amount of positive charge segments of METAC decreased the sensitivity and shifted the pK_a to low pH.

Crosslinker densities and species also affected the sensing performances of PSM2 (Figure 5C and 5D). Increasing the crosslinker densities decreased the sensitivity and shifted the pK_a to low values. Using SR454 instead of PEGDMA decreased significantly the sensitivity. This trend is consistent with those of the films with PSM1.

3.3. Dual color PSM1,2 sensor (F30, F31, F32)

A dual color sensor was prepared by copolymerization of SM1 and SM2 in the neutral PHEMA-co-PAM (80:15 by weight) film with 5% of PEGDMA, because the individual PSM1 (F5) and PSM2 (F16) have good sensitivity using the matrix with the above mentioned composition. Three films (F30, F31, and F32) with variable ratios of SM1 and SM2 were prepared (Table 3). Typical sensor’s responses of F32 to pH at the excitation wavelength of 488 nm were given in Figure 6A. The green emission at 520 nm decreased with the decrease of pH. The red emission at 612 nm increased with the decrease of pH value. At pH 9, mainly green emission was observed; while only the red emission is dominated at pH 3.

Table 3.

Comparison of the sensitivity and pK_a values of the films of PSM1,2 and their corresponding PSM1 and PSM2.

Films	Sensor	IpH=9/IpH=3 (520 nm) d)	IpH=3/IpH=9 (612 nm) e)	I520 at pH9/I612 at pH9f) [(I520/I612 at pH9)/(I520/I612 at pH3)]h)	I612 at pH3/I520 at pH3g) [(I612/I520 at pH3)/(I612/I520 at pH9)]i)	pKa [dynamic range]
F5	PSM1	102				6.58 [5.5 –7.5]
F16	PSM2		5.5			5.50 [4.0 –7.0]
F30 a)	PSM1,2	110	1.8	50 f)	4.3 g)	6.75 [6–8] d)
				[209] h)	[210] i)	5.41 [4.5–7] e)
						7.16 [6–8] f)
						4.60 [4–7] g)
						7.15 [5.5–8] h)
						4.67 [4–6] i)
F31 b)	PSM1,2	94	2.0	38 f)	5.6 g)	6.76 [6–8] d)
				[213] h)	[213] i)	5.26 [4.5–7] e)
						7.10 [6–8] f)
						4.72 [4–7] g)
						7.24 [5.5–8] h)
						4.60 [4–6] i)
F32 c)	PSM1,2	44	4.5	12 f)	13 g)	6.89 [6–8] d)
				[200] h)	[200] i)	5.85 [4.5–7] e)
						7.09 [6–8] f)
						5.01 [4–7] g)
						7.02 [5.5–8] h)
						4.69 [4–6] i)

^a)The weight ratio of SM1:SM2 is 1:1

^b)the weight ratio of SM1:SM2 is 1:5

^c)the weight ratio of SM1:SM2 is 1:10

^d)fluorescence intensity at 520 nm was used for ratio calculation

^e)fluorescence intensity at 612 nm was used for ratio calculation

^f)fluorescence intensity ratios were calculated by using I_520nm divided by I_612nm

^g)fluorescence intensity ratios were calculated by using I_612nm divided by I_520nm

^h)the ratios were calculated by using the fluorescence intensity ratios of I_520nm/I_612nm at pH 9 by I_520nm/I_{612 nm} at pH 3

ⁱ⁾the ratios were calculated by using the fluorescence intensity ratios of I_{612 nm}/I_{520 nm} at pH 3 by I_{612 nm}/I_{520 nm} at pH 9.

Figure 6.

A) pH responses excited at 488 nm; B) pH dependent fluorescence intensity ratios at 612 nm and 520 nm respectively; C) pH dependent fluorescence intensity ratios calculated by I₆₁₂/I₅₂₀ and reverse; D) pH dependent ratios of I₅₂₀/I₆₁₂ and reverse normalized at pH 3 or pH 9.

A few methods were used to calculate the pK_a and the application range of the dual color sensor and compare the sensitivity of the dual color sensor with its corresponding individual sensors. Method 1: Only the intensity at 520 nm was concerned. The intensity ratios by pH were plotted in Figure 6B. The pH sensitive range is from 6 to 8 with a pK_a of 6.89, which is close to the individual PSM1 sensor (F5). The comparison of the sensing performance is given in Table 3. Method 2: Only the intensity at 612 nm was considered. The intensity ratios by pH were plotted in Figure 6B. The pH sensitive range is from 4.5 to 7 with a pK_a of 5.85, which is close to the individual PSM2 sensor (F16). Method 3: Ratiometric approach using the ratios at 520 nm to 612 nm or reverse was used. The data was plotted in Figure 6C. By using the ratios of 520 nm divided by 612 nm, the pK_a of 7.09 was calculated with a sensitive range of 6 to 8; by using the ratios of 612 nm divided by 520 nm, pK_a of 5.01 was calculated with a sensitive range of 4 to 7. Because the dual color sensor is composted of the two individual PSM1 and PSM2 sensors, this sensor has a pH dynamic range from 4 to 8. This dynamic range is broader than those of the individual sensors.

In order to discern whether the dual color sensor has a higher sensitivity than its individual sensors, we used method 4 to perform the calculation. The intensity ratios of I₅₂₀/I₆₁₂ at various pH were plotted against the I₅₂₀/I₆₁₂ at pH3. This plot reflected the changes of the fluorescence intensity ratios by pH. It was found that the ratio at pH 9 was 200 folds of that at pH 3. The reverse order of I₆₁₂/I₅₂₀ generated similar results. The calculated results were plotted in Figure 6D. The data shown in Figure 6D indicated that the dual color sensor exhibited higher pH sensitivity than its individual sensors, showing the advantage of the dual color sensor. Most likely, this improvement of the sensitivity is due to the fact that the dual color sensor is an integration of two individual sensors with opposite pH responsive trends.

The comparison of the sensing performance of the three films with variable ratios of SM1 and SM2 in their matrices was given in Table 3. It was found the pK_a values calculated by the three methods as described above were not affected significantly by the compositions of the fluorophores of SM1 and SM2 in the dual color sensor films. This observation means most likely no significant fluorescence resonance energy transfer occurred between the two kinds of fluorophores (SM1 and SM2) in the thin films at our experimental conditions.

3.4 Colorimetric sensor

Because the colorimetric sensor possesses two emission colors at green and red spectral windows, changes in the pH dependent emission colors by pH were observed. Figure 7 showed the emission colors of the dual color sensor (F32) at different pH, taken by a camera (Nikon D3000) using 488 nm light as the excitation light source. Green emission dominated in the pH range of 7 to 9. Significant change of the color was observed from pH 7 to pH 4. For quantifying the pH depended emission color, we used Adobe Photoshop CS3 to analyze the pictures. Under the RGB model, the intensities of red (R) and green (G) from the pictures were obtained and their ratios were plotted against pH (Figure 7H). It was determined that the intensity ratios of G/R were sensitive to pH from 4 to 7, and that the ratios of R/G were sensitive to pH in the range of pH 3 to 6. Therefore, the data analysis indicated that the colorimetric sensor can be used to quantify pH from 3 to 7.

4. Conclusion

Two different monomeric pH probes were polymerized into polymer matrices to form thin film sensors. Influences of the matrices including the compositions of the matrices, cross-linkers, and charges, on the sensing performance were studied. Results showed that PHEMA-co-PAM with a weight ratio of 80:15 is the best matrix for the two sensors, which exhibit the highest sensitivity among the few compositions that we studied. Hydrophobic crosslinker SR454 decreased the sensitivity and shifted the pK_a to low value as compared with the hydrophilic crosslinker of PEGDMA we used. The matrices with more cross-linkers exhibited lower sensitivity. Charges also affected the sensitivity and pK_a values. The studies on the fundamental aspects about the influences of the compositions of the sensor films on the sensing performances may provide insightful information for sensor design.

Because of the two individual sensors (PSM1 and PSM2) possess different pH responsive trends and emission colors, their dual color sensor exhibited higher sensitivity to pH and broader responsive ranges than the individual sensors. The dual color sensor is also a colorimetric sensor. The colors can be easily visualized by the human eyes and commercially popular cameras, which showed plentiful and significant color differences in a pH range of 7 to 4. The color pictures can also be used to quantify pH values by the data analysis using the popular Photoshop program.

Biographies

Yanqing Tian received his B.S. and M.S. degrees from the Department of Organic Chemistry, Jilin University (China) in 1989 and 1992. In 1995, he received his Ph.D. degree in Polymer Chemistry and Physics from Jilin University. He is now working at the Center for Biosignatures Discovery Automation, Biodesign Institute at Arizona State University as an Assistant Research Professor. His research interests include synthesis and applications of optical sensors and block copolymers for bio-sensing, bio-imaging and drug delivery.

Emily Fuller is a current undergraduate student at Arizona State University studying Material Science and Engineering. She will be graduating in May of 2014. During her time at Center for Biosignatures Discovery Automation she worked under Dr. Yanqing Tian on various optical sensor studies.

Summar Klug will receive her BSE degree in bioengineering from Arizona State University in 2013. From 2011 to 2012 she has worked as a researcher on the development of optical sensors of pH for the Center for Biosignatures Discovery Automation at the Biodesign Institute at Arizona State University. She will commence additional education at Arizona State University in the field of electrical engineering in 2013.

Fred Lee is studying chemical engineering at Arizona State University. From 2012 he has been working as student researcher for the Center for Biosignatures Discovery Automation, Biodesign Institute at Arizona State University, focusing on the development and characterization of optical sensors of oxygen, pH and glucose. He will receive his BSE degree in chemical engineering in 2014.

Fengyu Su received her B.S. and M.S. degrees from the Department of Organic Chemistry, Jilin University (China) in 1990 and 1993, and her Ph.D. degree in Polymer Physics and Chemistry from Changchun Institute of Applied Chemistry, Chinese Academy of Sciences in 1997. She worked at Changchun Institute of Applied Chemistry, RIKEN Advanced Science Institute, Tokyo Metropolitan University, and University of Washington. She is now working at the Center for Biosignatures Discovery Automation of the Biodesign Institute at Arizona State University as an Associate Research Scientist. Her research interests include development of polymer hydrogels and sensors, and application of optical sensors for bio-sensing and bio-imaging.

Liqiang Zhang received his PhD degree in Biochemistry and Molecular Biology from the Institute of Biophysics, Chinese Academy of Sciences in 2006. He got his postdoctoral training in National Institutes of Health (NIH) in Bethesda, MD and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center in Omaha, NE. He started work at Biodesign Institute, Arizona State University in Tempe, AZ in 2010. He is now working at the Center for Biosignatures Discovery Automation, Biodesign Institute at Arizona State University as an Associate Research Scientist. His research interests include synthesis and application of biomaterials for bio-sensing, bio-imaging and drug delivery.

Shih-hui Chao received the MS degree in Aeronautics and Astronautics from the National Cheng Kung University, Tainan, Taiwan in 1992, the MS degree in Electrical Engineering from the University of Washington, Seattle, WA in 2001, and the PhD in Mechanical Engineering from the University of Washington in 2002. After accomplishing the milestone of the first nanoscale resolution magnetic resonant images for his PhD, he expanded his research subject emphasis to lab-on-a-chip systems. His main interests are bioinstrumentation and biomechatronic systems for single-cell analyses. Currently, he is a Research Assistant Professor in the Center for Biosignatures Discovery Automation, Biodesign Institute at Arizona State University.

Deirdre R. Meldrum received the B.S. degree in Civil Engineering from the University of Washington, Seattle, in 1983, the M.S. degree in electrical engineering from the Rensselaer Polytechnic Institute, Troy, NY, in 1985, and the Ph.D. degree in Electrical Engineering from Stanford University, Stanford, CA, in 1993. She is a graduate of the Stanford Executive Program 2009. As an Engineering Co-op Student at the NASA Johnson Space Center in 1980 and 1981, she was an Instructor for the astronauts on the Shuttle Mission Simulator. From 1985 to 1987, she was a member of the Technical Staff at the Jet Propulsion Laboratory working on the Galileo spacecraft, large flexible space structures, and robotics. From 1992 to 2006, she was a Professor of Electrical Engineering and Director of the Genomation Laboratory, University of Washington. She was Dean of the Ira A. Fulton School of Engineering from 2006 to 2010, and has been Professor of Electrical Engineering, and Director of the Center for Biosignatures Discovery Automation, Biodesign Institute, Arizona State University since 2006. Her research interests include genome automation, microscale systems for biological applications, ecogenomics, robotics, and control systems. Dr. Meldrum is a member of the American Association for the Advancement of Science (AAAS), ACS, AWIS, HUGO, Sigma Xi, and SWE. Her honors include an NIH Special Emphasis Research Career Award (SERCA) in 1993, a Presidential Early Career Award for Scientists and Engineers in 1996 for advancing DNA sequencing technology, Fellow of AAAS in 2003, Distinguished Lecturer for IEEE Robotics and Automation Society 2006–2009, Best Paper of the Year 2006 in the IEEE Transactions on Automation Science and Engineering, Director of an NIH Center of Excellence in Genomic Sciences called the Microscale Life Sciences Center from 2001 to 2011, Fellow of the IEEE 2004 Senior Editor for the IEEE Transactions on Automation Science and Engineering from 2003 to 2012.