Biocompatible Microporous Organically Modified Silicate Material with

Feb 23, 2018 - A new four-component organically modified silicate (ORMOSIL) material was developed with optical pH sensors in mind. Through a sol–ge...
17 downloads 7 Views 3MB Size
Download PDF
Article Cite This: ACS Sens. XXXX, XXX, XXX−XXX

pubs.acs.org/acssensors

Biocompatible Microporous Organically Modified Silicate Material with Rapid Internal Diffusion of Protons Christian Grundahl Frankær,*,†,‡ Kishwar J. Hussain,‡ Martin Rosenberg,† Anders Jensen,† Bo W. Laursen,† and Thomas Just Sørensen*,†,‡ †

Nano-Science Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø, Denmark FRS-systems ApS, Hovedgaden 20, 4621 Gadstrup, Denmark

‡

S Supporting Information *

ABSTRACT: A new four-component organically modified silicate (ORMOSIL) material was developed with optical pH sensors in mind. Through a sol−gel process, the porosity of an ORMOSIL framework was optimized to allow rapid diffusion of protons, ideal for fast response to pH in an optical sensor. The optically transparent material was produced by catalyzing the dual polymerization of 3-(glycidoxy)propyltrimethoxysilane (GPTMS) and propyltriethoxysilane (PrTES) with boron trifluoride diethyl etherate. The performance of the resulting material in fluorescence based optical pH sensors was evaluated by incorporation of active dye components in the inorganic polymer framework. It is demonstrated that the material has a short response time (t90 < 30 s) and high stability in medium and during storage, and resulting sensor spots are biocompatible. It is concluded that this ORMOSIL material has excellent properties for optical pH sensors. KEYWORDS: optical pH sensors, pH optode, porous sol−gels, ORMOSIL, sensor matrix, biocompatibility

A

chemical sensor is more than just a molecule.1 In addition to a responsive molecule with a stable and ratiometric readout that is proportional to the concentration of the target analyte, a chemical sensor requires a matrix material in which the responsive molecules are immobilized, and that allows efficient and selective diffusion of the target analyte. With the responsive molecule and matrix material at hand, a robust substrate on which the matrix with the responsive molecules adheres is required to create a chemical sensor spot. To complete the chemical sensor, hardware to generate a readout and software to interpret data are needed. Typically, the chemical information is obtained from the data via a calibration curve, enabling the plotting of the measured quantity on a monitor.1,2 First generation optical sensors for DO and pH are currently being introduced in biotechnological production platforms.3−9 The optical sensors solve critical challenges in the industry, and while the industry is pleased by the availability of optical sensors, they are less pleased by the quality of current optical pH sensors.10 Optical sensors are composed of five different elements, excluding possible fiber optical connectors: (i) a light source, (ii) a substrate, (iii) a polymer matrix, (iv) active dye components, and (v) detectors.11−15 Light sources and detectors are highly developed and are available at low cost; in this work, we are using a conventional fiber spectrometer. The substrate has to be chosen based on the platform in which the sensing will take place; typically a glass or a polymer support is chosen for easy incorporation in bioproduction © XXXX American Chemical Society

vessels. The key parameter regarding the substrate is that the matrix material has to be completely immobilized in or on the substrate. Here, we are using a microstructured polycarbonate substrate that gives physical stability to the optical sensor.16 The challenge of making a new optical sensor lies in the responsive molecules and in the matrix material. In particular, for the matrix material the demands are high: (i) The material has to allow the analyte concentration within the sensor spot to be in rapid equilibrium with that outside the sensor. (ii) The material has to fully encapsulate and retain the responsive molecules. (iii) The material has to be transparent and have a minimum of autofluorescence. (iv) The matrix material must tolerate various sterilization methods. (v) The material has to be chemically stable during storage for a minimum of three years. Finally, (vi) the material has to be stable in biological media for extended periods of time, even when exposed to sheer stress. The responsive molecules, often fluorescent dyes, can be a single ratiometric pH-responsive dye,17 or a set of a responsive dye and a reference dye with similar physicochemical properties. The latter is only possible if the physical stability of the polymer matrix ensures absolutely no loss of responsive molecules to the medium. Received: January 9, 2018 Accepted: February 23, 2018 Published: February 23, 2018 A

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

SevenCompact pH meter with an InLab Expert Pro electrode. This mixture was left stirring for 3−4 months, which allowed the polymerization process to proceed. The polymerization was regularly followed by infrared spectroscopy. Procedure for Preparation of GPTMS Gel Component. 6 mL of GPTMS (0.027 mol) was mixed with 11 mL of absolute ethanol (0.19 mol) while stirring. Amounts ranging from 1.0 to 5.0 mg red-emitting dye (1.3 to 6.6 μmol) were dissolved herein. Then 0.72 mL of cold boron trifluoride diethyl etherate (BF3·O(CH2CH3)2, 5.8 mmol) was added using a syringe pump at a rate of 7.6 mL h−1. The mixture was stirred for 30 min, while the temperature was monitored using a conventional glass alcohol thermometer. After 30 min the temperature of the mixture had dropped to room temperature, and 2 mL of Milli-Q water (0.11 mol) was added to the solution. The resulting mixture was left with stirring for 4 h. When the two gel components had been prepared, they were combined in (PrTES:GPTMS) molar ratios of 20:80, 30:70, 40:60, and 50:50 and left with agitation for a minimum of 3 days to allow cross-linking of the two networks. Deposition, Curing, and Washing. Optical pH sensor spots were prepared on both glass and polycarbonate substrates: The aged sol−gels were deposited onto polycarbonate surfaces by using drop deposition and hemiwicking. 16 After deposition, ethanol was evaporated from the samples for 30 min, before the samples were cured at 110 °C for 3−4 h using an electronically controlled oven. The sensor spots were washed by immersion in 0.020 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.6, for at least 1 h before testing. Infrared Spectroscopy. The sol−gels were analyzed by infrared spectroscopy. Using a benchtop FT-IR spectrometer (Agilent Cary 630) short acquisition times allowed spectra of the sol−gels without evaporation of solvent to be recorded. An average of 16 spectra was used. Response Time Test. Each sensor spot tested was glued to the outside bottom of a small polystyrene Petri dish (Ø 35 mm) using transparent two-component epoxy glue. A customized probe fixture was centered over the spot and glued to the inside of the Petri dish. The spot was mounted onto the optical probe and immersed into a reservoir of 250 mL 0.02 M HEPES buffer, pH 7.6. pH was externally monitored using a Mettler-Toledo SevenCompact pH meter with an InLab Expert Pro electrode. A home-built hardware setup was used in the performance tests. A white LED was used as light source. The light was filtered using a 550 nm short-pass filter (Omega Optical) before it was coupled into a QR600-7-VIS125BX optical probe (Ocean Optics). The Petri dish was fixed to the tip of the probe and the fluorescent light emitted from the sensor spot was collected through a 560 nm long-pass filter (Omega Optical) and analyzed using a fiber spectrometer (FREEDOM visNIR, Ibsen Photonics). An automated software routine allowed readout of 5 fluorescence spectra per minute. The signal from the sensor was monitored after inducing a significant (more than 4 pH units) jump in pH. Typically pH was altered between 3.0 and 8.0 by addition of aqueous HCl or NaOH. The time from injection to a partial response was reached (90%, 95%, and 99% compared to the equilibrium signal) was recorded. Sterilization and Biocompatibility Test. Sterilization tests were performed using e-beam by Sterigenics Denmark A/S. The biocompatibility of the sensor spots was determined according to ISO10993-part5 by Bioneer A/S. Spots made from matrix material with and without pH-responsive dye, and samples of only microstructured polycarbonate substrate were tested. All samples were sterilized by autoclave before the test. Two different mammalian cell models (CHO DG44 and L929) were used in the test, and both negative and positive control were included. The positive control consisted of two samples displaying respectively high and low cytotoxicity. A minimum of three replicates were performed for each sample.

Preparation of organically modified silicates (ORMOSILs)18−20 from alkyl and 3-glycidoxypropyl substituted trialkoxysilanes using various polymerization conditions has been reported.11,21−23 Lewis acids can catalyze the polymerization of 3-(glycidoxy)propyltrialkoxysilanes, accelerating both the polyether and the polysiloxane formation.21,22,24 In this work, boron trifluoride etherate was used.25 This Lewis acid has the added benefit of being incorporated in the sol−gel, possibly increasing the resulting porosity of the ORMOSIL material.26 The benchmark for matrix materials for optical sensors has been set in sensors, where fluorescein was used as the active dye component;14,15,27−29 despite the inherently poor photostability of fluorescein.15,30,31 The critical parameters when characterizing matrix materials for optical sensors are the response time of the sensor, the leakage of the dye, the stability of the signal, and finally the response to pH.32−34 While leakage of the highly water-soluble fluorescein has not been completely removed,35 other more lipophilic dyes have been successfully encapsulated in sol−gels.36,37 Here, we report on a new ORMOSIL matrix material. By covalent attachment of a polymerizable red-emitting pH-responsive dye bearing a silane group we document the properties of this new composite material. The material we report here is a single polymer made from four components: (1) 3-(glycidoxy)propyltrimethoxysilane or GPTMS monomers, (2) propyltriethoxysilane or PrTES monomers, (3) boron trifluoride diethyl etherate, and (4) red-emitting pH-responsive dye. The latter was covalently linked to the ORMOSIL matrix material through a silane group. The dye is included in order to evaluate the performance of the sensor, but does not influence the physicochemical properties of the material. Polymerization and sol−gel processes are often difficult to control, and for reproducible results the composition of reacted/unreacted groups in the sol−gel has to be followed throughout the process. We document that the sol−gel fabrication process gives rise to reproducible results, by following the thermal history of the exothermic GPTMS polymerization and the IR fingerprint changes during the condensation of alkoxysilanes. We can therefore make and test the matrix material for an optical sensor. Furthermore, we demonstrate that the matrix material has a rapid response to changes in pH, that it is biocompatible, that the matrix material is durable, and that it tolerates e-beam and repeated autoclave sterilization.

■

MATERIALS AND METHODS

All compounds were used as received. 3-(Glycidoxy)propyltrimethoxysilane (GPTMS; >98%), and boron trifluoride diethylethrate were purchased from Sigma-Aldrich. Propyl triethoxysilane (PrTES; 97%) was purchased from Alfa Aesar. Solvents used were analytical or HPLC grade. The red-emitting pH-responsive silane appended dye was purchased from KU dyes ApS.17 Preparation of Sol−Gels for Porous Matrix Material. The procedure included preparation of two separate sol−gels of two organically modified silanes: Propyltriethoxysilane (PrTES) and 3(glycidoxy)propyltrimethoxysilane (GPTMS). The PrTES sol−gel was prepared from polymerization of the silicon network in acidic conditions. The procedure is an adapted version of the procedure reported by Wencel et al.36,37 The GPTMS sol−gel is prepared from polymerization of the glycidylether catalyzed by the Lewis acid boron trifluoride diethyl etherate. Procedure for Preparation of PrTES Gel Component. 5 mL PrTES (0.022 mol) was dissolved in 8 mL absolute ethanol (0.14 mol) while stirring. Thereafter, 1.6 mL of 0.1 M HCl solution (0.16 mmol) was added dropwise. pH was measured to 1.6 using a Mettler-Toledo B

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

■

RESULTS AND DISCUSSION Matrix Material Synthesis. The organically modified silicate (ORMOSIL) matrix material is produced from four components or monomers, using hydrochloric acid and water as reagents. The four starting materials are shown in Scheme 1.

Propyltriethoxysilane (PrTES) is a monofunctional building block that can form up to three silicate Si−O−Si bridges by hydrolysis and subsequent condensation (see Scheme 1). 3(Glycidoxy)propyltrimethoxysilane (GPTMS) is a bifunctional building block that can form a polyether chain by reaction of the oxirane and take part in silicate networks (see Scheme 1). A small fraction of the dye functional triethoxysilane (DyeTES) can be included with the monomer. Finally, boron trifluoride diethyl etherate (BF3 etherate) is used as a catalyst and a building block. Upon hydrolysis boronic acid is formed and can act as a cross-linking agent generating kinetically inert crosslinks. The matrix material is formed in three polymerization steps. Two sol−gels are formed by polymerizing PrTES and GPTMS separately (see reactions in Scheme 1 and the cartoon representation of the resulting sol−gels in Scheme 2). The PrTES sol−gel is matured for weeks to form the right silicate clusters, while the BF3 etherate catalyzed polyether polymerization of GPTMS is completed in minutes. After a selected time the two sol−gels are mixed, forming the porous composite gel upon further silicate formation and boronic acid crosslinking. A cartoon of the porous ORMOSIL matrix material is included in Scheme 2. The details of each step in the matrix material are discussed below. Polymerization of PrTES. The PrTES monomer was hydrolyzed under acidic conditions, which initiated a series of condensation reactions creating a silicate network (Scheme 1). PrTES was dissolved in ethanol and 0.1 M hydrochloric acid was added resulting in a solution of a pH of ∼1.6. The initial step is a fast hydrolysis of PrTES to the corresponding silicic acid followed by a much slower condensation to form a silicate network. Figure 1 shows that the progress of the reaction can be followed by IR spectroscopy. The weak absorption band at 1165 cm−1 was attributed to the Si−OCH2CH3 group,38 and

Scheme 1. Matrix Material Starting Components

Scheme 2. Cartoon of Sol−Gel Componentsa

a PrTES: dark blue. GPTMS: light blue. Red-emitting pH-responsive dye: magenta. Spheres represent Si−Pr fragments from PrTES and GPTMS (dark and light blue, respectively).

C

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

decrease is coupled to an increasing intensity of the peak corresponding to the asymmetric Si−O−Si stretch at 1130 cm−1,39 we can conclude that we are indeed monitoring the reaction forming the ORMOSIL network. Figure 2 shows that this reaction is completed in 2−4 weeks. The data and observations have been reproduced in more than five individual experiments. Polymerization of GPTMS. For GPTMS the polymerization of the glycidyl ether has to take place before the ORMOSIL network is formed. The polymerization is one of several reactions taking place in the mixture, and is catalyzed by the slow addition of the Lewis acid boron trifluoride diethyl etherate. Boron trifluoride is added to a solution of GPTMS in absolute ethanol over a period of 5−6 min. The Lewis acid attacks the epoxy ring and allows for ring opening upon which another GPTMS molecule can react, initiating the chain polymerization reaction. For the first step, it is crucial that water-free conditions are maintained until all of the glycidylether residues of GPTMS have reacted. The ring opening reaction is exothermic, and the heat evolution can be followed as a function of time. Figure 3 shows

Figure 1. (a) IR-spectra of a PrTES sol−gel followed over a period of 104 days (blue to red). Arrows at 1165, 1130, and 915 cm−1, indicate appearance and disappearance of characteristic bands respectively assigned to the Si−OCH2CH3, Si−OH, and Si−O−Si groups.

used as a signature of the initial hydrolysis reaction. At pH ∼1.6 the hydrolysis occurs within the first hour, as seen by the disappearance of this band. The silanol Si−OH IR absorption band was observed at 915 cm−1.38 This band is intense from the beginning of the reaction as the hydrolysis takes place immediately after addition of HCl. The formation of the PrTES ORMOSIL network can be followed using IR until the point where steady state is reached: compare time points 21 days to 104 days in Figure 1. That the PrTES sol−gel continues to change in composition for a long period of time, and that further condensation occurs in this period can be seen by following the specific IR band of silanol groups. As the silanol groups are converted into Si−O− Si bonds in the ORMOSIL network, the peak intensity at 915 cm−1 continues to decrease for weeks (see Figure 2a). As the

Figure 3. Heat evolution of the ring opening polymerization reaction of the glycidylether of GPTMS. Addition of boron trifluoride is terminated after 5.5 min. Water is added after 30 min.

that heat evolves during the addition of boron trifluoride, which indicates that the polyether formation occurs in a fast reaction. The addition is completed after 5.5 min, and after ∼7 min a maximum temperature of 42 °C is reached. After 30 min the temperature of the reaction mixture has almost returned to room temperature; the temperature jump at 31 min in Figure 3 is due to addition of water to the reaction mixture. Water generates acid that catalyzes the hydrolysis and condensation reaction leading to formation of an ORMOSIL network (Scheme 1). The polyether formation and subsequent silicate formation was monitored by IR. In Figure 4a and b, the characteristic IR absorption bands for an epoxide can be seen prior to the reaction at, respectively, 3055 cm−1 (epoxide C−H-stretch) and 1254 cm−1 (ring breathing vibration).21 The band at 1254 cm−1 occurs in a region dominated by overlapping bands, but the details in Figure 4a and b show that the epoxide groups have reacted after 30 min, as the epoxide specific bands in the IR spectra have disappeared. The band at 1200 cm−1 was assigned to a C−O−C-stretching mode. This band is present in the spectra of unreacted GPTMS monomer,40 and was seen to increase upon polymerization. We conclude that this is due to an increase in the amount of C−O−C-groups through the formation of polyether chains. After complete polyether formation, water was added to initiate polymerization of a silicate network. As for PrTES, the IR spectra confirms that the formation of ORMOSIL networks

Figure 2. (a) Condensation of silanol groups followed by the absorption band at 915 cm−1 plotted as a function of time. (b) Formation of silicon oxide network followed by the absorption band at 1130 cm−1 plotted as a function of time. D

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

Figure 5. Combination of GPTMS and PrTES followed by IR. IR spectra of a mixture with PrTES:GPTMS ratios of 50:50 were followed during 3 days.

Figure 4. Polymerization of GPTMS followed by IR. (a,b) GPTMS solutions in EtOH before addition of boron trifluoride (black), and after 30 min reaction (light blue). Characteristic IR absorption bands for epoxide are indicated as dashed lines at 3055 and 1254 cm−1. (c) GPTMS solutions in EtOH 1−4 h after addition of water.

in the GPTMS sol−gel occurs through a condensation between silanol groups formed by rapid hydrolysis of the GPTMS Si− OCH3 group. The condensation is slow and only a small decrease of the amount of silanol groups was observed during the 4 h the reaction was allowed to proceed, monitored as the change in the 900−950 cm−1 region of the IR spectra (see Figure 4c). As expected, the decrease in this region is accompanied by an increase in the bands at 1030−1130 cm−1 that corresponds to vibrations in the Si−O−Si groups formed by the condensation reaction. The GPTMS ORMOSIL condensation reaction was not run to completion; after 4 h it was reacted with a PrTES sol−gel. The data and observations have been reproduced in more than five experiments. Combination of GPTMS and PrTES Sol−Gels. Typically, the PrTES sol−gel was allowed to react for 14 days. No difference in physicochemical properties of the resulting matrix material was observed, when PrTES sol−gels that had reacted between 7 and 21 days were used. A series of PrTES sol−gels that had reacted for 14 days was mixed with GPTMS sol−gels in molar ratios of 20:80, 30:70, 40:60, and 50:50 PrTES:GPTMS. The four resulting sol−gels were allowed to react for 3 days. The reaction between the two sol−gels was followed by IR (see Figure 5). By following the silanol signal at 915 cm−1, and the Si−O−Si stretch at 1130 cm−1, it is seen that the condensation was complete within 1 day. The IR spectra of four final sol−gels are shown in Figure 6. The absorption band observed at 1220 cm−1 is characteristic for the PrTES (see Figure 1), whereas the polyether C−O−Cvibrational band at 1200 cm−1 is unique for the GPTMS (see Figure 4). Thus, the ratio between the two confirms that the sol−gels have different ratios of GPTMS and PrTES. The four sol−gels were deposited on both flat glass and microstructured polycarbonate substrates and were cured at elevated temperatures. More than five replicas of each type of sensor spots were made and tested. The adhesion to both substrate types was excellent, and the cured sol−gels were found to be mechanically stable (hard and glass-like) and optically transparent. The sensor material resembled a smooth

Figure 6. Combination of GPTMS and PrTES followed by IR. IR spectra of four mixtures with PrTES:GPTMS ratios of 20:80, 30:70, 40:60, and 50:50, aged for 3 days.

glass surface. The properties of a similar matrix material has been thoroughly investigated using atomic force microscopy, scanning electron microscopy, thermogravimetric analysis, and IR and Raman spectroscopy.37 This study demonstrated that similar sol−gel processes led to an optically transparent and mechanically stable material with a smooth surface and uniform distribution of the sol−gel components, without describing the performance of the resulting sensor spot. Therefore, we chose to focus on the sensor spot performance, and no analysis of the physical appearance of the cured matrix material was performed. Matrix Material Properties. Sensor Spot Performance. The pH response from the red-emitting pH-responsive dye incorporated covalently in the PrTES-GPTMS matrix, hereafter denoted as the sensor spot, was tested. When protonated, fluorescence is emitted from the pH-responsive dye at 590 nm using an excitation wavelength of 505 nm. A series of emission spectra from the sensor spots were recorded consecutively and read out every 12 s. At a given time the pH was changed from 8.0 to 3.0 by rapid injection of HCl. To produce the readout from the sensor spot each spectrum was integrated from 580 to 630 nm and the integrated intensity normalized against the equilibrium intensity of the on-signal. The readout was then plotted as a function of time. The results from the four different sensors of different matrix material compositions are shown in Figure 7a. As seen, the read signal increased instantaneously when the pH was changed from 8.0 to 3.0. The real time response when pH is changed from 3.0 back to 8.0 is shown in Figure 7b. When increasing pH, the E

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

that a low PrTES content results in a more flexible and unstable matrix, from which diffusion of protons is slowed by reversible hydrolysis of the silicate polymer. We have not been able to find a standard for the response time of a pH sensor. The response time of a pH electrode is measured as the time it takes a pH electrode that was taken from a pH 7 buffer, wiped dry, and reimmersed in the same pH 7 buffer to stabilize. This test is not relevant for optical sensors. Sensor Spot Stability. The stability of the sensor spot with a PrTES:GPTMS ratio of 30:70 was tested in continued operation by cycling the pH. The result in Figure 8 shows that the sensor spot allows rapid diffusion of protons in and out of the matrix with a high reversibility. Further, the data show that, after the initial cycle, the sensor signal is constant at both low and high pH. The stability toward physical stress was tested by gluing a sensor spot (also with a PrTES:GPTMS ratio of 30:70) to the inside wall of a test tube. HEPES buffer was added and the solutions were stirred rigorously for 4 days. As no fluorescence was detected in solution, we conclude that no sensor dye has leached from the sensor spots (see SI for details). The matrix material hydrolyzes at pH > 9. In the operational window from pH 1 to 9, we were not able to challenge the stability of the matrix material enough to allow detection of fluorescence in solution. That included after e-beam and repeated autoclave sterilization. Finally, the shelf life of the sensor was evaluated using the response time, and it was found the no change in sensor performance could be determined between sensor spots that had just been produced and those that had been stored at ambient conditions for eight months. Sensor Spot Biocompatibility. Figure 9 shows the outcome of the biocompatibility test for the two cell model systems. Cursory inspection of Figure 9 reveal that there is no difference between sensor spots made from matrix material with and without dye, the microstructured polycarbonate substrate, and the negative control. The difference between the negative and the two positive controls verifies that both cell models are able to detect cytotoxic effects with a graded response, and allows the conclusion that the sensor spots are fully biocompatible.

Figure 7. Normalized response curves for all four sol−gel formulations with PrTES:GPTMS ratios of 20:80, 30:70, 40:60, and 50:50 where the integrated intensity of the emission spectra in the range 580 to 630 nm is plotted as a function of time. (a) pH change from 8.0 to 3.0, and (b) from 3.0 to 8.0. Injection of HCl or NaOH is indicated by a vertical line at t = 0 min.

response of the sensor is somehow slower. In either pH step the time it took for the reference pH electrode to stabilize was ∼60 s. The response time (t90, t95, t99) was evaluated as the time it takes to achieve, respectively, 90%, 95%, and 99% of the equilibrium signal. Average values and corresponding standard deviations for N measurements of each sol−gel composition are compiled in Table 1. The accessibility of the hydronium ions to the protonation sites of the pH-responsive dye is evaluated by the parameter xconv: xconv = [(IpH3 − IpH8)/IpH3]100%

(1)

■

which is summarized in Table 1 for all samples. IpH 3 and IpH 8 are the intensities of light emitted from the sensor spots at pH 3 and pH 8, respectively. In general, faster response time was observed for acid injections lowering pH from 8.0 to 3.0, as compared to base injections raising pH from 3.0 to 8.0. This implies that diffusion of protons into the porous material is faster than out of the material. For diffusion of protons into the material (response on acidification), the response time increases with increasing PrTES content and the fraction of accessible protonation sites of the pH-responsive dye decreases. Thus, higher PrTES content must reduce the porosity. For diffusion of protons out of the material (response on base injection) the response time slightly decreases with increasing PrTES fraction. We speculate

CONCLUSION We have developed a synthetic route to a transparent and nonfluorescent porous matrix material using sol−gel processes by using proton and boron trifluoride diethyl etherate mediated polymerization of two monomers, a monofunctionel propyltriethoxysilane, and a bifunctional 3-(glycidoxy)propyltrimethoxysilane. By incorporating a polymerizable responsive dye molecule in the matrix material, we were able to demonstrate the performance of the matrix material in an optical pH sensor. The results allow us to conclude that the new matrix material has a rapid diffusion of protons, is stable in operation, has the required shelf-life, and is fully biocompatible. We conclude that this matrix material, which is the result of

Table 1. Correlation between Response Time and Sol−Gel Composition PrTES:GPTMS

N

20:80 30:70 40:60 50:50

5 5 5 3

t90 pH 8 to 3 (s) 18 18 24 54

± ± ± ±

1 8 10 8

t90 pH 3 to 8 (s) 119 109 100 84

± ± ± ±

31 69 41 25

t95 pH 8 to 3 (s) 19 21 28 71

± ± ± ±

1 13 13 18

t95 pH 3 to 8 (s) 174 159 145 128

F

± ± ± ±

51 106 52 32

t99 pH 8 to 3 (s) 20 27 72 88

± ± ± ±

2 22 38 29

t99 pH 3 to 8 (s) 312 287 236 199

± ± ± ±

56 129 78 59

xconv (%) 59.8 56.9 46.7 32.4

± ± ± ±

8.3 5.9 7.9 2.1

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors

Figure 8. Response from a sensor spot with PrTES:GPTMS ratio of 30:70 exposed to six sequential stress cycles between pH 4.0 and 8.0, where the integrated intensity of the emission spectra in the range 580 to 630 nm are plotted as a function of time. An extra data point at pH 6.3 is recorded for the last cycle. Injection of acid or base is indicated by vertical lines.

a University of Copenhagen Spin-Out company based on the reserach findings disclosed in this manuscript.

■

ACKNOWLEDGMENTS The authors thank Lundbeckfonden (grant#2013-12793), Novo Nordisk Fonden (grant#4096), Carlsbergfonden, Villum Fonden (grant#14922), BIOPRO, Innovationsfonden (grant# 5179-00914B), UpX and the University of Copenhagen.

■

ABBREVIATIONS GPTMS, 3-(glycidoxy)propyltrimethoxysilane; PrTES, Propyltriethoxysilane; ORMOSIL, Organically Modified Silicate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LED, Light Emitting Diode

Figure 9. Number of cells after 3 days in culture. Twelve replicates were performed for the sensor, and three replicates for each of the other samples and controls. Two cell models were used: CHO DG44 (blue, left axis) and L929 (olive, right axis).

■

robust synthesis, can be used to make optical sensors that are highly stable, biocompatible, and have a rapid response to changes in pH.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.8b00024. Sensor terminology, stability test data, and data from 8months-old sensor spots (PDF)

■

REFERENCES

(1) Wolfbeis, O. S. Probes, Sensors, and Labels: Why is Real Progress Slow? Angew. Chem., Int. Ed. 2013, 52 (38), 9864−9865. (2) Wencel, D.; Abel, T.; McDonagh, C. Optical chemical pH sensors. Anal. Chem. 2014, 86 (1), 15−29. (3) Weichert, H.; Lüders, J.; Becker, M.; Adams, T.; Weyand, J. Integrated Optical Single-Use Sensors: Moving Toward a True SingleUse Factory for Biologics and Vaccine Production. BioProcess International 2014, 12, 20−24. (4) Janzen, N. H.; Schmidt, M.; Krause, C.; Weuster-Botz, D. Evaluation of fluorimetric pH sensors for bioprocess monitoring at low pH. Bioprocess Biosyst. Eng. 2015, 38 (9), 1685−1692. (5) Card, C.; Clark, K.; Furey, J. Adoption of Single-Use Sensors for BioProcess Operations. BioProcess International 2011, 9 (5), 36−42. (6) Kochmann, S.; Baleizao, C.; Berberan-Santos, M. N.; Wolfbeis, O. S. Sensing and imaging of oxygen with parts per billion limits of detection and based on the quenching of the delayed fluorescence of (13)C70 fullerene in polymer hosts. Anal. Chem. 2013, 85 (3), 1300− 1304. (7) Schaferling, M. The Art of Fluorescence Imaging with Chemical Sensors. Angew. Chem., Int. Ed. 2012, 51 (15), 3532−3554. (8) Dmitriev, R. I.; Papkovsky, D. B. Optical probes and techniques for O2 measurement in live cells and tissue. Cell. Mol. Life Sci. 2012, 69 (12), 2025−2039. (9) Qazi, H. H.; bin Mohammad, A.; Akram, M. Recent progress in optical chemical sensors. Sensors 2012, 12 (12), 16522−16556. (10) Diehl, B. H.; La Pack, M. A.; Wang, T. Y.; Kottmeier, R. E.; Kaneshiro, S. M.; Brandenstein, M. C.; Zhang, Y. C.; Chiu, Y. C.; Yoon, S.; Saucedo, V. M. A Biopharmaceutical Industry Perspective on Single-Use Sensors for Biological Process Applications. Biopharm Int. 2015, 28 (4), 28−31. (11) Jeronimo, P. C.; Araujo, A. N.; Conceicao, B. S. M. M. M. Optical sensors and biosensors based on sol-gel films. Talanta 2007, 72 (1), 13−27. (12) Borisov, S. M.; Wolfbeis, O. S. Optical Biosensors. Chem. Rev. 2008, 108, 423−461.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bo W. Laursen: 0000-0002-1120-3191 Thomas Just Sørensen: 0000-0003-1491-5116 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 the following competing financial interest(s): CGF, KJH and TJS are employed by FRS-systems ApS that is commercializing optical sensor technologies. BWL and TJS are founders and current owners of FRS-systems ApS, G

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX

Article

ACS Sensors (13) McDonagh, C.; Burke, C. S.; MacCraith, B. D. Optical Chemical Sensors. Chem. Rev. 2008, 108, 400−422. (14) Wolfbeis, O. S. Fiber-Optic Chemical Sensors and Biosensors. Anal. Chem. 2008, 80, 4269−4283. (15) Wang, X. D.; Wolfbeis, O. S. Fiber-optic chemical sensors and biosensors (2008−2012). Anal. Chem. 2013, 85 (2), 487−508. (16) Mikkelsen, M. B.; Marie, R.; Hansen, J. H.; Wencel, D.; McDonagh, C.; Nielsen, H. O.; Kristensen, A. Controlled deposition of sol−gel sensor material using hemiwicking. J. Micromech. Microeng. 2011, 21 (11), 115008. (17) Laursen, B. W.; Rosenberg, M.; Sørensen, T. J. Azatriangulenium salts as PET-quenched fluorescent probes; International Patent No. WO/ 2015/058777, 2015. (18) Mackenzie, J. D.; Bescher, E. P. Structures, properties and potential applications of Ormosils. J. Sol-Gel Sci. Technol. 1998, 13 (1− 3), 371−377. (19) Schottner, G. Hybrid sol-gel-derived polymers: Applications of multifunctional materials. Chem. Mater. 2001, 13 (10), 3422−3435. (20) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. Recent bioapplications of sol-gel materials. J. Mater. Chem. 2006, 16 (11), 1013− 1030. (21) Innocenzi, P.; Brusatin, G.; Guglielmi, M.; Bertani, R. New Synthetic Route to (3-Glycidoxypropyl)trimethoxysilane-Based Hybrid Organic-Inorganic Materials. Chem. Mater. 1999, 11, 1672−1679. (22) Innocenzi, P.; Brusatin, G.; Babonneau, F. Competitive Polymerization between Organic and Inorganic Networks in Hybrid Materials. Chem. Mater. 2000, 12, 3726−3732. (23) Jurmanović, S.; Kordić, Š.; Steinberg, M. D.; Steinberg, I. M. Organically modified silicate thin films doped with colourimetric pH indicators methyl red and bromocresol green as pH responsive sol-gel hybrid materials. Thin Solid Films 2010, 518 (8), 2234−2240. (24) Brusatin, G.; Innocenzi, P.; Guglielmi, M. Basic Catalyzed Synthesis of Hybrid Sol-Gel Materials Based on 3-Glycidoxypropyltrimethoxysilane. J. Sol-Gel Sci. Technol. 2003, 26, 303−306. (25) Sørensen, T. J.; Rosenberg, M.; Laursen, B. W. Sol-Gel Based Matrix; International Patent No. WO/2015/058778, 2015. (26) Xiang, W.; Zhong, J., Borosilicates Obtained by Sol-Gel Method. In Handbook of Sol-Gel Science and Technology, Klein, L., Ed.; Springer International Publishing, 2016; pp 1−45. (27) Lobnik, A.; Oehme, I.; Murkovic, I.; Wolfbeis, O. S. pH optical sensors based on sol-gels: Chemical doping versus covalent immobilization. Anal. Chim. Acta 1998, 367, 159−165. (28) Senarath-Yapa, M. D.; Saavedra, S. S. Dye leaching from a doped sol-gel is eliminated by conjugation to a dendrimer. Anal. Chim. Acta 2001, 432, 89−94. (29) Cajlakovic, M.; Lobnik, A.; Werner, T. Stability of new optical pH sensing material based on cross-linked poly(vinyl alcohol) copolymer. Anal. Chim. Acta 2002, 455, 207−213. (30) Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. Synthesis of fluorinated fluoresceins. J. Org. Chem. 1997, 62 (19), 6469−6475. (31) Holmehave, J.; Pedersen, S. K.; Jensen, H. H.; Ogilby, P. R. Aarhus Green: A tetrafluoro-substituted derivative of fluorescein. Arkivoc 2015, 52−64. (32) Makote, R.; Collinson, M. M. Organically modified silicate films for stable pH sensors. Anal. Chim. Acta 1999, 394, 195−200. (33) Skrdla, P. J.; Saavedra, S. S.; Armstrong, N. R. Reduction of Indicator Leaching from Doped Sol-Gels by Attachment of Macromolecular Carriers. Appl. Spectrosc. 1999, 53, 785−791. (34) Lobnik, A.; Turel, M.; Urek, Š. K. Optical Chemical Sensors: Design and Applications. In Advances in Chemical Sensors, Wang, P. W., Ed.; InTech, 2012; pp 3−28. (35) Adams, S. R.; Harootunian, A. T.; Buechler, Y. J.; Taylor, S. S.; Tsien, R. Y. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 1991, 349 (6311), 694−697. (36) Wencel, D.; MacCraith, B. D.; McDonagh, C. High performance optical ratiometric sol−gel-based pH sensor. Sens. Actuators, B 2009, 139 (1), 208−213.

(37) Wencel, D.; Barczak, M.; Borowski, P.; McDonagh, C. The development and characterisation of novel hybrid sol−gel-derived films for optical pH sensing. J. Mater. Chem. 2012, 22 (23), 11720− 11729. (38) Jiang, H. M.; Zheng, Z.; Wang, X. L. Kinetic study of methyltriethoxysilane (MTES) hydrolysis by FTIR spectroscopy under different temperatures and solvents. Vib. Spectrosc. 2008, 46 (1), 1−7. (39) Ou, D. L.; Seddon, A. B. Near- and mid-infrared spectroscopy of sol-gel derived ormosils: Vinyl and phenyl silicates. J. Non-Cryst. Solids 1997, 210 (2−3), 187−203. (40) Guo, R. L.; Hu, C. L.; Pan, F. S.; Wu, H.; Jiang, Z. Y. PVAGPTMS/TEOS hybrid pervaporation membrane for dehydration of ethylene glycol aqueous solution. J. Membr. Sci. 2006, 281 (1−2), 454−462.

H

DOI: 10.1021/acssensors.8b00024 ACS Sens. XXXX, XXX, XXX−XXX