Optical Chemical Sensor Using Intensity Ratiometric Fluorescence

Dec 28, 2018 - Lilly leads off JPMorgan Healthcare meeting with $8 billion acquisition of Loxo Oncology. The purchase is one of multiple deals focused...
0 downloads 0 Views 1MB Size
Download PDF
Subscriber access provided by Columbia University Libraries

Letter

An Optical Chemical Sensor using Intensity Ratiometric Fluorescence Signals for Fast and Reliable pH Determination Christian Grundahl Frankær, Kishwar J Hussain, Tommy C Dôrge, and Thomas Just Sørensen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01485 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

An Optical Chemical Sensor using Intensity Ratiometric Fluorescence Signals for Fast and Reliable pH Determination Christian G. Frankær†‡*, Kishwar J. Hussain‡, Tommy C. Dörge† and Thomas J. Sørensen†‡* †

Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø, Denmark

‡

FRS-systems ApS, Hovedgaden 20, 4621 Gadstrup, Denmark

KEYWORDS: optical pH sensors, chemical sensors, pH optode, ratiometric, ORMOSIL,

ABSTRACT: Optical pH sensors enable non-invasive monitoring of pH, yet in pure sensing terms, the potentiometric method of measuring pH is still vastly superior. Here, we report a full spectrometer-based optical pH sensor system consisting of sensor chemistry, hardware and software that for the first time is capable of challenging the performance of electrode-based pH meter in specific applications such as biopharmaceutical process monitoring and in single-use bioproduction. A highly photostable triangulenium fluorophore emitting at 590 nm was immobilized in an organically modified silicon matrix that allows for fast time-response by rapid diffusion of water in and out of the resulting composite polymer deposited on a polycarbonate substrate. Fluctuations from the fiber optical sensor hardware have been reduced by including a highly photostable terrylene-based reference dye emitting at 660 nm, thus enabling intensity-based ratiometric readouts. The dyes were excited by 505 nm light from a light emitting diode. The sensor was operational within a pH range of 4.6 and 7.6, and was characterized and demonstrated to have properties that are comparable to commercial pH electrodes considering time-response (t90 < 90 s), precision (0.03 pH-units) and drift.

Since the first commercialization of pH meters based on proton selective electrodes nearly a century ago, the potentiometric method has been the superior method of measuring pH.1 However, the importance of quantification of H+ activity is still growing and particularly the bioprocessing industry is looking for non-invasive alternatives to the pH-electrode.2 Optical pH sensors offer such opportunity as they can be integrated prior to sterilization in single-use equipment.2-6 Optical chemosensors transform changes in analyte concentration into changes in optical phenomena, for instance absorbance, reflectance, luminescence, fluorescence, and refractive index.7 Using fluorescence as the method of signal transduction has certain advantages: less dye is used and a higher sensitivity is obtained. The first generation of optical pH sensors has been commercially available for some time.8 These report the pH of a solution using frequency domain determination of fluorescence lifetime, reflectance, and dye absorption.9-11 Nevertheless, it seems that current optical pH sensor technologies are not acceptable to the industry.2-6 The industry requires robust optical pH sensors to replace conventional pH electrodes in single-use bioproduction, stem cell culture, and for monitoring extended biopharmaceutical processes.2-6 The competition from the familiar pH electrode has been too strong, and despite numerous reports on new fluorescent probes with promising sensor properties,12-19 the progress in development of real sensors is slow.20

Part of the reason to why the development is slow is that only few of the reported chemosensors constitute a full sensor system.20 An optical chemosensor is a multicomponent device capable at measuring a concentration of a specific analyte. Hence, a sensor is a system comprising an optode—the component in contact with the sample solution—and dedicated hardware and software that read the optical information from the optode and converted it into useful analytical quantities i.e. describing the concentration of the target analyte in the sample with a given precision. The performance of a chemosensor is determined by its limiting component, and it is important to characterize the sensor as a full sensor system. Here, we present an optical pH sensor based on a pH responsive triangulenium dye.21-23 We report a full fiber optical pH sensor system comprising sensor chemistry, dedicated hardware and software, see figure 1. The sensor is characterized and compared to conventional electrode-based pH meters. Where previously reported optical pH sensors suffers from significant drift,23 this sensor does not. This allows us to conclude that the optical pH sensor system can compete with the electrode-based pH meters in life science applications. There are two challenges in developing intensity based optical sensors. The first is fluctuations in the hardware, which we have eliminated by including a non-responsive reference dye. Thus, intensity ratiometric fluorescence signals are generated and can be used for the pH deter-

ACS Paragon Plus Environment

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mination. Figure 1 shows how the signals from the reference and pH responsive dye are read by fiber-based optics and analyzed by a home-built hardware platform. A second, major challenge in optical chemosensor development is the physical and chemical stability optode, and in particular the operational stability of sensor. The pH-responsive dyes may leak to the surroundings, and can bleach from repeated exposure to light during the required weeks of operation. Hence, the performance of the sensor suffers from drift or a limited number of reads. We have developed a composite polymer where the dyes are covalently attached to the composite matrix. This eliminates leaking, and the material was shown to allow fast diffusion of protons.21 Bleaching is avoided by using highly photostable triangulenium dyes.22-27 The responsive pH dyes are based on the red emitting diazaoxatriangulenium (DAOTA) fluorophore. This dye is exceptionally chemically and photochemically stable.22, 26, 28-30 We have previously reported optical pH sensors using triangulenium dyes as responsive fluorophores,22 and where both responsive and reference dyes were of the triangulenium type.23 The reference dye in the first sensor was not chemically stable. In the second sensor, the reference dye suffered from fluorescence quenching by water. Here, this has been overcome by physically separating the pH responsive and the reference dye as shown in Figure 1. The pH responsive dye is immobilized in a porous sensor matrix,21 which is deposited on the front of a microstructured poly-carbonate substrate.21, 22, 31 The reference dye is immobilized in a polystyrene thin film deposited on the back of the polycarbonate substrate.

Figure 1: Individual components and assembly of optical ratiometric fluorescence-based pH sensor system. Top: structure and optical characteristics of the pH-responsive dye 1 in the protonated form, and the reference dye 2. Center: Construction of the optode (sensor spot). Bottom: Mounting of the optode and hardware-setup.

Figure 1 shows the individual components of the optical pH-sensor system. The pH-responsive fluorophore is a diazaoxy-triangulenium (DAOTA) scaffold with a pH responsive phenol group and a siloxane-functionalized linker.21, 22, 32, 33 The siloxane enables covalent immobilization of the dye in a porous organically modified silicon (ORMOSIL) matrix.21 The ORMOSIL matrix was prepared by dissolving the pH-responsive DAOTA dye 1 and the (3glycidyloxypropyl)trimethoxysilane GPTMS monomer in ethanol. By adding a BF3·OEt2 catalyst, the ring opening polymerization of the glycidyl epoxide is initiated. After 30 minutes the reaction is complete resulting in a formation of a polyethylene glycol (PEG) network.34 Silyl ether hydrolysis and subsequent formation of a siloxane network is enabled by addition of water. After four hours, the partly polymerized GPTMS-DAOTA sol-gel was mixed with a sol-gel of PrTES that had been allowed to polymerize for 14 days.35, 36 The final sol-gel was allowed to polymerize for another three days. The procedure has been described in details elsewhere.21 For preparation of the optode, the sol-gel was deposited onto micro-pillar structured poly-carbonate using hemiwicking,31 cured at 110°C for four hours, and rinsed in neutral buffers to remove excess acid and uncured sensor material. The reference dye used is a bis(2,6diisopropylphenyl)terrylene diimide (2, TDI) which exhibit high fluorescence quantum yields, and high thermal, chemical and photochemical stability.37, 38 Water is an effective photoquencher of the rylene dyes. Thus, the reference dye was immobilized in polystyrene, and deposited on the back of the polycarbonate substrate. A thin film of TDI in polystyrene was made by spin-casting from chloroform after the fabrication of the pH-responsive sensor material. The full description of the optode preparation is included as Supplementary Information. The transmitter designed to control the optical measurement houses both the light source and detector, see Figure 1. The detector is a fiber spectrometer. The light source used to excite the dyes in the optode consists of a blueish green LED (505 nm), and the fluorescent light emitted from the optode was analyzed in a fiber spectrometer recording full spectra in the vis-NIR region (4751100 nm). The excitation light is sent through optical fibers and filtered through a 550 nm short-pass filter before it is coupled into the six 400 um fibers of the 6-1 bundle of optical fibers that are used as probe. The emission light collection is achieved by the central 400 um optical fiber of the 6-1 fiber bundle, and light is through a 560 nm long-pass filter guided to the 120 um entrance slit of the fiber spectrometer. The light-source and the spectrometer are controlled by a process control block (PCB) that can be addressed via a mini USB-connector. The PCB also reports the observed pH as a 4-20 mA signal and via Modbus. The sensor software allows control of on-line measurements via a PC or directly on the PCB. For the latter instrument control and output of observed pH and spectra occur via a touch-screen on the front of the hardware. The full description of the hardware design is included as Supplementary Information.

ACS Paragon Plus Environment

Page 2 of 7

Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors Figure 2 shows the excitation and emission spectra of the pH-responsive and the reference dye. Cursory inspection shows that both dyes absorb light at 505 nm, and that the emission maxima are 585 nm and 667 nm. A long pass filter with a cutoff at 560 nm positioned before the spectrometer ensured efficient acquisition of the emitted fluorescence from both dyes. The ratiometric signal can subsequently be calculated from the intensities, see SI for details. A spectrum recorded during characterisation of the sensor is included in Figure 2, the effect of changing pH-values on the recorded emission spectrum is readily observed.

operational range is defined by the pKa of the pH responsive dye and covers the region where the response is approximately linear, typically ~3 pH units,40 and is shown as grey areas. An operational range of 3 pH units is far less than the operational range of pH electrodes (pH 1 to 10, or pH 0 to 14 when compensating for acidic and alkaline errors)41. However, incorporation of more dyes with different pKa enables design of optical sensors with broader operational ranges.42

Figure 2: Top: Normalized excitation spectra (full line) and emission spectra (dashed) of the pH-responsive DAOTA dye, 1 (magenta), and the reference dye TDI 2 (blue). Bottom: Fluorescence spectra recorded using the sensor hardware at different pH values at steady-state conditions. The bands used for calculating the pH signal are highlighted, pHresponsive DAOTA dye, 1 (magenta), reference dye, 2 (blue).

A series of tests, each repeated several times, were performed in order to evaluate the sensor performance, the test setup is described in SI. The results are compiled in Figure 3 and Table 1. The response time determines when a reliable measurement can be read. For a measurement to be reliable, the response must exceed a conversion that matches the general standard deviation of a measurement at steadystate conditions, which for this sensor is ~ 1%. Hence, we evaluate the response time as t99. Figure 3A shows timeresponse curves for changes where pH is respectively increased and decreased. The response time depends on the direction of the pH change, and is consistent to what is reported earlier:21 t99 ~ 3 min for increasing pH, and t99 ~ 1 min for decreasing pH. In terms of t90, which is more frequently reported, values of 88 s and 25 s were measured. Compared to commercial pH electrodes that claims a reliable readout after 5-30 s, the time response is comparable or slightly slower. Figure 3B shows the sigmoidal calibration curve, which is the relation between pH and optical signal, S.39 The

Figure 3: Sensor characterization results. A, Time-response curves. B, Calibration curve relating the pH observed using a calibrated pH-meter with the optical signal, S. C, Evaluation function i.e. the inverted calibration function, relating the observed signals, S, to estimated pH values. Insert: Estimation of precision. Dashed lines in B and C indicate the standard deviation ±σ (i.e. 68.3% confidence interval). D, (t,S)curves from the sensor during 16-20 operation in buffers solutions at pH 3 and pH 8.

Table 1: Sensor properties Property

Na

This optical sensor

Commercial pH meter

Noise (% of signal span)

30

1.2

0.003

Response time t99 (pH 3 to 8) (s)

15

176 ± 75

5 – 30

Response time t99 (pH 8 to 3) (s)

15

50 ± 12

5 – 30

4.6 - 7.6

0 – 14

0.11 ± 0.03

59.16 ± 0.01

Operational range (pH) Sensitivity (signal units per pH)

ACS Paragon Plus Environment

3

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Precision (pH-units)

3

0.03 ± 0.01

0.02

Stability at pH = 3 (pH per hour)

5

0.008 ± 0.008

0.002

Stability at pH = 8 (pH per hour)

6

0.005 ± 0.003

0.002

a

N is the number of replica for each value. Standard deviations are given by ±.

The sensitivity, A, is the slope of a linearly approximated calibration curve within a given range. Optical sensors do not have linear response, but a linear approximation can be used. This affords a sensitivity of ~ 0.1 optical unit per pH unit. Sensitivity is a parameter that can be changed by changing the dye loading, but it is limited by the level of noise in the sensor system. It furthermore depends on the optical properties of the interface between sensor spot and probe. The precision of the sensor was estimated from the evaluation function, shown in Figure 3C. The evaluation function is calculated as the inverse of the calibration function and the precision was evaluated by calculating prediction intervals of 68.3% confidence, i.e. within ± one standard deviation, σ, assuming a normal distribution. In general, the precision varies with pH, but within the operational range of 3 pH units a mean precision of 0.03 pH unit was determined. This is comparable to commercial pH-meters, which claim to have a precision of ~ 0.02 pH units.

ment in sensitivity and accounting for temperature will reveal significant drift, if there is drift in the sensor. Finally, the sensor was tested in different buffers. Figure 4 shows the calibration curves sampled in four different buffers (four different optodes). Both pKa and sensitivity characteristics are similar, if not identical in all buffers. Note that the salinity changes from 0.02 M to 0.15 M during a calibration experiment, we do not observe any influence of this change in ionic strength on the sensor performace. In summary, an optical pH sensor based on a pHresponsive triangulenium dye immobilized in a porous ORMOSIL matrix was made. A terrylenediimide dye enabled intensity-based ratiometric readout of the pH signal reducing fluctuations originating in immobilization and from the hardware. The hardware, light source and photodetector, were controlled by dedicated software. The sensor was found to have precision of ~0.03 pH units, which is comparable to conventional electrode-based pH meters, within a 3 pH unit range of operation. The optical pH sensor was found to have an operational range from 4.6 to 7.6. Broader ranges can be accessed by incorporation of more dyes with sensitivities in other regions of the pH scale. The high photostability, and the covalent immobilization of the pH-responsive dye ensured excellent operational stability at both high and low pH. It was further demonstrated that the sensor was fully operational in different buffers, and by contrasting the properties of the developed sensor with those currently commercially available (See Table S2), we conclude that this may be the first example of an optical pH sensor that can compete with pH electrodes on equal terms.

ASSOCIATED CONTENT A full description of the sensor fabrication, performance test and data treatment protocols can be found as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected], [email protected]

Conflict of interest Figure 4: Calibration curves sampled in four different buffers viz. Citrate, phosphate, phthalate and HEPES (another sample than shown in Fig. 3B).

The long term operational stability at pH 3 and pH 8 was evaluated from the (t,S)-curves shown in Figure 3D. Six spots were tested for each pH value, and no significant drift was observed within 16-20 h. However, both positive and negative drift with magnitudes of 0.001-0.01 pH values per hour were observed during the individual experiments. This is comparable to drift in pH-electrodes. The standard deviations determined are of the same magnitude as the drift, thus we must conclude that the drift is insignificant for the optical pH sensor system. Improve-

CGF, KH, and TJS are employed by FRS-systems ApS that is commercializing optical sensor technologies. TJS is a founder and current owners of FRS-systems ApS, a University of Copenhagen Spin-Out company based on the research findings disclosed in this manuscript.

Funding Sources The work was supported by Lundbeckfonden (grant#201312793), Novo Nordisk Fonden (grant#4096), Carlsbergfondet, Villum Fonden (grant#14922), BIOPRO, Innovationsfonden (grant# 5179-00914B), UpX and the University of Copenhagen.

ACKNOWLEDGMENT

ACS Paragon Plus Environment

Page 4 of 7

Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors 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 HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, DAOTA: diazaoxatriangulenium, PrTES: propyltriethoxysilane, TDI: terrylenediimide, GPTMS: (3glycidyloxypropyl)trimethoxysilane, ORMOSIL: organically modified silicon, PCB: process control block, PEG: polyethyleneglycol, PS: poly-styrene

REFERENCES 1. Schulthess, C. P., Historical Perspective on the Tools That Helped Shape Soil Chemistry. Soil Sci Soc Am J 2011, 75 (6), 2009-2036. 2. 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. 3. Koch, A.; Pahl, I.; Tanner, M.; Burkhard, A.; Kubischik, J.; Peuker, T.; Eibl, D.; Kauling, J.; Meusel, W.; Kampeis, P.; Müller, D.; Böttcher, L.; Frense, D.; Jobst, G.; Moser, I.; Müller, H.; Nacke, T.; Preuß, K.; Tillich, D.; Weichert, H.; Eibl, R.; Muffler, K.; Neubauer, P.; Pörtner, R.; Ulber, R. Singleuse Technology in Biopharmaceutical Production; Technical Report for Dechema, Frankfurt am Main, Germany, March 2012, 4. Weichert, H.; Lüders, J.; Becker, M.; Adams, T.; Weyand, J., Integrated Optical Single-Use Sensors: Moving Toward a True Single-Use Factory for Biologics and Vaccine Production. BioProcess International 2014, 12 (Supp. 5), 20–24. 5. Janzen, N. H.; Schmidt, M.; Krause, C.; Weuster-Botz, D., Evaluation of fluorimetric pH sensors for bioprocess monitoring at low pH. Bioproc Biosyst Eng 2015, 38 (9), 1685–1692. 6. Card, C.; Clark, K.; Furey, J., Adoption of Single-Use Sensors for BioProcess Operations. BioProcess International 2011, 9 (5), 36–42. 7. Hulanicki, A.; Glab, S.; Ingman, F., Chemical Sensors Definitions and Classification. Pure Appl Chem 1991, 63 (9), 12471250. 8. PreSens GmbH website www.presens.de (accessed Dec. 19, 2018). 9. Kosch, U.; Klimant, I.; Werner, T.; Wolfbeis, O. S., Strategies to design pH optodes with luminescence decay times in the microsecond time regime. Anal Chem 1998, 70 (18), 38923897. 10. Wolfbeis, O. S.; Klimant, I.; Werner, T.; Huber, C.; Kosch, U.; Krause, C.; Neurauter, G.; Durkop, A., Set of luminescence decay time based chemical sensors for clinical applications. Sensor Actuat B-Chem 1998, 51 (1-3), 17-24. 11. Liebsch, G.; Klimant, I.; Frank, B.; Holst, G.; Wolfbeis, O. S., Luminescence lifetime imaging of oxygen, pH, and carbon dioxide distribution using optical sensors. Appl. Spec. 2000, 54 (4), 548-559. 12. Whitaker, J. E.; Haugland, R. P.; Prendergast, F. G., Spectral and Photophysical Studies of Benzo[C]Xanthene Dyes Dual Emission Ph Sensors. Anal Biochem 1991, 194 (2), 330-344. 13. Thomas, J. A.; Buchsbaum, R. N.; Zimniak, A.; Racker, E., Intracellular Ph Measurements in Ehrlich Ascites TumorCells Utilizing Spectroscopic Probes Generated Insitu. Biochem. 1979, 18 (11), 2210-2218.

14. Smith, D. G.; McMahon, B. K.; Pal, R.; Parker, D., Live cell imaging of lysosomal pH changes with pH responsive ratiometric lanthanide probes. Chem. Comm. 2012, 48 (68), 85208522. 15. Westerlund, F.; Hildebrandt, C. B.; Sørensen, T. J.; Laursen, B. W., Trihydroxytrioxatriangulene - An Extended Fluorescein and a Ratiometric pH Sensor. Chem Eur J 2010, 16 (10), 2992-2996. 16. Sørensen, T. J.; Shi, D.; Laursen, B. W., Tetramethoxy‐ Aminorhodamine (TMARh): A Bichromophore, an Improved Fluorophore, and a pH Switch. Chem Eur J 2016, 22 (21), 70467049. 17. Rink, T. J.; Tsien, R. Y.; Pozzan, T., Cytoplasmic Ph and Free Mg-2+ in Lymphocytes. J Cell Biol 1982, 95 (1), 189-196. 18. Baruah, M.; Qin, W.; Basarić, N.; De Borggraeve, W. M.; Boens, N., BODIPY-Based Hydroxyaryl Derivatives as Fluorescent pH Probes. J Org Chem 2005, 70 (10), 4152-4157. 19. Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S., Mitochondria-Immobilized pH-Sensitive Off-On Fluorescent Probe. J Am Chem Soc 2014, 136 (40), 14136-14142. 20. Wolfbeis, O. S., Probes, Sensors, and Labels: Why is Real Progress Slow? Angew Chem Int Ed 2013, 52 (38), 9864– 9865. 21. Frankær, C. G.; Hussain, K. J.; Rosenberg, M.; Jensen, A.; Laursen, B. W.; Sørensen, T. J., A Biocompatible Microporous Organically Modified Silicate Material with Rapid Internal Diffusion of Protons. ACS Sensors 2018, Submitted. 22. Rosenberg, M.; Laursen, B. W.; Frankær, C. G.; Sørensen, T. J., A Fluorescence Intensity Ratiometric Fiber Optics– Based Chemical Sensor for Monitoring pH. Adv Mater Technologies 2018, 2018, 1800205. 23. Sørensen, T. J.; Rosenberg, M.; Frankær, C. G.; Laursen, B. W., An optical pH sensor based on diazaoxatriangulenium and isopropyl-bridged diazatriangulenium covalently bound in a composite sol-gel. Adv Mater Technologies 2018. DOI: https://doi.org/10.1002/admt.201800561 24. Bosson, J.; Gouin, J.; Lacour, J., Cationic triangulenes and helicenes: synthesis, chemical stability, optical properties and extended applications of these unusual dyes. Chem Soc Rev 2014, 43 (8), 2824-2840. 25. Laursen, B. W.; Krebs, F. C., Synthesis of a triazatriangulenium salt. Angew Chem Int Ed 2000, 39 (19), 3432-+. 26. Laursen, B. W.; Krebs, F. C., Synthesis, structure, and properties of azatriangulenium salts. Chem-Eur J 2001, 7 (8), 1773-1783. 27. Sorensen, T. J.; Laursen, B. W.; Luchowski, R.; Shtoyko, T.; Akopova, I.; Gryczynski, Z.; Gryczynski, I., Enhanced fluorescence emission of Me-ADOTA(+) by self-assembled silver nanoparticles on a gold film. Chem Phys Lett 2009, 476 (1-3), 4650. 28. Dileesh, S.; Gopidas, K. R., Photoinduced electron transfer in azatriangulenium salts. J Photoch Photobio A 2004, 162 (1), 115-120. 29. Bora, I.; Bogh, S. A.; Rosenberg, M.; Santella, M.; Sorensen, T. J.; Laursen, B. W., Diazaoxatriangulenium: synthesis of reactive derivatives and conjugation to bovine serum albumin. Org Biomol Chem 2016, 14 (3), 1091-1101. 30. Bogh, S. A.; Simmermacher, M.; Westberg, M.; Bregnhoj, M.; Rosenberg, M.; De Vico, L.; Veiga, M.; Laursen, B. W.; Ogilby, P. R.; Sauer, S. P. A.; Sorensen, T. J., Azadioxatriangulenium and Diazaoxatriangulenium: Quantum Yields and Fundamental Photophysical Properties. ACS Omega 2017, 2 (1), 193-203. 31. Mikkelsen, M. B.; Marie, R.; Hansen, J. H.; Wencel, D.; McDonagh, C.; Nielsen, H. O.; Kristensen, A., Controlled

ACS Paragon Plus Environment

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

deposition of sol-gel sensor material using hemiwicking. J Micromechanics Microengineering 2011, 21 (11), 115008. 32. Laursen, B. V.; Rosenberg, M.; Sørensen, T. J. Patent WO/2015/058777, Azatriangulenium salts as PET-quenched fluorescent probes. 2015. 33. Sørensen, T. J.; Rosenberg, M.; Laursen, B. W. Patent WO/2015/058778, Sol-Gel Based Matrix. 2015. 34. Innocenzi, P.; Brusatin, G.; Guglielmi, M.; Bertani, R., New Synthetic Route to (3-Glycidoxypropyl)trimethoxysilaneBased Hybrid Organic-Inorganic Materials. Chem Mater 1999, 11, 1672–1679. 35. Wencel, D.; MacCraith, B. D.; McDonagh, C., High performance optical ratiometric sol–gel-based pH sensor. Sensor Actuat B-Chem 2009, 139 (1), 208–213. 36. Wencel, D.; Barczak, M.; Borowski, P.; McDonagh, C., The development and characterisation of novel hybrid sol–gelderived films for optical pH sensing. J Mater Chem 2012, 22 (23), 11720–11729.

37. Holtrup, F. O.; Muller, G. R. J.; Quante, H.; Defeyter, S.; DeSchryver, F. C.; Mullen, K., Terrylenimides: New NIR fluorescent dyes. Chem-Eur J 1997, 3 (2), 219-225. 38. Nolde, F.; Qu, J. Q.; Kohl, C.; Pschirer, N. G.; Reuther, E.; Mullen, K., Synthesis and modification of terrylenediimides as high-performance fluorescent dyes. Chem-Eur J 2005, 11 (13), 3959-3967. 39. Buck, R. P.; Lindner, E., Recomendations for nomenclature of ion-selective electrodes. Pure Appl Chem 1994, 66 (12), 2527-2536. 40. Wencel, D.; Abel, T.; McDonagh, C., Optical chemical pH sensors. Anal Chem 2014, 86 (1), 15–29. 41. Westcott, C. C., pH Measurements. Academic Press: New York, 1978. 42. Qi, J.; Liu, D. Y.; Liu, X. Y.; Guan, S. Q.; Shi, F. L.; Chang, H. X.; He, H. R.; Yang, G. M., Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore. Anal Chem 2015, 87 (12), 5897-5904.

ACS Paragon Plus Environment

Page 6 of 7

Page 7 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

for TOC only 86x40mm (275 x 275 DPI)

ACS Paragon Plus Environment