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Temperature Mapping in Hydrogel Matrices Using Unmodified Digital Camera Ghinwa H. Darwish, Hassan H. Fakih, and Pierre Karam J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11844 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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The Journal of Physical Chemistry

Temperature Mapping in Hydrogel Matrices Using Unmodified Digital Camera Ghinwa H. Darwish, Hassan H. Fakih, and Pierre Karam* Department of Chemistry, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon *To whom correspondence should be addressed: Phone: 961-1-350000 Ext 3989 Fax: 961-1-350000 Ext 3970 Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: We report a simple, generally applicable and noninvasive fluorescent method for mapping thermal fluctuations in hydrogel matrices using an unmodified commercially available digital single-lens reflex camera (DSLR). The nanothermometer is based on the complexation of short conjugated polyelectrolytes, poly-(phenylene ethynylene) carboxylate, with an amphiphilic polymer, polyvinylpyrrolidone, which is in turn trapped within the porous network of a gel matrix. Changes in the temperature lead to a fluorescent ratiometric response with a maximum relative sensitivity of 2.0% and 1.9% at 45.0 °C for 0.5% agarose and agar, respectively. The response was reversible with no observed hysteresis when samples were cycled between 20 °C and 40 °C. As a proof of concept, the change in fluorescent signal/color was captured using a digital camera. The images were then dissected into their red-green-blue (RGB) components using a Matlab routine. A linear correlation was observed between the hydrogel temperature and the green and blue intensity channels. The reported sensor has the potential to provide a wealth of information when thermal fluctuations mapped in soft gels matrices are correlated with chemical or physical processes.


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INTRODUCTION Hydrogel materials, given their unique physical and chemical properties and the easiness of molding them into complex shapes, made them a popular choice in a wide range of applications, including, but not limited to, tissue engineering, drug delivery, DNA and protein electrophoresis techniques and cellular immobilization.1-6 Diffusion reaction framework is also an example where hydrogel matrices unraveled an exquisite time lapse of many chemical reaction processes like the ability to form and sustain thermodynamically unstable product such as alpha-Co(OH)2.7 While these soft materials provide a unique support, they create a physical barrier to probe parameters of specific importance such as pH, concentration gradient, temperature etc. Indeed, probing temperatures within these materials will prove instrumental to enhance our understanding of many of these supported biological and chemical processes. For instance, growing a healthy organ in a 3-dimentional (3D) soft scaffold requires accurate temperature control throughout the matrix.8-9 DNA folding/diffusion also requires a precise spatial thermal information.9-11 Many approaches have been developed to measure the temperature in solutions at the macroand nano-scale level.12-15 However, little work have been done to accurately map temperature changes in hydrogel materials where fluctuations are more likely to be perceived and samples are inherently heterogeneous.16 Small thermocouples, for instance, were used to monitor thermal cycling for rapid PCR-Based DNA analysis in a microfluidic device.17 However, the intrusive nature of these probes can be disruptive to the matrix of interest. In addition, the reported temperature is only in close vicinity of the thermocouple which negates the accurate reporting of any temperature gradient or fluctuation in the system. Thermal imaging, while an attractive option, lacks the possibility to map temperature in 3D matrices and is not optically compatible



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with other well established spectroscopic probing methods. There is therefore a need to develop a probe capable of mapping the thermal distribution with high spatial resolution in hydrogels materials. Fluorescence spectroscopy based imaging techniques has a clear advantage when it comes to probing events of interest in a transparent matrix. It is now possible to monitor molecular and supramolecular interactions with a three dimension spatial resolution of only few nanometers.1820

In addition, fluorescent based methods to probe and monitor a full spectrum of analytes have

been widely established. Our group, has recently explored the thermal sensitivity of short conjugated polyelectrolytes (CPEs), poly-(phenylene ethynylene) (PPE-CO2-7) in complex with an amphiphilic macromolecule, polyvinylpyrrolidone (PVP).21 The probe was tested between 10 °C and 70 °C and has shown a high relative sensitivity of 2.7% for PPE-CO2-7/PVP-360K at 35 °C.22 We showed that in the presence of PVP, the PPE-CO2-7 aggregates are destabilized and a small change in the solution temperature disaggregates the CPE in the excited state.22 This shifted the equilibrium between two emission spectra; 520 nm at low temperatures and a brighter peak (455 nm) overlapping with the emission of individual chains at higher temperatures. The probe sensitivity increased with increasing the amphiphilic polymer molecular weight and concentration. Its ratiometric signal was reversible with a fast response time. Based on the previously reported system, we aimed at mapping thermal fluctuations in hydrogel matrices. As such, in this work we embedded PPE-CO2-7/PVP hybrid macromolecules within the porous structure of two widely used soft gels materials agar and agarose. These biocompatible materials were chosen given their extensive use in various applications and their chemical functionality.23 Chemically, the two gels share the same galactose-based water soluble


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backbone; however, agar contains agaropectin which is decorated with sulfate (1-6%) and pyruvates side groups24-25 which makes it the material of choice for microbiological studies for bacteria and fungi growth.26 Agarose, on the other hand, given its relative neutrality and inertness, has greater gelling abilities and is the preferred matrix for working with small biological molecules such as nucleic acids and proteins.27-28

METHODS Materials. Poly-(phenylene ethynylene) carboxylate (PPE-CO2-7), with an average of 7 monomer repeating units, was synthesized as previously described.29-30 Polyvinylpyrrolidone (PVP) 360K was purchased from Sigma Aldrich. Agar and agarose were purchased from Becton and Lonza, respectively. Encapsulation of the Nanothermometer. Five microliters of 2 mg.mL-1 PPE-CO2-7 was added to 2000 µL of 10 mM HEPES and 150 mM NaCl (pH = 7.3) buffer, followed by the addition of 50 µL of 100 mg.mL-1 PVP (360K). The solution was then placed in a thermostat and stirred at 400 rpm until the inner temperature reached 45 °C. 200 µL of 5% agar or agarose was then added to the PPE-CP2-7/PVP solution at 45 °C. Stirring was then stopped and the temperature was lowered to 10 °C. Thermal Sensing. Steady-state fluorescence spectroscopy was carried out using a Thermo Fisher Lumina spectrometer equipped with a temperature controller unit (T3 Quantum Northwest). Fluorescent emission spectra were acquired upon excitation at 420 nm. Before each measurement, the solution was allowed to stabilize for five minutes.



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For practical purposes, the temperature sensitivities were calculated by first integrating the fluorescence signal between 435 nm and the isoemission point, which is denoted as IBlue. A second integration was calculated between the isoemission point and 650 nm (IRed). The ratio ( =

) of the integrated signal was then calculated and plotted against the change in

temperature. Given the lack of a physical model that can fully describe the ratiometric signal readout, the thermal response was fitted into a second order polynomial function. The relative sensitivity (S) was calculated using equation (1) for the fitted polynomial:12 (% = × )

Thermal Imaging. An agarose (0.5 %) sample that embeds PPE-CO2-7/ PVP was prepared and molded into a cuboid shape (2.2 x 2.2 x 1 cm3). The hydrogel, separated by a thin glass slide (0.5 mm), was placed on a thermoelectric cooler (TEC1-12710, Imax: 10.5 A, Umax: 15.4 V, R: 1.08 ohm, 127 couples, ∆T max.=68 °C, and Qmax (∆T=0):100.0 W). The temperature was regulated by a DC power supply connected to the thermoelectric cooler by changing the applied voltage between 0 V and 6 V with 2 V intervals. A K-type thermocouple (chromel/Alumel; 200 °C to 1260 °C; 36 gauge, HosKins Mfg.Co., Hamburg, MI) was inserted inside the hydrogel and used to measure the temperature changes (Figure 6). The PPE-CO2-7/PVP was excited using a BLAK-RAY high intensity ultraviolet inspection lamp (UV-pland 95-0127-01, model B-100 AP, 100-watt, 365 nm long wave). A series of photographic images were acquired using the DSLR camera equipped with a 50-80 mm Lens, and saved as RAW files. The exposure time (1/3 s), ISO 100 and the f-number (f/5.6) were kept constant throughout the experiment. A matlab


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routine served to dissect each picture into its three color components Red-Green-Blue. The average intensity of each channel was then plotted versus temperature.

RESULTS AND DISCUSSION Our previously reported results showed that the nanothermometer sensitivity and performance greatly depends on the probe environment.22 Given the structural and chemical complexity of the agar and agarose, it was a challenge for us to trap the probe while preserving its thermal properties. If the PPE-CO2-7/PVP complex dissociates or the conjugated polyelectrolytes disaggregate, the ratiometric response of the probe is lost. Trapping the Nanothermometer. We faced three main challenges while designing our experiments. (i) The hydrogel matrix needs to be transparent for fluorescent measurements. Fortunately, both materials were previously used to trap and image fluorescently labeled proteins, macromolecules and bacteria.10,

31-33

(ii) The probe needs to be smaller than the

microporous cavities so it can uniformly distribute and diffuse freely. Agar and agarose matrices are highly porous with a pore size that ranges between 100 nm and 900 nm when prepared at 0.5%.34-35 The calculated radius of gyration of PVP used in this study with an average molecular weight of 360K was estimated to be equal to 35 nm.36 PVP is believed to be efficiently complexing PPE-CO2-7 aggregates.21-22 Given the matrix pore size, the probe radius, and its solution dispersity, we expect the nanothermometer to distribute uniformly in the hydrogel. (iii) Minimize potential interactions between the host and the probe. This turned out to be the most challenging point. When the probe was added to a hot solution of 0.5% agar or agarose at 85 °C or even at 45 °C, the CPE exhibited an emission peak at 455 nm with no observed thermal response (Fig. S1). We therefore experimented by changing the mixing temperature of the probe



and the hydrogel. We found that by keeping the probe solution at 45 °C (10 mM HEPES and 150 mM NaCl at pH=7.3, PPE-CO2-7 (5 µg.mL-1), PVP-360K (2.5 mg.mL-1)) and adding to it a concentrated agar or agarose solution prepared at high temperatures (85 °C), this ensured that the nanothermometer complex is in its active and sensitive form. The hydrogel final solution concentration was set at 0.5%. It is important to mention that the experiment reproducibility is highly dependent on the accurate preparation of the gel sample (See supporting information). The ratio between the conjugated polyelectrolyte and the amphiphilic macromolecule and its molecular weight were previously optimized to be 1:3.5 (CPE: PVP) polymer ratio and 360K, respectively.22 The probe-hydrogel mixture was stirred at 400 rpm for only few minutes to avoid gel cracking and formation of air bubbles before lowering the temperature to 10 °C. Steady-state fluorescence spectroscopy was carried out using Thermo Fisher Lumina spectrometer equipped with a temperature controller unit (T3 Quantum Northwest). Fluorescence emission spectra were acquired upon excitation at 420 nm.

Figure 1 summarizes the

fluorescence emission of the nanothermometer probe in buffer and when trapped in agar and agarose at 20.0 °C. Fluoresence Intensity (a.u.)

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5x10

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4

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0 450

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Figure 1: PPE-CO2-7 fluorescence under different experimental conditions. Fluorescence emission intensity of PPE-CO2-7 (5 µg.mL-1) complexed with 360K PVP (2.5 mg.mL-1) in (●)


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10 mM HEPES and 150 mM NaCl (pH= 7.3) buffer, (■) Agar (0.5 g.mL-1), and (▲) Agarose (0.5 g.mL-1) acquired at 20.0 °C when excited at 420 nm. The probe emission intensity profile was not affected when trapped in either agar or agarose; indeed, a small peak at 525 nm from aggregated species, another peak at 485 nm from the destabilized aggregates and a shoulder at 455 nm for the disaggregated polymer were observed in the three tested environments (buffer, agar and agarose) (Figure 1).21 The intensity is, however, observed to be enhanced by 17% and 37% in agarose and agar, respectively. Conjugated polyelectrolyte photophysics is highly dependent on their micro-environment.37-45 The galactosebased backbone chain in agarose and the added sulfonate side groups in agar could be playing a role in this enhancement. Thermal Sensitivity. To evaluate the thermal sensitivity of the probe in the soft gel materials, we followed the changes in the fluorescent emission of PPE-CO2-7/PVP between 15.0 °C and 70.0 °C at a 5.0 °C incremental interval. Before each measurement, the gel was allowed to stabilize for three to four minutes at the set temperature. Figure 2.B shows the emission intensity of PPE-CO2-7/PVP in 0.5% agarose as function of temperature. Upon increasing the temperature, the emission peak at 525 nm steadily decreased concomitant with an increase in the intensity peak at 455 nm. An isoemission point is observed at ca. 500 nm. This ratiometric behavior is what we have previously reported for PPE-CO2-7/PVP in solution.21-22 The temperature sensitivity was calculated by first integrating the fluorescence signal between 435 nm and the isoemission point (IBlue). A second integration was calculated between the isoemission point and 650 nm (IRed). The ratio ( =

) of the integrated signal was then

calculated and plotted against the change in temperature (Figure 2.C). The temperature response was reproducible with relatively small errors as calculated from the standard deviation of 4 runs.



The thermal response was fitted into a second order polynomial function and the relative sensitivity

(%S)

was

calculated

using

the

following

equation:

(% = × ) The maximum relative sensitivity is reported as the highest computed relative sensitivity within the studied range and was equal to 2.0% at 45 °C for agarose (Figure 2.D). o

B

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Fluoresence Intensity (a.u.)

A

450

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Wavelength (nm)

C

D

1.5

2.2 2.0

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%S

IBlue/IRed

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Figure 2: Thermal response of PPE-CO2-7/PVP-360K in agarose. (A) chemical structure of agarose. (B) The fluorescent emission intensity of PPE-CO2-7 (5 µg.mL-1)/PVP-360K (2.5 mg.mL-1) trapped in agarose upon excitation at 420 nm when monitored between 435 nm and 650 nm acquired between 15 °C and 70 °C at 5 °C incremental intervals. (C) Integrated fluorescent ratio Q calculated as =

; where IBlue is the fluorescent signal between 435 nm


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and the isoemission point and IRed is calculated between the isoemission point and 650 nm. The error bars represent the standard deviation of four independent measurements. (D) Percent relative sensitivity %S calculated from (C). The thermal sensitivity was also tested when PPE-CO2-7/PVP is trapped in agar (Figure 3.B). A ratiometric response was also observed over the tested window and a calculated maximum relative sensitivity was found to be equal to 1.9% at 45.0 °C (Figure 3.D). When compared to the relative sensitivity of the probe in buffer (2.7% at 35 °C), both values in agar and agarose were found to be compromised. The difference might be due to the temperature sensitivity of the polysaccharides which unwind and gelate with the temperature fluctuations (Figures 2.A and 3.A).23 This can in turn affect the probe performance. As a control, PPE-CO2-7 polymer only solution, was trapped in agarose and agar. As the temperature increased, the fluorescent intensity of the sample decreased accompanied by a blue shift in the emission with no observed ratiometric thermal sensitivity (Fig. S2).



o

15 C o 20 C o 25 C o 30 C o 35 C o 40 C o 45 C o 50 C o 55 C o 60 C o 65 C o 70 C

Fluoresence Intensity (a.u.)

B

Agar

A

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500

550

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650

Wavelength (nm)

C

D

1.6

2.1

1.8 1.2

%S

IBlue/IRed

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1.5

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Temperature (°C)

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Figure 3: Thermal response of PPE-CO2-7/PVP-360K in agar. (A) chemical structure of agar which is comprised of a bland of agarose and agaropectin. (B) The fluorescent emission intensity of PPE-CO2-7 (5 µg.mL-1)/PVP-360K (2.5 mg.mL-1) trapped in agar upon excitation at 420 nm when monitored between 435 nm and 650 nm acquired between 15 °C and 70 °C at 5 °C incremental intervals. (C) Integrated fluorescent ratio Q. The error bars represent the standard deviation of four independent measurements. (D) The relative sensitivity %S calculated from (C). Probe Stability. To gain meaningful information on the thermal fluctuation of any studied system, the probe must be able to endure continues monitoring and repetitive cycling.46 To this end, the hydrogels samples were cycled for 16 times between 20 °C and 40 °C and the fluorescent ratio was calculated (Figure 5). Both samples showed no sign of hysteresis.47


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However, the agar was less reproducible than agarose and both less consistent than the free probe in a buffer solution.22 Agarose had an average value of 0.656 ± 0.012 at 40 °C (Figure 4.A), while agar had an average value of 0.959 ± 0.031 at the same temperature (Figure 4.B). This fluctuation in signal repeatability is understandable given that the hydrogel micro-environment is temperature sensitive and local degradation of the matrix is expected with these temperature cycles.48 The fluorescence emission intensity was however severely affected but the ratiometric response nature of the probe negated this effect. For instance, the intensity decreased by 21% in agarose and 32% in agar between the first and last cycle at 20 °C (Fig. S4). We speculate that this decrease is due to the photobleaching of the fluorescent polymer, unlike the solution-based experiments, the probe does not replenish by diffusing to the focal point.

A

Agarose

0.8

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Agar

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IBlue / IRed

0.7

IBlue / IRed

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Figure 4: Probe stability. Cycling an (A) agarose and (B) agar samples between 20.0 °C (in blue) and 40.0 °C (in red) embedded with PPE-CO2-7 (5 µg.mL-1)/PVP-360 K (2.5 mg.mL-1). The lines connecting the experimental points are for visual aid. Thermal Imaging. To showcase the potential applicability of the noninvasive hydrogel temperature sensor, we acquired, at variable temperatures, a series of images using a commercially available 5100 Nikon DSLR camera for the agarose hydrogel matrix. The two emission peaks at 455 nm and 525 nm coincide with the blue and green regions of the red-green-



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blue (RGB) channels in commercial “complementary metal-oxide semiconductor” (CMOS) sensors.49 An agarose (0.5%) sample that embeds PPE-CO2-7/ PVP was prepared and molded into a cuboid shape. The hydrogel, separated by a thin glass slide (0.5 mm), was placed on a thermoelectric cooler. The temperature was regulated by a DC power supply connected to the thermoelectric cooler. A K-type thermocouple was inserted inside the hydrogel and used to measure the temperature changes (Figure 5). The four applied voltages lead to a measured temperature of 20 °C, 29 °C, 38 °C, and 52 °C. The PPE-CO2-7/PVP was excited using a BLAKRAY high intensity ultraviolet inspection lamp. A series of photographic images were acquired using the DSLR camera.

Figure 5: Schematic illustration for the imaging setup. The molded hydrogel is placed on a thermoelectric cooler (Peltier device) regulated by a DC power supply. The hydrogel temperature is measured with a thermocouple. The nanothermometer is illuminated with a UV lamp and the images are captured using a DSLR Nikon 5100 camera.


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Figure 6: Thermal probing using a DSLR. (A) series of photos captured with a DSLR camera (exposure time 1/3 s, f/5.6, ISO 100) while increasing the temperature. (B) Cropped images (49x15 pixels) from (A) selected from the thermocouple vicinity where temperature was reported to increase from 20 °C to 52 °C. (C) The green and blue intensity channels distribution for the cropped images in (B) plotted on 256-level gray scale. (C) A calibration plot of the average blue and green intensities versus temperature. The linear fit is described by Blue/Green= 0.0255(Temperature) + 0.462. (E) Temperature mapping of the image in (A) acquired at the highest temperature calculated pixel by pixel.



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The change in color with increasing the hydrogel temperature was evident to the naked eye (Figure 6.A) and changed from dominantly green to a blue color. To accurately map the temperature of the prepared hydrogel, we first analyzed the area in direct contact with the inserted thermocouple (49 pixel x 15 pixels; Figure 6.B). The previous calibration reported in figure 2.b would not have been applicable since the defined spectral window for RGB, and the detector quantum yield is different than the clear cut windows 495-500 nm / 500-650 nm, defined earlier. The 4 images were dissected to their red, green and blue components using Matlab, and binned on a scale from 0 to 256. Since the red component was minimal, it was discarded (Figure 6.C). The image analysis revealed a strong correlation between the average intensity, in both the green and the blue channels, and the external applied temperature. An increase in temperature led to a decrease in the green average intensity from 102 ± 9 to 73 ± 8 and an increase in the blue channel from 99 ± 6 to 128 ± 7, between the reported temperature of 20 °C and 52 °C. The ratio of the blue to green was calculated and plotted versus the change in temperature. The thermal response was linear over the tested range with a response of Blue/Green=0.0255(Temperature) + 0.462.


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Figure 7: Temperature mapping of the image in Figure 6.A acquired at the highest temperature calculated pixel-by-pixel. The result is in agreement with the steady-state spectroscopic measurements where the increase in temperature resulted in a ratiometric response between these two chromatic colors. Subsequently, the image captured at the highest temperature was mapped pixel by pixel. The blue to green ratio was deconvoluted and their ratios calculated. Using the thermal response (Figure 6.D), the temperature of the entire agar gel was mapped (Figure 7). It revealed a concentric temperature profile with much cooler edges. The result could be explained by (i) faster heat dissipation at the cuboid edges and (ii) the temperature profile of the thermocouler used in this study whose heat is more concentrated in the center. This result offers a simple way to detect temperature changes in solid and liquid matrices using a simple digital camera.

CONCLUSIONS



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We have successfully reported a simple and non-invasive tool to detect temperature changes in hydrogel systems. The probe was quickly prepared in a one-pot reaction mixture and it showed an excellent thermal sensitivity between 15 °C and 70 °C with a maximum relative sensitivity of 1.95% and 2.03% for agar and agarose, respectively. The temperature fluctuation was evident to the naked eye and a commercially available digital camera allowed us to accurately measure these variations. The reported contactless temperature sensor with its high sensitivity will be instrumental for the active ongoing research in hydrogel materials applied to the different fields of chemistry, physics, and biology. Unlike, thermal cameras, the reported system can potentially unravel temperature distribution in a three dimensional matrix and could be complemented with other fluorescent probes to simultaneously probe for instance, pH changes and biomarkers of interest.

Supporting Information: Probe preparation, thermal sensitivity and photostability are available in the supporting documents. Acknowledgment: The authors are thankful to Dr. Michel Kazan from the physics department at the American University of Beirut for providing the thermocouple and thermoelectric cooler. This work was supported by Lebanese National Council for Scientific Research (CNRS #102901) and the University Research Board (URB #102848 and #103009) at the American University of Beirut. The authors are also thankful for the Kamal A. Shair Central Research Science Lab (KAS CRSL) of the Faculty of Arts and Sciences at AUB for providing access to their facilities.


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