In situ observation of sol-gel transition of agarose aqueous solution by fluorescence measurement

Accepted Manuscript In situ observation of sol-gel transition of agarose aqueous solution by fluorescence measurement

Zheng Wang, Kun Yang, Haining Li, Chaosheng Yuan, Xiang Zhu, Haijun Huang, Yongqiang Wang, Lei Su, Yapeng Fang PII: DOI: Reference:

S0141-8130(17)34693-7 https://doi.org/10.1016/j.ijbiomac.2018.02.020 BIOMAC 9074

To appear in: Received date: Revised date: Accepted date:

26 November 2017 25 January 2018 4 February 2018

Please cite this article as: Zheng Wang, Kun Yang, Haining Li, Chaosheng Yuan, Xiang Zhu, Haijun Huang, Yongqiang Wang, Lei Su, Yapeng Fang , In situ observation of solgel transition of agarose aqueous solution by fluorescence measurement. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), https://doi.org/10.1016/j.ijbiomac.2018.02.020

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ACCEPTED MANUSCRIPT In situ observation of sol-gel transition of agarose aqueous solution by fluorescence measurement

Zheng Wanga,b, Kun Yangb, Haining Lia,b, Chaosheng Yuanb, Xiang Zhub, Haijun Huanga,*,

School of Sciences, Wuhan University of Technology, Wuhan, Hubei 430070, China

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Yongqiang Wangb, Lei Sub,c,*, and Yapeng Fangd,*

Center for High Pressure Science and Technology Research, Zhengzhou University of Light

Key Laboratory of Photochemistry, Institute of Chemistry, University of Chinese Academy of

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c

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Industry, Zhengzhou, 450002, China

Sciences,Chinese Academy of Sciences, Beijing, 100190, China d

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Department of Food and Pharmaceutical Engineering, Glyn O Phillips Hydrocolloids Research

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Centre, Hubei University of Technology, Wuhan, 430068, China

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ACCEPTED MANUSCRIPT Abstract Sol-gel transition behavior of agarose aqueous solution was investigated by using rheology and fluorescence measurement. On heating, the storage modulus G′ decreased gradually, then deviated abruptly at the temperature of about 65℃, and

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finally decreased slowly again. For fluorescence measurement, the phase transition point kept almost at the temperature of 65℃, which was consistent with that in

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rheology measurement. Upon compression, it was indicated that the fluorescence

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lifetime for the probe in the agarose aqueous solution showed a dramatic change in the vicinity of the phase transition point. T vs. P phase diagram of agarose aqueous

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solution was constructed, which showed that the melting point was an increasing

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function of pressure. Based on the phase diagram, the agarose gels were prepared by cooling under atmospheric pressure and the pressure of 300MPa, respectively. From the result of the recovered samples studied by optical rheometry, it was found that

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agarose gel prepared under high pressure had a higher elasticity and lower viscosity index, compared with that under atmospheric pressure. It could be speculated that such kinds of properties might be attributed to the smaller pore size during gelation

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under high pressure.

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Keywords: Phase transition; Agarose; Fluorescence

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ACCEPTED MANUSCRIPT 1. Introduction Agarose is a main ingredient of agar-agar, which is a linear polymer composed of D-galactose and 3, 6-anhydro-L-galactose [1]. Agarose gel is typically rigid and prone to the phenomenon of spontaneous loss of water on standing that is known as

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syneresis [2]. Gel formation is accompanied by a large, sharp and sigmoidal increase

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in the magnitude of the negative optical rotation. Sol-gel transition and gel topology

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involve many conceptual aspects, such as phase transition and scaling, and therefore are of high intrinsic interest. The structure of agarose gel is expected to tend to a more

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crystalline state in very concentrated gels, and the properties of the concentrated gels

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are very important from the practical point of view [3-10]. For the gelation mechanism of agarose, Bulone D et al studied the gelation

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kinetics of agarose aqueous solution or in the presence of NaCl using dynamic light

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scattering and rheology methods. The results showed that a region three to four times larger than the aggregate became depleted of agarose as the gelation proceeds, and the

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aggregation process froze spatial ordering rapidly, resulting in fragile macroscopic

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gels [5]. San Biagio et al discussed the self-assembly of agarose gels which was performed with the use of a variety of techniques allowing identification of the initial break of symmetry and the actual path leading to self-assembly at concentrations well below the random percolation threshold. The analogous role of either permanent or transient demixing of the sol in providing preferential paths for cross-links and promoting gelation at moderate and low concentrations has been established also in a variety of other biopolymeric systems [11]. Matsuo et al studied the gelation 3

ACCEPTED MANUSCRIPT mechanism of agarose aqueous solutions by using polarized light scattering in terms of the liquid-liquid phase separation [12-13]. Boral et al studied the self-assembly of agar molecules in detail, which clearly indicated simultaneous growth of self-similar aggregates and larger macrodomains due to spinodal demixing. And it was observed

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that microdomains existed even in sol state at temperature far away from Tg. The

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results presented provide a significant insight into the distinctive microstructural

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features of agar sol that dynamically evolve and form a gel, and it made an attempt to give some foundation to its understanding [14]. And then Boral et al discussed the

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differential microscopic structures of agar solutions and gels by small angle neutron

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scattering. The results revealed that the persistence length of fibre bundles was found to be 3 nm implying change in the specificity of fibre-solvent interactions. The

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scattering profile of agar solution was observed to be different from that of its gel

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phase. However, the scattering profile for the gel was observed to be anomalous, and close to that predicted for statistical gels [15]. Recently we examined the

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thermoreversible sol−gel transitions of agarose solution on isobaric cooling or heating

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under high pressure up to 400 MPa by in situ observations of optical transmittance. The measurements of optical transmittance and the falling ball proved to be simple yet useful and powerful to understand this interplay. The results were discussed in terms of the pressure effects on ΔV and ΔH associated with the sol−gel transitions along with the transformation of the hydration structure by compression [16]. In order to further clarify the structure of agarose gel, in this paper, in situ observation of sol-gel transition of agarose aqueous solution was studied by rheology 4

ACCEPTED MANUSCRIPT and fluorescence measurement. The phase diagram of agarose gel under the pressure up to 300 MPa was investigated in detail. Furthermore, a novel method to prepare agarose gel under high pressure was also reported, and the properties of agarose gel

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had been studied by optical rheometry.

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2. Experimental procedure

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The agarose used in our experiments was supplied by Wako Pure Chemical Industries, Ltd. (Japan), with a molecular weight of about 120,000. The structure was

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shown in Fig.1. The dried specimens were swollen in water at 40 °C over night,

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preheated at 70 °C for 1 h and then heated at 100 °C for 30 min. The gels were kept at 4 °C for 1 d to mature and then kept at the temperature of measurement for 1 h before

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measurements were taken.

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Dynamic viscoelastic measurements were carried out using a Rheostress 600 (Haake, Thermo Electron, Germany) with a cone plate geometry. The exposed surface

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of the sample was covered with silicone oil to avoid evaporation of water in the

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solution, immediately after the sample solution was set on the lower plate. The temperature dependence of the storage (G′) and the loss (G″) shear moduli was examined at a rate of 0.5 °C/min and at an angular frequency of 1.0 rad/s. For fluorescence measurements, the fluorescence probe 9-(dicyanovinyl)julolidine, DCVJ (Invitrogen, USA), was used as received, which is well-established as an indicator. A sample-filled internal cell made of quartz (the optical path length was 5 mm) which had a section of stretch plastic tube was inserted in a stainless steel 5

ACCEPTED MANUSCRIPT (JIS SKD-62) high-pressure cell with four sapphire windows sealed with o-rings and gasket rings, which was connected to a high-pressure pump (Hikari High Pressure Machinery, Hiroshima, Japan). The internal cell was compressed through the pressure medium of the silicone oil (Shin-Etsu Chem. Co. Ltd., Japan). The cell body had a

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tubelike flow channel, in which the constant-temperature water was circulated to

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maintain the temperature. Fluorescence lifetime were measured using time-correlated

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single-photon counting (TCSPC) techniques. The excitation source was a BDL-405 diode laser (Becker and Hickl GmbH), which provided output pulses of 60 ps duration

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with a wavelength of 405 nm and a repetition rate of 10 MHz. The emission from the

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sample was detected by a PMC-100 detector head (Becker and Hickl GmbH) Emission decay curves were recorded on a SPC-130 TCSPC module (Becker and

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Hickl) at each pressure. Fluorescence lifetime were determined by multiple

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exponential decay fitting. Agarose aqueous solutions were subjected to pressures up to 450MPa at the temperature range from 15℃ to 75℃. According a formula log Ф=

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C + x log η [17], where Ф is the fluorescence quantum yield, C and x are constants

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(for DCVJ, x has been reported to be 0.6) [18]. And, it should be noted that dramatic change in fluorescence lifetime is an effective criterion to judge the sol-to-gel phase transition. Details of the theoretical framework and analytical method are the same as those described in previous report [19-20]. In order to study the influence of pressure on the structure of agarose gels, these two samples of agarose aqueous solution were prepared under high pressure and atmospheric pressure respectively. A set of piston-cylinder made of tungsten carbide 6

ACCEPTED MANUSCRIPT with a resistance coil heater was used to produce high pressure and high temperature. The sample was filled in an aluminum container and compressed to a predetermined pressure level, then the temperature was raised to a predetermined level, which was higher than its gelation temperature under this pressure. After equilibrium was

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established, the sample cooled naturally by turning off the power to room temperature

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and finally decompressed slowly. Another sample was directly cooled naturally to

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room temperature under atmospheric pressure. Diffusing wave spectroscopy (DWS) based micro-rheology was used to measure the rheological behaviour of agarose

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aqueous solutions. The samples were contained in glass cells of width 10 mm, height

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50 mm and length 4 mm, and were illuminated with a 35 mW HeNe laser operating at wavelength with 633 nm. The laser beam was expanded to approximately 8 mm on and backwards was

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the surface of the cell. The light scattered both forwards

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separately detected using single mode optical fibres (P1-3224-PC-5, Thorlabs Inc., Germany) fitted with GRIN lenses (F230FC-B FC, Thorlabs Inc., Germany). The

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optical fibres were connected to a Hamamatsu HC120-08 PMT photomultiplier tube

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module and the intensity autocorrelation functions of the scattered light were obtained using a Malvern 7132 correlator.

3. Results and Discussion Fig. 2 showed storage modulus G′ and loss modulus G″ as a function of temperature in heating processes for agarose aqueous solution. The change in G′ during the whole heating process could be divided into three distinct stages. The 7

ACCEPTED MANUSCRIPT storage modulus G′ decreased slowly in the first stage, and then decreased suddenly at the temperature of about 65℃, and finally decreased slowly again. The temperature at which G′ deviated from the base line was defined as the melting temperature Tmelt or the gelation temperature Tgel hereafter [16], and it indicated that agarose gel melt

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above this temperature. Besides 0.5wt% agarose aqueous solution, 1wt% and 2wt%

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agarose aqueous solution were also studied in details. The results showed that Tgel of

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1wt% and 2wt% agarose aqueous solution almost kept at 65℃ with increasing concentration of agarose aqueous solution. It could be seen that the same behavior had

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been found for all these three systems and were represented by typical curves shown

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in Fig. 2. Evingur et al discussed the mechanical properties of swollen PAAm-NIPA copolymers by the compressive testing technique and speculated that the entropic

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model for copolymer elasticity was a reasonable approximation [21]. It was

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understood that the compressive elastic modulus was found to increase by increasing NIPA contents, and the critical exponent of elasticity above the critical NIPA

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concentration was found to be as 0.74, which was consistent with the suggestions of

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percolation for superelastic percolation network. For agarose gel, entropic elasticity occurred when flexible objects were involved, and these flexible objects could be the chains connected at the junctions of a network. At high concentrations, agarose gels could be described with the usual equilibrium thermodynamics for multiphasic systems. As long as no transformation took place, the different phases should keep the same composition at a given temperature independent of the agarose concentration. Evidently, it was no longer so in the vicinity of the critical gelation concentration, as 8

ACCEPTED MANUSCRIPT the composition of the dilute phase varied when altering the agarose concentration. Agarose aqueous solution had also been studied by fluorescence measurement at the temperature range from 15 ℃ to 75 ℃ by using a quartz cell. The microdomains were probed by 9-dicyanovinyl julolidine (DCVJ) for which the

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monomer emission intensity (Ie) provides relative measures of the fluorescence

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lifetime in the immediate probe surroundings. Fig. 3 shows the temperature

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dependence of the fluorescence lifetime for the probe in the agarose aqueous solution with different concentrations. From this figure, it could be seen that, with increasing

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temperature, the fluorescence lifetime for the probe in the agarose aqueous solution

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increased gradually in the initial stage, and then decreased suddenly at the temperature of 65℃, and finally increased with the increasing temperature. It was indicated that

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the fluorescence lifetime of agarose aqueous solution showed a dramatic decrease in

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the vicinity of the phase transition point. And the result suggested that the gel-to-sol transition point of the agarose aqueous solution was about 65℃. In addition, with

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increasing concentration of agarose aqueous solution, the phase transition point kept

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almost at the temperature of 65℃. Pekcan et al studied thermoreversible phase transitions of high and low melting point agarose gels by using the UV–vis and fluorescence spectroscopy techniques [22]. They thought that, at the beginning of the heating process (lower temperature region), the gel structure consisted of double helix aggregates and water trapped in the gel network, however, at the high temperature region, the amplitudes of molecular vibrations started to increase, resulting in the destruction of the double helix 9

ACCEPTED MANUSCRIPT aggregates. And critical temperatures could be produced from the inflection points of the sigmoidal curves of thermal phase transitions. According to the previous theoretical framework and analytical method: log Ф= C + x log η and Ф= Kτ, where Ф is the fluorescence quantum yield, τ is the lifetime, and, C and x are constants

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[19-20]. For Fig. 3, it shall be stated that microviscosity is a local solvent property in

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the vicinity of the solute, and it can be distinguished from the bulk viscosity by

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considering some heterogeneity on a molecular scale. When the solution is homogeneous, the local viscosity will be characteristic of the macroviscosity, and

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when the sample is inhomogeneous, the local viscosity will not be characteristic of the

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macroviscosity. In this study, at the beginning of the heating process (lower temperature region), the gel structure consisted of double helix aggregates and water

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trapped in the gel network, and the DCVJ went into the water rich region, so it

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detected only the viscosity of water. With the temperature increasing, the amplitudes of molecular vibrations started to increase, resulting in the destruction of the double

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helix aggregates. Under such a situation, the microdomains around DCVJ were

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composed of water and some aggregates, and microviscosity increased gradually with the increasing temperature. In the view of this point, when the microviscosity showed a dramatic change with the temperature increasing, it meant that there would be a phase transition in the vicinity of this inflection point. And then, more and more the double helix aggregates were destroyed, the microdomains around DCVJ were composed of water and more aggregates, and the microviscosity (the viscosity of the microdomain) increased agian. Of course, when all of the double helix aggregates 10

ACCEPTED MANUSCRIPT were destroyed, the solution was homogeneous, and the local viscosity would be characteristic of the macroviscosity. At that time, the microviscosity (macroviscosity) would decrease with the increasing temperature. From the results above, whether rheology or fluorescence measurement, the gel-sol critical transition temperatures

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(Tgs) were determined from the peak positions of the first derivatives of the curves in

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Fig. 2 and Fig. 3. It could be seen that both the temperature values obtained from the

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Storage modulus G′ (closed) and loss modulus G″ as a function of temperature in heating processes and the fluorescence lifetime for the probe in agarose solutions

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were in accord with each other. The result showed that both the rheology and

critical transition temperatures.

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fluorescence monitoring techniques supported each other in the determination of

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According to the same method mentioned above, pressure dependence of the

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fluorescence lifetime for the probe in agarose aqueous solution was also carried out by the compressing process at 75 ℃. Fig. 4 showed the pressure dependence of the

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fluorescence lifetime for the probe in 1wt% agarose aqueous solution at the

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temperature of 75 ℃, which was above the melt point of agarose gel. Compression of agarose aqueous solution was measured under pressures up to 450MPa by using a high pressure cell. From Fig. 4, it could be seen that the fluorescence lifetime for the probe in agarose aqueous solution increased gradually in the initial stage, and then showed a dramatic increase with increasing pressure, at last remained a certain value again. And it could be speculated that the sol-to-gel transition point of agarose aqueous solution is about 300MPa at the temperature of 75 ℃. On the basis of the 11

ACCEPTED MANUSCRIPT results above, the TSG-P phase diagram of the agarose aqueous solution was shown in Fig. 5. TSG meant the sol-to-gel phase transition temperature of agarose aqueous solution. From the sol-gel equilibrium line, it could be seen that the gelation temperature increased with the increasing pressure, which meant that the thermal

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stability of the gel phase was enhanced as the pressure increased.

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Based on the TSG-P phase diagram of the agarose aqueous solution, two

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experiments of the agarose gel were performed with the piston-cylinder apparatus. As shown in Fig. 5, sample 1 was melted at 95 ℃ (above its TSG of 65 ℃) under the

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atmospheric pressure and then cooled naturally by turning off the power to room

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temperature. Sample 2 was melted at 95 ℃ for 10 min under the atmospheric pressure, and then was compressed to the pressure of 300 MPa, finally, it was cooled

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naturally to room temperature and then decompressed slowly. Tab. 1 showed elasticity

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index and viscosity index of the agarose gel prepared at atmospheric pressure and high pressure. The viscosity of a material is a measure of its resistance to gradual

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deformation by shear stress or tensile stress. And the elasticity means continuum

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mechanics of bodies that deform reversibly under stress. From the result of the recovered samples studied by optical rheometry, which was shown in Tab. 1, it could be seen that the elasticity index of agarose gel prepared under high pressure was higher than that prepared under atmospheric pressure, while the viscosity index of agarose gel prepared under high pressure was lower than that prepared under atmospheric pressure. Kanaya et al studied the structure of poly (viny1 alcohol) gels by wide- and 12

ACCEPTED MANUSCRIPT small- angle neutron scattering techniques [23]. They inferred that the cross-linking points of the gels were crystallites, and the correlation length was assigned to the average distance between the neighboring crystallites. It was also found that the distance distribution function was useful to intuitively understand the distribution of

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these crystallites. Xiong et al discussed the evolution of the agarose gel topology and

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the gelation mechanism [24]. They thought that aggregation of agarose chains was

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promoted in the polymerrich phase and such kind of effect was evident from the increasing length ratio of the fiber bundles upon gelation, and continuously increasing

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pore size during gelation might be attributed to the coagulation of the local

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polymerrich phase so as to approach the global minimum of the free energy of the gelling system. It could be speculated that these excellent properties might be

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attributed to the smaller pore size during gelation under high pressure. From the

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discussion above, the gelation process could be divided into three distinct stages: induction stage, gelation stage, and pseudoequilibrium stages, and the cross-linking

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points formed rapidly in induction stage. Under high pressure, upon quenching,

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agarose gelation was initiated through a nucleation and growth mechanism, leading to many nuclei composed of polymer-rich phases, and the tendency of aggregation of the polymer monomers increased. That was, the number of cross-linking points per unit area increased (As shown in Fig. 6). Therefore, the intermolecular force increased and the elasticity index of agarose gel prepared under high pressure became larger. On the contrary, the fluidity was limited and the viscosity index of agarose gel prepared under high pressure changed into smaller one. 13

ACCEPTED MANUSCRIPT Macromolecular order of biological significance found in hydrogels formed by biopolymers was of interest from both theoretical and practical aspects. For the mechanism of sol-gel phase transition of agarose aqueous solution, San Biagio thought that the overall process was seen to occur through the following sequence:

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break of symmetry in the sol, causing the spontaneous generation of mesoscopic

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polymer-rich and solvent-rich regions; percolation, or nearly percolation of

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polymer-rich regions through the sample, still in the sol state; start of polymer cross-linking within polymer-rich regions; progress of cross-link percolation,

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channeled along the pathways of polymer-rich regions [11]. Recently we examined

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the thermoreversible sol-gel transition of agarose on cooling or heating under high pressure by in situ observations of optical transmittance and falling-ball experiments.

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It was found that the agarose underwent gelation almost in parallel with phase

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separation on cooling under both ambient and high-pressure conditions [16]. In this work, for the sol-gel phase transition of agarose, the measurements were used to

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identify the induction time for the nucleation process from Fig. 4 and Fig. 5. It

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suggested that the gelation process could also be divided into three distinct stages: induction stage, gelation stage, and pseudoequilibrium stages. And, from the results in Tab.1, it was speculated that aggregation of agarose chains was promoted in the polymer rich phase and it was evident from the increasing length ratio of the fiber bundles upon gelation. In summary, sol-gel transition behavior of agarose aqueous solution was investigated by using rheology and fluorescence measurements. The results showed 14

ACCEPTED MANUSCRIPT that the phase transition point of agarose aqueous solution was 65℃ at atmospheric pressure, and its gelation point was 300MPa at the temperature of 75℃ upon compression. The agarose gel prepared under high pressure had a higher elasticity and lower viscosity index, comparing with that prepared under atmospheric pressure. Such

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kinds of properties might be attributed to the smaller pore size during gelation under

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high pressure. The results indicated that the gel formation under high pressure might

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be another promising way to prepare hydrogels with excellent properties.

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Acknowledgments

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This work is supported by the National Natural Science Foundation of China (No.21273206, No.21503194 and No.41322028), New Century Excellent Talents in

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University (No.1209090), and the Scientific Research Key Project Foundation of

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Henan Province (No.15A140016, No.12B140018 and No.152102210143).

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ACCEPTED MANUSCRIPT References [1] M. Watase, K. Nishinari, Macromolecular Chemistry and Physics 188 (1987) 1177. [2] S. Boral, A. Saxena, H. B. Bohidar, International journal of biological

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macromolecules 46 (2010) 232.

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[3] K.C. Labropoulos, D.E. Niesz, S.C. Danforth, P.G. Kevrekidis, Carbohydrate

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Polymers 50 (2002) 393.

[4] T. Fujii, T. Yano, H. Kumagai, O. Miyaw, Food Hydrocolloids 14 (2000) 359.

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[5] D. Bulone, D. Giacomazza, V. Martorana, J. Newman, P.L. Biagio, Physical

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Review E 69 (2004) 041401.

[6] V. Normand, D.L. Lootens, E. Amici, K.P. Plucknett, P. Aymard.

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[7] E. Amici, A.H. Clark, V. Normand, N.B. Johnson, Biomacromolecules 1 (2000) 721.

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[8] P. Aymard, D.R. Martin, K.P. Plucknett, T.J. Foster, A.H. Clark, I.T. Norton,

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Biopolymers 59 (2001) 131. [9] K. Nishinari, M. Watase, K. Kohyama, N. Nishinari, D. Oakenfull, S. Koide, K. Ogino, P.A. Williams, G.O. Phillips, Polymer Journal 24 (1992) 871. [10] K. Nishinari, M. Watase, E. Miyoshi, T. Takaya, D. Oakenfull, Food Technology Chicago 49 (1995) 90. [11] P.I. San Biagio, D. Bulone, A. Emanuele, M.B. Palma-Vittorelli, M.U. Palma, Food Hydrocolloids 10 (1996) 91. 16

ACCEPTED MANUSCRIPT [12] M. Manno, A. Emanuele, V. Martorana, D. Bulone, P.L. San Biagio, M.B. Palma-Vittorelli, M.U. Palma, Physical Review E 59 (1999) 2222. [13] M. Matsuo, T. Tanaka, L. Ma, Polymer 43 (2002) 5299. [14] S. Boral, A. Saxena, H. B. Bohidar, J. Phys. Chem. B 112 (2008) 3625.

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[15] S. Boral, H. B. Bohidar, Polymer 50 (2009) 5585.

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[16] N. Kometani, M. Tanabe, L. Su, K. Yang, K. Nishinari, J. Phys. Chem. B 119

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(2015) 6878.

[17] M. Haidekker, T. Ling, M. Anglo, H. Stevens, J. Frangos, E. Theodorakis,

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Chemistry & Biology 8 (2001) 123.

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[18] T. Förster, G.Z. Hoffmann, Physical Chemistry 75 (1971) 63. [19] G. Hungerford, A. Allison, D. McLoskey, M.K. Kuimova, G. Yahioglu, K.

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Suhling, The Journal of Physical Chemistry B 113 (2009) 12067.

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[20] L. Su, Z. Wang, K. Yang, Y. Minamikawa, N. Kometani, K. Nishinari, International Journal of Biological Macromolecules 64 (2014) 409.

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[21] G. A. Evingur, O. Pekcan, Eur. Phys. J. Appl. Phys. 69 (2015) 11201.

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[22] E. Arda, S. Kara, O. B. Mergen, O. Pekcan, Polym. Bull. 72 (2015) 157. [23] T. Kanaya, M. Ohkura, K. Kaji, M. Furusaka, M. Misawa, Macromolecules 27 (1994) 5609.

[24] J.Y. Xiong, J. Narayanan, X.Y. Liu, T.K. Chong, S.B. Chen, T.S. Chung, J. Phys. Chem. B 109 (2005) 5638.

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Fig. 1. Structural representation of agarose

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Fig. 2. Storage modulus G′ (closed) and loss modulus G″ (open) as a function of temperature in heating processes for agarose solution at atmospheric pressure (a-0.5wt% agarose solution; b-1wt% agarose solution; c-2wt% agarose solution) 19

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Fig. 3. Temperature dependence of the fluorescence lifetime for the probe in agarose solution with different concentration at atmospheric pressure during heating process.(a-0.5wt% agarose solution; b-1wt% agarose solution; c-2wt% agarose solution) 20

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Fig. 4. Pressure dependence of the fluorescence lifetime for the probe in 1wt%

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agarose solution at temperature of 75 ℃

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Fig. 5. The TSG-P phase diagram of agarose gel and the schematic diagram of sample

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preparation.

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Fig. 6. Schematic representation of the network structure of agarose gel prepared

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under (a) atmospheric pressure and (b) high pressure.

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ACCEPTED MANUSCRIPT Tab.

1

Elasticity index

and

viscosity index

of

the

agarose

gel

prepared at atmospheric pressure and high pressure

Elasticity Index (nm-2)

Viscosity Index (nm-2 s-1)

Sample 1

1.16E0

7.51E0

Sample 2

1.39E0

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Sample

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3.54E0

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