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Abstract
Whispering gallery mode (WGM) resonators are compact and ultrasensitive devices, which enable label-free sensing at the single-molecule level. Despite their high sensitivity, WGM resonators have not been thoroughly investigated for use in dynamic biochemical processes including molecular diffusion and polymerization. In this work, the first report of using WGM sensors to continuously monitor a chemical reaction (i.e. gelation) in situ in a hydrogel is described. Specifically, we monitor and quantify the gelation dynamics of polyacrylamide hydrogels using WGM resonators and compare the results to an established measurement method based on rheology. Rheology measures changes in viscoelasticity, while WGM resonators measure changes in refractive index. Different gelation conditions were studied by varying the total monomer concentration and crosslinker concentration of the hydrogel precursor solution, and the resulting similarities and differences in the signal from the WGM resonator and rheology are elucidated. This work demonstrates that WGM alone or in combination with rheology can be used to investigate the gelation dynamics of hydrogels to provide insights into their gelation mechanisms.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Label-free biosensors are at the forefront of a new generation of technologies for small molecule detection. Labeling a molecule can adversely affect its chemical structure and function, and also quantitative analysis from fluorescent labels can be difficult due to photobleaching and autofluorescence [1–3]. Whispering gallery mode (WGM) resonators have emerged as a promising technology for compact and ultrasensitive optical, label-free biochemical sensing over the past two decades [4–8]. WGM resonators can detect, at a single-particle level, molecules such as viruses, proteins, nucleic acids, and even atomic ions [9–13]. Although many prior works have focused on the detection of particles and chemical moieties, WGM resonators can also have broader applications in investigating the dynamic processes that occur in the molecules of interest, such as protein conformational changes [14] and anomalous diffusion in polymers [15]. One potential application of WGM resonators that is yet to be investigated is characterizing dynamic material processes, such as the polymerization of hydrogels.
Hydrogels are a class of biomaterials with a broad range of applications in areas such as biochemistry and biopharmaceutics; in these fields, hydrogels are implemented in protein and nucleic acid assays [16, 17], drug delivery [18–21], and cell culture [22–26]. The structural and chemical properties of hydrogels, including degradability, mechanical stability, and drug release mechanisms, depend heavily on the gelation conditions and dynamics [27]. Thus, measuring and understanding the gelation kinetics, efficiency and mechanism for hydrogels are important for understanding their structure-property relationships.
Due to its significance, several methods have been developed to monitor gelation. Nuclear magnetic resonance (NMR) can track changes in chemical structure as a solution polymerizes and has been used to follow gelation processes; however, it is very expensive, requires specialized equipment and is hampered by low resolution in aqueous environments leading to overlapping broad peaks [28, 29]. Another common technique is the inverted tube method, which is simple but solely based on observation, and hence, prone to subjective and variable quantitative data [30–32]. Other specialized techniques to measure gelation include calorimetry, which is sensitive to heat-induced structural changes in the material upon gelation [33], optical rotation measurements, which are sensitive to the degree of helical conformation [34], and ultrasound, which is sensitive to the low-frequency changes in the material viscoelastic properties [35]. Rheology and micro-rheology are also commonly employed techniques to characterize the gelation of hydrogels [36, 37]. In rheology, the rheometer applies an oscillating shear stress to the hydrogel while measuring its resulting strain. Through this force-based measurement, the viscous and elastic properties of the materials are measured as the loss (G”) and storage (G’) moduli, respectively. When a hydrogel precursor solution begins to polymerize, it becomes more elastic and the G’ surpasses the G”; the crossover point is termed the “gel point”, indicating the formation of a critical number of crosslinks [38]. However, rheology cannot be easily implemented to study the gelation time of rapidly gelling or mechanically weak materials that could be disrupted by external stresses [39]. In general, there is no universal method capable of measuring gelation directly for any material and any gelation kinetics; hence, new and improved methods for gelation measurements are sought after.
In this work, we report the first demonstration of using WGM sensors to continuously monitor a chemical reaction (i.e. gelation) in situ in a hydrogel. Specifically, we measure the gelation dynamics for polyacrylamide (PA) hydrogels using WGM sensors, which are sensitive to the changes in refractive index of the material upon gelation [8]. PA hydrogels were chosen as a model system, because their properties have been extensively studied and they are routinely used in a variety of applications including protein and DNA separation [40], drug delivery [41], and toxin removal [42]. The WGM measurements were validated by rheology, which, as mentioned above, is a well-accepted method to measure hydrogel gelation by characterizing changes in precursor solution viscoelasticity as gelation progresses. However, unlike rheology, the WGM technology does not work by applying force on the hydrogels, and thus, can be used to study mechanically weak hydrogels. It is also capable of measuring fast dynamics with high sensitivity, on the order of micro- to nano-seconds, rendering it superior to many alternative methods.
2. Hydrogel
We studied the gelation kinetics of PA gels fabricated through the co-polymerization of various concentrations and ratios of acrylamide monomer and bis-acrylamide crosslinker. The different concentrations are summarized in Tables 1 and 2. We define the following variables [43]:
where %T is the total percent of monomer, MAcr is the mass of acrylamide, MBis is the mass of bis-acrylamide, and VSol is the total volume of solution. The reaction is a free radical polymerization initiated by ammonium persulfate (APS) and catalyzed by tetramethylethylenediamine (TEMED) as shown in Fig. 1. To vary the gelation conditions, we first increased %T, while keeping %C constant as presented in Table 1. Next, we increased %C, while keeping %T constant as presented in Table 2.
Table 1. Gel composition of PA precursor solution with increasing %T
Table 2. Gel composition of PA precursor solutions with increasing %C
Fig. 1 Gelation mechanism of polyacrylamide hydrogels. Ammonium persulfate (APS) acts as a free-radical initiator, while Tetramethylethylenediamine (TEMED) catalyzes the polymerization. The bis-acrylamide crosslinks the polyacrylamide chains to form a hydrogel network.
2.1 Hydrogel preparation
Hydrogel precursor solutions were prepared by combining acrylamide (Acr) (40% w/v; Bio-Rad, Hercules, CA), bis-acrylamide (Bis) (2% w/v; Bio-Rad), and de-ionized (DI) water at specific ratios (Tables 1 and 2) in a 15 mL conical. A 10% w/v stock solution of ammonium persulfate (APS; Bio-Rad) was prepared and 10 µL was added to the precursor solutions (0.1% w/v final APS concentration) prior to purging with argon for 1 min. Afterwards, 1 µL of tetramethylenediamine (TEMED; Bio-Rad) was added at a 0.1% v/v final concentration and a timer was immediately started to denote the start of the gelation reaction. The contents of the conical were mixed gently by pipetting up and down to avoid oxygen infiltration. The precursor solution was then pipetted directly onto either the WGM resonator stage or the rheometer stage. The time between adding TEMED to the precursor (i.e. start of the gelation reaction) and the start of recording the gelation curves was noted as the “lag time,” and this “lag time” was appended to the reported gelation time. Typical lag times were around 50 s for WGM measurement and 20 s for rheology measurement.
2.2 Rheology
After the precursor solutions were prepared as described previously, they were pipetted onto the rheometer Peltier plate (AR 2000ex rheometer, TA Instruments, New Castle, DE). A time sweep was performed using a 20 mm parallel plate geometry and the rheometer properties were set as follows: 0.01% strain, 25°C, 1 rad/s angular frequency. The time sweep was run until saturation in the storage modulus (G’) was reached.
3. WGM sensor design and characterization
To characterize the gelation, we prepared and used bottle WGM resonators, as seen in the optical microscope image in Fig. 2 inset. Bottle WGM resonators have been demonstrated to be an excellent platform for enhanced light-matter interaction, as well as a convenient platform that can be accessed through a tapered optical fiber [44–46]. The experimental setup for optical characterization is presented in Fig. 2. The bottle resonator was sandwiched between two glass slides, and a tunable laser scanning across a 0.12 nm spectral range around 765 nm was used to probe the WGMs of the resonator. A thermistor was placed close to the WGM resonator to investigate the contribution of temperature to the WGM signal. The thermistor was added because temperature affects the WGMs of the resonator [47] and PA gelation is an exothermic reaction [48]. Hydrogel precursor solutions were then pipetted into the gap between the two glass slides and the transmission spectrum of the bottle resonator was monitored in situ during hydrogel gelation.
Fig. 2 Schematic drawing of the optical characterization setup. Inset: micrograph of a typical bottle WGM resonator.
3.1 Bottle resonator fabrication
Bottle resonators were fabricated from standard silica optical fibers by tapering the fiber at two locations, creating a bulge in the center [45,46]. The tapering was done by pulling the fiber while heating it with a graphite filament. We utilized an automated glass processor to finely control the shape of the resonator and the fabrication of multiple resonators with similar geometries, with a typical Q > 107 in air. The diameter of a typical bottle resonator at its center was ~70 – 80 µm.
3.2 Optical characterization
The bottle WGM resonator was placed between two glass slides, with an approximately 2 mm gap between the two glass slides. A fiber taper, fabricated with the heat-and-pull technique [49], was placed through the gap for optical coupling to the resonator. This fiber taper was placed in gentle contact with the bottle resonator. To monitor the WGMs in the bottle resonator, an external cavity diode laser (Newport Velocity TLB-6700) was kept scanning across a 0.12 nm range around λ = 765 nm, and the transmission through the resonator was monitored using a photodiode. This wavelength was chosen rather than the commonly used 1550 nm wavelength band since at 1550 nm, the absorption loss of water is significant, leading to a much lower quality factor [50].
A cross-correlation algorithm was used to calculate the “average” wavelength shift of the WGMs present in the transmission spectrum. Cross-correlation was calculated for each successive frame of measurement and the lag that resulted in the maximum cross-correlation was taken as the wavelength shift between each successive frame, as in
Here, and are the observed transmission spectrum at time and , is the wavelength shift between these two frames, and denotes cross-correlation. To calculate the wavelength shift as a function of time, the wavelength shift between each consecutive frames are added together and the cumulative wavelength shift is presented.4. In situ measurement of hydrogel gelation
WGM sensors measure the change in refractive index of the hydrogel surrounding the resonator through its evanescent field and translate any change in the refractive index of the gel to a shift in the WGM resonance wavelength [8]. In general, the refractive index of a polymer is influenced by several factors: formation of chemical bonds, change in gel density, and the introduction of additional stresses and strains within the gel [51–53]. The measurement of this refractive index change can be done in many ways, including a prism-based refractive index technique [54] and several techniques based on optical fibers [53, 55, 56], but WGM sensing is expected to have the highest sensitivity among these techniques. Typically, the refractive index of a precursor solution increases as monomers are polymerized, and this is thought to be predominantly caused by an increase in the mass density of the gel [51–53, 56]. The chemical reaction itself may also be exothermic, as is the case for PA gels, which increases the temperature of the hydrogel and in turn influences its refractive index [57]. The direction and magnitude of WGM wavelength shift upon temperature increase then depend on the thermo-optic coefficient of the gel, as well as that of the resonator [47, 58].
4.1 WGM wavelength shift upon hydrogel gelation
Figure 3(a) shows a representative transmission spectrum of a typical bottle resonator embedded in a PA hydrogel. Due to the bottle resonator’s large radius of curvature in the axial direction, which leads to a weak confinement of light in this direction, numerous WGMs are supported in a given spectral range [45, 59]. WGM sensing typically tracks the wavelength shift of one WGM using a peak-finding algorithm. However, continuously measuring a single WGM was found to be unreliable in the context of hydrogel measurements. We noted that the shrinkage of the PA gel during gelation could physically move the relative position of the fiber taper and the resonator, thus changing the coupling between the fiber taper and WGMs. Interestingly, we observed that all WGMs that appear in the transmission spectrum shifted together during hydrogel gelation, with less than 10% difference in the total wavelength shift amongst different modes, as shown in Fig. 3(b). In general, different WGMs have slightly different penetrations of the evanescent fields into the gel, and thus, their wavelengths shift by different amounts. However, our observations suggested that for the WGMs of a bottle resonator efficiently coupled to a fiber taper at a particular position, the difference in sensitivity among these WGMs was small.
Fig. 3 (a) Transmission spectrum of a bottle resonator embedded in a PA-08-37 gel. (b) Wavelength shift of five different WGMs, which are indicated in (a), compared with the wavelength shift obtained from a cross-correlation method. (c) Temperature change within the gel measured by a thermistor. (d) Wavelength shift during gelation (blue, solid curve) measured by a WGM resonator, and storage modulus change during gelation (red, dotted curve) measured by rheology. The gelation times (tgelation), for both the WGM curve and the rheology curve, are indicated in the figure.
Taking advantage of this observation, we used a cross-correlation-based algorithm to track the “average” shift of all modes present in the transmission spectrum. Figure 3(b) presents the wavelength shift of five WGMs recorded using a peak-finding algorithm in comparison to the shift obtained from the cross-correlation algorithm. As evident from the wavelength shift curves, the result obtained from the cross-correlation algorithm agreed with the wavelength shift of each individual WGM. The advantage in using the cross-correlation method as opposed to the single-mode-tracking method was improved data reliability and repeatability. For example, in the curve for “WGM 1” in Fig. 3(b), there was a jump in an otherwise continuous curve that was caused by the difficulty in tracking the original mode due to coupling change. This effect of coupling change was circumvented by using the cross-correlation method. In addition, the cross-correlation method also worked for the sometimes non-ideal, Fano-resonance-like lineshapes of the WGMs [60,61], which could be difficult to analyze using the single-mode-tracking method.
As expected from the exothermic nature of the PA polymerization reaction [48], we found that there was significant temperature increase during PA gelation, as shown in Fig. 3(c). The temperature increased by ~2 – 3 °C during the initial phase of gelation (it is important to note that the temperature increase is dependent on monomer concentration), followed by a gradual cooling to room temperature. We suggest that this change in temperature was responsible for a small blue shift at the onset of gelation as described later. To validate the WGM measurements, we used rheology to follow PA hydrogel gelation and compared gelation measurements from both methods. This result is shown in Fig. 3(d). Unlike WGM sensing, rheology measures the gel’s G’ and G”, rendering it sensitive to changes in PA viscoelasticity upon gelation. Thus, the origin of the signal for the WGM sensing and rheology are fundamentally different. Despite this difference, the overall pattern of the gelation curve obtained with the two methods agreed well; both showed an initial lag followed by rapid gelation and eventual saturation, which is typical for PA gelation [62]. In order to obtain quantitative measures for the gelation kinetics of each gel composition, we calculated two parameters: the gelation time and total change in the measured signal (resonance wavelength shift for WGM or storage modulus for rheology). These parameters were found from fitting the experimental gelation curve to Hill equations [62]:
where and are the final steady-state storage modulus and resonance wavelength shift, respectively; is the gelation half-time, and this value was taken as the measure of the characteristic gelation time for a particular gel. Finally, m is the Hill coefficient, related to the slope of the gelation curve at gelation half-time. Note that the total resonance wavelength shift of the WGM sensor corresponded to refractive index change, which predominantly resulted from the change in the density of the gel [63].We noted from Fig. 3(d) that for the WGM measurements, there was a small blue-shift (dip) at the onset of gelation, while for the rheology measurement no such dip was observed. We suggest that this blue-shift measured with WGM was a result of an increase in temperature during gelation. Similar trends in the curing of polymers have been previously observed in PA gels using an ultrasound technique [56] and in resin systems using optical fiber-based techniques [53, 55] Note that silica (the bottle resonator) has a positive thermo-optic coefficient [47], so a red-shift could be expected in WGM wavelength upon temperature increase. However, heat from the exothermic polymerization reaction was generated in the PA gel surrounding the resonator. We assumed that the thermo-optic coefficient of the curing PA gel was dominated by that of water [64], so the PA gel itself had a negative thermo-optic coefficient. Since the evanescent field of the WGM penetrates into the gel surrounding the resonator, the final wavelength shift of the resonator depends on the contribution from both the silica resonator itself, as well as that of the surrounding gel. We suggest that upon heat generation in the PA gel, the gel heated up first without transferring the heat fast enough to the silica resonator, which resulted in a blue-shift in the WGM wavelength initially. Eventually, the silica resonator heated up sufficiently to exhibit an overall red-shift. However, further work might be necessary to confirm the exact origin of the initial blue-shift.
4.2 WGM sensing and rheology with different hydrogel composition
To further validate the suitability of the WGM resonator technique to measure hydrogel gelation, we varied PA gelation kinetics by changing %T and %C and followed the gelation reaction via WGM sensing and rheology, as shown in Fig. 4. All results were reported as mean values ± standard deviation of triplicates performed in three independent experiments. The two methods to measure gelation time were compared using a two-tailed Student’s t-test. Differences were considered significant when p < 0.05. With increasing %T, the gelation time decreased, ranging from ~600-220 s, as shown in Fig. 4(a). Overall, WGM appeared to measure a slightly shorter gelation time than rheology, albeit the difference was only significant for one gel composition (PA-08-25). This difference in measured gelation times could be due to the differential changes in hydrogel density (refractive index) and modulus with the progression of the gelation reaction. For PA gels, the first step of gelation is the formation of micro-gel particles, which do not substantially contribute to the gel elasticity but contribute to gel density, followed by the formation of crosslinks between the micro-gel particles, which contribute to both gel elasticity and density [62]. We suggest that the formation of these micro-gel particles resulted in an earlier density (refractive index) change measured by WGM and a later elasticity (storage modulus) change measured by rheology.
Fig. 4 Gelation kinetics of PA hydrogels prepared with varying amounts of acrylamide and bis-acrylamide. (a) Increase in %T led to a decrease in gelation time measured by WGM and rheology. (b) Increase in %T caused an increase in both the final steady-state resonance wavelength shift and storage modulus. (c) Increase in %C led to a decrease in gelation time measured by WGM and rheology. (d) Increase in %C caused an increase in final steady-state storage modulus, but did not lead to a change in resonance wavelength shift.
Both the final steady-state WGM wavelength shift and storage modulus increased as %T increased, as shown in Fig. 4(b). This suggested that as %T increased, a stiffer PA gel was formed (a higher storage modulus as measured by rheology) with a greater density (i.e. higher refractive index). The decrease in gelation time and increase in storage modulus of PA hydrogels with increasing %T was expected [26, 65, 66]. As %T was increased from 3 to 15% (5-fold change), the storage modulus increased from 0.11 to 27.0 kPa (~245-fold change) and the resonance wavelength shift increased from 31.6 to 224.4 pm (~7-fold change).
Next, we varied %C to influence the gelation kinetics. It has been shown that an increase in %C leads to a decrease in the gelation time of PA hydrogels, albeit to a lesser extent than an increase in %T [67, 68] As expected, in Fig. 4(c) we observed a small but significant decrease in PA gelation time with an increase in %C, where gelation time varied in the range of ~460-250 s. Again, WGM appeared to measure a slightly shorter gelation time than rheology, where the difference was significant for three gel compositions and most pronounced for the softest, PA-08-01 gel (i.e. the lowest %C).
As shown in Fig. 4(d), an increase in the storage modulus was observed as %C was increased, which was expected because %C is directly correlated to hydrogel stiffness [66, 69, 70]. As %C was increased from 0.125% to 3.750% (30-fold change), the storage modulus increased from 0.73 to 8.51 kPa (~12-fold change). Unlike the storage modulus, the WGM wavelength shift was essentially uncorrelated to %C change. Thus, unlike %T, which affected both the density and elasticity of the hydrogel, the change in %C seemed to predominantly affected the hydrogel elasticity due to the formation of additional crosslinks [62]. Our results highlighted the similarities and differences in the signal obtained from WGM and rheology and demonstrated that the two techniques provide complementary information on hydrogel gelation and the underlying changes in hydrogel properties.
5. Conclusion
In conclusion, we developed a novel WGM sensing technique to characterize hydrogel gelation. The WGM wavelength shift was linked to the density and refractive index changes of the hydrogel as gelation progressed. This novel method was validated by rheology, which measures changes in hydrogel elasticity upon gelation. Both WGM sensing and rheology characterized gelation, but with slight differences; for instance, a small blue shift was observed at the onset of gelation with WGM sensing but not with rheology, likely attributed to the different sensitivity to temperature change between the two methods. Estimates of gelation time through rheology appeared slightly higher compared to estimates through WGM, especially at low %C and %T, due to differential changes in hydrogel microstructure, density, and crosslinking as a function of monomer and/or crosslinker concentration. Our study shows that WGM alone or in combination with rheology can be used to investigate the gelation dynamics of other hydrogels and provide insight into their gelation mechanisms.
Funding
ARO grant W911NF-17-1-0189; Startup funds from Saint Louis University.
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