Research Article www.acsami.org
Nonradiation Cellular Thermometry Based on Interfacial Thermally Induced Phase Transformation in Polymer Coating of Optical Microfiber Yunyun Huang, Tuan Guo, Zhuang Tian, Bo Yu, Mingfei Ding, Xiangping Li, and Bai-Ou Guan* Guangdong Provincial Key Laboratory of Optical Fiber Sensing and Communications, Institute of Photonics Technology, Jinan University, Guangzhou 510632, China S Supporting Information *
ABSTRACT: A nonradiation approach based on thermoresponsive polymer coated silica microfibers has been developed. A highly thermoresponsive and biocompatible poly(Nisopropylacrylamide) (pNIPAM) was surface functionlized to conjugate to the tapered silica microfiber with waist diameter of 7.5 μm. The interfacial phase transtition of coating triggered by the lower critical solution temperature (LCST) causes a drastic molecular morphological change in the body temperature range of 35−42 °C. This surface morphological change strongly modulates optical path difference between the higher order and the fundamental mode propagating in the microfiber because of the evanescent-field interaction and, therefore, shifts the intermodal interference fringe. Owing to the nonradiation-based nature, the thermoresponsive polymer coated microfiber enables an improved thermal sensitivity of 18.74 nm/°C and, hence, a high-temperature resolution of millidegree. Furthermore, the micrometer-sized footprint enables its easy implantation in human organs for cellular thermometry and for the potential of in vivo applications. KEYWORDS: cellular thermometry, interface, thermoresponsive polymer, coating, silica microfiber, sensor
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INTRODUCTION Sensitive thermometry at the micro- and nanoscales represents an outstanding challenge in various principles of modern science and technology.1−3 In particular, the development of ultrasensitive thermometers capable of millidegree temperature resolution at cellular levels within a living system ushers in an emerging research field, which could underpin many significant biological and life science research including gene expression,4,5 tumor metabolism,6 and pathogenesis of disease.7 The considerable demand has evoked onrushing research on single-cell nanothermal probes such as organic dyes,8 fluorescent polymers,9,10 green fluorescent proteins,11 and nanoparticles,12−14 which exhibit strong temperature-dependent fluorescent radiations in quantum yield, peak position, and lifetime. Although promising, the nature of temperaturedependent features in the fluorescent radiation associated with the electron−phonon interaction on excited-state relaxation sets up a fundamental physical limit on the thermal sensitivity to ∼0.1 nm/°C,12,13 inherently leading to a lowtemperature resolution of ∼0.5 °C within in the temperature change range of human organs of 35−42 °C.10,15 In real-world applications, it is essential to develop nonradiation thermometries, which are biocompatible and which have miniaturization and ultrasensitivity. Here, we developed a nonradiation-based approach by thermoresponsive polymer coated abruptly tapered silica microfiber which serves as interferometer and which produces interferometric fringe in the transmission spectrum. A highly thermoresponsive and © XXXX American Chemical Society
biocompatible poly(N-isopropylacrylamide) (pNIPAM) was first aminated and then surface functionalized to conjugate to the silica microfiber (Figure 1a). The phase transition of pNIPAM triggered by the lower critical solution temperature (LCST) causes a drastic molecular morphological change in the interface of silica fiber and detected environment. Because of the restriction of the optical microfiber surface, the phase transformation of polymer coating occurs in the body temperature range of 35−42 °C. The interfacial morphological change was captured by the tapered silica microfiber because of the strong evanescent-field interaction and was translated into a significant wavelength shift in the interferometric fringe (Figure 1b). Owing to the nonradiation-based nature, the thermoresponsive polymer coated microfiber enables an improved thermal sensitivity of 18.74 nm/°C which is 2 orders higher than typical radiation-based thermometry and, hence, a hightemperature resolution of millidegree. Furthermore, the micrometer-sized footprint enables its easy implantation in human organs for cellular thermometry and the potential of in vivo applications (Figure 1c).
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RESULTS AND DISCUSSION As a highly thermoresponsive smart polymer with biocompatibility and nontoxity, the pNIPAM has been widely used as a Received: January 2, 2017 Accepted: February 22, 2017 Published: February 22, 2017 A
DOI: 10.1021/acsami.7b00049 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. (a−c) The schematic of the temperature-induced morphological change of the pNIPAM skin in water. (d−f) The height images and (g−i) phase images of AFM of pNIPAM chain coating on the microfiber surface; a, d, g, below 35 °C; b, e, h, above 35 °C; and c, f, i, above 42 °C (after drying).
Figure 1. (a) The scheme of surface polymer functionalized microfiber (inset: scanning electron microscope (SEM) image of it), (b) the morphological change induced immense shifts in the interferometric fringe of the transmission spectrum, and (c) photo of the microfiber biosensor needle (inset: SEM image of it).
°C, pNIPAM chains shrunk and were further dehydrated (Figure 2c), becoming poorly solvated random coils,21 and formed a lumpy skin on the fiber surface as shown in Figure 2f and i. During this process, water was squeezed out from the pNIPAM skin of the microfiber surface. The thickness of the pNIPAM skin on the microfiber was gradually reduced14 until the final collapsed one with less than 50% of that of the swollen microgels.20 Consequently, this dramatic interfacial morphology change was captured by the tapered silica microfiber because of the evanescent-field interaction. The abruptly tapered silica microfiber excites the higher order mode (HE12) which interferes with the fundamental mode (HE11) and which creates interferometric fringe in the fiber transmission spectrum. As the HE12 mode spreads into the pNIPAM coating, it feels the change of the pNIPAM morphology, and translates it into a wavelength shift in the interference fringe. Figure 3d shows the transmission spectra of the pNIPAM coated tapered microfiber at different temperatures in the range of 30−42 °C in water. Figure 3e shows the transmission dip wavelength as a function of temperature. When the temperature was below 35 °C, the dip wavelength was insensitive to temperature. The field emission scanning electron microscope (SEM) image (Figure 3a) reveals a smooth and even pNIPAM coating on the microfiber (the pNIPAM chains are extended, becoming wellsolvated random coils and forming a smooth surface on the microfiber) at temperature below 35 °C. However, when the temperature was increased to above 35 °C, the temperature sensitivity was significantly improved to 18.74 nm/°C in the temperature range of 35−42 °C, which is 2 orders of magnitude higher than that of the typical fluorescent radiation based methods such as quantum dots of 0.1 nm/°C.13 The SEM images illustrate an uneven surface at 35 °C (Figure 3b) and a lumpy surface on microfiber at 42 °C (Figure 3c). They prove that the pNIPAM chains undergo a drastic phase change in a physiological temperature range from 35 to 42 °C. The thermal sensitivity of the naked microfiber without the pNIPAM skin is only 0.17 nm/°C within the temperature range of 30−42 °C (Figure S4). In essence, it is the surface refractive index (sRI) change induced by the interfacial pNIPAM phase
promising material for temperature-modulating controlled release,16−18 which is employed here as the coating material. The abruptly tapered silica microfiber with waist diameter of 7.5 μm (Figure S1) was fabricated by flame-brushing technique. To conjugate to the surface of the silica microfiber, electrostatic attraction was employed in the surface functionalization as follows: the end of the pNIPAM chain was modified by amino (Figure S2), while the surface of the silica microfiber was functionalized by hydroxyl group, and then the aminated pNIPAM was attached on the hydroxylated surface of the microfiber by electrostatic attraction, as shown in Figure 1a. The key to the ultrahigh thermal sensitivity of the thermoresponsive polymer coated microfiber thermometer is the phase transition of pNIPAM and the consequent morphological change when the temperature increases above the LCST. The LCST of our synthesized pNIPAM solution without conjugating to the silica microfiber is 31 °C, which was obtained from the temperature dependence of elastic light scattering intensity as shown in Figure S3. It was previously demonstrated that the LCST could be raised when one end of the pNIPAM chain was restricted.19,20 Here, after being conjugated to the surface of the silica microfiber, the LCST of interfacial pNIPAM was raised to ∼35 °C, which is close to the physical temperature. As shown in Figure 2a, at temperature below 35 °C, the pNIPAM chains on the microfiber are extended, becoming well-solvated random coils which exhibit little change with increasing temperature. This was further corroborated by the atomic force microscope (AFM) images in Figure 2d and g where the morphology of pNIPAM chains presented smooth skinning on the microfiber surface. However, when temperature slightly increased to above 35 °C, the pNIPAM chains began to dehydrate (Figure 2b). Their conformation underwent a “coil−globule” transition and started to aggregate.19 The swollen chains were dehydrated, shrunk gradually, and gave rise to collapsed microgels, wrapping on the microfiber surface. Such a dramatic phase behavior change is evidenced in Figure 2e and h. After continuous heating to 42 B
DOI: 10.1021/acsami.7b00049 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 3. SEM images of microfiber with pNIPAM-coating at (a) 30, (b) 35, and (c) 42 °C; (d) transmission spectra of the pNIPAM-coating silica microfiber interferometer at 30, 35, and 42 °C; (e) measured wavelength shift of the pNIPAM-coating silica microfiber interferometer as a function of temperature (30−42 °C, in simple condition) (dots: measured results; curve: linear fitting).
transformation that enables the high thermal sensitivity of the polymer coated tapered microfiber. At temperature below the LCST, as there was a large amount of water between pNIPAM chains, it can be deduced that the sRI is close to the solution refractive index at 35 °C. With the increase of temperature, the pNIPAM chains were gradually dehydrated and finally became a dried skin tightly coated onto the microfiber surface at 42 °C. This indicates that sRI of the microfiber coated with pNIPAM undergoes a dramatic change as temperature increases from 35 to 42 °C. The tapered microfiber induces interference between the HE12 and the HE11 mode. The HE12 mode is highly sensitive to the sRI because its mode energy spreads into the pNIPAM coating. As a result, the dramatic sRI change induced by the pNIPAM morphology transformation modulates the phase difference between the HE12 and HE11 mode and, therefore, the interference pattern. For optical wavelength encoded sensors, the resolution is determined by both the response sensitivity of the sensor element and the wavelength resolution of the readout unit. Typically, the commercial optical spectrum analyzers could achive a wavelength resolution of 0.02 nm. This indicates that the pNIPAM coated microfiber thermometer could enable a temperature resolution up to 1 × 10−3 °C (temperature resolution = R/S; R is the spectrum resolution of the optical spectrum analyzer (OSA); S is the temperature sensitivity). This provides the potential for biological research and life science applications requiring fine monitoring of cellular temperature. Figure 4 presents the reversibility of temperature response in cooling−heating cycles. The heating and cooling were realized by a water bath. The sensor was employed under a simple condition (nonbiological condition) just as that in Figure 3e. The pNIPAM coating exhibits excellent reversibility between the swollen and dehydrated states without any fatigue. The reversible collapse and swelling of the pNIPAM coating realize the reversibility of the sensor’s temperature detecting in the range from 35 to 42 °C. The micrometer-sized footprint of coated microfiber enables the easy implantation for ultrasensitive cellular temperature sensing. Figure 5 demonstrates the capability of cellular temperature sensing by the pNIPAM coated microfiber. The coated microfiber was implanted into a cluster of rat breast carcinoma cells, and the cellular temperature information can be captured by the interfacial polymer phase transformation
Figure 4. Reversibility of temperature response over several cooling− heating (35−42 °C) cycles.
and can be translated to the optical signal. In the temperature range from 35 to 42 °C, the temperature sensitivity of the sensor was 14.32 nm/°C. It was slightly lower than that in the simple distilled water condition because there might be some loss caused by cells and the culture fluid. Nevertheless, this realworld application sensitivity is still 2 orders of magnitude higher than that of typical fluorescent radiation based methods.13 The temperature resolution reached 1 × 10−3 °C, which seems high enough for further applications in temperature fluctuation at cellular levels within a living system.
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CONCLUSIONS A nonradiation-based approach by thermoresponsive polymer coated silica microfibers tapered to micrometer has been developed. A highly thermoresponsive and biocompatible pNIPAM was conjugated to the tapered microfiber surface. Interfacial phase transition of pNIPAM triggered by the LCST causes obvious sRI change in the temperature range of 35−42 °C. As a consequence, the intermodal coupling between the fundamental and the higher-order modes at tapered regions can produce a large phase abruptly, allowing immense shifts in the interferometric fringe of the transmission spectrum. Owing to the nonradiation-based nature, the thermoresponsive polymer coated microfiber enables a 2-order magnitude improved sensitivity of 18.74 nm/°C and, hence, a high-temperature resolution of millidegrees. Moreover, the micrometer-sized footprint enables its easy implantation in human organs for C
DOI: 10.1021/acsami.7b00049 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 5. (a) Schematic illustration, (b) optical microscope photo, and (c) measured wavelength shift of cellular temperature sensing by the pNIPAM coated microfiber.
cellular thermometry and for the potential of in vivo applications.
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EXPERIMENTAL SECTION Materials and Characterization. The materials and characterization is provided in the Supporting Information. Briefly, aminated pNIPAM (NH2-pNIPAM) was prepared following our previous work19 (as shown in Figure S2) and was dissolved in diluted water to form a solution. Nonadiabatic Tapered Microfiber Sensor. The fabrication of nonadiabatic tapered microfiber was as previously described by our group.22,23 Briefly, a double-cladded singlemode fiber was abruptly tapered down to micrometer scale in diameter by using the flame-brushing method. The flame with a width of 5 mm scanned across the fiber once while slowly stretching the fiber with two linear stages. The geometrical parameters including the diameter of the fiber and the length of the transition regions were mainly determined by the moving speeds of the flame and the stages. Conjugating of pNIPAM onto Microfiber Surface. The conjugating method is provided in the Supporting Information. Experimental Setup and Optical Configuration. The experimental setup was configured to allow it to operate in the transmission mode. During the experiments, the sensor was fixed in a polydimethylsiloxane (PDMS)-based microfluidic channel designed specifically for the temperature-sensing tests. Water was injected into the microfluidic chip to form a temperature probe. Heatings and coolings by water bath realized the temperature changing from 26 to 50 °C and from 50 to 26 °C. The temperature interval was 1 °C. The sensing taper was excited by a broad-band source (BBS) with the beam ranging from 1250 to 1650 nm. The transmission spectra were monitored by an OSA with minimum wavelength resolution of 0.02 nm. The measurements were recorded continuously at a rate of 1 spectrum every 30 s. Temperature Sensing of Rat Breast Carcinoma. The probe was placed in the culture fluid of the rat breast carcinoma cells and was surrounded by cells. A water bath was employed to realize the temperature from 26 to 50 °C.
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of the pNIPAM-NH2 fabrication process, thermal property of pure pNIPAM, and thermal response of naked silica microfiber (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +86-20-8522206. ORCID
Yunyun Huang: 0000-0001-7528-1001 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51403077), the National Science Fund for Distinguished Young Scholars of China (No. 61225023), the Guangdong Natural Science Foundation (Nos. S2013030013302, 2014A030313387), and the Youth Science and Technology Innovation Talents of Guangdong (No. 2014TQ01X539).We acknowledge Otsuka Electronics Co., Ltd for the measurement of the RI of pure pNIPAM, and we thank Prof. D. Ma in Jinan University for offering the cells.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00049. Expermential details about materials and reagents, characterization, conjugating of pNIPAM onto microfiber surface, schematic geometry and transmission spectrum of silica microfiber, schematic representation D
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DOI: 10.1021/acsami.7b00049 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
