Biosensors and Bioelectronics 65 (2015) 220–225
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Enzyme incorporated microfluidic device for in-situ glucose detection in water-in-air microdroplets Yunxian Piao a,n, Dong Ju Han b, Mohammad Reza Azad b, Minsu Park b, Tae Seok Seo b,nn a
Key Laboratory of Groundwater Resources and Environment, Ministry of Education, Jilin University, Changchun 130021, China Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2014 Received in revised form 9 October 2014 Accepted 13 October 2014 Available online 18 October 2014
Droplet generating microfluidic systems can provide miniaturized bioanalytical tools by using the homogenous and high-throughput droplets as nanoreactors. In this study, we demonstrated a sensitive and in-situ glucose monitoring system using water-in-air droplets in an enzyme incorporated microfluidic device. A thin film structure of a glucose oxidase (GOx) enzyme immobilized hydrogel was constructed in the middle of the microfluidic channel, and nanoliter scaled water-in-air droplets which contain a glucose sample, horseradish peroxidase (HRP), and an Amplex Red substrate were generated by flow focusing of water phase with air. Once the droplets passed through the enzyme trapped hydrogel, the droplets temporarily halted and a GOx mediated catalytic reaction with glucose proceeded, resulting in producing fluorescent resorufin products in the droplets. With optimized conditions such as the thickness of a hydrogel film and the size and flowing rate of droplets, fluorescence intensities of the released droplets linearly increased in proportional to the glucose concentration up to 3 mM, and the limit of detection was calculated as 6.64 mM. A spiked glucose in a real urine sample was also successfully analyzed, and the functionality of the proposed enzyme immobilized microfluidic chip was maintained for at least two weeks without loss of enzymatic activity and detection sensitivity. Thus, our methodology suggests a novel droplet based glucose sensing chip which can monitor glucose in a real-time and high-throughput manner. & Elsevier B.V. All rights reserved.
Keywords: Droplet Glucose Enzyme Microfluidics Hydrogel film Urine
1. Introduction Numerous efforts have been dedicated for the development of in vitro monitoring of glucose level in blood or urine samples by using a miniaturized analytical system, which is significant for the treatment and control of diabetes (Heo and Crooks, 2005; Lankelma et al., 2012; Martinez et al., 2007; Yu et al., 2011; Zhang et al., 2004). Among them, enzyme-based glucose sensing has been widely adopted due to high selectivity, sensitivity and rapidity. GOx is the most commonly used, and it converted the glucose to gluconolactone, while GOx itself is reduced. The reduced GOx reacts with oxygen to generate hydrogen peroxide (H2O2), which is detectable by electrochemical or optical methods. For example, Tang et al. developed an organic electrochemical n
Corresponding author. Fax: +86 431 8850 2606. Corresponding author. Fax: +82 42 350 3910. E-mail addresses: [email protected] (Y. Piao), [email protected] (D.J. Han), [email protected] (M.R. Azad), [email protected] (M. Park), [email protected] (T.S. Seo). nn
http://dx.doi.org/10.1016/j.bios.2014.10.032 0956-5663/& Elsevier B.V. All rights reserved.
transistor with a GOx-immobilized platinum gate electrode to detect H2O2 by amperometric responses (Tang et al., 2011). Kang et al. presented a GOx–graphene–chitosan modified electrode for direct and sensitive electrochemical glucose detection (Kang et al., 2009). Gu et al. established a droplet-based microfluidic device with incorporation of platinum-black microelectrodes for detecting glucose in the droplet with an improved current response (Gu et al., 2014). On the other hand, Wu et al. has shown that a novel glucose sensor which was made of Mn-doped ZnS quantum dots conjugated with GOx revealed the optically quenched phosphorescence of quantum dots by the generated H2O2 (Wu et al., 2010). Kim et al. used a CdTe quantum dot embedded peptide hydrogel matrix for optical glucose sensing. The photoluminescence of the CdTe was reduced as the glucose concentration increased (Kim et al., 2011). In case of the enzyme mediated glucose detection, the immobilization of enzymes in a solid matrix such as nanomaterials (Zhang et al., 2004) and polymers (Ivekovic et al., 2004) has been investigated to achieve the enhanced enzymatic activity by avoiding the enzyme aggregation and the improved enzyme stability. Hydrogels are well known for providing biocompatible
Y. Piao et al. / Biosensors and Bioelectronics 65 (2015) 220–225
environments for immobilizing enzymes in a desired composite structure. While the entrapped enzymes inside the hydrogel matrix are too large to escape from the matrix, the glucose and products are small enough to enter or release from the hydrogel. Regarding miniaturized analytical systems, microfluidics has garnered their attention over decades due to many advantages such as low sample consumption, rapidity, high detection sensitivity, high integration and portability. In particular, droplet based microfluidics became popular by serving as uniform nanoliterscale chemical and biochemical reactors in an extraordinary highthroughput manner. In addition, droplet fusion, breakage, and mixing can be performed by simple operation in the microfluidics (Song et al., 2006). The droplet size can be tuned by changing the flow rate of the two different phases (i.e., oil and liquid phase), and the composition of the reagents in the droplet can be varied depending on the input solution. Utilizing such advantages of the microfluidic droplets, there have been a number of reports for droplet based chemical and biomolecular assays, including gene amplification by polymerase chain reaction, homogeneous nanoparticle synthesis, protein crystallization and single cell analysis (Jung et al., 2012; Theberge et al., 2010). However, the glucose sensing in droplets has been rarely studied in spite of their great potentials for continuous and real-time monitoring of glucose. In this study, we report sensitive and consistent droplet based glucose detection in the microfluidics by an enzyme catalytic reaction. A GOx incorporated hydrogel (hydrogel/GOx) was formed inside the microchannel, which catalytically converted glucose and oxygen to gluconolactone and hydrogen peroxide, respectively, and in the presence of hydrogen peroxide, the HRP changed the Amplex Red substrate to resorufin in the nanolitered droplets. The resultant resorufin product in the droplets was monitored by an optical fluorescence microscope to identify the glucose level quantitatively. In order to maintain the biological stability of GOx, we employed water-in-air droplets instead of the conventional water-in-oil droplets, since continuously passing hydrophobic oil phase might disrupt 3D structure of the immobilized enzymes in the hydrogel. Thus, we combined the generation of nanoliter-scale water-in-air droplet reactors with the enzyme immobilized
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hydrogel composite film in the microchannel, and explored the applicability of the proposed microdevice for fluorescently quantitative and real-time glucose detection.
2. Materials and methods 2.1. Chemicals and materials Poly (ethylene glycol) diacrylate (PEG-DA, MW 575), 2-hydroxy-2-methylpropiophenone (HMPP), 3-(trimethoxysilyl) propyl methacrylate (TPM), glucose oxidase obtained from Aspargilus niger, D-glucose and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (MO, USA). Amplex Red and HRP were obtained from Molecular Probes (Invitrogen, CA, USA). 2.2. Design and fabrication of a microfluidic chip The design of the water-in-air droplet generating microfluidic chip is presented in Fig. 1. The microdevice is composed of four parts: a flow-focusing area for generating water-in-air droplets, two serpentine-shaped microchannels for mixing and slowing down the flowing rate of droplets by creating a smooth pressure gradient inside the channel, an enzyme incorporated hydrogel film between the two serpentine microchannels, and a detection region. The chip consists of a channel patterned PDMS layer and a flat PDMS layer, and the patterned PDMS layer was fabricated using conventional soft lithographic techniques (Xia and Whitesides, 1998). Briefly, SU-8 was photopatterned on a silicon wafer (IDB Technologies Ltd., North Somerset, UK) to form a master. After treatment with a hexamethyldisilazane (HMDS) solution, a PDMS mixture including a 10:1 weight ratio of the base and the curing agent (Dow corning, Seneffe, Belgium) was poured on the master and cured at 65 °C for 4 h to yield a 2.5-mm thick PDMS channel layer. The cured PDMS was subsequently peeled off from the master and the inlet and outlet reservoirs were formed using a 1-mm dia. biopsy punch (Nu-Care Products, Bedfordshire, UK).
Fig. 1. (A) Schematics of the water-in-air droplet generating microfluidic chip which integrated a hydrogel/GOx film in the microchannel for glucose sensing. (B) Biochemical reaction for glucose detection.
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Fig. 2. Fabrication and characterization of the hydrogel/GOx film in the microfluidic channel. (A) Schematics of constructing a hydrogel/GOx film in the microchannel. (B) A digital image of the microdevice and a microscopic image of the produced hydrogel/GOx film inside the microchannel. Scale bar: 500 mm. (C) Fluorescence (left panel) and merged (right panel) images of the cross-sectional hydrogel/GOx films which were produced with different UV exposure times. Fluorescence signal of the film was attributed to the FITC dye that was labeled to the incorporated GOx. Scale bar: 100 mm. (D) Thickness profile of the hydrogel/GOx film depending on the UV exposure times (n ¼5).
A 1-mm thick flat PDMS layer was used as a bottom substrate. The two PDMS layers were permanently bonded together by O2 plasma treatment.
filtration column. The conjugation of FITC to the GOx was confirmed by a UV–vis spectrophotometer (UV-2450, Shimadzu, Japan).
2.3. Construction of a hydrogel/GOx film
2.5. Droplet generation
PDMS microfluidic channels were treated with O2 plasma for 1 min, and immediately functionalized with TPM, which further reacted with PEG-DA to ensure adhesion of the hydrogel film to the PDMS (Revzin et al., 2001). PEG-DA was mixed with 1 wt% of HMPP initiator and then stored at 4 °C until needed. A photomask with a rectangular hole (2 mm length and 1 mm width) was placed above the PDMS microchannels (500 mm width and 50 mm height) of the assembled microdevice (Fig. 2A). Then, a hydrogel precursor solution consisting of 67 vol% PEG-DA and 3.33 mg/mL GOx in a Tris buffer (pH 7.4) was injected from the outlet with a flow rate of 20 mL/h. A hydrogel/GOx film was formed by exposing the precursor solution to UV light (EFOS Lite E3000, Ontario, Canada) for 10 s. After photopolymerization of hydrogel in the channel, the remaining hydrogel precursor solution was flushed out by injecting a phosphate buffer (50 mM, pH 7.4) with a flow rate of 200 mL/h. The thickness of the produced hydrogel/GOx films was measured by calculating the cross-section height of the films using a fluorescence image analysis software.
To produce nanolitered water-in-air droplets, deionized water or buffer was used as a water phase, and ambient air was used as an air phase. Injection of water and air into the channel was performed using gas-tight glass syringes (Hamilton, Switzerland) and syringe pumps (SPLG110, WPI, FL, USA). The droplet generation was monitored by using a fluorescence microscope (Nikon, ECLIPSE, TE 2000-U, Japan). The ejection rate of the droplet whose volume was 250 nL was 3∼4 droplets for 5 min. Since we needed an incubation time for the enzyme reaction to occur and tried to quantify the fluorescence signal of the droplets in the microfluidics, we set up a slow droplet generation speed.
2.4. Labeling of FITC to GOx To monitor the formation of a hydrogel film in the microfluidic channel, the enzymes to be trapped in the hydrogel was initially fluorescently labeled. Free amino-groups of GOx were linked with isothiocyanate reactive groups of FITC, forming a stable thiourea bond. 4 mg/mL GOx was reacted with 50 mg/mL FITC in a 0.1 M carbonate-bicarbonate buffer (pH 9.0) for 2 h in a dark room at room temperature. Then, the labeled GOx was isolated by a gel
2.6. Glucose sensing For glucose detection, a water phase solution consisting of 100 mM Amplex Red, 0.2 U/mL HRP, and a certain amount of D-glucose in a 50 mM phosphate buffer (pH 7.4) was injected into the channel with a flow rate of 200 nL/min, while the air was also infused with a flow rate of 20 mL/min. The produced water-in-air droplets containing a glucose sample passed through the hydrogel/GOx film and catalytic reaction with the immobilized GOx enzymes proceeded, converting Amplex Red substrates to the fluorescent resorufin products. Then, the resultant droplets were monitored by using a confocal laser microscope inside the microchannel (Nikon, DECLIPSE, C1si, Japan). The droplets were also collected from the outlet and immersed in an oil phase (n-hexadecane with 5 wt% span-80 surfactant), and the fluorescence images of droplets were analyzed by the confocal laser
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microscope. The fluorescence signal quantification was conducted by calculating the average fluorescence intensity per unit area (μm2) in the droplets.
3. .3. Results and discussion 3.1. Fabrication of a hydrogel/GOx film To successively monitor the glucose level in the droplets, a biocompatible hydrogel and GOx composite film was constructed in the middle of the microfluidic channel. The hydrogel precursor solution containing enzymes was photocurable under fluidized conditions, and the flow rate of the precursor solution was controlled to form a defined hydrogel structure. If the flow rate was too fast, a smeared and thin film was produced, while slow flow rate induced the blocking of the channel that hinders the formation and stability of droplets. When the flow rate was fixed at 20 mL/h, the hydrogel/GOx film was reproducibly generated at the end of the spiral section of the microchannel. Fig. 2B shows a digital image of a real chip filled with a red dye solution, and an optical microscope image of the fabricated hydrogel/GOx composite film (width length: 1.7 mm 400 mm) inside the microchannel. The thickness of the film was tuned by UV exposure time. To visualize the film thickness depending on the UV time, we first conjugated the FITC dye to the GOx, and confirmed the linkage by observing two absorption peaks at 280 nm from the enzyme and at 495 nm from FITC (Fig. S1). FITC labeled GOx was added in the PEG-DA polymerization, and the UV exposure time was controlled from 0 to 60 s. The fluorescence images of the cross-sectional film formed on the top of the channel were shown in Fig. 2C. Longer UV exposure time produced thicker films, and the thicknesses were 29.4 72.6, 34.8 73.3, and 44.8 77.3 mm as the UV times were 10, 30, and 60 s, respectively (Fig. 2D). In order to minimize the photodamage of the enzymatic activity and to retain the droplet stability during passing through the composite film, we used UV exposure time of 10 s to generate the hydrogel/GOx matrix (dimension of width length height: 1.7 mm 400 mm 29 mm). 3.2. Droplet generation On contrary to the conventional droplets in which the oil is used as a continuous phase, we employed air as a continuous phase to generate a water-in-air droplet. In order to maintain the enzymatic activity of GOx in the hydrogel during glucose sensing, the air would be ideal rather than the hydrophobic oil phase. Water-in-air droplet was generated by flow focusing of water fluid with two air phase fluids, where water acts as a discontinuous phase and air serves as a continuous phase (Fig. 1). Air was injected into the channel in two different directions: one is perpendicular and the other is almost parallel to the water fluidic direction. In the flow focusing area, the perpendicular air flow facilitated the process of necking and breaking the water phase stream to produce discrete droplets, while the parallel air flow was applied to control the flow rate of the resultant droplets. Fig. S2 shows time-lapse digital images at flow rates of water and air with 200 nL/min and 20 mL/min, respectively. Initially, the water formed an arc shape (t ¼0 s) and gradually grew like a blowing bubble with semicircular morphology (t¼ 13 s). Then, the water droplet was changed to a band structure (t ¼14 s), and finally separated from the flow focusing region (t ¼19 s). At t ¼53 s, the droplet started to contact with a hydrogel film, and after 88 s, the droplet passed through the hydrogel film without breakage. Then, next droplet is generated at the flow focusing region again with a droplet-producing cycle time of 88 s. These results show that the
Fig. 3. (A) Microscope images of the water-in-air droplets which were generated under the different flow rates of the water and the air phases. Scale bar: 500 mm. (B) Volume profiles of the generated droplets, n¼ 3. The air phase flow rate was set to 50 mL/min and the water phase flow rate was changed to (a) 1 mL/min, (b) 500 nL/min, and (c) 200 nL/min, respectively. (d) The air phase flow rate was 20 mL/min, and the water flow rate was 200 nL/min.
droplets were continuously produced in every 88 s and successfully passed through the hydrogel film inside the microchannel. The size of the water-in-air droplet was controlled by changing the flow rates of the water and air phase (Fig. 3A). At a fixed flow rate of 50 mL/min of air phase, the water phase rate was changed with 1 mL/min, 500 nL/min, and 200 nL/min, and accordingly, the volume of the droplets was defined as 12.5 107 mm3 (125 nL), 7.9 107 mm3 (79 nL), and 4.6 107 mm3 (46 nL), showing that the slower flow rate of water phase resulted in smaller droplets (Fig. 3B). When the air flow rate was reduced to 20 mL/min with the water flow rate of 200 nL/min, the velocity of the droplet decreased with a droplet volume of 6.2 107 mm3 (62 nL). For successful glucose sensing, there should be enough time for droplet to reside in a hydrogel/GOx film to induce catalytic reactions. Thus, we chose the air flow rate of 20 mL/min and the water flow rate of 200 nL/min for further experiments, in which the velocity of droplet passing through a hydrogel/GOx film was evaluated to be around 120 nL/min.
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Fig. 4. Fluorescence (left) and merged (right) images of the water-in-air droplets. While the droplet did not include glucose in the top panel, the droplet in the bottom panel contained glucose, which revealed an orange fluorescent signal after passing through the hydrogel/GOx film in the microfluidic channel. Scale bar: 500 mm.
3.3. Glucose detection Fig. 1 shows the reaction scheme for sensing glucose in the nanolitered droplets via the enzyme which was immobilized in the hydrogel film in the microfluidic channel. Under the optimized retention time of the droplet which contains the glucose, Amplex Red, and HRP as described above, the GOx catalytic reaction with glucose and oxygen occurred in the hydrogel film to produce gluconolactone and H2O2, The resultant hydrogen peroxide (MW: 34 Da) is small enough to move from the hydrogel matrix into the contacted droplets, and further reacts with Amplex Red substrates in the presence of HRP to generate a fluorescent product, resorufin, in the droplets. Thus, the glucose could be monitored by observing the resorufin fluorescence in the output droplet at the wavelength of 590 nm. For the continuous monitoring of glucose in the droplets, the activity of the entrapped enzymes in the hydrogel film should be maintained, and serial mass transfer of the substrates and products between the droplet and the hydrogel film should be available. To demonstrate this hypothesis, we, first, tested the fluorescence signal of the output droplet which contained 100 mM Amplex Red, 0.2 U/mL HRP with or without glucose (1 mM) after passing through a hydrogel/GOx film in the microfluidic channel. Fig. 4 (top panel) shows that the droplet without glucose addition did not reveal any fluorescence, meaning no catalytic reaction occurred. In contrast, the droplet which contained glucose shows strong orange fluorescence (Fig. 4, bottom panel). These results indicated that the catalytic reaction was conducted by the active enzymes entrapped in hydrogel film, and rapid mass transfer of the substrate and fluorescent products through the hydrogel was valid. To quantify the glucose level in the droplet, the resultant droplets were collected from the outlet and the fluorescence intensities of the droplets were analyzed. The typical diameter of the collected droplets was 225 mm and a relative standard deviation was 17.06%. As shown in Fig. 5A, the fluorescence intensities of the droplets were gradually elevated as the glucose concentration increased from 0 to 3 mM. The average fluorescence intensities of the total amount of droplets versus the glucose concentrations
Fig. 5. (A) Quantitative analysis of glucose. Fluorescence intensities of the resultant droplets collected in an oil solution were augmented as the glucose concentration increased. Scale bar: 100 mm. (B) Linear relationship between the glucose concentration and the fluorescence intensity of droplets. The standard deviations were obtained from the triplicate experiments (n¼ 3).
were linearly correlated with an equation of y¼0.0849x þ7.3306 (R² ¼0.9931). The lowest concentration of glucose to be experimentally detectable was 10 mM, in which the fluorescence intensity of the droplet was higher than that of the blank by 13% (Fig. 5B). The limit of detection was calculated as 6.64 mM, which was lower than the camera phone detection method (0.5 mM) (Martinez et al., 2008), the paper-based electrochemical method (0.21 mM) (Dungchai et al., 2009), and the paper-based electrochemical flow-injection method (0.2 mM) (Lankelma et al., 2012). The normal physiological level of glucose in urine should be less than 0.3 mg/mL (namely, 1.67 mM), while the glucose concentration more than 2 mM in urine is considered abnormal (Lankelma et al., 2012). Thus, the proposed microdevice can function as an advanced glucose monitoring assay to determine the physiological glucose level in urine. After washing the hydrogel/GOx film with a phosphate buffer (PB) with a flow rate of 200 mL/h for 3 min, the proposed biosensor was reused from 5 to 10 times. Fig. S3 illustrated that the fluorescence image of the droplet, which passed through the film five times, was almost equivalent to that of the first use, demonstrating that the enzymes trapped in the hydrogel/GOx film were still active for reusability. We believe that such an enzyme activity could be retained, because water and air phases in the microchannel were biocompatible and the shear stress of the fluids on the enzymes would be minimized by encapsulating enzymes in the hydrogel matrix. Furthermore, it turned out that the glucose sensing capability of the hydrogel/GOx film in the microchennel was maintained at least for 2 weeks when stored at 4 °C.
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To evaluate real sample analysis, we spiked glucose in the urine samples and detected it on the droplet based glucose sensor with the hydrogel/GOx film. Urine samples were obtained from healthy men and diluted 10 times with a PB solution (50 mM, pH 7.4). After spiking the diluted urine with glucose (1 mM), the fluorescence of the recovered droplets was measured. Fig. S4 shows that the resultant fluorescence intensities in the urine sample and the buffer solution were 103.87 8.1 and 100.7 711.1 (n ¼3), respectively, indicating 102.99% of signal recovery. These results demonstrated that urine contents have negligible negative effects on the glucose measurement in the propose system, which might be due to high specificity of the glucose oxidase enzyme to the glucose substrate. Thus, these results suggested that the droplet and hydrogel/GOx film based glucose sensor in microfluidic channel has great potential for glucose monitoring even with real biological samples.
Acknowledgments
4. Conclusions
References
In summary, we have shown the success of the nanoliter scaled droplet based glucose sensing by integrating the hydrogel/GOx film in the microfluidic channel. Instead of the conventional waterin-oil droplets, we utilized the water-in-air droplets which were critical to maintain the enzyme activity. The incorporation of the GOx into the biocompatible hydrogel matrix also contributed to minimizing the shear stress on the GOx. The size and flow rate of the water-in-air droplets were controlled by tuning the flow rate of the water and air phase, and the droplets containing glucose, HRP, and Amplex Red could contact the biocompatible hydrogel/ GOx film for sufficient time, leading to the catalytic reaction between the entrapped enzymes in the hydrogel film and the target glucose in the droplet as well as between the HRP and the substrate in the droplet. As a result, the fluorescence signal was generated in the output droplets, and these fluorescent intensities were linearly proportional to the glucose concentrations up to 3 mM with a detection limit of 10 mM. Reusability of the hydrogel/ GOx film in the microfluidic channel was also demonstrated, and the glucose analysis in the real urine samples was quite convincing. Since the droplet based bioassay can provide incomparable high-throughput capability with tiny amount of samples, our novel methodology can be utilized as an advanced glucose monitoring system.
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This work was supported by the Center for BioNano Health-Guard funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea as Global Frontier Project (H-GUARD_2013M3A6B2078964), The Scientific Research Foundation for A Talent by Jilin University (419080500134) and the Engineering Research Center of Excellence Program of Korea Ministry of Science, ICT and Future Planning (MSIP)/ National Research Foundation of Korea (NRF) (Grant NRF-2014009799).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at 10.1016/j.bios.2014.10.032.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Enzyme incorporated microfluidic device for in-situ glucose detection in water-in-air microdroplets Yunxian Piao a,n, Dong Ju Han b, Mohammad Reza Azad b, Minsu Park b, Tae Seok Seo b,nn a
Key Laboratory of Groundwater Resources and Environment, Ministry of Education, Jilin University, Changchun 130021, China Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2014 Received in revised form 9 October 2014 Accepted 13 October 2014 Available online 18 October 2014
Droplet generating microfluidic systems can provide miniaturized bioanalytical tools by using the homogenous and high-throughput droplets as nanoreactors. In this study, we demonstrated a sensitive and in-situ glucose monitoring system using water-in-air droplets in an enzyme incorporated microfluidic device. A thin film structure of a glucose oxidase (GOx) enzyme immobilized hydrogel was constructed in the middle of the microfluidic channel, and nanoliter scaled water-in-air droplets which contain a glucose sample, horseradish peroxidase (HRP), and an Amplex Red substrate were generated by flow focusing of water phase with air. Once the droplets passed through the enzyme trapped hydrogel, the droplets temporarily halted and a GOx mediated catalytic reaction with glucose proceeded, resulting in producing fluorescent resorufin products in the droplets. With optimized conditions such as the thickness of a hydrogel film and the size and flowing rate of droplets, fluorescence intensities of the released droplets linearly increased in proportional to the glucose concentration up to 3 mM, and the limit of detection was calculated as 6.64 mM. A spiked glucose in a real urine sample was also successfully analyzed, and the functionality of the proposed enzyme immobilized microfluidic chip was maintained for at least two weeks without loss of enzymatic activity and detection sensitivity. Thus, our methodology suggests a novel droplet based glucose sensing chip which can monitor glucose in a real-time and high-throughput manner. & Elsevier B.V. All rights reserved.
Keywords: Droplet Glucose Enzyme Microfluidics Hydrogel film Urine
1. Introduction Numerous efforts have been dedicated for the development of in vitro monitoring of glucose level in blood or urine samples by using a miniaturized analytical system, which is significant for the treatment and control of diabetes (Heo and Crooks, 2005; Lankelma et al., 2012; Martinez et al., 2007; Yu et al., 2011; Zhang et al., 2004). Among them, enzyme-based glucose sensing has been widely adopted due to high selectivity, sensitivity and rapidity. GOx is the most commonly used, and it converted the glucose to gluconolactone, while GOx itself is reduced. The reduced GOx reacts with oxygen to generate hydrogen peroxide (H2O2), which is detectable by electrochemical or optical methods. For example, Tang et al. developed an organic electrochemical n
Corresponding author. Fax: +86 431 8850 2606. Corresponding author. Fax: +82 42 350 3910. E-mail addresses: [email protected] (Y. Piao), [email protected] (D.J. Han), [email protected] (M.R. Azad), [email protected] (M. Park), [email protected] (T.S. Seo). nn
http://dx.doi.org/10.1016/j.bios.2014.10.032 0956-5663/& Elsevier B.V. All rights reserved.
transistor with a GOx-immobilized platinum gate electrode to detect H2O2 by amperometric responses (Tang et al., 2011). Kang et al. presented a GOx–graphene–chitosan modified electrode for direct and sensitive electrochemical glucose detection (Kang et al., 2009). Gu et al. established a droplet-based microfluidic device with incorporation of platinum-black microelectrodes for detecting glucose in the droplet with an improved current response (Gu et al., 2014). On the other hand, Wu et al. has shown that a novel glucose sensor which was made of Mn-doped ZnS quantum dots conjugated with GOx revealed the optically quenched phosphorescence of quantum dots by the generated H2O2 (Wu et al., 2010). Kim et al. used a CdTe quantum dot embedded peptide hydrogel matrix for optical glucose sensing. The photoluminescence of the CdTe was reduced as the glucose concentration increased (Kim et al., 2011). In case of the enzyme mediated glucose detection, the immobilization of enzymes in a solid matrix such as nanomaterials (Zhang et al., 2004) and polymers (Ivekovic et al., 2004) has been investigated to achieve the enhanced enzymatic activity by avoiding the enzyme aggregation and the improved enzyme stability. Hydrogels are well known for providing biocompatible
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environments for immobilizing enzymes in a desired composite structure. While the entrapped enzymes inside the hydrogel matrix are too large to escape from the matrix, the glucose and products are small enough to enter or release from the hydrogel. Regarding miniaturized analytical systems, microfluidics has garnered their attention over decades due to many advantages such as low sample consumption, rapidity, high detection sensitivity, high integration and portability. In particular, droplet based microfluidics became popular by serving as uniform nanoliterscale chemical and biochemical reactors in an extraordinary highthroughput manner. In addition, droplet fusion, breakage, and mixing can be performed by simple operation in the microfluidics (Song et al., 2006). The droplet size can be tuned by changing the flow rate of the two different phases (i.e., oil and liquid phase), and the composition of the reagents in the droplet can be varied depending on the input solution. Utilizing such advantages of the microfluidic droplets, there have been a number of reports for droplet based chemical and biomolecular assays, including gene amplification by polymerase chain reaction, homogeneous nanoparticle synthesis, protein crystallization and single cell analysis (Jung et al., 2012; Theberge et al., 2010). However, the glucose sensing in droplets has been rarely studied in spite of their great potentials for continuous and real-time monitoring of glucose. In this study, we report sensitive and consistent droplet based glucose detection in the microfluidics by an enzyme catalytic reaction. A GOx incorporated hydrogel (hydrogel/GOx) was formed inside the microchannel, which catalytically converted glucose and oxygen to gluconolactone and hydrogen peroxide, respectively, and in the presence of hydrogen peroxide, the HRP changed the Amplex Red substrate to resorufin in the nanolitered droplets. The resultant resorufin product in the droplets was monitored by an optical fluorescence microscope to identify the glucose level quantitatively. In order to maintain the biological stability of GOx, we employed water-in-air droplets instead of the conventional water-in-oil droplets, since continuously passing hydrophobic oil phase might disrupt 3D structure of the immobilized enzymes in the hydrogel. Thus, we combined the generation of nanoliter-scale water-in-air droplet reactors with the enzyme immobilized
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hydrogel composite film in the microchannel, and explored the applicability of the proposed microdevice for fluorescently quantitative and real-time glucose detection.
2. Materials and methods 2.1. Chemicals and materials Poly (ethylene glycol) diacrylate (PEG-DA, MW 575), 2-hydroxy-2-methylpropiophenone (HMPP), 3-(trimethoxysilyl) propyl methacrylate (TPM), glucose oxidase obtained from Aspargilus niger, D-glucose and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (MO, USA). Amplex Red and HRP were obtained from Molecular Probes (Invitrogen, CA, USA). 2.2. Design and fabrication of a microfluidic chip The design of the water-in-air droplet generating microfluidic chip is presented in Fig. 1. The microdevice is composed of four parts: a flow-focusing area for generating water-in-air droplets, two serpentine-shaped microchannels for mixing and slowing down the flowing rate of droplets by creating a smooth pressure gradient inside the channel, an enzyme incorporated hydrogel film between the two serpentine microchannels, and a detection region. The chip consists of a channel patterned PDMS layer and a flat PDMS layer, and the patterned PDMS layer was fabricated using conventional soft lithographic techniques (Xia and Whitesides, 1998). Briefly, SU-8 was photopatterned on a silicon wafer (IDB Technologies Ltd., North Somerset, UK) to form a master. After treatment with a hexamethyldisilazane (HMDS) solution, a PDMS mixture including a 10:1 weight ratio of the base and the curing agent (Dow corning, Seneffe, Belgium) was poured on the master and cured at 65 °C for 4 h to yield a 2.5-mm thick PDMS channel layer. The cured PDMS was subsequently peeled off from the master and the inlet and outlet reservoirs were formed using a 1-mm dia. biopsy punch (Nu-Care Products, Bedfordshire, UK).
Fig. 1. (A) Schematics of the water-in-air droplet generating microfluidic chip which integrated a hydrogel/GOx film in the microchannel for glucose sensing. (B) Biochemical reaction for glucose detection.
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Fig. 2. Fabrication and characterization of the hydrogel/GOx film in the microfluidic channel. (A) Schematics of constructing a hydrogel/GOx film in the microchannel. (B) A digital image of the microdevice and a microscopic image of the produced hydrogel/GOx film inside the microchannel. Scale bar: 500 mm. (C) Fluorescence (left panel) and merged (right panel) images of the cross-sectional hydrogel/GOx films which were produced with different UV exposure times. Fluorescence signal of the film was attributed to the FITC dye that was labeled to the incorporated GOx. Scale bar: 100 mm. (D) Thickness profile of the hydrogel/GOx film depending on the UV exposure times (n ¼5).
A 1-mm thick flat PDMS layer was used as a bottom substrate. The two PDMS layers were permanently bonded together by O2 plasma treatment.
filtration column. The conjugation of FITC to the GOx was confirmed by a UV–vis spectrophotometer (UV-2450, Shimadzu, Japan).
2.3. Construction of a hydrogel/GOx film
2.5. Droplet generation
PDMS microfluidic channels were treated with O2 plasma for 1 min, and immediately functionalized with TPM, which further reacted with PEG-DA to ensure adhesion of the hydrogel film to the PDMS (Revzin et al., 2001). PEG-DA was mixed with 1 wt% of HMPP initiator and then stored at 4 °C until needed. A photomask with a rectangular hole (2 mm length and 1 mm width) was placed above the PDMS microchannels (500 mm width and 50 mm height) of the assembled microdevice (Fig. 2A). Then, a hydrogel precursor solution consisting of 67 vol% PEG-DA and 3.33 mg/mL GOx in a Tris buffer (pH 7.4) was injected from the outlet with a flow rate of 20 mL/h. A hydrogel/GOx film was formed by exposing the precursor solution to UV light (EFOS Lite E3000, Ontario, Canada) for 10 s. After photopolymerization of hydrogel in the channel, the remaining hydrogel precursor solution was flushed out by injecting a phosphate buffer (50 mM, pH 7.4) with a flow rate of 200 mL/h. The thickness of the produced hydrogel/GOx films was measured by calculating the cross-section height of the films using a fluorescence image analysis software.
To produce nanolitered water-in-air droplets, deionized water or buffer was used as a water phase, and ambient air was used as an air phase. Injection of water and air into the channel was performed using gas-tight glass syringes (Hamilton, Switzerland) and syringe pumps (SPLG110, WPI, FL, USA). The droplet generation was monitored by using a fluorescence microscope (Nikon, ECLIPSE, TE 2000-U, Japan). The ejection rate of the droplet whose volume was 250 nL was 3∼4 droplets for 5 min. Since we needed an incubation time for the enzyme reaction to occur and tried to quantify the fluorescence signal of the droplets in the microfluidics, we set up a slow droplet generation speed.
2.4. Labeling of FITC to GOx To monitor the formation of a hydrogel film in the microfluidic channel, the enzymes to be trapped in the hydrogel was initially fluorescently labeled. Free amino-groups of GOx were linked with isothiocyanate reactive groups of FITC, forming a stable thiourea bond. 4 mg/mL GOx was reacted with 50 mg/mL FITC in a 0.1 M carbonate-bicarbonate buffer (pH 9.0) for 2 h in a dark room at room temperature. Then, the labeled GOx was isolated by a gel
2.6. Glucose sensing For glucose detection, a water phase solution consisting of 100 mM Amplex Red, 0.2 U/mL HRP, and a certain amount of D-glucose in a 50 mM phosphate buffer (pH 7.4) was injected into the channel with a flow rate of 200 nL/min, while the air was also infused with a flow rate of 20 mL/min. The produced water-in-air droplets containing a glucose sample passed through the hydrogel/GOx film and catalytic reaction with the immobilized GOx enzymes proceeded, converting Amplex Red substrates to the fluorescent resorufin products. Then, the resultant droplets were monitored by using a confocal laser microscope inside the microchannel (Nikon, DECLIPSE, C1si, Japan). The droplets were also collected from the outlet and immersed in an oil phase (n-hexadecane with 5 wt% span-80 surfactant), and the fluorescence images of droplets were analyzed by the confocal laser
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microscope. The fluorescence signal quantification was conducted by calculating the average fluorescence intensity per unit area (μm2) in the droplets.
3. .3. Results and discussion 3.1. Fabrication of a hydrogel/GOx film To successively monitor the glucose level in the droplets, a biocompatible hydrogel and GOx composite film was constructed in the middle of the microfluidic channel. The hydrogel precursor solution containing enzymes was photocurable under fluidized conditions, and the flow rate of the precursor solution was controlled to form a defined hydrogel structure. If the flow rate was too fast, a smeared and thin film was produced, while slow flow rate induced the blocking of the channel that hinders the formation and stability of droplets. When the flow rate was fixed at 20 mL/h, the hydrogel/GOx film was reproducibly generated at the end of the spiral section of the microchannel. Fig. 2B shows a digital image of a real chip filled with a red dye solution, and an optical microscope image of the fabricated hydrogel/GOx composite film (width length: 1.7 mm 400 mm) inside the microchannel. The thickness of the film was tuned by UV exposure time. To visualize the film thickness depending on the UV time, we first conjugated the FITC dye to the GOx, and confirmed the linkage by observing two absorption peaks at 280 nm from the enzyme and at 495 nm from FITC (Fig. S1). FITC labeled GOx was added in the PEG-DA polymerization, and the UV exposure time was controlled from 0 to 60 s. The fluorescence images of the cross-sectional film formed on the top of the channel were shown in Fig. 2C. Longer UV exposure time produced thicker films, and the thicknesses were 29.4 72.6, 34.8 73.3, and 44.8 77.3 mm as the UV times were 10, 30, and 60 s, respectively (Fig. 2D). In order to minimize the photodamage of the enzymatic activity and to retain the droplet stability during passing through the composite film, we used UV exposure time of 10 s to generate the hydrogel/GOx matrix (dimension of width length height: 1.7 mm 400 mm 29 mm). 3.2. Droplet generation On contrary to the conventional droplets in which the oil is used as a continuous phase, we employed air as a continuous phase to generate a water-in-air droplet. In order to maintain the enzymatic activity of GOx in the hydrogel during glucose sensing, the air would be ideal rather than the hydrophobic oil phase. Water-in-air droplet was generated by flow focusing of water fluid with two air phase fluids, where water acts as a discontinuous phase and air serves as a continuous phase (Fig. 1). Air was injected into the channel in two different directions: one is perpendicular and the other is almost parallel to the water fluidic direction. In the flow focusing area, the perpendicular air flow facilitated the process of necking and breaking the water phase stream to produce discrete droplets, while the parallel air flow was applied to control the flow rate of the resultant droplets. Fig. S2 shows time-lapse digital images at flow rates of water and air with 200 nL/min and 20 mL/min, respectively. Initially, the water formed an arc shape (t ¼0 s) and gradually grew like a blowing bubble with semicircular morphology (t¼ 13 s). Then, the water droplet was changed to a band structure (t ¼14 s), and finally separated from the flow focusing region (t ¼19 s). At t ¼53 s, the droplet started to contact with a hydrogel film, and after 88 s, the droplet passed through the hydrogel film without breakage. Then, next droplet is generated at the flow focusing region again with a droplet-producing cycle time of 88 s. These results show that the
Fig. 3. (A) Microscope images of the water-in-air droplets which were generated under the different flow rates of the water and the air phases. Scale bar: 500 mm. (B) Volume profiles of the generated droplets, n¼ 3. The air phase flow rate was set to 50 mL/min and the water phase flow rate was changed to (a) 1 mL/min, (b) 500 nL/min, and (c) 200 nL/min, respectively. (d) The air phase flow rate was 20 mL/min, and the water flow rate was 200 nL/min.
droplets were continuously produced in every 88 s and successfully passed through the hydrogel film inside the microchannel. The size of the water-in-air droplet was controlled by changing the flow rates of the water and air phase (Fig. 3A). At a fixed flow rate of 50 mL/min of air phase, the water phase rate was changed with 1 mL/min, 500 nL/min, and 200 nL/min, and accordingly, the volume of the droplets was defined as 12.5 107 mm3 (125 nL), 7.9 107 mm3 (79 nL), and 4.6 107 mm3 (46 nL), showing that the slower flow rate of water phase resulted in smaller droplets (Fig. 3B). When the air flow rate was reduced to 20 mL/min with the water flow rate of 200 nL/min, the velocity of the droplet decreased with a droplet volume of 6.2 107 mm3 (62 nL). For successful glucose sensing, there should be enough time for droplet to reside in a hydrogel/GOx film to induce catalytic reactions. Thus, we chose the air flow rate of 20 mL/min and the water flow rate of 200 nL/min for further experiments, in which the velocity of droplet passing through a hydrogel/GOx film was evaluated to be around 120 nL/min.
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Fig. 4. Fluorescence (left) and merged (right) images of the water-in-air droplets. While the droplet did not include glucose in the top panel, the droplet in the bottom panel contained glucose, which revealed an orange fluorescent signal after passing through the hydrogel/GOx film in the microfluidic channel. Scale bar: 500 mm.
3.3. Glucose detection Fig. 1 shows the reaction scheme for sensing glucose in the nanolitered droplets via the enzyme which was immobilized in the hydrogel film in the microfluidic channel. Under the optimized retention time of the droplet which contains the glucose, Amplex Red, and HRP as described above, the GOx catalytic reaction with glucose and oxygen occurred in the hydrogel film to produce gluconolactone and H2O2, The resultant hydrogen peroxide (MW: 34 Da) is small enough to move from the hydrogel matrix into the contacted droplets, and further reacts with Amplex Red substrates in the presence of HRP to generate a fluorescent product, resorufin, in the droplets. Thus, the glucose could be monitored by observing the resorufin fluorescence in the output droplet at the wavelength of 590 nm. For the continuous monitoring of glucose in the droplets, the activity of the entrapped enzymes in the hydrogel film should be maintained, and serial mass transfer of the substrates and products between the droplet and the hydrogel film should be available. To demonstrate this hypothesis, we, first, tested the fluorescence signal of the output droplet which contained 100 mM Amplex Red, 0.2 U/mL HRP with or without glucose (1 mM) after passing through a hydrogel/GOx film in the microfluidic channel. Fig. 4 (top panel) shows that the droplet without glucose addition did not reveal any fluorescence, meaning no catalytic reaction occurred. In contrast, the droplet which contained glucose shows strong orange fluorescence (Fig. 4, bottom panel). These results indicated that the catalytic reaction was conducted by the active enzymes entrapped in hydrogel film, and rapid mass transfer of the substrate and fluorescent products through the hydrogel was valid. To quantify the glucose level in the droplet, the resultant droplets were collected from the outlet and the fluorescence intensities of the droplets were analyzed. The typical diameter of the collected droplets was 225 mm and a relative standard deviation was 17.06%. As shown in Fig. 5A, the fluorescence intensities of the droplets were gradually elevated as the glucose concentration increased from 0 to 3 mM. The average fluorescence intensities of the total amount of droplets versus the glucose concentrations
Fig. 5. (A) Quantitative analysis of glucose. Fluorescence intensities of the resultant droplets collected in an oil solution were augmented as the glucose concentration increased. Scale bar: 100 mm. (B) Linear relationship between the glucose concentration and the fluorescence intensity of droplets. The standard deviations were obtained from the triplicate experiments (n¼ 3).
were linearly correlated with an equation of y¼0.0849x þ7.3306 (R² ¼0.9931). The lowest concentration of glucose to be experimentally detectable was 10 mM, in which the fluorescence intensity of the droplet was higher than that of the blank by 13% (Fig. 5B). The limit of detection was calculated as 6.64 mM, which was lower than the camera phone detection method (0.5 mM) (Martinez et al., 2008), the paper-based electrochemical method (0.21 mM) (Dungchai et al., 2009), and the paper-based electrochemical flow-injection method (0.2 mM) (Lankelma et al., 2012). The normal physiological level of glucose in urine should be less than 0.3 mg/mL (namely, 1.67 mM), while the glucose concentration more than 2 mM in urine is considered abnormal (Lankelma et al., 2012). Thus, the proposed microdevice can function as an advanced glucose monitoring assay to determine the physiological glucose level in urine. After washing the hydrogel/GOx film with a phosphate buffer (PB) with a flow rate of 200 mL/h for 3 min, the proposed biosensor was reused from 5 to 10 times. Fig. S3 illustrated that the fluorescence image of the droplet, which passed through the film five times, was almost equivalent to that of the first use, demonstrating that the enzymes trapped in the hydrogel/GOx film were still active for reusability. We believe that such an enzyme activity could be retained, because water and air phases in the microchannel were biocompatible and the shear stress of the fluids on the enzymes would be minimized by encapsulating enzymes in the hydrogel matrix. Furthermore, it turned out that the glucose sensing capability of the hydrogel/GOx film in the microchennel was maintained at least for 2 weeks when stored at 4 °C.
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To evaluate real sample analysis, we spiked glucose in the urine samples and detected it on the droplet based glucose sensor with the hydrogel/GOx film. Urine samples were obtained from healthy men and diluted 10 times with a PB solution (50 mM, pH 7.4). After spiking the diluted urine with glucose (1 mM), the fluorescence of the recovered droplets was measured. Fig. S4 shows that the resultant fluorescence intensities in the urine sample and the buffer solution were 103.87 8.1 and 100.7 711.1 (n ¼3), respectively, indicating 102.99% of signal recovery. These results demonstrated that urine contents have negligible negative effects on the glucose measurement in the propose system, which might be due to high specificity of the glucose oxidase enzyme to the glucose substrate. Thus, these results suggested that the droplet and hydrogel/GOx film based glucose sensor in microfluidic channel has great potential for glucose monitoring even with real biological samples.
Acknowledgments
4. Conclusions
References
In summary, we have shown the success of the nanoliter scaled droplet based glucose sensing by integrating the hydrogel/GOx film in the microfluidic channel. Instead of the conventional waterin-oil droplets, we utilized the water-in-air droplets which were critical to maintain the enzyme activity. The incorporation of the GOx into the biocompatible hydrogel matrix also contributed to minimizing the shear stress on the GOx. The size and flow rate of the water-in-air droplets were controlled by tuning the flow rate of the water and air phase, and the droplets containing glucose, HRP, and Amplex Red could contact the biocompatible hydrogel/ GOx film for sufficient time, leading to the catalytic reaction between the entrapped enzymes in the hydrogel film and the target glucose in the droplet as well as between the HRP and the substrate in the droplet. As a result, the fluorescence signal was generated in the output droplets, and these fluorescent intensities were linearly proportional to the glucose concentrations up to 3 mM with a detection limit of 10 mM. Reusability of the hydrogel/ GOx film in the microfluidic channel was also demonstrated, and the glucose analysis in the real urine samples was quite convincing. Since the droplet based bioassay can provide incomparable high-throughput capability with tiny amount of samples, our novel methodology can be utilized as an advanced glucose monitoring system.
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This work was supported by the Center for BioNano Health-Guard funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea as Global Frontier Project (H-GUARD_2013M3A6B2078964), The Scientific Research Foundation for A Talent by Jilin University (419080500134) and the Engineering Research Center of Excellence Program of Korea Ministry of Science, ICT and Future Planning (MSIP)/ National Research Foundation of Korea (NRF) (Grant NRF-2014009799).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at 10.1016/j.bios.2014.10.032.
