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    osensors and Bielecrenks 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 Downes Yunxian Piao**, Dong Ju Han”, Mohammad Reza Azad, Minsu Park”, Tae Seok Seo”** “Key laboratory of Groundwater Resouces and Envionment Ministry of ducaton, lin University, Changchun 12021, Ching » Departmen 9) Chmicl ond Biomolecular Engineering (BK21 PLUS Program) Korea Advanced Insta Science an Tecloy (KAT 281 Data, ARTICLE INFO ABSTRACT Acceped 15 October 2014 Droplet generating micoflidic systems can provide miniaturized bioanalytical tools by using the homogenous and high-throughput droplets as nanorescors, In ths study. we demonstrated a sensitive and in-situ glucose monitoring system using water-in-ait droplets in an enzyme incorporated micro Audie device. thin fim structure of a glucose oxidase (GOX) enayme immobilized hydrogel was con structed in the mide ofthe microfluidic channel, and nanoliter sealed watersin-air droplets which contain a glucose sample, horseradish peroxidase (HRP). and an Amplex Red substrate were generated by Keyword: fw focusing of water phase with ai. Once the droplets passed through the enzyme trapped hyeroge Droplet the droplets temporarily halted and a GOx mediated catalytic reaction with glucose proceeded esting lure in producing forescent resorafin prodts in the droplets. With optimized conditions such as the Micrftidics thickness of a hydrogel film and the size and flowing rate of droplets, fluorescence intensities of the toro ln feleased droplets linearly increased in proportional tothe glicose concentration up to 3 mM, and the one limit of detection was caleulated as 8.64 jM.A spiked glicose in a ral urine sarape was also successfull analyzed, and the functionality ofthe proposed enzyme immobilized microfluidic chip was maintained for a least two weeks without loss of enzymatic activity and detection sensitivity. Thus, our metho~ ology suggests a novel droplet based glucose sensing chip which can monitor glucose in areaLtime and high-throughput manner. © 2014 Elsevier BY. Al rights reserved 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; Lan- kelma et al, 2012; Martinez et al, 2007; Yu et al, 2011; Zhang ce al, 2004). Among them, enzyme-based glucose sensing has been widely adopted due to high selectivity, sensitivity and ra- Pidity. Ox 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 (H,0;), which is detectable by electrochemical or optical methods. For example, Tang et al. developed an organic electrochemical sneazadoiastacle (MA Azad, voareDhastacs (M Pas * transistor with 2 GOx-immobilized platinum gate electrode to etect H303 by amperometric responses (Tang. al, 2011). Kang et al. presented a GOx-graphene-chitosan modified electrode for direct and sensitive electrochemical glucose detection (Kang et al 2008). Gu et al. established a droplet-based microfluidic device with incorporation of platinum-black microelectrodes for éetect- ing glucase in the droplet with an improved current response (Cit ¢ a1, 2014), On the other han, Wut etal, has shown that a novel licose sensor which was made of Mn-doped ZnS quantum dots Conjugated with GOx revealed the optically quenched phosphor- escence of quantum dots by the generated H,0» (Wu et al. 2010). Kim et al. used a CTe quantum dot embedded peptide hydrogel ‘matrix for optical glucose sensing. The photoluminescence of the Cafe was reduced as the glucose concentration increased (Kim etal, 2011). n 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 etal, 2004) has been investigated to achieve the enhanced enzymatic activity by avoiding the enzyme aggregation and the improved enzyme sta- bility. Hydrogels are well known for providing biocompatible 1. Pa eta /Bosensore and Bottoms 65 (2015) 220-225. aa environments for immobilizing enzymes in a desired composite structure, While the entrapped enzymes inside the hydrogel ma- trix are too large to escape from the matrix, the glucose and products ate small enough to enter or release from the hydrogel, Regarding miniaturized analytical systems, microfluidics has gamered their attention over decades due to many advantages such as low sample consumption, rapidity, high detection sensi- tivity, high integration and portability. In particular, droplet based ‘microfluidics became popular by serving as uniform nanoliter scale chemical and biochemical reactors in an extraordinary high- throughput 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 ow rate ofthe two different phases (ie, oil and liquid phase), and ‘the composition of the reagents in the droplet can be varied de- pending on the input solution. Utilizing stich advantages of the ‘microfluidic droplets, there have been a number of reports for Groplet based chemical and biomolecular assays, including gene amplification by polymerase chain reaction, homogeneous nano- particle synthesis, protein crystallization and single cell analysis Gung 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 sluicose detection in the microfiuiics by an enzyme catalytic re- action, 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 Quotescence 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 A a de | a“ Detection region Sons, (E100 atm b00 on HydrogeGOx film hydrogel composite film in the microchannel, and explored the applicability of the proposed microdevice for fuorescently quan- titative and real-time glucose detection 2. Materials and methods 24. Chemicals and materials Poly (ethylene glycol) diacrylate (PEG-DA, MW 575), 2-hydro- xy-2-methylpropiophenone (HMPP), 3-(erimethoxysilyl) propyl ‘methacrylate (TPM), glucose oxidase obtained from Aspargilus ni ger, o-giuicase and fluorescein isothiocyanate (FITC) were pur chased from Sigma-Aldrich (MO, USA), Amplex Red and HRP were ‘obtained from Molecular Probes (Invitrogen. CA, USA). 22. 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: 2 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 re- sion. 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 White- sides, 1998). Briefly, SU-8 was photopatterned on a silicon wafer (ADB Technologies Usd, North Somerset, UK) to form a master, After treatment with a hexamethyldsilazane (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 4h 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 I-mm dia. biopsy punch (Nu-Care Products, Bedfordshire, UK) HOH HoH oe +0, G%y ° HO, Glucose ‘Amplex Red Resorutin B Gluconolactone Fig 1. (A) Serato the wate dope generating micoflde cip whch incgrateds hydoge]COx la the merchanne fr glucose sesing (8) Biochem reac for lucse detection 7 TPM modiied POMS s. ete Cc eel eS Fig. 2. Fabrication an charateiation ofthe hyéraeel/GOx film in he micofidc channel (A) Schemas of enstacing a er e\GOx film nthe microchannel Photomask Hydroge#Ox fim ¥. Pao ec a sensors and bieleewoncs 5 (2015) 220-225 B ° Thickness (um) rr) LUV Exposure Time (see) ‘digital image ofthe microdevice and a microscopic maze of the produced hydrozel/GOx film inside the microchannel, Seale bar: 500 ym, (C) Fluorescence (eft pane!) and ‘mereed ight pane image of the eroe-setiona hydro ‘A I-mnm thick flat PDMS layer was used as a bottom substrate. The two PDMS layers were permanently bonded together by ©; plasma treatment, 23. Construction ofa hydrogel/GOx film PDMS microfluidic channels were treated with O plasma for ‘Lmin, and immediately functionalized with TPM, which further reacted with PEG-DA to ensure adhesion of the hydrogel film to the PDMS (Revzin etal, 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 (2mm length and 1mm width) was placed above the PDMS microchannels (500 um width and $0 ym 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 74) was injected from the outlet with a Nlow rate of 20 L/h, A hydrogel/GOx film was formed by exposing the precursor solution to UV light (EFOS Lite £3000, Ontario, Canada) for 10s, After photopolymerization of hydrogel in the channel, the remaining hydrogel precursor solution was flushed ‘out by injecting a phosphate buffer (50 mM, pil 74) with a flow rate of 200 lh. The thickness of the produced hydrogel/GOx films ‘was measured by calculating the cross-section height ofthe films using a fluorescence image analysis software. 24. Labeling of FITC to Cox ‘To monitor the formation of a hydrogel film in the microfluidic channel, the enzymes to be trapped in the hydrogel was initially {vorescently 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 ug/ml. FITC in a 0. M carbonate-bicarbonate buffer (pH 9.0) for 2h in a dark room at room temperature. Then, the labeled GOx was isolated by a gel Aitration column. The conjugation of FITC to the GOx was con- firmed by a UV-vis spectrophotometer (UV-2450, Shimadzu, Japan) 25. Droplet generation ‘To produce nanolitered water-in-air droplets, deionized water or buffer was used as a water phase, and ambient ait was used as an ait phase, Injection of water and air into the channel was per- formed using gas-tight glass syringes (Hamilton, Switzerland) and syringe pumps (SPLGHIO, WPI, FL, USA). The droplet generation ‘was monitored by using a fluorescence microscope (Nikon, ECLIPSE, TE 2000-U, Japan), The ejection rate ofthe droplet whose volume was 250 nl was 3~4 droplets for 5 min. Since we needed an incubation time for the enzyme reaction to occut and tried to ‘quantify the fuorescence signal of the droplets in the micro- Auidies, we set up a slow droplet generation speed, 26. Glucose sensing For glucose detection, 2 water phase solution consisting of 100j.M Amplex Red, 0.2 UjmL HRP, and a certain amount of b-glucose in a 50 mM phosphate buffer (pHi 74) was injected into the channel with a flow rate of 200 nmin, while the air was also infused with a flow rate of 20 sL/min. The produced water-in-air Groplets containing a glucose sample passed through the hydro- sel/GOx film and catalytic reaction with the immobilized GOx enzymes proceeded. converting Amplex Red substrates to the fuorescent resorufin products. Then, the resultant droplets were monitored by using a confocal laser microscope inside the mi- ctochannel (Nikon, DECUPSE, Ctsi, Japan). The droplets were also collected from the outlet and immersed in an oil phase (n-hex- adecane with $ wt span-80 surfactant), and the fluorescence Images of droplets were analyzed by the confocal laser 1. Pa eta / sensor and Bottoms 65 (2015) 220-225. m microscope. The fluorescence signal quantification was conducted by calculating the average fluorescence intensity per unit area (um?) in the droplets, 3. 3. Results and discussion 3.1, Fabrication of « hydrogel/GOx film ‘To successively monitor the glucose level in the droplets, a biocompatible hydrogel and GOx compasite film was constructed ‘in the middle of the microfluidic channel, The bydrogel precursor solution containing enzymes was photocurable under fluidized conditions, and the flow rate of the precursor solution was con- trolled to form a defined hydrogel structure. Ifthe 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 forma- tion and stability of droplets. When the flow rate was fixed at 20 ulJh, the hydrogel/GOx film was reproducibly generated at the fend of the spiral section of the microchannel, Fig, 2B shows a ital image of a real chip filled with a red dye solution, and an optical microscope image of the fabricated hydrogel/GOx compo- site film (width x length: 1.7 mm x 400 um) inside the micro- channel. The chickness 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. $1), FTC labeled GOx was added in the PEG-DA polymerization, and the UV exposure time was controlled from 0 to 60s. The fluorescence images af the cross-sectional film formed on the top ofthe channel were shown in Fig 2C. Longer UV exposure time produced thicker films, and the thicknesses were 294426, 348-33, and 448 «73 ym as the UV times were 10, 30, and 605, respectively (Fix. 2D), In order to minimize the photadamage of the enzymatic activity and to retain the droplet stability during passing through the composite film, we used UV exposure time of 10 to generate the hydrogel/GOx matrix (di- ‘mension of width « length x height: 1.7 mm x 400 ym x 29 ym). 32, 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 duting 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 in- jected into the channel in two different directions: one is per pendicular and the other is almost parallel to the water fluidic Gitection. In the flow focusing area, the perpendicular ait 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 low rates of water and air with 200 nb/min and 20 yLjmin, respectively. Initially, the water formed an are shape (t=0s) and gradually grew like blowing bubble with semicircular morphology (t=13 5). Then, the water droplet was changed to a band structure (t= 14s) and finally separated from the flow focusing region (¢=19's). At f=53s, the droplet started to contact with a hydrogel film, and after 88 5, the droplet passed through the hydrogel flm without breakage. Then, next droplet is generated at the flow focusing region again with a

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