Nitric oxide sensitive fluorescent poly(ethylene glycol) hydrogel microstructures

Sensors and Actuators B 115 (2006) 503–509

Nitric oxide sensitive fluorescent poly(ethylene glycol) hydrogel microstructures Jeanna Zguris a , Michael V. Pishko b,∗ a

Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802-4400, United States b Departments of Chemical Engineering, Chemistry, and Materials Science and Engineering, The Pennsylvania State University, 104 Fenske Laboratory, University Park, PA 16802-4400, United States Received 16 March 2005; received in revised form 21 September 2005; accepted 13 October 2005 Available online 6 December 2005

Abstract Here we discuss a nitric oxide (NO) sensor which utilizes 4-amino-5-methylamino-2 ,7 -difluorofluorescein (DAF-FM) entrapped in a poly(ethylene glycol) (PEG) hydrogel microstructure prepared by photolithography. The sensor can be used to detect cellular quantities of dissolved nitric oxide produced in cultured mammalian cells without directly interacting with the cells. The NO-sensitive fluorescence of the dye was characterized in solutions of Phenol Red free Dulbecco’s modified Eagle’s medium (DMEM), Phenol Red DMEM, and phosphate buffered saline (PBS). The nitric oxide concentration range that was detected was 1–100 ␮M with only DAF-FM in solution. Hydrogel microstructures encapsulating this dye were 500 ␮m diameter cylinders and were prepared on glass substrate. The poly(ethylene glycol) hydrogel was derived by cross-linking poly(ethylene glycol) diacrylate (molecular weight 575) with a photoinitiator using 365 nm ultraviolet light. The sensor response was linear in Phenol Red free DMEM, Phenol Red DMEM, and PBS, though the sensitivity of the sensor was dependent on the media that is used. The nitric oxide concentration range that was investigated was 0.5–8 ␮M in PBS for the hydrogel microstructures with DAF-FM. © 2005 Elsevier B.V. All rights reserved. Keywords: Nitric oxide; DAF-FM; Hydrogels

1. Introduction Nitric oxide (NO) has been determined to play many roles as both an inter- and extra-cellular messenger molecules [1] and has been shown to be involved with cardiovascular and metabolic homeostasis [2]. Nitric oxide is produced by a number of different cellular phenotypes, including endothelial cells, leukocytes, and macrophages. These cells all utilize nitric oxide synthases to convert l-arginine and oxygen to nitric oxide and lcitrulline [3]. The regulation of NO production is associated with disorders such as atherosclerosis, diabetes, cigarette smoking, hypertension, stroke, and heart failure [4]. NO can be produced in cytotoxic concentrations, like those that are often found in inflammatory diseases [3]. The maximum biological concentrations of nitric oxide are 1–5 ␮M in in vivo conditions [5]. It has also been shown that nitric oxide is also present before apoptosis and necrosis because the cell is stressed or under infection [6].

∗

Corresponding author. Tel.: +1 814 863 4810; fax: +814 865 7846. E-mail address: [email protected] (M.V. Pishko).

0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.10.032

Due to the prevalence of nitric oxide as a component of cellular health in a variety of cellular phenotypes, it could be used as an indicator for abnormal cell behavior. NO has a very short lifetime within tissues (half-life around 5–6 s [7]) before it is oxidized to form nitrite (NO2 − ) and nitrate (NO3 − ). The detection of nitric oxide or its oxidation products has been studied for a number of years. The most common technique to determine nitric oxide production is the Greiss Reaction [8]. This colorimetric reaction detects the NO oxidation products, nitrate and nitrite as an indirect determination of nitric oxide concentration. This method cannot determine the real time production of nitric oxide, and is reagent intensive. There have been many sensors that have been made to detect nitric oxide; some of the different types have been fluorescent [9–11], chemiresistive microsensor [12], colorimetric [13], quartz crystal microbalance [14] and electrochemical [15] methods. Many of these sensors are designed for detection of gas phase nitric oxide [14] rather than dissolved NO in aqueous media. A number of researchers have sought to create NO sensors for life science applications. The colorimetric sensor developed by Dacres and Narayanaswamy uses Cu(II) complex of eriochrome

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cyanine R dispersed in toluene/methanol mixture immobilized in a silicone rubber membrane to detect NO in cell culture [13]. Barker and coworkers’ fluorescent-based sensor [9,10] utilizes a dye-labeled cytochrome c , which has a reversible sensing chemistry in solution but not when it is entrapped in a polymer matrix. Barker and Kopelman also developed a gold adsorbed fluorophore-based nitric oxide sensor [10]. Both of these sensors require the use of Ringer’s buffer for sensing, which does not contain glucose. As glucose is necessary for normal cellular health, this will likely alter normal cellular function and represent NO levels inconsistent with the normal metabolism of the cells in question. We previously demonstrated, using poly(ethylene glycol) and photolithography, that an array can be created with hydrogel microstructures by injecting the precursor solution into a microfluidic device and polymerizing it to create parallel sensors with different sensing chemistry [16]. Here we describe the use of 4-amino-5-methylamino-2 ,7 -difluorofluorescein (DAFFM) entrapped in a hydrogel microstructure to develop a sensor to monitor the nitric oxide concentration in a cell culture environment. This dye was developed by Kojima et al. [17] to analyze real time production of intra-cellular nitric oxide in mammalian cells. DAF-FM is entrapped in a hydrogel microstructure made of poly(ethylene glycol), which has generally accepted biocompatibility, is non-toxic, and resists protein adhesion [18–20]. The goal of this work is to develop NO detection systems that can be easily integrated into bioreactors or cell-containing microanalytical devices. 2. Experimental section 2.1. Reagents A 5 mM stock solution of 4-amino-5-methylamino-2 ,7 difluorofluorescein (DAF-FM) (Molecular Probes, Eugene, OR) dissolved in dimethyl sulfoxide (DMSO) (VWR International, West Chester, PA) was used in the following experiments. Diethylamine nitric oxide, sodium salt (DEANO) was obtained from Molecular Probes (Eugene, OR). DEANO was the source of nitric oxide (NO) used to activate DAF-FM. The decomposition of DEANO results in two molecules of nitric oxide and a molecule of diethylamine for every DEANO molecule, with a half-life of 2 min in pH 7.4 PBS at 37 ◦ C [21,22]. DEANO was placed into a stock solution at least 15 min prior to the use in an experiment. The DEANO stock solution was then used to create the desired concentrations by serial dilutions. Poly(ethylene glycol) diacrylate (PEG-DA) (MW 575), anhydrous carbon tetrachloride, n-heptane, 3-(trichlorosilyl)propyl methacrylate (TPM), and 6 N sulfuric acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sodium hydroxide was obtained from VWR International (West Chester, PA) to create a 1 M solution with deionized water. The water used was 18 M
cm deionized water (Barnstead, Dubuque, IW). Dulbecco’s modified Eagle’s medium (DMEM) with and without Phenol Red was obtained from Aldrich Chemical Co. (Milwaukee, WI).

2.2. Preparation of PEG hydrogel microstructures The hydrogel microstructures were made using a similar method as reported previously [23]. In short the hydrogel arrays were patterned photolithographically with PEG-DA on glass substrates. To prepare the substrates for the gel microstructures, an oxidized surface was created using a sulphuric acid wash for at least 4 h, followed by a sodium hydroxide 1 M wash for at least 4 h. The oxidized glass was then treated with 3(trichlorosilyl)propyl methacrylate in a 3:1 hexane to carbon tetrachloride ratio to form a self-assembled monolayer (SAM) with pendant methacrylate groups. A solution containing PEGDA, DAF-FM and Darocur 1173, (30 ␮L) was subsequently placed onto the methacrylated substrate. The polymer layer was then covered with a photomask and exposed to ultraviolet light at 365 nm, 300 mW/cm2 (EFOS Ultracure l00ss Plus, UV spot lamp, Mississauga, Ontario) region for 1–2 s. Areas exposed to UV light cross-link via a free radical mechanism and the resulting hydrogel network was fixed in place while the remaining unreacted macromer was washed away with water. 2.3. Preparation of PEG hydrogel spheres Hydrogel spheres were prepared by following a previously described procedure [24]. A PEG precursor solution (2 ␮L of fully activated DAF-FM, 2 ␮L of PBS, 2 mL of PEG-DA and 20 ␮L of Duracur 1173) was extruded through a 16-gauge syringe into a graduated cylinder of mineral oil. As the droplets descended down the column of mineral oil, they passed through a focused beam of UV light and were photogelled with a 365 nm, 300 mW/cm2 light source (EFOS Ultracure l00ss Plus, UV spot lamp, Mississauga, Ontario). The spheres were collected and washed repeatedly with water. 2.4. Fluorometric analysis Fluorescent spectra of the dye in aqueous solution were acquired using a fluorescence spectrometer (QM-1, Photon Technology International). The fluorescent images of the hydrogel microstructures were taken with an Axiovert Zeiss 200M fluorescent microscope with a mercury light source and a FITC filter set (exciter filter 480 ± 20 nm, emitter filter 535 ± 25 nm and long pass beamsplitter starting at 505 nm, Zeiss, Thornwood, NY). The hydrogel microstructures were soaked in respective solution (over 2 h) prior to running an experiment. The images of the hydrogel microstructures were taken at a constant exposure of 800 ms for PBS and DMEM. The images were analyzed using Zeiss Image Version 3.0 (Thornwood, NY), which breaks the images into its three composite colors (red, blue and green). Statistical analysis was conducted using paired t-test using Sigma Plot 8.02. 3. Results and discussion The characterization of the dye in aqueous solution was needed to compare the fluorescence of the dye to that of DAF-FM immobilized in the hydrogel matrix. The changes in NO con-

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Fig. 1. The emission spectra of a 0.016 ␮M concentration of DAF-FM in varying solutions of PBS, Phenol Red free DMEM, and Phenol Red containing DMEM activated with 0.1 mM concentration of NO. In Phenol Red free DMEM the peak is broader than in PBS. The peak for Phenol Red containing DMEM has two peaks, one associated with DAF-FM and one associated with Phenol Red in the cell culture media.

centration were determined with DAF-FM in solutions of PBS, Phenol Red free DMEM, and Phenol Red containing DMEM. The detectable nitric oxide concentration range was 12–119 ␮M. The emission scans for the three solutions were all unique; Phenol Red free DMEM had a broader peak than the peak obtained

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in PBS. The emission scan in DMEM with Phenol Red had two peaks, one associated with the DAF-FM and another smaller one corresponding to Phenol Red in the media (Fig. 1). The initial spectra of DAF-FM in both types of the DMEM differ from the spectra of DAF-FM in PBS; indicating media induced activation of the dye. The dye fluoresced in the presence of the cell culture media, indicating that the data obtained when working with cell culture media must be corrected for the induced fluorescence. The induced fluorescence by the media is thought to be due to the amines that are present in the amino acids in the DMEM. This was determined by examining the spectra of PBS solution with DAF-FM before addition of the lysine and after the addition, the peak increased (data not shown). The peak of the emission scan intensity corresponded linearly to DAF-FM activation of varying concentrations of nitric oxide in all three solutions (Fig. 2(a) PBS, (b) Phenol Red DMEM, and (c) Phenol Red free DMEM), though sensitivity varied depending on the solution noted by the broadening of the emission peak due to the optically active components in cell culture media. This data corresponds to a compilation of fluorophores in solution in which they were exposed to one single concentration of nitric oxide and then was disposed. The variations in the linear graph correspond to pippetting error, due to small variations in volume per each sample, which represents a point. Each batch of solution cannot be compiled onto one graph but the linear trend is

Fig. 2. Emission intensity at 515 nm of a 0.016 ␮M concentration of DAF-FM in varying solutions of (a) PBS P < 0.01 and R2 = 0.9258, (b) Phenol Red DMEM P < 0.01 and R2 = 0.8965, and (c) Phenol Red free DMEM P < 0.01 and R2 = 0.9214 vs. varying concentrations of nitric oxide.

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Fig. 3. Mean intensity of the green component of pictures taken over time of already activated hydrogel microstructures in PBS.

preserved between batches and so a calibration curve for each batch is necessary. After finding the response of the fluorophore in solution, DAF-FM was immobilized in a matrix of poly(ethylene glycol) to create NO-sensitive gel microstructures. The hydrogel microstructures were made with a precursor solution with a 100:1:1 volume ratio of PEG-DA to Darocur 1173 to 5 mM DAF-FM in DMSO. The hydrogel microstructures entrapped DAF-FM within the gel matrix. To determine if the fluorophore leached from the hydrogel matrix, DAF-FM was fully activated by using DEANO before the structures were made and placed in water. The fluorescent images of hydrogel microstructures on substrate were taken over a period of time and analyzed to quantify the emission intensity of the dye. The intensity initially decreased and then reached steady state after 2.5 h (Fig. 3). The decrease in fluorescent intensity was likely due to leaching of the free dye from the hydrogel. This was confirmed by placing pre-activated DAF-FM hydrogel spheres in an aqueous media and acquiring fluorescence spectra of the media periodically. The fluorescent intensity in the media increased with time, indicating that the free dye was leaching out of the sphere to the water (Fig. 4) instead of photobleaching. One possibility is that

Fig. 4. Samples taken over time of the surrounding liquid of PEG hydrogel spheres with DAF-FM entrapped in them. The intensity increases over time indicating leaching of the dye. The emission wavelength is 515 nm.

free dye may be able to escape from the gel because the precursor solution had limited solvent. Thus the hydrogel swelled with water until the equilibrium volume had been reached, allowing some of the dye to escape because of the increase in free volume in the gel. Additionally photopolymerized hydrogels in general do not have uniform polymer mesh sizes, i.e. some regions of the gel are less cross-linked than others [25,26]. These were likely the regions where leaching occurred. The mesh size of the remaining gel was sufficiently small as to prevent leaching [27]. The free dye lost to solution did not significantly alter the fluorescence of the microstructure, due to its dimensions causing limitations on the transfer (diameter 500 ␮m, height 1 mm). It was demonstrated when these sensors are significantly smaller in height the leaching of the fluorophore becomes a more significant issue (data not shown). Other avenues, which would influence the leaching or swelling of the hydrogel microstructures are individual solutions themselves with varying ionic strengths. The hydrogel microstructures were then soaked for 2.5 h before experiments were performed on them to remove the dye that leaches out of the hydrogel microstructure due to swelling. The hydrogel microstructures were soaked in PBS, Phenol Red free DMEM, and Phenol Red DMEM. The hydrogel microstructures did not activate in the presence of PBS, though they did activate partially in the presence of the Phenol Red free DMEM and DMEM containing Phenol Red (Fig. 5). The selectivity of diaminofluoroesceins (DAFs) has been previously shown to react with dehydroascorbic acid and ascorbic acid [28] and was probably activated due to one of the amino acids in DMEM. Since the hydrogel microstructure containing DAF-FM were activated in DMEM, the NO-specific emission needed to be normalized to this background dye activation. Structures were imaged after soaking in DMEM for 2.5 h and images of the same structure were acquired after exposure to NO. The images were broken down and the initial green pixel count was subtracted from pixel count obtained after soaking the structures in the nitric oxide solution. Pre-soaked hydrogel microstructures were placed in solutions with varying nitric oxide concentrations. Images of the structures were taken (response time of 6 min) and were analyzed by decomposing the image into its RGB components. The initial intensity was normalized to zero nitric oxide concentration to more accurately characterize the response. The nitric oxide concentration range that was investigated was 0–1740 ␮M in PBS (Fig. 6). Sensitivity of the camera on the microscope limits our ability to look at physiologically relevant nitric oxide concentrations but thus shows that the response time and the higher end range of the sensor. This graph represent sensor made in the same batch of precursor solutions and so can be compared to each other. The sensors are batch dependent but the trend is preserved. The figure is a representative of one of the experiments. It should be noted that the solution was static, if the solution had some velocity it would help in decreasing the response time of the sensor. Another avenue that would help in lowering the response time of the sensor would be to reduce the volume to surface area ratio of the structure though this is not a recommended avenues due to the leaching of the fluorophore at

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Fig. 5. Fluorescent images of gel microstructures taken after soaking and after total activation of DAF-FM with NO in PBS, and Phenol Red free, DMEM. (a) Image of hydrogels soaked after 3 h in Phenol Red free DMEM, (b) image of hydrogel activated with nitric oxide after soaking in Phenol Red free DMEM, (c) image of hydrogels soaked after 3 h in PBS, and (d) image of hydrogel activated with nitric oxide after soaking in PBS.

smaller volumes. It should be noted that elevated levels of nitric oxide production by mammalian cells is generally significantly longer than 6 min. To look at more appropriate ranges NO-sensitive hydrogel spheres were made to be analyze with a fluorescent spectrometer for more sensitive readings. The spheres were pre-soaked in PBS for 3 h. The range that was investigated was 0–9 ␮M nitric oxide in PBS. The NO-sensitive hydrogels were statistically linear in the range of 0.5 ␮M nitric oxide in PBS and higher with a P-value <0.0001 and R2 = 92.2% when performing a paired t-test using Sigma Plot 8.02. The lower limit of the sensor that was investigated was approximately 0.5 ␮M nitric oxide in PBS (Fig. 7). The data had to be placed into log of the mean intensity 513–517 nm. This is thought to be due to transfer of the nitric oxide into the hydrogel microstructure. This is still below relevant physiological levels of nitric oxide. The variation in the data is likely due to variations in the individual samples’ orientation of spherical sensors. As noted before there is batch-to-batch

variation but the trend is preserved from one batch to another indicating that a calibration curve is needed. One of the limitations of DAF-FM is that it is not reversible in nature so a dynamic response is not achievable. This means that the sensor will be only able to be used once. The fabrication technique can control the feature size and the amount of needed material, which can still make this sensor economical. This can be used as a disposable sensor for the detection of nitric oxide due to its size and affordable fabrication cost. These nitric oxide sensitive structures can be used in the detection of cellular nitric oxide concentrations, due to the nature of the DAF-FM, which was developed and is used for the analysis of real time production of intra-cellular nitric oxide in mammalian cells [17]. The limit of the sensor corresponds to previous reported nitric oxide levels reported [29–33] if using a large volume of cells, such as in a bioreactor. For a better characterization technique of the sensor a fluorescent spectrometer was used. Using this device our sensor can show a response between 0.5 and 5 ␮M

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and can be used as a disposable sensor. The response of the NOsensitive hydrogels depends on the media that is used as well, though the response in Phenol Red free DMEM, Phenol Red DMEM, and PBS are all linear. The sensor does need to respond to the cell culture media before being used due to a component in the media that reacted with DAF-FM. This sensor could be used inside a microfluidic device to detect nitric oxide production, with its ease in fabrication and affordability to help in creating a total analysis device with whole cell biosensor for use in toxin detection. This sensor could also be used in a paralleled sensor array with other sensing chemistry to create a total analysis system for monitoring cellular health in a bioreactor (i.e. NO, pH, dissolved oxygen and glucose). Acknowledgment We thank NASA (contract BIOTECH-01-0023-0131) for their kind support. Fig. 6. Change in fluorescence response of hydrogel microstructures with DAFFM in water with varying NO concentrations. The graph is the modified mean response of the green pixels of the images over time in seconds.

Fig. 7. Change in fluorescence response of NO-sensitive PEG hydrogel microstructures exposed to nitric oxide using fluorescent spectrometer. P < 0.0001, R2 = 0.92, log(mean intensity) = 0.1078 + 5.6935[NO].

nitric oxide, which is more applicable to physiological levels, 1–5 ␮M. 4. Conclusion Here we described hydrogel microstructures with DAF-FM entrapped in the matrix to be used as a biosensor for nitric oxide. The sensor has a lower detection range of 0.5 ␮M of nitric oxide in solution and is applicable for nitric oxide production in certain mammalian cells, such as macrophages [33]. The poly(ethylene glycol) matrix makes the sensor inert to the cells, so fouling of the microstructures is not a concern. DAF-FM hydrogel microstructures can be used to detect the concentration of nitric oxide but the dye capabilities are not dynamic in response because of the irreversibility of the dye. This would limit a DAF-FM hydrogel microstructure to one time usage, but it is inexpensive to make

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Biographies Jeanna Zguris was born on 27 October 1978 and received her Ph.D. in Chemical Engineering from the Pennsylvania State University in 2005. She is currently a Staff Scientist at Johnson and Johnson Consumer and Personal Products Worldwide in Skillman, NJ. Michael Pishko was born on 25 April 1964 and received his Ph.D. in Chemical Engineering from the University of Texas at Austin in 1992. He is currently a Professor of Chemical Engineering at Penn State University, with joint appointments in the Department of Materials Science & Engineering and in the Department of Chemistry at Penn State. He is engaged in interdisciplinary research in the biomaterials and biosensors areas. He has received an NSF CAREER award, was named an Alfred P. Sloan Research Fellow, and is an Associate Editor for the IEEE Sensors Journal. Dr. Pishko has co-authored over 80 publications and is a co-inventor of 19 issued U.S. patents.