sensors Article
Hydrogel-Based Fluorescent Dual pH and Oxygen Sensors Loaded in 96-Well Plates for High-Throughput Cell Metabolism Studies Shanshan Wu 1,2,† , Siying Wu 1,† , Zheyuan Yi 1 , Fei Zeng 1 , Weizhen Wu 1 , Yuan Qiao 1 , Xingzhong Zhao 2 , Xing Cheng 1, * ID and Yanqing Tian 1, * ID 1
2
* †
Department of Materials Science and Engineering, Southern University of Science and Technology, No 1088 Xueyuan Road, Xili, Nanshan District, Shenzhen 518055, China;
[email protected] (Sh.W.);
[email protected] (Si.W.);
[email protected] (Z.Y.);
[email protected] (F.Z.);
[email protected] (W.W.);
[email protected] (Y.Q.) Department of Physics, Wuhan University, Wuhan 430072, China;
[email protected] Correspondence:
[email protected] (X.C.);
[email protected] (Y.T.); Tel.: +86-755-8801-8997 (Y.T.) These authors contribute equally to this work.
Received: 4 January 2018; Accepted: 10 February 2018; Published: 13 February 2018
Abstract: In this study, we developed fluorescent dual pH and oxygen sensors loaded in multi-well plates for in-situ and high-throughput monitoring of oxygen respiration and extracellular acidification during microbial cell growth for understanding metabolism. Biocompatible PHEMA-co-PAM materials were used as the hydrogel matrix. A polymerizable oxygen probe (OS2) derived from PtTFPP and a polymerizable pH probe (S2) derived from fluorescein were chemically conjugated into the matrix to solve the problem of the probe leaching from the matrix. Gels were allowed to cure directly on the bottom of 96-well plates at room-temperature via redox polymerization. The influence of matrix’s composition on the sensing behaviors was investigated to optimize hydrogels with enough robustness for repeatable use with good sensitivity. Responses of the dual sensing hydrogels to dissolved oxygen (DO) and pH were studied. These dual oxygen-pH sensing plates were successfully used for microbial cell-based screening assays, which are based on the measurement of fluorescence intensity changes induced by cellular oxygen consumption and pH changes during microbial growth. This method may provide a real-time monitoring of cellular respiration, acidification, and a rapid kinetic assessment of multiple samples for cell viability as well as high-throughput drug screening. All of these assays can be carried out by a conventional plate reader. Keywords: dual oxygen-pH sensing plates; oxygen respiration; extracellular acidification; high throughput analysis
1. Introduction Dissolved oxygen (DO) measurement has attracted great attention because oxygen is a critical indicator for monitoring the cellular microenvironment, especially in understanding metabolism [1], tumor respiration studies [2], neuron cell research [3], and ecology applications [4]. Many kinds of diseases and cancers are associated with the mutual effect of superoxide dismutase (SOD) and reactive oxygen species (ROS) [5]. ROS are originated as a natural by-product of metabolism processes involving DO [6]. Further, cellular respiration is the process both prokaryotic and eukaryotic cells use to convert the energy contained in the chemical bonds of nutrients into ATP energy. Besides the significance of DO in vitro, monitoring and regulating the pH in culture medium are key steps in the successful operation of bioreactions as well. Hydrogen ion concentration (pH value)-responsive permeable factors on the cell surface are commonly investigated at cancer drug therapy [7]. Cellular
Sensors 2018, 18, 564; doi:10.3390/s18020564
www.mdpi.com/journal/sensors
Sensors 2018, 18, 564
2 of 13
pHs are closely related with cell metabolism, disease occurrence, the proliferation and apoptosis of embryonic cells and stem cells. It has been reported that the extracellular pH in certain cases becomes more acidic than the neutral pH of around 7.2 for normal tissues, to pH values or around 6.5 or even 5.5 [8]. On the other hand, the metabolic switch from oxidative phosphorylation to aerobic glycolysis in cancer cells—the Warburg effect—is one of the emerging hallmarks of cancer [9,10]. These two processes of oxidative phosphorylation and aerobic glycolysis are directly relevant to DO and pH levels. There are several types of methods known for DO and pH value determination, including pressure, electrochemistry, and optical approaches. Optical assays are frequently used for rapid analysis in clinical setups and in laboratories for its convenient features. Fluorescence analysis is one of the major optical approaches, providing non-invasive, easily operated, disposable, and low cost assays [11] which plays a critical role in bioprocess monitoring and diagnosis. Some review articles have summarized current knowledge about pH sensors or oxygen sensors, including dual pH/oxygen sensors [12–14] Although many suitable fluorescent materials are available and some are even commercially available for DO and pH measurement [15–29], few of them were demonstrated to be capable or applicable for high throughput and in-situ monitoring of cell respiration and acidification using the widely applied multi-well plates used in many bio-labs [18]. Kocincova et al. reported the use of methacrylate-based pH-sensitive microbeads stained with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) as pH probes and oxygen-sensitive ruthenium-tris-4,7-diphenyl-1,10-phenanthrolinedi-(trimethylsilylpropanesulfonate) (Ru(dpp)3 (TMS)2 ) microparticles in organosilica doped in polyurethane hydrogel as a dual pH and oxygen sensing material and deposited such dual sensors in 24 well plates for monitoring bacterial growth [18]. Further, Seahorse Bioscience has developed its XF96 metabolism instrument for oxygen respiration and extracellular acidification measurements [25]. The sensors in the XF96 instrument are a kind of polymeric gel-based sensor. For measuring cell respiration and acidification, Seahorse’s XF instrument uses a specific chip design to achieve its goal for high-throughput analysis of extracellular pH and oxygen measurements in their sophisticated and advanced instrument. Our purpose is to prepare suitable hydrogels for dual pH and oxygen sensing to enable their direct use in 96 well plates acting as a type of representative functional multi-well plates as continuation of our previous work on the development of hydrogel-based sensors using thermal and/or redox polymerizations of polymerizable oxygen and pH probes [30–33]. Previously, we have developed some hydrogel sensors for individual pH [30] or oxygen [31,32], or dual pH and oxygen analysis [33]. Most of these were unfortunately unsuitable for direct use in the bottom of 96-well plates. Herein, we finely tuned the monomer mixture with a minimum amount of DMF and applied redox polymerization [34] for gelation of the monomer mixtures at room temperature on the bottom of 96 well plates for in-situ assays. Through careful control of the matrix’s composition, uniform sensing thin films were deposited on the bottoms of 96-well plates. The sensing performance was characterized in detail. Further the functionalized plates were used to monitor microbial cell respiration and extracellular acidification in E. coli with and without antibiotic drug treatments, demonstrating the capability of the application of the devices using an ordinary plate reader. 2. Materials and Methods 2.1. Materials and Reagents All chemicals and solvents were of analytical grade and were used without further purification. Glucose, glucose oxidase, ethanol, DMF, dimethyl sulfoxide (DMSO), 2-hydroxyethyl methacrylate (HEMA), acrylamide (AM), poly(ethylene glycol) dimethacrylate (PEGDMA, Mn ≈ 550), and 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) were commercially available from Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. The oxygen probe (OS2) and pH probe (S2) were prepared according to known procedures [31,33] and their structures are given in Figure 1. Doubly distilled water was used for the preparation of buffer solutions. A series
Sensors 2018, 18, 564
3 of 13
of standard Britton-Robison pH buffers (BR, H3 PO4 -HOAc-H3 BO3 ) ranging from pH 3.0–9.0 were prepared with 40 mM H3 PO4 , HOAc, and H3 BO3 adjusted by NaOH (200 mM). Calibration gases (nitrogen and oxygen, each of 99.99% purity) were purchased from Shenzhen Huatepeng Special Gases Cooperation (Shenzhen, China). E. coli K12 was purchased from ATCC (25404, ATCC, Manassas, VA, USA). 2.2. Instruments A digital pH meter (Thermo Electron Corporation, Beverly, MA, USA) was used for pH measurements. A gas manipulator (Alicat Scientific Instrument, Tucson, AZ, USA, measuring error: ±1%) was used for controlling the exact gas percentage. All measurements of sensing behaviors were carried out at an atmospheric pressure (101 kPa) and room temperature (22 ± 1 ◦ C). All the performances of multi-well plate sensing hydrogels were read by a plate reader (Cytation 3, BioTek, Winooski, VT, USA). A Lambda 25 UV/Vis spectrophotometer (PerkinElmer, Shelton, CT, USA) was used to read the OD values of E. coli at 600 nm for density determination. 2.3. Fabrication of Dual-Sensing Films in 96-Well Plates In this work, the hydrogel matrices consist of 2-hydroxyethyl methacrylate (HEMA), acrylamide (AM), and poly(ethylene glycol) (PEGDMA) which acts as a cross-linker for gelation. OS2 and S2 were chosen as an oxygen probe and a pH probe, respectively. Platinum(II) porphyrin (OS2) is a representative emission-based oxygen probe with four methacrylate units for being easily immobilized into polymer matrices. OS2 is a derivative of the highly efficient oxygen probe of Pt(II)meso-tetra(pentafluorophenyl)porphine (PtTFPP) with high photo-stability and sufficient triplet-triplet energy transfer to oxygen molecules. Fluorescein derivative (S2) exhibits pH-dependent fluorescence changes due to its multiple pH-dependent ionic equilibria, where only fluorescein dianion and monoanion emit strong fluorescence under basic conditions and the intra-ester isomer formed under acidic conditions does not emit. According to our previous experience, probe concentrations of 0.5 and 1.0 mg/g in matrixes did not obviously affect the sensing performance [33]. Herein, the pH probe S2 (0.9 mg) was dissolved in the 1 mL mixture of HEMA/AM/PEGDMA with proper ratios (S2 stock solution); the oxygen probe of OS2 was dissolved in DMF with the concentration of 3.6 mg/mL (OS2 stock solution); K2 S2 O8 in H2 O (30 mg/mL) was prepared as an initiator for redox polymerization (initiator stock solution); 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) was used as the catalyst in this system. Then pH probe S2 stock solution, oxygen sensor OS2 stock solution, initiator stock solution, and catalyst were mixed in suitable proportions to make the final stock solutions of the dual probes with concentration of S2 at 0.5 mg/g and OS2 at 1 mg/g in the matrices. In optimized conditions, the ratios of OS2 stock:S2 stock:initiator stock:HMTETA were 16:9:9:4 by volume. The well-mixed solution was dispensed by a micropipette onto the walls of a 96-well plate (40 µL for each well). To get a flat and uniform film, a layer of butanol (50 µL), which is almost immiscible with the gel precursors, was added on the top of the sensor solution with two functions: (1) the butanol layer acts as a sealing layer to avoid the interaction of the sensor precursors with air to speed up the radical polymerization and (2) the thin layer of butanol eliminates the tension effect of the sensor mixture in the well to get a smooth film. The mixed monomers were then allowed to cure for 1 h at room temperature. After that the butyl alcohol was removed by pipetting and the wells were washed with ethanol and then BR buffers (pH 3.0 and pH 7.0) before use. 2.4. Optimization of the Sensing Films To enhance the sensing activities of the hydrogel films, we have explored the effects of the matrix’s components, mainly HEMA, AM, and PEGDMA, on the sensitivity through an optimization of their ratios. Hydrogel sensing films were prepared in 96-well plates as mentioned above in Section 2.3. Five films (F1–F5) with variable weight ratios of HEMA and AM (Table 1) were prepared with a fixed
Sensors 2018, 18, 564
4 of 13
percentage (10%) of the PEGDMA crosslinker. Oxygen and pH titrations were used for optimization of the ratios of HEMA and AM. Another four films (F6–F9) were also prepared by changing the weight ratios of PEGDMA with the fixed optimized proportion of HEMA and AM for further optimizing sensing matrix. Table 1. Compositions of sensing hydrogel films of F1–F9 and their sensitivity.
Films
HEMA a
AM a
PEGDMA in the Total Weight of HEMA and AM
F1 F2 F3 F4 F5 F6 F7 F8 F9
100 95 85 75 65 85 85 85 85
0 5 15 25 35 15 15 15 15
10 10 10 10 10 2.5 5.0 15 20
Film-Forming yes yes yes yes no no yes yes yes
Sensitivity to DO b (I0 /I100 )
Sensitivity to Ph c (IpH9.0 /IpH3.0 )
5.73 5.95 5.83 1.54 4.28 3.39 2.31
17.3 18.0 21.4 11.3 17.6 22.3 24.5
a
Weight ratios of HEMA/AM in the gel films. b Fluorescence intensity ratios between those with 100% nitrogen and with 100% of oxygen. I0 is the emission intensity at 645 nm under deoxygenated condition; I100 is the emission intensity under oxygenated condition. c Fluorescence intensity ratios between those at pH 9.0 BR buffer and at pH 3.0 BR buffer. IpH9 is the emission intensity of 525 nm at pH 9; IpH3 is the emission intensity of 525 nm at pH 3.
2.5. Thickness Measurements of the Sensing Hydrogels Thickness measurements for the gels were carried out under both wet and dry conditions by using a microcaliper. When measured wet, films were removed from the plate after polymerization, and their thickness was measured immediately. These fresh films were then put in a Petri dish at room-temperature for 3 days to dry slowly. Then the thicknesses were measured again. All the measurements were taken at the center of each film. Five samples were tested at each a condition. Average thicknesses and standard deviations are given in Figure 2. 2.6. Characterization of the Sensing Behaviors of the Hydrogels in 96-Well Plates All the hydrogels were washed adequately with pH 7.0 BR buffer before the measurements. Fluorescence property of the hydrogels in multi-well plate was read by a plate reader. Emission spectra collected from 520 nm to 680 nm were measured at the excitation wavelengths of 405 nm (for oxygen probe) and of 488 nm (for pH probe), respectively. 2.7. Photostability Test and Ionic Influences The photostability of O2 probes and pH probes were monitored at 645 nm with an excitation of 405 nm and at 525 nm under the excitation of 488 nm, respectively. As shown in Figure S1 in the Supplementary Materials, after four hours of continuously exposure, good stability with less than 10% decomposition was observed. The influence of ions on emission changes of the two probes was tested by adding a few kinds of biologically relevant cations at their physiological concentrations to the liquids. We did not observe any influence on the oxygen concentrations and pHs (Figure S2 in the Supplementary Materials). 2.8. pH Responses pH values from 3.0 to 9.0 were chosen to characterize the pH responses of the sensing films. Before titration, each well was previously repeatedly washed by pH 3.0 and pH 9.0 buffer for several times. The titration was carried out from basic to acidic conditions. Each well was washed at least three times when changing buffers. Titrations were performed in duplicate to get the average value. The hydrogel was excited at 488 nm, and the emission spectra were measured from 510 to 660 nm.
Sensors 2018, 18, 564
Sensors 2018, 18, x FOR PEER REVIEW
5 of 13
5 of 13
2.9. Oxygen Responses DO concentrations were adjusted by saturating the buffers or medium using the gas bubbling approach. we collected theadjusted spectra ofby the hydrogelsthe in buffers the 96 well plates using thethe plate DOWhen concentrations were saturating or medium using gasreader, bubbling weapproach. found the When intensities at 645 nm changed because of theinoxygen exchange air,the which we collected the spectrafast of the hydrogels the 96 well plateswith using platemay reader, result in significant experimental calibration errors. Thus we did not collect the spectra of the oxygen we found the intensities at 645 nm changed fast because of the oxygen exchange with air, which may probe, and we experimental just measuredcalibration the intensities at 645 nmwe to did avoid errors.of the oxygen result ininstead, significant errors. Thus notexperimental collect the spectra probe, and instead, we just measured the intensities at 645 nm to avoid experimental errors. 2.10. E. Coli Culture 2.10. E. coli Culture E. coli K12 was cultured in liquid Lysogeny Broth (LB) medium at 37 °C with shaking at 200 rpm coli K12 cultured Broth (LB) medium at 37 ◦ C the withoptical shaking at 200at rpm for 12 h.E.The cell was density of E. in coliliquid in LBLysogeny broth was estimated by measuring density for 12 h. The cell density of E. coli in LB broth was estimated by measuring the optical density 600 nm, which is linearly proportional to E. coli cell density when OD600 is in the range of 0.1 to 1.0. A at 600of nm, which is alinearly proportional to8E. coli cell densityunit when the range based of 0.1 to value 1 indicated cell density of 5.0 × 10 colony-forming perOD milliliter (CFU/mL), on1.0. 600 is in 8 colony-forming unit per milliliter (CFU/mL), based on A value ofof1 the indicated a cell density of 5.0 × 10Appropriate calibrations UV–vis spectrophotometer. dilutions were made by using fresh LB the UV–vis spectrophotometer. Appropriate dilutions were by using fresh LB to to calibrations achieve theofdesired initial cell densities for oxygen respiration andmade cellular acidification achieve the desired initial cell densities for oxygen respiration and cellular acidification experiments. experiments. 3. Results Discussion 3. Results andand Discussion 3.1. Sensor Preparation in Multi-Well Plates 3.1. Sensor Preparation in Multi-Well Plates A redox polymerization [34] method was used to prepare the multi-well hydrogel sensor plates, A redox polymerization [34] method was used to prepare the multi-well hydrogel sensor plates, and no thermal treatment is needed for this polymerization. We also finely tuned the sensor monomer and no thermal treatment is needed for this polymerization. We also finely tuned the sensor monomer composition to minimize the use of DMF, which is a necessary solvent for OS2. By minimizing the use composition to minimize the use of DMF, which is a necessary solvent for OS2. By minimizing the use of DMF, the precursors for sensing film formation have no influence on the optical properties of the of DMF, the precursors for sensing film formation have no influence on the optical properties of the polystyrene of the 96-well plate bottoms. A schematic procedure for the hydrogel preparation in a polystyrene of the 96-well plate bottoms. A schematic procedure for the hydrogel preparation in a 96-well plate was given in Figure 1. The concentrations for oxygen and pH probes for sensor hydrogel 96-well plate was given in Figure 1. The concentrations for oxygen and pH probes for sensor hydrogel preparation were 1 mg/g and 0.5 mg/g, respectively, in the gel precursors. Premixed hydrogel preparation were 1 mg/g and 0.5 mg/g, respectively, in the gel precursors. Premixed hydrogel precursor solutions (40 µL) were added to each micro-well and polymerized for 1 hour at room precursor solutions (40 μL) were added to each micro-well and polymerized for 1 hour at room temperature. Two groups of cured gels in a 96-well plate were presented, which were polymerized temperature. Two groups of cured gels in a 96-well plate were presented, which were polymerized with and without covering a layer of butyl alcohol, respectively. Gels appeared to have a lot of winkles with and without covering a layer of butyl alcohol, respectively. Gels appeared to have a lot of winkles in the micro-wells when polymerized without the butanol layer, while the films prepared with a layer in the micro-wells when polymerized without the butanol layer, while the films prepared with a layer of butyl alcohol were flat and uniform. The thickness of the flat films is about 0.675 ± 0.005 mm in wet of butyl alcohol were flat and uniform. The thickness of the flat films is about 0.675 ± 0.005 mm in wet condition and 0.58 ± 0.003 mm in dry condition (Figure 2). condition and 0.58 ± 0.003 mm in dry condition (Figure 2).
Figure 1. A1.simple schematic drawing for for the the preparation of 96-well sensing gel gel plate andand the the photos Figure A simple schematic drawing preparation of 96-well sensing plate photos of the formed gelsgels in ain96-well plate. BUOH indicates butanol or butyl alcohol. of the formed a 96-well plate. BUOH indicates butanol or butyl alcohol.
TheThe fluorescence properties of aofgroup of sensing gelsgels (nine gelsgels prepared in different wells) fluorescence properties a group of sensing (nine prepared in different wells) excited at 488 nm for pH sensor’s emissions only and excited at 405 nm for both the pH sensor andand excited at 488 nm for pH sensor’s emissions only and excited at 405 nm for both the pH sensor oxygen sensor areare shown in Figure 3. Results showed thatthat thethe fluorescence properties of the nine oxygen sensor shown in Figure 3. Results showed fluorescence properties of the nine different films were almost the same, indicating the high reliability and repeatability of this approach and the potential high throughput applications of the sensor gels in 96 well plates.
Sensors 2018, 18, 564
6 of 13
different films were almost the same, indicating the high reliability and repeatability of this approach and theSensors potential 2018, 18,high x FORthroughput PEER REVIEW applications of the sensor gels in 96 well plates. 6 of 13 Sensors 2018, 18, x FOR PEER REVIEW
6 of 13
Figure 2. Thickness of five membranesininwet wet and and dry dry conditions. Figure 2. Thickness of five membranes conditions. Figure 2. Thickness of five membranes in wet and dry conditions.
B) 6.0x10 4 B) 6.0x10 4
Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.)
Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.)
A) A)
2000 2000 1500 1500 1000 1000 500 500 0 0500 500
550 600 550 600 Wavelength (nm) Wavelength (nm)
650 650
4
4.0x10 4 4.0x10
4
2.0x10 4 2.0x10
0.0 0.0500 500
550 600 550 600 Wavelength (nm) Wavelength (nm)
650 650
Figure 3. Fluorescence properties of nine fresh sensing gels in a 96-well plate in pH 7.0 BR buffer.
Figure 3. Fluorescence properties of nine freshsensing sensing gels gels in in a a 96-well plate ininpH 7.07.0 BRBR buffer. Figure (A) 3. Excited Fluorescence properties of nine fresh 96-well pH at 405 nm and (B) Excited at 488 nm. The emission peak at 525 nmplate was attributed to the buffer. pH (A) Excited at 405 nm and (B) Excited at 488 nm. The emission peak at 525 nm was attributed to the pHpH (A) Excited nmemission and (B) peak Excited at 488 nm.from The the emission nm was attributed to the probeatof405 S2; the at 645 nm was oxygenpeak probeatof525 OS2. probe of S2; the emission peak at 645 nm was from the oxygen probe of OS2. probe of S2; the emission peak at 645 nm was from the oxygen probe of OS2. 3.2. Optimization of Sensing Hydrogels 3.2. Optimization of Sensing Hydrogels 3.2. Optimization of Sensing Hydrogels To achieve sensitive sensing hydrogel films for biological studies, biocompatible copolymers of To achieve sensitive sensing hydrogel films for biological studies, biocompatible copolymers of PHEMA-co-PAM were chosen as the matrices [35,36]. For achieving stable hydrogel, a ToPHEMA-co-PAM achieve sensitivewere sensing hydrogel films for biological biocompatible copolymers chosen as the matrices [35,36]. studies, For achieving stable hydrogel, a of dimethacrylate moieties-containing PEGDMA was used as a cross-linker to make the films robust. dimethacrylate moieties-containing PEGDMA was For usedachieving as a cross-linker to make the films robust. PHEMA-co-PAM were chosen as the matrices [35,36]. stable hydrogel, a dimethacrylate Since the matrix’s compositions may potentially affect the sensors’ behaviors, some optimization Since the matrix’s compositions mayas potentially affectto the sensors’ behaviors, some optimization moieties-containing PEGDMA was used a cross-linker make the films robust. Since the matrix’s experiments were performed to get the best proportions of HEMA, AM and PEGDMA for achieving experiments were performed to getthe thesensors’ best proportions of HEMA, AM and PEGDMA for achieving compositions may potentially affect behaviors, some optimization experiments were high sensitivity. high sensitivity. performed Firstly, to get the AM and PEGDMA achieving fivebest filmsproportions (F1–F5, Tableof1)HEMA, with variable weight ratios of for HEMA and AMhigh weresensitivity. prepared Firstly, five films (F1–F5, Table 1) with variable weight ratios of HEMA and AM were prepared Firstly, films (F1–F5,(10%) Tableof 1) PEGDMA. with variable weight ratios of were HEMA and AM were prepared with afive fixed percentage Uniform films F1–F4 obtained, while with the with a fixed percentage (10%) of PEGDMA. Uniform films F1–F4 were obtained, while with the increase amount of AM to 35%, the film’ quality for F5 was poor. Most likely, the high composition with a fixed percentage (10%) of PEGDMA. Uniform films F1–F4 were obtained, while with the increase increase amount of AM to 35%, the film’ quality for F5 was poor. Most likely, the high composition of of PAM resulted in crystallization filmpoor. hindering hydrogel formation. Additionally, amount AMeasily to 35%, the film’ quality forof F5the was Mostthe likely, the high composition of PAM of PAM easily resulted in crystallization of the film hindering the hydrogel formation. Additionally, the oxygen sensitivity represented by using I 0/I100 among the three films F1, F2, and F3 for DO were easily resulted in crystallization of the film hindering the hydrogel formation. Additionally, the oxygen the oxygen sensitivity represented by using I0/I100 among the three films F1, F2, and F3 for DO were all round 5.7~5.95 (Table 1), where I0 is the emission intensity at 645 nm under deoxygenated sensitivity represented using /I100 among three films F1, F2, andnm F3 for DOdeoxygenated were all round all round 5.7~5.95by (Table 1),I0where I0 is thethe emission intensity at 645 under condition and I100 is the emission intensity under oxygenated condition. Among the films of F1–F4, condition andwhere I100 is the intensity under oxygenated Among the films of F1–F4,and 5.7~5.95 (Table 1), I0 isemission the emission intensity at 645 nmcondition. under deoxygenated condition F4 showed the lowest sensitivity to DO with I0/I100 at 1.54, which is also the worst one for pH sensing F4 showed theintensity lowest sensitivity to DO with Icondition. 0/I100 at 1.54, Among which is the alsofilms the worst one forF4 pH sensing I100 is the emission under oxygenated of F1–F4, showed by comparing the value of IpH9.0/IpH3.0, showing that the film with higher AM composition is not the by comparing the value of I pH9.0/IpH3.0, showing that the film with higher AM composition is not lowest better sensitivity to DO with permeability I0 /I100 at 1.54, which is also theSince worst sensing comparing for higher oxygen or ion permeability. theone F3 for filmpH showed the by highest pH better for higher oxygen permeability orfilm ion permeability. Since the F3 film showed the highest pH the value of I /I , showing that the with higher AM composition is not better for higher sensitivity, the later studies, the ratios of HEMA and AM of F3 were fixed at 85% and 15% by pH9.0for pH3.0 sensitivity, for the later studies, the ratios of HEMA and AM of F3 were fixed at 85% and 15% by oxygenweight permeability or ion permeability. Since the F3 found film showed the highest pHformed sensitivity, when optimizing PEGDMA (F6–F9). It was that films could not be when for the the weight when optimizing PEGDMA (F6–F9). It was found that films could not be formed when the cross-linker PEGDMA was less than wt.F3 % were of thefixed sum of and AM. sensitivity later studies, the ratios of HEMA and AM5 of at HEMA 85% and 15% byDO weight whenincreased optimizing cross-linker PEGDMA was less than 5 wt. % of the sum of HEMA and AM. DO sensitivity increased with(F6–F9). increasing PEGDMA from 5% to 10%, andnot decreased whenwhen PEGDMA was more than 10%. Thiswas PEGDMA It was found that films could be formed the cross-linker PEGDMA with increasing PEGDMA from 5% to 10%, and decreased when PEGDMA was more than 10%. This observation indicated that likely theAM. oxygen limited by the gel density and also less than 5 wt. % of the sum ofmost HEMA and DOpermeability sensitivity is increased with increasing PEGDMA observation indicated that most likely the oxygen permeability is limited by the gel density and also the compositions of PEG segments. However, the sensing ability to pH was continuously from 5% 10%, and decreased when PEGDMA was thanability 10%. This indicated thetocompositions of PEG segments. However, themore sensing to pHobservation was continuously strengthened with the increase of the cross-linker PEGDMA from 5% to 20 wt%, showing the ion strengthened the increase of the cross-linker PEGDMA from 5% and to 20also wt%, showing the ion of that most likely thewith oxygen permeability is limited by the gel density the compositions permeability was not inhibited by the PEG segment or crosslinking degrees. Considering the sensing permeability was not inhibited by the PEG segment or crosslinking degrees. Considering the sensing PEG segments. However, the sensing ability to pH was continuously strengthened with the increase
Sensors 2018, 18, 564
7 of 13
of the Sensors cross-linker PEGDMA from 5% to 20 wt. %, showing the ion permeability was not inhibited 2018, 18, x FOR PEER REVIEW 7 of 13 by the PEG segment or crosslinking degrees. Considering the sensing abilities of both DO and pH, abilities of both and pH, the compositions of 10% F3 with 85% HEMA, 15% AM, and 10% as of the the compositions of F3DO with 85% HEMA, 15% AM, and of PEGDMA by weight was chosen PEGDMA by weight was all chosen as the optimized one (Table 1). Thus,toallthe theprotocol hydrogels were optimized one (Table 1). Thus, the hydrogels were prepared according of F3 in the prepared according to the protocol of F3 in the follow-up work. follow-up work. 3.3. Typical Oxygen Sensing for DO 3.3. Typical Oxygen Sensing for DO A mixture of oxygen and nitrogen gas was used to saturate aqueous solutions to tune DO
A mixture of oxygen and nitrogen gas was used to saturate aqueous solutions to tune DO concentrations by bubbling into a plate reader. Fluorescence intensities at 645 nm were measured at concentrations by bubbling into a plate reader. Fluorescence intensities at 645 nm were measured at different DO concentrations. Figure 4a shows DO responses of F3 with 0% of oxygen (deoxygenated different DO concentrations. Figure 4a shows DO of F3 with 0% of oxygen condition) to 21% of oxygen (8.6 ppm in water at responses 23 °C, air saturated condition). Intensity(deoxygenated ratios (I0/I) ◦ C, air saturated condition). Intensity ratios (I /I) condition) to 21% of oxygen (8.6 ppm in water at 23 2 of the F3 followed the Stern-Volmer equation with a correlation coefficient (R ) exceeding 0.997 as 0 of the given F3 followed the (1): Stern-Volmer equation with a correlation coefficient (R2 ) exceeding 0.997 as in Equation given in Equation (1): I0 (1) (1) sv[pO pO2] = 1 1+ Ksv 2 I where KSV is the Stern-Volmer quenching constant. I0 and I are the steady-state fluorescence intensity KSV is theinStern-Volmer are the steady-state fluorescence quenching constant. I0 and Isolutions, at 645where nm measured deoxygenated and oxygen containing respectively. [pO2 ] is the intensity at 645 nm measured in deoxygenated and oxygen containing solutions, respectively. [pO2] oxygen partial pressure in the nitrogen and oxygen mixture gas. is the oxygen partial pressure in the nitrogen and oxygen mixture gas. Fluorescence intensities of the OS2 probe decreased with the increase in DO concentration. It is Fluorescence intensities of the OS2 probe decreased with the increase in DO concentration. It is well-known that PHEMA-based hydrogels have excellent oxygen permeability for contact lenses [35,36]. well-known that PHEMA-based hydrogels have excellent oxygen permeability for contact lenses The DO can interact efficiently with the sensor molecules in the gel as it swells significantly in water [33]. [35,36]. The DO can interact efficiently with the sensor molecules in the gel as it swells significantly in It waswater found that the found fresh that filmthe has a better sensitivity than that of that rehydrated film film afterafter oneone week. [33]. It was fresh film has a better sensitivity than of rehydrated This isweek. mostThis likely due to the fact that the gel might keep on continuously crosslinking during is most likely due to the fact that the gel might keep on continuously crosslinking during the slowlythe drying process, wesince onlywe let only the gels drygels slowly in air at temperature without using a slowly drying since process, let the dry slowly in room air at room temperature without a strong dryingTherefore, condition. Therefore, fresh hydrogels for further studies. strongusing drying condition. we used we theused freshthe hydrogels for further studies.
Figure 4. Response of the sensing gels to dissolved oxygen in a plate reader. Oxygen partial pressure
Figure 4. Response of the sensing gels to dissolved oxygen in a plate reader. Oxygen partial pressure was changed from 0 to 21% of atmosphere pressure. (a) fresh gel; (b) rehydrated gel (after one week). was changed from 0 to 21% of atmosphere pressure. (a) fresh gel; (b) rehydrated gel (after one week). The intensity (I) was measured at 645 nm. The intensity (I) was measured at 645 nm.
3.4. pH Sensing
3.4. pH Sensing
pH responses were studied from pH 3 to 9 using the fresh and rehydrated films. Figure 5A
showed the pH responses offrom a fresh at the 488fresh nm for S2 rehydrated and 540 nm films. for OS2. The emission pH responses were studied pHfilm 3 toexcited 9 using and Figure 5A showed peaks of the two probes are well separated, showing that there were no cross-talks between pHpeaks and of the pH responses of a fresh film excited at 488 nm for S2 and 540 nm for OS2. The emission oxygen probes. This alleviates the interferences between the two probes. The sensing performance the two probes are well separated, showing that there were no cross-talks between pH and oxygen followed the Boltzmann responses (Equation (2) and Figure 5B). Green emission decreased with the probes. This alleviates the interferences between the two probes. The sensing performance followed decease of pHs, similar to those of other fluorescein-containing probes. As shown in the insert figure the Boltzmann responses (Equation (2) and Figure 5B). Green emission decreased with the decease of of Figure 5A, oxygen probe’ emission was not affected by the pH values. The pKas for the sensing pHs, similar to those other fluorescein-containing Asfor shown in the insert of Figure hydrogels were of calculated to be 6.14 for fresh filmprobes. and 6.19 the rehydrated filmfigure after one week 5A, oxygen probe’ emission was not affected by the pH values. The pKa s for the sensing hydrogels were calculated to be 6.14 for fresh film and 6.19 for the rehydrated film after one week drying at room
Sensors 2018, 18, x FOR PEER REVIEW
8 of 13
Sensors 2018, 18, x FOR PEER REVIEW Sensors 2018, 564 drying at18,room temperature. Similar
8 of813of 13 to the oxygen responses, rehydrated films showed slightly
decreased sensitivity (Figure 5B): drying at room temperature. Similar to the oxygen responses, rehydrated films showed slightly temperature. to the oxygen showed I responses, = Imax + Imin rehydrated × [10pKa-pH/(1films + 10pKa-pH )], slightly decreased sensitivity (2) decreased Similar sensitivity (Figure 5B): (Figure 5B): maxpKa-pH is the maximum signal at pH 9.0, and Imin is the where I is the fluorescent intensity 525 I[10 pKa-pH)], pKa-pH =atIImax + Inm, min ×pKa-pH I = ImaxI + /(1/(1+ +1010 )], (2)(2) min × [10 minimum signal at pH 3. maxthe is the maximum at pH minthe is the where is the fluorescent at 525 nm, where I is Ithe fluorescent at 525 nm, ImaxIis maximum signal at pH 9.0, 9.0, and Imin Iis The insert in Figureintensity 5Bintensity showed the emission color changes at asignal few typical pH and values. The minimum signal at pH 3. with green emissions and oxygen probes with red emissions. At pH 9, minimum signal at pH 3.0. hydrogels contain pH probes The insert in Figure 5Bshowed showed the emission emission color changes at pH The Thegreen insertemission in Figure the color changes at aa few fewtypical typical pHvalues. values. strong was5B observed; while at pH 3, because of the disappearance of green emission, hydrogels contain pH probes with green emissions and oxygen probes with red emissions. At pH The pH probes with green emissions and oxygen probes with red emissions. At pH 9, 9, redhydrogels emission contain was observed. strong green emission observed; while at pH 3, because of the disappearance of green emission, strong green emission waswas observed; while at pH 3, because of the disappearance of green emission, red emission was observed. red emission was observed.
Figure 5. pH titration of the film F3 in a 96-well plate via a plate reader. (A) Fluorescence scan of F3 in a series of BR buffers excited at 488 nm; the insert figure in A is for the emissions from the oxygen Figure 5. pH titration of the a 96-well a plate reader. (A) Fluorescence scan F3 Figure 5. excited pH titration of nm. the film F3 inF3a in 96-well plateplate avia plate reader. (A) Fluorescence scanweek). of F3of inThe a in probes at 540 (B)film Boltzmann curves ofvia fresh and rehydrated gels (after one series of BR buffers excited at 488 nm; the insert figure in A is for the emissions from the oxygen probes a series of BR buffers excited at 488 nm; the insert figure in A is for the emissions from the oxygen insert in B shows the color changes at different pHs illuminated by a 365 nm LED light and taken by excited atcamera. 540 nm.Two (B)540 Boltzmann curves of were fresh andofrehydrated gels one probes excited at nm. (B) Boltzmann curves freshusing and rehydrated gelsweek). (after The one insert week).inThe a color parallel experiments performed fresh(after hydrogels. B shows changes at different pHs illuminated by a 365 nmbyLED light color by insert the in Bcolor shows the color changes at different pHs illuminated a 365 nmand LEDtaken light by anda taken camera. Two parallel experiments were performed using fresh hydrogels. a color camera. Two parallel experiments were performed using fresh hydrogels. 3.5. Biocompability Test of the Sensor Hydrogels in the Wells
For bioapplications, toxicity study in was 3.5.3.5. Biocompability TestTest of the Sensor Hydrogels the Wells Biocompability of athe Sensor Hydrogels inrequired the Wells for proving the biocompatibility. Parallel experiments were conducted in the multi-well plate under the same conditions with and without the ForFor bioapplications, a toxicity waswas required for for proving biocompatibility. Parallel bioapplications, a toxicity required proving the biocompatibility. Parallel 7 and sensing hydrogels. One hundred μLstudy ofstudy E. coli K12 with densities of 2.5the × 10 5 × 107 CFU/mL in LB experiments were conducted in the multi-well plate under the same conditions with and without theat experiments were conducted in the multi-well plate under the same conditions with and without the broth were seeded into each micro-well with and without sensing gels, and cultured in the plate 7 and 77 CFU/mL 7 and sensing hydrogels. One hundred μL of E. coli K12 with densities of 2.5 × 10 5 × 10 CFU/mL in sensing hydrogels. One hundred µL of E. coli K12 with densities of 2.5 × 10 5 × 10 in 37 °C for 5 h. The absorbance was periodically monitored at a wavelength of 600 nm (OD600) by LB a broth were seeded seeded into eachmicro-well micro-well withand andwithout without sensing gels, and plate LB broth each with sensing and cultured in in thethe plate at at plate reader. Resultsinto showed that time dependent OD600 were notgels, affected bycultured the hydrogel-based ◦37 °C 5for h. wells The absorbance was monitored at a wavelength ofhydrogels’ 600 600 nm) by (OD ) by a 37sensors C for h. 5the The absorbance was6)periodically monitored atshowing a wavelength of 600 nm (OD a 600 plate in (Figure for periodically 5 h’ cell culture, the sensing excellent plate reader. Results showed that time dependent OD 600 were not affected by the hydrogel-based reader. Results showed that time dependent OD were not affected by the hydrogel-based sensors in 600 biocompatibility. in the6)wells (Figure 6) forshowing 5 h’ celltheculture, theexcellent sensingbiocompatibility. hydrogels’ excellent thesensors wells (Figure for 5 h’ cell culture, sensingshowing hydrogels’ biocompatibility. 0.20 0.18 0.20 OD 600nm OD 600nm
0.16 0.18 0.14 0.16
7
0.12 0.14 0.10 0.12 0 0.10
60 0
60
5x10 CFU/mLwith F3 7 5x10 CFU/mL without F3 7 2.5x10 7 CFU/mL with F3 5x107 CFU/mLwith F3 2.5x10 7 CFU/mL without F3 5x10 CFU/mL without F3 7 2.5x10 CFU/mL with F3 7 120 2.5x10 180 240without300 CFU/mL F3 Time (minute) 120
180
240
300
2.5××10 1077 and andTime × 10 Figure6.6.Time Time dependent OD (minute) Figure dependent OD 55 × 1077CFU/mL CFU/mLofofE.E.coli coliwith withand andwithout withoutF3 F3film filmat 600600ofof2.5 ◦ at37 37°C. C. Figure 6. Time dependent OD600 of 2.5 × 107 and 5 × 107 CFU/mL of E. coli with and without F3 film at 37 °C.
Sensors 2018, 18, 564
Sensors 2018, 18, x FOR PEER REVIEW
9 of 13
9 of 13
3.6. In-Situ Monitoring of the pH and Oxygen Changes during the Growth of E. coli K12 3.6. In-Situ Monitoring of the pH and Oxygen Changes during the Growth of E. coli K12 E. coli K12 was cultured in LB broth at 37 ◦ C with shaking at 200 rpm for 12 h. Cells were E. coli K12 was cultured in LB broth at 737 °C with shaking at7 200 rpm for 12 h. Cells were gradually gradually diluted with LB broth to 5.0 × 10 CFU/mL, 2.5 × 10 CFU/mL, 1.25 × 107 CFU/mL and 7 7 7 diluted with LB broth to 5.0 × 10 CFU/mL, 2.5 × 10 CFU/mL, 1.25 × 10 CFU/mL and 6.25 × 106 CFU/mL. 6 6.25 × 10 CFU/mL. Then 100 µL of each diluted sample in LB medium was seeded into the well with Then 100 μL of each diluted sample in LB medium was seeded into the well with sensing gels, and the sensing gels, and the same volume of LB broth without cells was set as the blank control. In order same volume of LB broth without cells was set as the blank control. In order to prevent the exchange of to prevent the exchange of oxygen in the media with air, 100 µL of mineral oil was used to seal the oxygen in the media with air, 100 μL of mineral oil was used to seal the well to avoid the exchange of well to avoid the exchange of oxygen in cell culture medium with air. The use of mineral oil for oxygen in cell culture medium with air. The use of mineral oil for oxygen consumption is popularly oxygen consumption is popularly applied [37]. Emission measurements at 645 nm and 525 nm were applied [37]. Emission measurements at 645 nm and 525 nm were carried out to record the oxygen carried out to record the oxygen and pH changes during the cell growth with excitation wavelengths and pH changes during the cell growth with excitation wavelengths of 405 nm for oxygen probe and of 405 nm for oxygen probe and 488 nm for pH probe, respectively. It was observed that the oxygen 488 nm for pH probe, respectively. It was observed that the oxygen probes’ emissions increased probes’ emissions increased much faster at higher cell densities, indicating that the oxygen was much faster at higher cell densities, indicating that the oxygen was consumed much faster at these consumed much faster at these higher densities (Figure 7A). After the DO in the medium was consumed higher densities (Figure 7A). After the DO in the medium was consumed completely, the completely, the fluorescence intensities did not change further. A slight decrease of the fluorescence fluorescence intensities did not change further. A slight decrease of the fluorescence intensities at 645 nm intensities at 645 nm at the early stage of the experiments was observed, which was attributed to the at the early stage of the experiments was observed, which was attributed to the temperature effect. temperature effect.
Figure pH monitoring of E. coli K12ausing a sensing 96-well gel sensing plate. Fluorescence Figure 7. 7. DODO andand pH monitoring of E. coli K12 using 96-well plate. gel Fluorescence intensity intensity changes were monitored at 645 nm with an excitation at 405 nm (oxygen probe, A),under and at changes were monitored at 645 nm with an excitation at 405 nm (oxygen probe, A), and at 525 nm 525 nm under of 488 B) nmduring (pH probe, B) during of E. coli initial with different initial the excitation ofthe 488excitation nm (pH probe, the growth of E.the coligrowth with different inoculum at 6, 1.25 × 7, 2.5 × 10 7 CFU/mL. (C,D): The calculated time and 5 × 10(C,D): inoculum at × 0 (blank), 0 (blank), 6.25 106 , 1.256.25 × 10×7 10 , 2.5 × 107 10 and 5 × 1077 CFU/mL. The calculated time dependent dependent pHs from A andbyB,referring respectively by referring their corresponding DOs and pHs DOs from and A and B, respectively their corresponding calibration curves ascalibration showing in Figures and 5B. Theonly blank is from a well only with sensors without E. coli. incurves Figuresas4showing and 5B. The blank is4 from a well with sensors without E. coli.
A very significant temperature effect was observed for the pH probe. Clear decreases of the A very significant temperature effect was observed for the pH probe. Clear decreases of the fluorescence intensities at 525 nm were observed within the first 1 h of the experiments. After that a fluorescence intensities at 525 nm were observed within the first 1 h of the experiments. After that a slight fluorescence decrease was observed for the blank sample, along with significant decreases of slight fluorescence decrease was observed for the blank sample, along with significant decreases of the fluorescence intensities with cells. Samples with higher cell densities produced faster intensity the fluorescence intensities with cells. Samples with higher cell densities produced faster intensity decreases, showing faster acidification rates. It was observed the pH changes were much slower than decreases, showing faster acidification rates. It was observed the pH changes were much slower than oxygen changes. This is due to that the fact LB medium has significant buffering capability to delay oxygen changes. This is due to that the fact LB medium has significant buffering capability to delay the the pH changes. On the other hand, pH changes were observed after the DO was consumed pH changes. On the other hand, pH changes were observed after the DO was consumed completely, completely, showing cells can still undergo metabolism. This is because that H+ ions were translocated outwards across the plasma membrane of E. coli by a respiratory pulse in the anaerobic conditions [38].
Sensors 2018, 18, 564
10 of 13
showing cells can still undergo metabolism. This is because that H+ ions were translocated outwards across the plasma membrane of E. coli by a respiratory pulse in the anaerobic conditions [38]. Sensors 2018, 18, x FOR PEER REVIEW 10 of 13 The fluorescence signals as shown in Figure 7A,B were transferred to DO concentrations and pHs by referring their corresponding calibration curves shown in Figures and 5B, whichand were The fluorescence signals as shown in Figure 7A,B as were transferred to DO4 concentrations given in Figure 7C,D. Therefore, the hydrogel-based 96 wells with sensors showed the capability pHs by referring their corresponding calibration curves as shown in Figures 4 and 5B, which were for simultaneously monitoring the pH and changes96atwells cellular Since the given in Figure 7C,D. Therefore, theoxygen hydrogel-based withmicroenvironments. sensors showed the capability forgels simultaneously oxygen changes at cellular microenvironments. Since the were incorporated at monitoring the bottomthe of pH the and 96 wells, high throughput analysis of cellular environment were incorporated at the bottom 96 wells,temperature high throughput can be gels easily achieved. However, because of of thethe significant effect analysis and alsoofthecellular possible environment can be easily achieved. However, because of the significant temperature andpH alsoand photo-bleaching effect for the pH probes based on fluorescein, for accurate analysiseffect of the the possible photo-bleaching effect for the pH probes based on fluorescein, for accurate analysis of oxygen changes, new sensor formats including the probes, matrices, and/or thicknesses of films with the pH and oxygen changes, new sensor formats including the probes, matrices, and/or thicknesses less temperature effects and better photostability needed to be considered and chosen. of films with less temperature effects and better photostability needed to be considered and chosen.
3.7. Sensing Films for Antibiotic Test
3.7. Sensing Films for Antibiotic Test
The influence of antibiotics on the cellcell metabolic oxygenconsumption consumption The influence of antibiotics on the metabolicactivity, activity,herein herein oxygen andand pH,pH, was investigated by using ampicillin as an Ampicillin is aaβ-lactam β-lactamantibiotic antibiotic which was investigated by using ampicillin as example. an example. Ampicillin(AMP) (AMP) is which could act as aact competitive inhibitorinhibitor of the enzyme for cell-wall-forming, and ultimately could as a competitive of the transpeptidase enzyme transpeptidase for cell-wall-forming, and ultimately leads to cell For this study, coli K12 cellscultured were cultured an initial OD of 0.1 leads to cell lysis [31]. Forlysis this[31]. study, E. coli K12E.cells were withwith an initial OD 600600of 0.1 under various concentrations of ampicillin (12.5, 25 and 50 μg/mL) under aerobic condition at 37 °C under various concentrations of ampicillin (12.5, 25 and 50 µg/mL) under aerobic condition at with shaking shaking in in the the 96-well 96-well sensing sensing plate. plate. AMP inhibition of of oxygen 37 ◦ C with AMP concentration concentrationdependent dependent inhibition oxygen consumption was observed (Figure 8A,C). Higher AMP concentration resulted in more significant consumption was observed (Figure 8A,C). Higher AMP concentration resulted in more significant oxygen consumption inhibition. A similar effect was also observed for the pH changes (Figures oxygen consumption inhibition. A similar effect was also observed for the pH changes (Figure 8B,D). 8B,D). Higher AMP concentration resulted in more significant cellular acidification inhibition. HigherHence, AMP our concentration resulted in more significant cellular acidification inhibition. Hence, studies indicated our sensing materials have potentials for understanding of the our studies indicated ouronsensing materials have potentials for understanding of the antibiotics antibiotics influence cell metabolic activities and the evaluation of the antibiotics activities. influence on cell metabolic activities and the evaluation of the antibiotics activities.
Figure 8. Antibiotic (ampicillin, AMP) test against E. coli K12 with sensing films in a 96-well plate.
Figure 8. Antibiotic (ampicillin, AMP) test against E. coli K12 with sensing films in a 96-well plate. Fluorescence intensity changes monitored at 645 nm with an excitation at 405 nm (oxygen probe, A) Fluorescence intensity changes monitored at 645 nm with an excitation at 405 nm (oxygen probe, A) and at 525 nm under the excitation of 488 nm (pH probe, B). DO concentrations and pH changes and at 525 nm under the excitation of 488 nm (pH probe, B). DO concentrations and pH changes corresponding to A and B, respectively, which was calculated by referring their individual corresponding to curves A and as B, plotted respectively, which was byfrom referring individual calibration in Figures 4 and 5B.calculated The blank is a well their only with sensorscalibration without curves as plotted in Figures 4 and 5B. The blank is from a well only with sensors without E. coli. Control E. coli. Control is a well with E. coli however without AMP. is a well with E. coli however without AMP. 4. Conclusions
Sensors 2018, 18, 564
11 of 13
4. Conclusions New hydrophilic PHEMA-co-PAM hydrogel-based oxygen/pH dual sensing films were fabricated on the bottom of commercial multi-well plates through redox polymerization. The sensors’ sensitivity was optimized by adjusting the proportions of the hydrogel matrix components PHEMA, PAM, and the cross-linker PEGDMA. The final composition for good sensing performance was found to be a weight ratio of HEMA:AM:PEGDMA of 85:15:10. To get flat and uniform hydrogel films in the 96 well pates, a thin layer of butyl alcohol was placed on the top of the mixed monomeric precursors for gel formation in each micro-well. The sensor films with the aid of butyl alcohol are quite flat and smooth. The hydrogels could be reused and were robust enough to be washed, dried, rehydrated and stored in air for at least 1 week. Such functional 96-well plates were used for simultaneously monitoring the changes of pH and oxygen during the growth of E. coli. Clear cell density dependent oxygen consumption and extracellular acidification rates were observed by using the sensor gels. Further, the sensors provided the capability for studying the effects of antibiotics on cell oxygen respiration and cellular acidification. Although the current sensor formats still exhibited photo-instability and temperature effects during the measurements, our results demonstrated herein a new avenue to broaden the oxygen and pH sensor’s applications in biological fields for in-situ and high throughput analysis of pH and dissolved oxygen in cellular micro-environments. It is expected that this type dual-functional biocompatible sensing hydrogel can be used for understanding of cell growth, proliferation, and cell metabolism. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/18/2/564/s1, Figure S1: The photostability of O2 sensor and pH sensor. Emission intensity of oxygen probes was monitored at 645 nm with an excitation of 405 nm; emission intensity of pH probes was monitored at 525 nm with an excitation of 488 nm., Figure S2: dissolved oxygen changes (A), and pH changes (B) for the sensing films in the presence of various biological cations at their physiological concentrations in B.R. buffer (pH 7.4): KCl (5.0 mM), NaCl (15 mM), MgSO4 (2.5 mM), CaCl2 (0.5 mM), FeCl2 (18 mM), CuCl2 (16 mM) and MnCl2 (0.9 mM). Acknowledgments: This work was supported by National Science Foundation of China (21574061 and 21604036), Shenzhen fundamental research programs (JCYJ20150630145302243), start fund of SUSTech (FRG-SUSTC1501A-01, Y01256009) and Shenzhen Science and Technology Innovation Committee for key laboratory (ZDSYS20140509142721431). Author Contributions: Siying Wu and Shanshan Wu designed and performed most of the experiments, and wrote the draft; Zheyuan Yi and Weizhen Wu helped the film preparation; Fei Zeng helped the cell culture; Yuan Qiao helped some cell respiration measurements; Xingzhong Zhao and Xing Cheng participated the discussions; Yanqing Tian revised this manuscript and guided the design and discussion. Conflicts of Interest: The authors declare no conflict of interest.
References 1. 2.
3.
4. 5. 6. 7.
Beal, M.F.; Hyman, B.T.; Koroshetz, W. Do defecs in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci. 1993, 16, 125–131. [CrossRef] Rosas, C.; Martinez, E.; Gaxiola, G.; Brito, R.; Sánchez, A.; Soto, L.A. The effect of dissolved oxygen and salinity on oxygen consumption, ammonia excretion and osmotic pressure of Penaeus setiferus (Linnaeus) juveniles. J. Exp. Mar. Biol. Ecol. 1999, 234, 41–57. [CrossRef] Morrison, S.J.; Csete, M.; Groves, A.K.; Melega, W.; Wold, B.; Anderson, D.J. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J. Neurosci. 2000, 20, 7370–7376. [PubMed] Amon, R.; Benner, R. Photochemical and microbial consumption of dissolved organic carbon and dissolved oxygen in the Amazon River system. Geochim. Cosmochim. Acta 1996, 60, 1783–1792. [CrossRef] Bandyopadhyay, U.; Das, D.; Banerjee, R.K. Reactive oxygen species: Oxidative damage and pathogenesis. Curr. Sci. 1999, 77, 658–666. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 373–399. [CrossRef] [PubMed] Su, J.; Chen, F.; Cryns, V.L.; Messersmith, P.B. Catechol polymers for pH-responsive, targeted drug delivery to cancer cells. J. Am. Chem. Soc. 2011, 133, 11850–11853. [CrossRef] [PubMed]
Sensors 2018, 18, 564
8.
9. 10. 11. 12. 13. 14.
15. 16.
17. 18.
19.
20.
21.
22. 23. 24. 25. 26. 27.
28. 29.
12 of 13
Hansen, G.; Johansen, C.L.; Wilmes, J.; Jespersen, L.; Arneborg, N. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of lactococcus lactis in batch fermentations. Appl. Microbiol. Biotechnol. 2016, 100, 5965–5976. [CrossRef] [PubMed] Hainaut, P.; Plymoth, A. Targeting the hallmarks of cancer: Towards a rational approach to next-generation cancer therapy. Curr. Opin. Oncol. 2013, 25, 50–51. [CrossRef] [PubMed] Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell 2011, 144, 646–674. [CrossRef] [PubMed] Wang, X.D.; Wolfbeis, O.S. Optical methods for sensing and imaging oxygen: Materials, spectroscopies and applications. Chem. Soc. Rev. 2014, 43, 3666–3761. [CrossRef] [PubMed] Wolfbeis, O.S. Materials for fluorescence-based optical chemical sensors. J. Mater. Chem. 2005, 15, 2657–2669. [CrossRef] Wolfbeis, O.S. Fiber-optic chemical sensors and biosensors. Anal. Chem. 2000, 72, 81–90. [CrossRef] Zou, X.; Pan, T.T.; Chen, L.; Tian, Y.Q.; Zhang, W.W. Luminescence materials for pH and oxygen sensing in microbial cells-structures, optical properties, and biological applications. Crit. Rev. Biotechnol. 2017, 37, 723–738. [CrossRef] [PubMed] Nagl, S.; Wolfbeis, O.S. Optical multiple chemical sensing: Status and current challenges. Analyst 2007, 132, 507–511. [CrossRef] [PubMed] Vasylevska, G.S.; Borisov, S.M.; Krause, C.; Wolfbeis, O.S. Indicator-loaded permeation-selective microbeads for use in fiber optic simultaneous sensing of pH and dissolved oxygen. Chem. Mater. 2006, 18, 4609–4616. [CrossRef] Schröder, C.R.; Polerecky, L.; Klimant, I. Time-resolved pH/pO2 mapping with luminescent hybrid sensors. Anal. Chem. 2007, 79, 60–70. [CrossRef] [PubMed] Kocincová, A.S.; Nagl, S.; Arain, S.; Krause, C.; Borisov, S.M.; Arnold, M.; Wolfbeis, O.S. Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH. Biotechnol. Bioeng. 2008, 100, 430–438. [CrossRef] [PubMed] Lu, H.G.; Jin, Y.G.; Tian, Y.Q.; Zhang, W.W.; Holl, M.R.; Meldrum, D.R. New ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activity in cyanobacteria. J. Mater. Chem. 2011, 21, 19293–19301. [CrossRef] [PubMed] Wang, X.D.; Stolwijk, J.A.; Lang, T.; Sperber, M.; Meier, R.J.; Wegener, J.; Wolfbeis, O.S. Ultra-small, highly stable, and sensitive dual nanosensors for imaging intracellular oxygen and pH in cytosol. J. Am. Chem. Soc. 2012, 134, 17011–17014. [CrossRef] [PubMed] Meier, R.J.; Schreml, S.; Wang, X.D.; Landthaler, M.; Babilas, P.; Wolfbeis, O.S. Simultaneous photographing of oxygen and pH in vivo using sensor films. Angew. Chem. Int. Ed. 2011, 50, 10893–10896. [CrossRef] [PubMed] Zhou, X.F.; Su, F.Y.; Tian, Y.Q.; Meldrum, D.R. Dually fluorescent core-shell microgels for ratiometric imaging in live antigen-presenting cells. PLoS ONE 2014, 9, e88185. [CrossRef] [PubMed] Borchert, N.B.; Ponomarev, G.V.; Kerry, J.P.; Papkovsky, D.B. O2 /pH multisensor based on one phosphorescent dye. Anal. Chem. 2011, 83, 18–22. [CrossRef] [PubMed] PreSens Precision Sensing. Available online: https://www.presens.de/ (accessed on 11 February 2018). Agilent Trusted Answers. Available online: http://cn.agilent.com/en-us/products/cell-analysis-(seahorse) /how-seahorse-xf-analyzers-work (accessed on 11 February 2018). Wang, X.D.; Meier, R.J.; Wolfbeis, O.S. Fluorescent pH-sensitive nanoparticles in an agarose matrix for imaging of bacterial growth and metabolism. Angew. Chem. Int. Ed. 2013, 52, 406–409. [CrossRef] [PubMed] Birmele, M.; Roberts, M.; Garland, J. Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates. J. Microbiol. Meth. 2006, 67, 619–623. [CrossRef] [PubMed] Chen, X.; Zhong, Z.M.; Li, Z.; Jiang, Y.Q.; Wang, X.R.; Wong, K. Characterization of ormosil film for dissolved oxygen-sensing. Sens. Actuators B Chem. 2002, 87, 233–238. [CrossRef] Moßhammer, M.; Strobl, M.; Kühl, M.; Klimant, I.; Borisov, M.S.; Koren, K. Design and application of an optical sensor for simultaneous imaging of pH and dissolved O2 with low cross-talk. ACS Sens. 2016, 1, 681–687. [CrossRef]
Sensors 2018, 18, 564
30.
31.
32.
33.
34. 35. 36. 37. 38.
13 of 13
Tian, Y.Q.; Su, F.Y.; Weber, W.; Nandakumar, V.; Shumway, B.R.; Jin, Y.G.; Zhou, X.F.; Holl, M.; Johnson, R.H.; Meldrum, D.R. A series of naphthalimide derivatives as intra and extracellular pH sensors. Biomaterials 2010, 31, 7411–7422. [CrossRef] [PubMed] Tian, Y.Q.; Shumway, B.R.; Gao, W.M.; Youngbull, C.; Holl, M.R.; Johnson, R.H.; Meldrum, D.R. Influence of matrices on oxygen sensing of three sensing films with chemically conjugated platinum porphyrin probes and preliminary application for monitoring of oxygen consumption of Escherichia coli (E. coli). Sens. Actuators B Chem. 2010, 150, 579–587. [CrossRef] [PubMed] Tian, Y.Q.; Shumway, B.R.; Meldrum, D.R. A new cross-linkable oxygen sensor covalently bonded into poly(2-hydroxyethyl methacrylate)-co-polyacrylamide thin film for dissolved oxygen sensing. Chem. Mater. 2010, 22, 2069–2078. [CrossRef] [PubMed] Tian, Y.Q.; Shumway, B.R.; Youngbull, A.C.; Li, Y.Z.; Jen, A.K.-Y.; Johnson, R.H.; Meldrum, D.R. Dually fluorescent sensing of pH and dissolved oxygen using a membrane made from polymerizable sensing monomers. Sens. Actuators B Chem. 2010, 147, 714–722. [CrossRef] [PubMed] Sarac, A.S. Redox polymerization. Prog. Polym. Sci. 1999, 24, 1149–1204. [CrossRef] Fornasiero, F.; Krull, F.; Prausnitz, J.M.; Radke, C.J. Steady-state diffusion of water through soft-contact-lens materials. Biomaterials 2005, 26, 5704–5716. [CrossRef] [PubMed] Wang, Y.; Tan, G.; Zhang, S.; Guang, Y. Influence of water states in hydrogels on the transmissibility and permeability of oxygen in contact lens materials. Appl. Surf. Sci. 2008, 255, 604–606. [CrossRef] O’Riordan, T.C.; Buckley, D.; Ogurtsov, V.; O’Connora, R.; Papkovsky, D.B. A cell viability assay based on monitoring respiration by optical oxygen sensing. Anal. Biochem. 2000, 278, 221–227. [CrossRef] [PubMed] West, I.C.; Mitchell, P. Proton/sodium ion antiport in Escherichia coli. Biochem. J. 1974, 144, 87–90. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
