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Forward osmosis in a portable device for automatic osmolality adjustment of environmental water samples evaluated by cell-based biosensors
Journal of Membrane Science 454 (2014) 470–477
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Forward osmosis in a portable device for automatic osmolality adjustment of environmental water samples evaluated by cell-based biosensors Sara Talaei a,n, Yusaku Fujii a,1, Frederic Truffer b, Peter D. van der Wal a, Nico F. de Rooij a a Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Microengineering (IMT), Sensors, Actuators and Microsystems Laboratory (SAMLAB), Rue Jaquet Droz 1, 2000 Neuchatel, Switzerland b University of Applied Sciences of Western Switzerland (HES-SO Valais), Rte du Rawyl 47, 1950 Sion, Switzerland
art ic l e i nf o
a b s t r a c t
Article history: Received 1 February 2013 Received in revised form 29 October 2013 Accepted 16 December 2013 Available online 25 December 2013
Forward osmosis (FO) is a well-established process that has been used for different applications like desalination of water, concentration of foods or drugs, and energy harvesting. We exploited this process in a fully automatic system to adjust osmolality of environmental water samples that are to be tested by cell-based biosensors. In cell-based biosensors, samples are brought into contact with living cells. Therefore, the samples0 osmolality and pH should be in a range that is tolerable for the cells. Controlling these parameters has been a significant challenge especially in environmental monitoring, where the biosensors are required to work on-site. In this paper, we introduce a low-cost portable fluidic system that works automatically, and adjusts the osmolality and pH of environmental samples without diluting or denaturizing the ingredients of the samples. We report the performance of this system in adjusting the osmolality and pH of Swiss environmental waters with a natural osmolality of 47 1 mmol/kg and a pH of 7.847 0.02. & 2013 Elsevier B.V. All rights reserved.
Keywords: Sample pretreatment Cell-based biosensors Forward osmosis Osmolality/pH adjustment
1. Introduction For more than 30 years whole living cells have been used as analytical tools [1]. The most popular applications of whole cell-based biosensors are for detecting environmental pollutions [2–5], pharmacological screening [6–8] and food analysis [9,10]. The principle is to monitor and evaluate the response of the cells as they come into contact with samples. The most important point that has to be kept in mind before exposing a cell-based biosensor to samples is to adjust the chemical and physical parameters of the samples to the standard values of the appropriate cell-culture medium. In general, each type of cell requires a specific well-controlled microenvironment. Two important parameters of a cell-culture medium in in-vitro cells monitoring that have to be controlled are osmolality and pH [5,11]. By keeping the cells in their standard cell-culture conditions, variable
Abbreviations: FO, Forward osmosis; CP, Concentration polarization; MgCl2, Magnesium chloride; SRSF, Specific reverse salt flux; CLS, Capacitive liquid level sensor; FR, Feed reservoir; LED, Light emitting diode; EPROM, Erasable programmable read only memory; CA, Cellulose acetate; AFM, Atomic force microscopy; PMMA, Poly methyl methacrylate; SEM, Scanning electron microscopy; DMEM, High Glucose Dulbecco0 s Modified Eagle Medium n Corresponding author. Tel.: þ 41 32 720 5432; fax: þ 41 32 720 5711. E-mail address: sara.talaei@epfl.ch (S. Talaei). 1 Present address: University of Tokyo, Institute of Industrial Science, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan 0376-7388/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.memsci.2013.12.041
responses of the cells subjected to different analytes can be evaluated. Changes in the metabolism of the cells [12,13], action potentials [4,5] and their bioelectrical impedance [14] are amongst the studied cell responses. The cell membrane is permeable to water. Therefore, the osmolality difference between the inside and outside of the membrane can cause shrinkage or swelling of the cells [15,16]. Consequently, any changes in the size and morphology of the cells can cause unwanted interferences in the response of the cell-based biosensor [5]. The other critical parameter in cell-cultures is the pH. Cells contain enzymes for performing their metabolic activities. Changes in the pH of the cells microenvironment can degenerate the enzymes, and deform the structure of the proteins. It can also perturb the cells electrical activity [5,11], ion transport through the membrane channels as well as intercellular communication through the gap junctions [5,17]. Adjusting properties such as osmolality and pH of samples has been a limitation for using cell-based biosensors for on-site environmental monitoring. Occasionally, these parameters have been measured in a laboratory equipped with bulky expensive osmometers, and bench-top pH meters, and then according to the measured value, the samples0 osmolality and pH have been adjusted by adding a solution containing the necessary ions or molecules. This type of adjustment is not convenient for on-site monitoring, and it dilutes the concentration of the substances
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inside the sample. For increasing the osmolality of the samples, evaporative concentration has been another solution. However, heating can cause degradation of the sample ingredients [18–21]. For on-line measurement of osmolality and pH, adapted on-line osmometers and pH sensors have been introduced [2,22]. However, the fabrication of these sensors requires clean-room facilities which are costly. Moreover, adjusting of the parameters still results in dilution of the sample. In this article we present a novel method for the on-line adjustment of osmolality and pH of samples without requiring osmometers or pH sensors. Using this method, the treated samples will not be diluted, and the structure of the ingredients will not be deformed. The design principle is based on exploiting an FO process. In this process, when two solutions with different osmolalities are separated by an osmotic membrane, water molecules will pass through the membrane due to the osmotic pressure from the low concentrated solution (low osmotic pressure) towards the high concentrated side (high osmotic pressure) [23,24]. An osmotic membrane is a specific type of membrane through which in the ideal case only water molecules can pass [23,25]. The solution on one side of the membrane which provides water molecules for the other side is called the feed solution, and the solution which absorbs the water molecules at the other side is called the draw solution [23]. In our system, for adjusting osmolality and pH of a sample, first the sample is mixed with a concentrated solution containing all the necessary ions and molecules (e.g. buffers, nutrients, etc.), and then using FO, water molecules are selectively removed in order to return to the initial concentration of the sample ingredients. In other words, the osmolality of the sample is adjusted by balancing the concentration of water in the two sides of the osmotic membrane upon the FO process while the addition of the buffering substances adjusts the sample0 s pH to the desired value. A general introduction of this system was briefly presented elsewhere [26].
2. Theoretical background FO has been reported as a useful technique for water treatment [27–29], energy supply [30,31], drug delivery [32], concentrating food [18,33] or pharmaceutical products [34], etc. Compared to reverse osmosis, where an external high pressure is applied to drive water through the membrane, in FO, osmotic pressure is the driving force for the water transport. Therefore, FO has a lower fouling potential, and as the whole process is performed at low pressure and low temperature, no degradation of the sample ingredients will occur [19]. FO membranes are asymmetric. They consist of a dense active side for facing the solution with the lower osmotic pressure (feed solution), and a porous supporting side for facing the solution with the higher osmotic pressure (draw solution) [23]. The important issue in FO that determines the efficiency of the process is the water flux (Jw) which can be calculated from the general Eq. (1) [23,24]: J w ¼ AsΔπ
ð1Þ
where A and s are the water permeability constant and the reflection coefficient of the membrane respectively, and Δπ is the differential of the osmotic pressure across the membrane. According to Eq. (1), for a high water flux, the osmotic pressure difference across the membrane should be high. Concentration polarization (CP) is an important limiting factor which decreases the osmotic pressure difference across the membrane. Using asymmetric membranes, CP occurs in two types: external CP and internal CP [23,24]. The external CP has two features: concentrative external CP and dilutive external CP. When the active side of the membrane
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faces the feed solution, as it is the case in FO applications, the concentrative external CP occurs due to the accumulation of the solutes at the active side, and the dilutive external CP takes place due to the dilution of the draw solution at the support side of the membrane [23,24]. Depending on the orientation of the membrane0 s active side, the internal CP also has two types: dilutive internal CP and concentrative internal CP. When the active side faces the feed solution, water from the feed side passes the dense active substructure of the membrane, and penetrates into the porous support substructure. Thus, the draw solution within the membrane is diluted. This is called the dilutive internal CP. When the orientation of the membrane is reversed, water and solutes from the feed side penetrate the porous substructure of the membrane facing the feed solution. The accumulation of the solutes in the porous substructure leads to the phenomenon called the concentrative internal polarization [23,24,29,35]. Neglecting the effects of the external CP, in an FO membrane when the active side of an asymmetric membrane faces the solution with the lower osmotic pressure, the water flux can be calculated using the following equation [23]: Jw ¼
1 B þ Aπ Hi ln K B þ J w þ Aπ Low
ð2Þ
where πHi and πLow are the osmotic pressure of the bulk draw solution and feed solution respectively. K is the resistance to solute diffusion within the membrane porous support layer, and B is the solute permeability coefficient of the active layer of the membrane. K and B can be calculated using Eqs. (3) and (4) respectively. K¼
tτ εDs
ð3Þ
B¼
AΔπðR 1Þ R
ð4Þ
where t, τ and ε are the membrane thickness, tortuosity and porosity respectively. Ds is the diffusion coefficient of the solute, and R is the salt rejection. One of the parameters that can be manipulated for increasing the water flux is the osmotic pressure of the draw solution. Petrotos et al. [18] demonstrated that the square root of the draw solution0 s viscosity is inversely proportional to the water flux. A higher viscosity implies having a smaller Ds and a larger K (Eq. (3)). Therefore a lower viscosity of the draw solution can increase the flux (Eq. (2)). Cath et al. [23] compared the osmotic pressure of 8 different solutions as a function of their concentration at 25 1C. They reported that at a same concentration, magnesium chloride (MgCl2) has a relatively higher osmotic pressure compared to the 7 other tested solutions (CaCl2, NaCl, KCl, sucrose, MgSO4, KNO3 and NH4HCO3). Ideally, only water molecules are able to pass through an osmotic membrane. That is when 100% of the solutes are rejected by the membrane (ơ ¼1). However, due to the imperfect nature of the membrane solutes in both draw and feed solutions also diffuse through the membrane in opposite directions (ơo1). Therefore, in addition to producing a higher water flux, for a faster and more efficient process a draw solution should have a low solute flux towards the feed solution as well. Hancock et al. [36] compared the ratio of the reverse solute flux (i.e. flux of the solutes from the draw into the feed solution) to the water flux of three different draw solutions (NaCl2, MgCl2 and NH4HCO3) through 2 different cellulose acetate osmotic membranes. They called this ratio as the specific reverse salt flux (SRSF), and demonstrated that at the same osmotic pressures, MgCl2 has the minimum SRSF (at least 55% less than NaCl) while NH4HCO3 has the maximum one (at least 10 times more than NaCl).
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Another technique for increasing the flux is to minimize the effect of the external CP. Using flow mode in an FO process is an established approach recommended for diminishing this effect [23]. In this mode, the solutions at both sides of the membrane should be pumped at the same flow rates. Loeb et al. [37] proposed that counter flow is an important factor for increasing the flux by forming a higher osmotic pressure difference at all points along the membrane. It is also reported that increasing the flow-rate can help preventing the accumulation of the solutes in the feed side, and water in the draw side of the membrane. It can also lower the fouling effect. Therefore, the water flux can be enhanced by increasing the flow-rate [38]. Increasing the temperature can also increase the flux by decreasing the viscosity of the draw solution and increasing the diffusion coefficient of the water passing through the membrane [18]. A drawback of increasing the temperature is that it can cause a decrease in the reflection coefficient of the membrane [39]. Thus, some solutes which were rejected by the membrane at lower temperatures will pass through the membrane at higher temperatures.
3. Theory of operation Considering all the factors described in the theoretical background, we developed an osmosis-based system. The schematic design of the system is presented in Fig. 1. The operation of all components shown in Fig. 1 is controlled by a microcontroller. In addition, a graphical user interface is programmed with Labview to provide an easy control over all actuators and sensors of the system. A photo of the whole automated system is shown in Fig. 2. Before an environmental sample can be applied into the system, the sample needs to be filtered to remove bacteria, viruses, microorganism and unwanted particles. This filtration can be done using a 0.2 mm syringe filter. The filtered water sample and a concentrated standard cell-culture medium are pumped into the feed reservoir (FR). These two solutions are mixed together drop-by-drop at the inlet of the FR. The volume ration of the two solutions can be regulated by controlling the flow-rate of the corresponding pumps. The custom-made liquid trackers ensure the presence of liquid in both tubes entering the FR before being mixed. The working principle of these sensors is based on measuring the intensity of the transmitted light through the tube using an optical set-up
consisting of a light emitting diode (LED) and a photodiode. If there is no liquid detected by at least one of the liquid trackers within 5 min after starting the pumps (1) and (2), the process will stop, and an error will appear on the graphical user interface indicating that at least one of the reservoirs is empty. Upon accumulation of the mixed solution inside the FR, the attached CLS measures the liquid level inside the reservoir, and sends the value to the microcontroller. As the feed solution reaches a certain volume, the microcontroller stops pumps (1) and (2). Simultaneously, it opens valve (1), and pump (3) starts pumping the solution from the FR towards the osmotic chamber. At the same time, using pump (4), the draw solution is directed through valve (2) to the opposite side of the membrane, and exits the osmotic chamber towards the waste container. In this paper, the aim was to obtain a final osmolality of 330710 mmol/kg, and an osmolality of 220 mmol/kg was chosen as the initial value for the feed solution. The designed osmotic chamber contains two identical fluidic channels that are separated by the osmotic membrane. This device is explained in Section 3.1. MgCl2, one molar (1 M), is the draw solution, and the feed solution is the mixture of the water sample and concentrated cell-culture medium being accumulated in the FR. Both solutions are pumped at equal flow-rates, and they flow in opposite directions inside the chamber. Due to osmosis water transfers from the low concentrated side towards the high concentrated side. Valve (3) redirects the feed solution back into the FR. The feed solution circulates inside the osmotic chamber until the level of the feed solution inside the FR equals the initial volume of the water sample before mixing with the concentrated solution. At this stage, the excess water that was added to the sample in the mixing process is transferred to the draw side, and the pretreatment is terminated. Subsequently, valve (3) directs the treated sample towards the final reservoir, and valve (2) directs air into the draw side of the chamber to pump out the remaining draw solution inside the osmotic chamber. Prior to introducing the treated sample into the cell-based analyzing biosensor, the temperature of the sample should be adjusted to the appropriate value for the living cells. In order to use the system without a computer, a default procedure with automatic switching of the valves and predefined values for the pumping rate and the CLS level is stored in an integrated erasable programmable read only memory (EPROM). This procedure can be started and terminated by pressing the “Start” and “Stop” buttons shown in Fig. 2.
Fig. 1. Schematic design of the system. The pumps and the valves are shown with different signs and named by numbers. The arrows indicate the direction of the flow through the system. The red line inside the osmotic chamber represents the osmotic membrane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Photo of the complete automated system. Colored solutions are used for demonstrating different liquid reservoirs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Herein, we present an example to describe the calculations required for defining the system0 s set up for the osmolality adjustment of a sample obtained from the Lake of Neuchatel in Switzerland. The Swiss environmental waters have a negligible osmolality of below 10 mmol/kg. In order to adjust the osmolality of a sample with such low osmolality to a standard value of 330 (required for Caco-2 cell cultures), the sample is mixed with a concentrated culture medium to first obtain an osmolality of 220 mmol/kg for the feed solution. The following equation was applied to determine the ration of the sample and the concentrated cell-culture medium. ðV s þ V c Þ 220 ¼ V c N 330;
N41
ð5Þ
or Vs ¼
3 N1 Vc 2
ð6Þ
where Vs and Vc are the volumes of the applied sample and the added concentrated culture medium, respectively. N represents the concentration coefficient of the added concentrated culture medium. For example, using a double concentrated solution (N ¼2), Eq. (6) results in: V s ¼ 2V c
ð7Þ
Thus, using a double concentrated culture medium, for the osmolality adjustment of a 4 ml sample, first it has to be mixed with a 2 ml concentrated culture medium forming the feed solution accumulated in the FR. Afterwards, the feed solution has to circulate in the osmotic chamber until 2 ml water is transferred to the draw side upon the FO process. Removing this 2 ml water prevents the dilution of the sample ingredients such as potentially present toxicants that are needed to be detected by the cell-based biosensor.
3.1. Osmotic chamber As it is described above, the heart of the system is the osmotic chamber where the water concentration of the sample is adjusted.
An osmotic chamber integrating a commercial osmotic membrane was designed for this system. Two parallel fluidic channels were formed inside the osmotic chamber by mechanical machining in two identical poly methyl methacrylate (PMMA) blocks. Each PMMA block integrates one fluidic channel (4 mm width, 10 cm length). The two parallel fluidic channels are positioned against each other holding the osmotic membrane in between, and fixed with screws. To improve the sealing, gaskets were applied on both sides of the membrane. A schematic design of one of the PMMA blocks and a photo of the chamber after assembly with the PMMA blocks are shown in Fig. 3. The surface area between the draw and feed solutions in the osmotic chamber is 400 mm2. The osmotic membrane has an asymmetric structure with a polyester screen mesh that strengthens the membrane. Therefore it could be applied for long term experiments (i.e. up to 4–6 h), and even reused for several times.
4. Materials and compartments The commercial osmotic membrane (HTI OsMem™ CTA-ES) used in the osmotic chamber was provided by Hydration Technology Innovations (USA). The CLS (S 15N, BA 116329), the 3-way "Y" solenoid valves (LFYA1203032H) and the piezoelectric pumps (mp6, 2011510163) were purchased from Sensortechnics (Germany), Lee (USA) and Bartels Mikrotechnik (Germany) respectively. The LED used for the liquid trackers was LRP47F purchased from Osram (Germany), and the photodiode was TEMD 7000 01 from Vishay Semiconductors (USA). The cell-culture medium was High Glucose Dulbecco0 s Modified Eagle Medium (DMEM) purchased from Life Technologies (USA). The concentrated cell-culture medium was prepared by removing water from the standard one using FO in the second generation of the osmotic chamber. For this concentration, the standard medium was applied as the feed solution, and MgCl2 (1 M) was applied as the draw solution. MgCl2 6H2O, D(þ)-glucose anhydrous and CA were purchased from Merck (Germany), Sigma-Aldrich (Switzerland) and Eastman Kodak (USA) respectively. The microcontroller was MPS430 16 bit from Texas Instruments (USA), and EPROM 24LC256 was purchased from
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Fig. 4. Effect of flow-rate on water-flux when both feed and draw solutions were pumped at equal flow-rates. The maximum water-flux was obtained at 400 ml/min.
Fig. 3. Second generation of the osmotic chamber. (a) Schematic design of each PMMA block of the osmotic chamber. (b) Photo of the assembled osmotic chamber.
Microchip (USA). Sylgards 184 was purchased from Dow Corning (USA), and SU-8 50 was purchased from Microchem (USA). Osmolality and pH of the samples were measured by Vapros 5600 vapor pressure osmometer (Wescor Inc., USA) and Orion model 610 pH meter (USA) respectively.
5. Results and discussions For characterization of the system, 4 ml filtered water samples (osmolality: 4 mmol/kg, pH of 7.8), and 2 ml two times concentrated cell-culture medium (osmolality: 660 mmol/kg, pH: 8.5) were applied, and mixed in a ratio of 2:1. This mixture with osmolality of 220 mmol/kg formed the feed solution. The draw solution was MgCl2 with the osmolality of 3500 mmol/kg. In the following, we present the results obtained using the second generation of the osmotic chamber. The water samples were obtained from the Lake of Neuchatel in Switzerland. 5.1. Effect of flow-rate As it is explained in the theoretical background (Section 2), in FO the use of counter flow and increasing the flow-rate are effective techniques for increasing the water flux through the membrane. However, in small systems, when the feed and draw solutions0 velocity is at least ten times higher than the pure water flux across the membrane, the influence of the flow directions is insignificant. In order to determine the maximum water flux in our system, the water flux was measured in a series of experiments
Fig. 5. Monitoring osmolality and pH of the feed solution over time when both feed and draw solutions were pumped with the flow-rate of 400 ml/min. The osmolality of the feed solution increases over time while the pH remains almost stable.
upon pumping both feed and draw solutions at equal flow-rates in opposite directions. The results are shown in Fig. 4. The error bars in all the graphs presented in this paper demonstrate the standard deviation of three measurements around the average value. As one can see in this graph, the flux does not increase in a linear manner. Applying the flow, washes away the accumulated solutes and water at the feed and draw side of the membrane respectively, and causes an increase in the osmotic pressure difference across the membrane. When the flow rate is high enough to avoid this accumulation, the water flux reaches a saturation point. In the presented system, the maximum water flux of 11.1 L m 2 h 1 was obtained at flow rates of 400 ml/min which is equal to 0.006 ml/s. The surface area of the membrane between the two solutions is 400 mm2. This results in a maximum water flux of 0.001 ml/s across the membrane when both solutions are flowing at 0.006 ml/s. Therefore, the velocity is only 6 times (i.e. o10 times) higher than the water flux. This implies that the effect of the flow direction is not completely insignificant in the presented device, and counter flow can still have an influence on improving the water flux.
5.2. Osmolality and pH fluctuations over time Applying the flow-rate of 400 ml/min (in which the maximum water flux was observed), the osmolality and pH variations of the feed solution were measured over time. As it is shown in Fig. 5, the
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osmolality increased from 220 mmol/kg to 350 mmol/kg in 48 min. However, the pH remained almost constant. The stability of the pH shows that mixing the sample with the concentrated cell-culture medium adds enough buffering substances to the feed solution for keeping the pH stable. As the required osmolality value for the specific cell-based system was 330 mmol/kg, Fig. 5 determines that using the presented osmoticbased system and a double concentrated culture medium, the pretreatment of a 4 ml sample can be performed in 30 min. The applied draw solution is almost 15 times more concentrated than the feed solution. Therefore, according to the FO process, continuously, the feed solution loses water to the draw side. However, due to the fouling of the membrane, and the increase in the osmotic pressure of the feed side, the water flux is decreased over time. 5.3. Influence of temperature Another factor that was studied for increasing the water flux is the temperature. As we already mentioned in Section 2, increasing the temperature increases the water flux. In cell-based sensors, depending on the cells0 type, usually, the final treated sample should be at a temperature of 37 1C when being brought into contact with the living cells. Therefore, the impact of temperature on the water flux, osmolality and pH of the final treated samples was studied by performing the whole process of treatment both at room temperature (25 1C) and 37 1C. The results are compared in Fig. 6. Fig. 6a demonstrates that increasing the temperature from 25 1C to 37 1C increased the water flux by 35%. On the other hand, the pH values of the final treated samples were almost equal to the pH value of the standard cell-culture medium. In Fig. 6b the osmolality of the feed solution and the final treated samples at both temperatures are compared. As we can see in this graph, after mixing the sample with the concentrated cell-culture medium, the osmolality of the feed solution was reduced to 220 mmol/kg. Ultimately, at both temperatures the osmolality of the final treated samples was adjusted to the osmolality value of the standard cellculture medium (330 mmol/kg). Overall, the required time for the whole process of treating 1 ml of sample was 5.5 min and 7.5 min at 37 1C and 25 1C, respectively. An important issue that was observed upon increasing the temperature was a decline in the reflection coefficient of the membrane. As it is also mentioned in the theoretical background (Section 2) the reflection coefficient of osmotic membranes can change with temperature. At higher temperatures although a higher water flux will be obtained, at the end of the process, the concentrations of the solutes in the feed and draw solutions will not be the same as the ones treated at a lower temperature. In the sample pretreatment for cell-based studies it is more critical to adjust the osmolality to the desired value rather than
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controlling the concentration of specific ingredients precisely. A cell-culture medium is a combination of many chemical components. As it was not practical to monitor the changes in the concentration of all components, the concentration of glucose as one of the main ingredients was studied. It was observed that at 37 1C, the concentration of glucose in the final treated sample was lower than the one treated at 25 1C. However, the osmolality of the final treated samples at both 25 1C and 37 1C was 330 mmol/kg. Obtaining the same osmolalities despite the higher diffusion of glucose at 37 1C from the feed solution to the draw solution is possibly due to a similar higher diffusion of MgCl2 from the draw solution into the feed solution at this temperature. The glucose concentration of the treated samples was measured by our previously reported glucose sensor [40] and compared to the glucose concentration of the standard cell-culture medium. The results are shown in Fig. 7. This graph illustrates that, respectively, 9% and 22% of the feed0 s glucose concentration was lost for the sample treatment at 25 1C and 37 1C. Occasionally, for adjusting the osmolality of the samples without using this system, 10 times concentrated cell-culture medium was added to the sample with the ratio of 1:9, that results in 10% dilution of sample ingredients [41]. Using the osmosis-based system less dilution occurred at 25 1C by adding only 2 times concentrated cell-culture medium. Using higher concentrated media, less water has to be removed from the feed solution in the treatment process. This will lead to a faster process, and therefore, less diffusion of the solutes through the membrane. Therefore, despite of the imperfect quality of the osmotic membrane, and the unwanted diffusion of the solutes through the membrane, we claim that using the osmosis-based system can avoid the sample dilution significantly.
Fig. 7. Effect of temperature on the final concentration of glucose in the final treated sample.
Fig. 6. Effect of temperature. (a) Effect of temperature on the water flux and pH of the final treated samples and (b) effect of temperature on the osmolality of the final treated sample.
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5.4. Multitask system The presented example for adjusting a sample osmolality was performed for a Swiss environmental sample with an initial osmolality close to zero. The negligible osmolality value helped estimating the initial osmolality value of the feed solution as the sample was mixed with a known concentration of the concentrated culture medium. For using this system, it is not essential to know the exact osmolality of the sample. In general, for adjusting the osmolality of a feed solution, a solution with the designated osmolality value can be applied as the draw solution. By keeping the osmolality of the draw side of the membrane constant, due to the FO process, the osmolality of the feed side will be adjusted to the desired value over time. When the CLS determines that the total volume of the feed side remains stable, it indicates that the osmolality process is accomplished. In cases that the osmolality of the sample is close to zero, in order to obtain a high osmolality (4 20 mmol/kg) it is required to add solutes into the sample. Therefore, although the exact value of the sample osmolality is not essential, it is necessary to know the approximate value. For example, when the final required value for the osmolality is around 330 mmol/kg, knowing the approximate value of the sample osmolality (e.g. o10 mmol/kg, between 10 and 200 mmol/kg or higher than 300 mmol/kg) helps programming the volume and concentration of the concentrated culture medium that has to be mixed with the sample. In order to reduce the required time for the process, the osmolality of the feed solution needs to be adjusted to a value higher than 50% of the final designated value (e.g. for the final value of 330 mmol/kg, we chose an osmolality of 220 mmol/kg for the feed solution). In the presented example the osmolality of the applied sample was close to zero, and as a result the osmolality of the feed solution was known. Thus, measuring the volume of the feed solution upon the FO process provides the possibility of estimating the osmolality of the feed solution. Therefore, in order to decrease the process duration, a solution with a high osmotic pressure could be used as the draw solution (e.g. MgCl2, 1 M). Overall, the presented system has a high flexibility to be programmed for pretreatment of samples with various initial osmolalities. In addition to adjusting the osmolality of the samples, due to the presence of the buffering substances in the concentrated solution, the pH is also adjusted to the required value. Furthermore, by changing some parameters, the operation of the osmosisbased system is flexible to be adapted for other applications as well. For example, by pumping the standard cell-culture medium as the feed solution and MgCl2 as the draw solution, we used the same system for concentrating the cell-culture medium. For two times concentration, the microprocessor was programmed to stop the circulation of the feed solution in the osmotic chamber as soon as the CLS detected the liquid level corresponding to the half volume of the initial feed solution. A similar process can be applied for pre-concentration of different samples. To avoid the loss of any ingredients of the feed solution due to the unwanted diffusion, changing the formulation of the draw solution can be helpful. In cases that the concentration of the ingredient is known, by having the same or smaller concentration of the ingredient in the draw solution, the diffusion of that specific substance will be avoided or reduced.
6. Conclusions In this paper we introduced a portable low-cost system for adjusting the osmolality and pH of an aqueous sample, on-line and automatically. In this system forward osmosis was exploited as a
well-established process to manipulate only the water content of the sample without denaturizing the ingredients after adding all the necessary substances. We demonstrated the application of this system for adjusting the osmolality and pH of Swiss environmental water samples that are to be evaluated by a cell-based biosensor. Controlling the osmolality and pH of such samples is imperative before applying them to a cell-culture. We studied the influence of flow-rate and temperature on the water flux that ultimately changes the process duration. The maximum water flux was provided by applying the feed and draw solutions at 400 ml/ min in opposite directions. By increasing the temperature from 25 1C to 37 1C, the water flux was increased by 2.9% per degree Celsius while the pH was stable. Using this system the turnaround time for pretreatment of 1 ml sample was 7.5 min and 5.5 min at 25 1C and 37 1C, respectively. At the higher temperature, a decline in the reflection coefficient of the membrane was observed. However, although the quality of the osmotic membrane is not perfect, the presented osmosis-based system prevents the sample dilution significantly compared to the conventional sample pretreatment techniques.
Acknowledgements Special thanks to Martial Geiser from HES-SO Valais, Sion Switzerland, Jules Hajj from the University of Saint-Joseph, Beirut Lebanon and Edward G. Beaudry from the Hydration Technology Innovations, Oregon, USA for their valuable discussions. The osmotic membranes were kindly provided by the Hydration Technology Innovations. The authors gratefully thank the staff at the Microsystems Technology Division of the CSEM for their technical assistance. This work was supported by the NanoTera “Livesense” project funded by the “Swiss National Science Foundation” (SNSF).
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Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Forward osmosis in a portable device for automatic osmolality adjustment of environmental water samples evaluated by cell-based biosensors Sara Talaei a,n, Yusaku Fujii a,1, Frederic Truffer b, Peter D. van der Wal a, Nico F. de Rooij a a Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Microengineering (IMT), Sensors, Actuators and Microsystems Laboratory (SAMLAB), Rue Jaquet Droz 1, 2000 Neuchatel, Switzerland b University of Applied Sciences of Western Switzerland (HES-SO Valais), Rte du Rawyl 47, 1950 Sion, Switzerland
art ic l e i nf o
a b s t r a c t
Article history: Received 1 February 2013 Received in revised form 29 October 2013 Accepted 16 December 2013 Available online 25 December 2013
Forward osmosis (FO) is a well-established process that has been used for different applications like desalination of water, concentration of foods or drugs, and energy harvesting. We exploited this process in a fully automatic system to adjust osmolality of environmental water samples that are to be tested by cell-based biosensors. In cell-based biosensors, samples are brought into contact with living cells. Therefore, the samples0 osmolality and pH should be in a range that is tolerable for the cells. Controlling these parameters has been a significant challenge especially in environmental monitoring, where the biosensors are required to work on-site. In this paper, we introduce a low-cost portable fluidic system that works automatically, and adjusts the osmolality and pH of environmental samples without diluting or denaturizing the ingredients of the samples. We report the performance of this system in adjusting the osmolality and pH of Swiss environmental waters with a natural osmolality of 47 1 mmol/kg and a pH of 7.847 0.02. & 2013 Elsevier B.V. All rights reserved.
Keywords: Sample pretreatment Cell-based biosensors Forward osmosis Osmolality/pH adjustment
1. Introduction For more than 30 years whole living cells have been used as analytical tools [1]. The most popular applications of whole cell-based biosensors are for detecting environmental pollutions [2–5], pharmacological screening [6–8] and food analysis [9,10]. The principle is to monitor and evaluate the response of the cells as they come into contact with samples. The most important point that has to be kept in mind before exposing a cell-based biosensor to samples is to adjust the chemical and physical parameters of the samples to the standard values of the appropriate cell-culture medium. In general, each type of cell requires a specific well-controlled microenvironment. Two important parameters of a cell-culture medium in in-vitro cells monitoring that have to be controlled are osmolality and pH [5,11]. By keeping the cells in their standard cell-culture conditions, variable
Abbreviations: FO, Forward osmosis; CP, Concentration polarization; MgCl2, Magnesium chloride; SRSF, Specific reverse salt flux; CLS, Capacitive liquid level sensor; FR, Feed reservoir; LED, Light emitting diode; EPROM, Erasable programmable read only memory; CA, Cellulose acetate; AFM, Atomic force microscopy; PMMA, Poly methyl methacrylate; SEM, Scanning electron microscopy; DMEM, High Glucose Dulbecco0 s Modified Eagle Medium n Corresponding author. Tel.: þ 41 32 720 5432; fax: þ 41 32 720 5711. E-mail address: sara.talaei@epfl.ch (S. Talaei). 1 Present address: University of Tokyo, Institute of Industrial Science, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan 0376-7388/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.memsci.2013.12.041
responses of the cells subjected to different analytes can be evaluated. Changes in the metabolism of the cells [12,13], action potentials [4,5] and their bioelectrical impedance [14] are amongst the studied cell responses. The cell membrane is permeable to water. Therefore, the osmolality difference between the inside and outside of the membrane can cause shrinkage or swelling of the cells [15,16]. Consequently, any changes in the size and morphology of the cells can cause unwanted interferences in the response of the cell-based biosensor [5]. The other critical parameter in cell-cultures is the pH. Cells contain enzymes for performing their metabolic activities. Changes in the pH of the cells microenvironment can degenerate the enzymes, and deform the structure of the proteins. It can also perturb the cells electrical activity [5,11], ion transport through the membrane channels as well as intercellular communication through the gap junctions [5,17]. Adjusting properties such as osmolality and pH of samples has been a limitation for using cell-based biosensors for on-site environmental monitoring. Occasionally, these parameters have been measured in a laboratory equipped with bulky expensive osmometers, and bench-top pH meters, and then according to the measured value, the samples0 osmolality and pH have been adjusted by adding a solution containing the necessary ions or molecules. This type of adjustment is not convenient for on-site monitoring, and it dilutes the concentration of the substances
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inside the sample. For increasing the osmolality of the samples, evaporative concentration has been another solution. However, heating can cause degradation of the sample ingredients [18–21]. For on-line measurement of osmolality and pH, adapted on-line osmometers and pH sensors have been introduced [2,22]. However, the fabrication of these sensors requires clean-room facilities which are costly. Moreover, adjusting of the parameters still results in dilution of the sample. In this article we present a novel method for the on-line adjustment of osmolality and pH of samples without requiring osmometers or pH sensors. Using this method, the treated samples will not be diluted, and the structure of the ingredients will not be deformed. The design principle is based on exploiting an FO process. In this process, when two solutions with different osmolalities are separated by an osmotic membrane, water molecules will pass through the membrane due to the osmotic pressure from the low concentrated solution (low osmotic pressure) towards the high concentrated side (high osmotic pressure) [23,24]. An osmotic membrane is a specific type of membrane through which in the ideal case only water molecules can pass [23,25]. The solution on one side of the membrane which provides water molecules for the other side is called the feed solution, and the solution which absorbs the water molecules at the other side is called the draw solution [23]. In our system, for adjusting osmolality and pH of a sample, first the sample is mixed with a concentrated solution containing all the necessary ions and molecules (e.g. buffers, nutrients, etc.), and then using FO, water molecules are selectively removed in order to return to the initial concentration of the sample ingredients. In other words, the osmolality of the sample is adjusted by balancing the concentration of water in the two sides of the osmotic membrane upon the FO process while the addition of the buffering substances adjusts the sample0 s pH to the desired value. A general introduction of this system was briefly presented elsewhere [26].
2. Theoretical background FO has been reported as a useful technique for water treatment [27–29], energy supply [30,31], drug delivery [32], concentrating food [18,33] or pharmaceutical products [34], etc. Compared to reverse osmosis, where an external high pressure is applied to drive water through the membrane, in FO, osmotic pressure is the driving force for the water transport. Therefore, FO has a lower fouling potential, and as the whole process is performed at low pressure and low temperature, no degradation of the sample ingredients will occur [19]. FO membranes are asymmetric. They consist of a dense active side for facing the solution with the lower osmotic pressure (feed solution), and a porous supporting side for facing the solution with the higher osmotic pressure (draw solution) [23]. The important issue in FO that determines the efficiency of the process is the water flux (Jw) which can be calculated from the general Eq. (1) [23,24]: J w ¼ AsΔπ
ð1Þ
where A and s are the water permeability constant and the reflection coefficient of the membrane respectively, and Δπ is the differential of the osmotic pressure across the membrane. According to Eq. (1), for a high water flux, the osmotic pressure difference across the membrane should be high. Concentration polarization (CP) is an important limiting factor which decreases the osmotic pressure difference across the membrane. Using asymmetric membranes, CP occurs in two types: external CP and internal CP [23,24]. The external CP has two features: concentrative external CP and dilutive external CP. When the active side of the membrane
471
faces the feed solution, as it is the case in FO applications, the concentrative external CP occurs due to the accumulation of the solutes at the active side, and the dilutive external CP takes place due to the dilution of the draw solution at the support side of the membrane [23,24]. Depending on the orientation of the membrane0 s active side, the internal CP also has two types: dilutive internal CP and concentrative internal CP. When the active side faces the feed solution, water from the feed side passes the dense active substructure of the membrane, and penetrates into the porous support substructure. Thus, the draw solution within the membrane is diluted. This is called the dilutive internal CP. When the orientation of the membrane is reversed, water and solutes from the feed side penetrate the porous substructure of the membrane facing the feed solution. The accumulation of the solutes in the porous substructure leads to the phenomenon called the concentrative internal polarization [23,24,29,35]. Neglecting the effects of the external CP, in an FO membrane when the active side of an asymmetric membrane faces the solution with the lower osmotic pressure, the water flux can be calculated using the following equation [23]: Jw ¼
1 B þ Aπ Hi ln K B þ J w þ Aπ Low
ð2Þ
where πHi and πLow are the osmotic pressure of the bulk draw solution and feed solution respectively. K is the resistance to solute diffusion within the membrane porous support layer, and B is the solute permeability coefficient of the active layer of the membrane. K and B can be calculated using Eqs. (3) and (4) respectively. K¼
tτ εDs
ð3Þ
B¼
AΔπðR 1Þ R
ð4Þ
where t, τ and ε are the membrane thickness, tortuosity and porosity respectively. Ds is the diffusion coefficient of the solute, and R is the salt rejection. One of the parameters that can be manipulated for increasing the water flux is the osmotic pressure of the draw solution. Petrotos et al. [18] demonstrated that the square root of the draw solution0 s viscosity is inversely proportional to the water flux. A higher viscosity implies having a smaller Ds and a larger K (Eq. (3)). Therefore a lower viscosity of the draw solution can increase the flux (Eq. (2)). Cath et al. [23] compared the osmotic pressure of 8 different solutions as a function of their concentration at 25 1C. They reported that at a same concentration, magnesium chloride (MgCl2) has a relatively higher osmotic pressure compared to the 7 other tested solutions (CaCl2, NaCl, KCl, sucrose, MgSO4, KNO3 and NH4HCO3). Ideally, only water molecules are able to pass through an osmotic membrane. That is when 100% of the solutes are rejected by the membrane (ơ ¼1). However, due to the imperfect nature of the membrane solutes in both draw and feed solutions also diffuse through the membrane in opposite directions (ơo1). Therefore, in addition to producing a higher water flux, for a faster and more efficient process a draw solution should have a low solute flux towards the feed solution as well. Hancock et al. [36] compared the ratio of the reverse solute flux (i.e. flux of the solutes from the draw into the feed solution) to the water flux of three different draw solutions (NaCl2, MgCl2 and NH4HCO3) through 2 different cellulose acetate osmotic membranes. They called this ratio as the specific reverse salt flux (SRSF), and demonstrated that at the same osmotic pressures, MgCl2 has the minimum SRSF (at least 55% less than NaCl) while NH4HCO3 has the maximum one (at least 10 times more than NaCl).
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Another technique for increasing the flux is to minimize the effect of the external CP. Using flow mode in an FO process is an established approach recommended for diminishing this effect [23]. In this mode, the solutions at both sides of the membrane should be pumped at the same flow rates. Loeb et al. [37] proposed that counter flow is an important factor for increasing the flux by forming a higher osmotic pressure difference at all points along the membrane. It is also reported that increasing the flow-rate can help preventing the accumulation of the solutes in the feed side, and water in the draw side of the membrane. It can also lower the fouling effect. Therefore, the water flux can be enhanced by increasing the flow-rate [38]. Increasing the temperature can also increase the flux by decreasing the viscosity of the draw solution and increasing the diffusion coefficient of the water passing through the membrane [18]. A drawback of increasing the temperature is that it can cause a decrease in the reflection coefficient of the membrane [39]. Thus, some solutes which were rejected by the membrane at lower temperatures will pass through the membrane at higher temperatures.
3. Theory of operation Considering all the factors described in the theoretical background, we developed an osmosis-based system. The schematic design of the system is presented in Fig. 1. The operation of all components shown in Fig. 1 is controlled by a microcontroller. In addition, a graphical user interface is programmed with Labview to provide an easy control over all actuators and sensors of the system. A photo of the whole automated system is shown in Fig. 2. Before an environmental sample can be applied into the system, the sample needs to be filtered to remove bacteria, viruses, microorganism and unwanted particles. This filtration can be done using a 0.2 mm syringe filter. The filtered water sample and a concentrated standard cell-culture medium are pumped into the feed reservoir (FR). These two solutions are mixed together drop-by-drop at the inlet of the FR. The volume ration of the two solutions can be regulated by controlling the flow-rate of the corresponding pumps. The custom-made liquid trackers ensure the presence of liquid in both tubes entering the FR before being mixed. The working principle of these sensors is based on measuring the intensity of the transmitted light through the tube using an optical set-up
consisting of a light emitting diode (LED) and a photodiode. If there is no liquid detected by at least one of the liquid trackers within 5 min after starting the pumps (1) and (2), the process will stop, and an error will appear on the graphical user interface indicating that at least one of the reservoirs is empty. Upon accumulation of the mixed solution inside the FR, the attached CLS measures the liquid level inside the reservoir, and sends the value to the microcontroller. As the feed solution reaches a certain volume, the microcontroller stops pumps (1) and (2). Simultaneously, it opens valve (1), and pump (3) starts pumping the solution from the FR towards the osmotic chamber. At the same time, using pump (4), the draw solution is directed through valve (2) to the opposite side of the membrane, and exits the osmotic chamber towards the waste container. In this paper, the aim was to obtain a final osmolality of 330710 mmol/kg, and an osmolality of 220 mmol/kg was chosen as the initial value for the feed solution. The designed osmotic chamber contains two identical fluidic channels that are separated by the osmotic membrane. This device is explained in Section 3.1. MgCl2, one molar (1 M), is the draw solution, and the feed solution is the mixture of the water sample and concentrated cell-culture medium being accumulated in the FR. Both solutions are pumped at equal flow-rates, and they flow in opposite directions inside the chamber. Due to osmosis water transfers from the low concentrated side towards the high concentrated side. Valve (3) redirects the feed solution back into the FR. The feed solution circulates inside the osmotic chamber until the level of the feed solution inside the FR equals the initial volume of the water sample before mixing with the concentrated solution. At this stage, the excess water that was added to the sample in the mixing process is transferred to the draw side, and the pretreatment is terminated. Subsequently, valve (3) directs the treated sample towards the final reservoir, and valve (2) directs air into the draw side of the chamber to pump out the remaining draw solution inside the osmotic chamber. Prior to introducing the treated sample into the cell-based analyzing biosensor, the temperature of the sample should be adjusted to the appropriate value for the living cells. In order to use the system without a computer, a default procedure with automatic switching of the valves and predefined values for the pumping rate and the CLS level is stored in an integrated erasable programmable read only memory (EPROM). This procedure can be started and terminated by pressing the “Start” and “Stop” buttons shown in Fig. 2.
Fig. 1. Schematic design of the system. The pumps and the valves are shown with different signs and named by numbers. The arrows indicate the direction of the flow through the system. The red line inside the osmotic chamber represents the osmotic membrane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Photo of the complete automated system. Colored solutions are used for demonstrating different liquid reservoirs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Herein, we present an example to describe the calculations required for defining the system0 s set up for the osmolality adjustment of a sample obtained from the Lake of Neuchatel in Switzerland. The Swiss environmental waters have a negligible osmolality of below 10 mmol/kg. In order to adjust the osmolality of a sample with such low osmolality to a standard value of 330 (required for Caco-2 cell cultures), the sample is mixed with a concentrated culture medium to first obtain an osmolality of 220 mmol/kg for the feed solution. The following equation was applied to determine the ration of the sample and the concentrated cell-culture medium. ðV s þ V c Þ 220 ¼ V c N 330;
N41
ð5Þ
or Vs ¼
3 N1 Vc 2
ð6Þ
where Vs and Vc are the volumes of the applied sample and the added concentrated culture medium, respectively. N represents the concentration coefficient of the added concentrated culture medium. For example, using a double concentrated solution (N ¼2), Eq. (6) results in: V s ¼ 2V c
ð7Þ
Thus, using a double concentrated culture medium, for the osmolality adjustment of a 4 ml sample, first it has to be mixed with a 2 ml concentrated culture medium forming the feed solution accumulated in the FR. Afterwards, the feed solution has to circulate in the osmotic chamber until 2 ml water is transferred to the draw side upon the FO process. Removing this 2 ml water prevents the dilution of the sample ingredients such as potentially present toxicants that are needed to be detected by the cell-based biosensor.
3.1. Osmotic chamber As it is described above, the heart of the system is the osmotic chamber where the water concentration of the sample is adjusted.
An osmotic chamber integrating a commercial osmotic membrane was designed for this system. Two parallel fluidic channels were formed inside the osmotic chamber by mechanical machining in two identical poly methyl methacrylate (PMMA) blocks. Each PMMA block integrates one fluidic channel (4 mm width, 10 cm length). The two parallel fluidic channels are positioned against each other holding the osmotic membrane in between, and fixed with screws. To improve the sealing, gaskets were applied on both sides of the membrane. A schematic design of one of the PMMA blocks and a photo of the chamber after assembly with the PMMA blocks are shown in Fig. 3. The surface area between the draw and feed solutions in the osmotic chamber is 400 mm2. The osmotic membrane has an asymmetric structure with a polyester screen mesh that strengthens the membrane. Therefore it could be applied for long term experiments (i.e. up to 4–6 h), and even reused for several times.
4. Materials and compartments The commercial osmotic membrane (HTI OsMem™ CTA-ES) used in the osmotic chamber was provided by Hydration Technology Innovations (USA). The CLS (S 15N, BA 116329), the 3-way "Y" solenoid valves (LFYA1203032H) and the piezoelectric pumps (mp6, 2011510163) were purchased from Sensortechnics (Germany), Lee (USA) and Bartels Mikrotechnik (Germany) respectively. The LED used for the liquid trackers was LRP47F purchased from Osram (Germany), and the photodiode was TEMD 7000 01 from Vishay Semiconductors (USA). The cell-culture medium was High Glucose Dulbecco0 s Modified Eagle Medium (DMEM) purchased from Life Technologies (USA). The concentrated cell-culture medium was prepared by removing water from the standard one using FO in the second generation of the osmotic chamber. For this concentration, the standard medium was applied as the feed solution, and MgCl2 (1 M) was applied as the draw solution. MgCl2 6H2O, D(þ)-glucose anhydrous and CA were purchased from Merck (Germany), Sigma-Aldrich (Switzerland) and Eastman Kodak (USA) respectively. The microcontroller was MPS430 16 bit from Texas Instruments (USA), and EPROM 24LC256 was purchased from
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Fig. 4. Effect of flow-rate on water-flux when both feed and draw solutions were pumped at equal flow-rates. The maximum water-flux was obtained at 400 ml/min.
Fig. 3. Second generation of the osmotic chamber. (a) Schematic design of each PMMA block of the osmotic chamber. (b) Photo of the assembled osmotic chamber.
Microchip (USA). Sylgards 184 was purchased from Dow Corning (USA), and SU-8 50 was purchased from Microchem (USA). Osmolality and pH of the samples were measured by Vapros 5600 vapor pressure osmometer (Wescor Inc., USA) and Orion model 610 pH meter (USA) respectively.
5. Results and discussions For characterization of the system, 4 ml filtered water samples (osmolality: 4 mmol/kg, pH of 7.8), and 2 ml two times concentrated cell-culture medium (osmolality: 660 mmol/kg, pH: 8.5) were applied, and mixed in a ratio of 2:1. This mixture with osmolality of 220 mmol/kg formed the feed solution. The draw solution was MgCl2 with the osmolality of 3500 mmol/kg. In the following, we present the results obtained using the second generation of the osmotic chamber. The water samples were obtained from the Lake of Neuchatel in Switzerland. 5.1. Effect of flow-rate As it is explained in the theoretical background (Section 2), in FO the use of counter flow and increasing the flow-rate are effective techniques for increasing the water flux through the membrane. However, in small systems, when the feed and draw solutions0 velocity is at least ten times higher than the pure water flux across the membrane, the influence of the flow directions is insignificant. In order to determine the maximum water flux in our system, the water flux was measured in a series of experiments
Fig. 5. Monitoring osmolality and pH of the feed solution over time when both feed and draw solutions were pumped with the flow-rate of 400 ml/min. The osmolality of the feed solution increases over time while the pH remains almost stable.
upon pumping both feed and draw solutions at equal flow-rates in opposite directions. The results are shown in Fig. 4. The error bars in all the graphs presented in this paper demonstrate the standard deviation of three measurements around the average value. As one can see in this graph, the flux does not increase in a linear manner. Applying the flow, washes away the accumulated solutes and water at the feed and draw side of the membrane respectively, and causes an increase in the osmotic pressure difference across the membrane. When the flow rate is high enough to avoid this accumulation, the water flux reaches a saturation point. In the presented system, the maximum water flux of 11.1 L m 2 h 1 was obtained at flow rates of 400 ml/min which is equal to 0.006 ml/s. The surface area of the membrane between the two solutions is 400 mm2. This results in a maximum water flux of 0.001 ml/s across the membrane when both solutions are flowing at 0.006 ml/s. Therefore, the velocity is only 6 times (i.e. o10 times) higher than the water flux. This implies that the effect of the flow direction is not completely insignificant in the presented device, and counter flow can still have an influence on improving the water flux.
5.2. Osmolality and pH fluctuations over time Applying the flow-rate of 400 ml/min (in which the maximum water flux was observed), the osmolality and pH variations of the feed solution were measured over time. As it is shown in Fig. 5, the
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osmolality increased from 220 mmol/kg to 350 mmol/kg in 48 min. However, the pH remained almost constant. The stability of the pH shows that mixing the sample with the concentrated cell-culture medium adds enough buffering substances to the feed solution for keeping the pH stable. As the required osmolality value for the specific cell-based system was 330 mmol/kg, Fig. 5 determines that using the presented osmoticbased system and a double concentrated culture medium, the pretreatment of a 4 ml sample can be performed in 30 min. The applied draw solution is almost 15 times more concentrated than the feed solution. Therefore, according to the FO process, continuously, the feed solution loses water to the draw side. However, due to the fouling of the membrane, and the increase in the osmotic pressure of the feed side, the water flux is decreased over time. 5.3. Influence of temperature Another factor that was studied for increasing the water flux is the temperature. As we already mentioned in Section 2, increasing the temperature increases the water flux. In cell-based sensors, depending on the cells0 type, usually, the final treated sample should be at a temperature of 37 1C when being brought into contact with the living cells. Therefore, the impact of temperature on the water flux, osmolality and pH of the final treated samples was studied by performing the whole process of treatment both at room temperature (25 1C) and 37 1C. The results are compared in Fig. 6. Fig. 6a demonstrates that increasing the temperature from 25 1C to 37 1C increased the water flux by 35%. On the other hand, the pH values of the final treated samples were almost equal to the pH value of the standard cell-culture medium. In Fig. 6b the osmolality of the feed solution and the final treated samples at both temperatures are compared. As we can see in this graph, after mixing the sample with the concentrated cell-culture medium, the osmolality of the feed solution was reduced to 220 mmol/kg. Ultimately, at both temperatures the osmolality of the final treated samples was adjusted to the osmolality value of the standard cellculture medium (330 mmol/kg). Overall, the required time for the whole process of treating 1 ml of sample was 5.5 min and 7.5 min at 37 1C and 25 1C, respectively. An important issue that was observed upon increasing the temperature was a decline in the reflection coefficient of the membrane. As it is also mentioned in the theoretical background (Section 2) the reflection coefficient of osmotic membranes can change with temperature. At higher temperatures although a higher water flux will be obtained, at the end of the process, the concentrations of the solutes in the feed and draw solutions will not be the same as the ones treated at a lower temperature. In the sample pretreatment for cell-based studies it is more critical to adjust the osmolality to the desired value rather than
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controlling the concentration of specific ingredients precisely. A cell-culture medium is a combination of many chemical components. As it was not practical to monitor the changes in the concentration of all components, the concentration of glucose as one of the main ingredients was studied. It was observed that at 37 1C, the concentration of glucose in the final treated sample was lower than the one treated at 25 1C. However, the osmolality of the final treated samples at both 25 1C and 37 1C was 330 mmol/kg. Obtaining the same osmolalities despite the higher diffusion of glucose at 37 1C from the feed solution to the draw solution is possibly due to a similar higher diffusion of MgCl2 from the draw solution into the feed solution at this temperature. The glucose concentration of the treated samples was measured by our previously reported glucose sensor [40] and compared to the glucose concentration of the standard cell-culture medium. The results are shown in Fig. 7. This graph illustrates that, respectively, 9% and 22% of the feed0 s glucose concentration was lost for the sample treatment at 25 1C and 37 1C. Occasionally, for adjusting the osmolality of the samples without using this system, 10 times concentrated cell-culture medium was added to the sample with the ratio of 1:9, that results in 10% dilution of sample ingredients [41]. Using the osmosis-based system less dilution occurred at 25 1C by adding only 2 times concentrated cell-culture medium. Using higher concentrated media, less water has to be removed from the feed solution in the treatment process. This will lead to a faster process, and therefore, less diffusion of the solutes through the membrane. Therefore, despite of the imperfect quality of the osmotic membrane, and the unwanted diffusion of the solutes through the membrane, we claim that using the osmosis-based system can avoid the sample dilution significantly.
Fig. 7. Effect of temperature on the final concentration of glucose in the final treated sample.
Fig. 6. Effect of temperature. (a) Effect of temperature on the water flux and pH of the final treated samples and (b) effect of temperature on the osmolality of the final treated sample.
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5.4. Multitask system The presented example for adjusting a sample osmolality was performed for a Swiss environmental sample with an initial osmolality close to zero. The negligible osmolality value helped estimating the initial osmolality value of the feed solution as the sample was mixed with a known concentration of the concentrated culture medium. For using this system, it is not essential to know the exact osmolality of the sample. In general, for adjusting the osmolality of a feed solution, a solution with the designated osmolality value can be applied as the draw solution. By keeping the osmolality of the draw side of the membrane constant, due to the FO process, the osmolality of the feed side will be adjusted to the desired value over time. When the CLS determines that the total volume of the feed side remains stable, it indicates that the osmolality process is accomplished. In cases that the osmolality of the sample is close to zero, in order to obtain a high osmolality (4 20 mmol/kg) it is required to add solutes into the sample. Therefore, although the exact value of the sample osmolality is not essential, it is necessary to know the approximate value. For example, when the final required value for the osmolality is around 330 mmol/kg, knowing the approximate value of the sample osmolality (e.g. o10 mmol/kg, between 10 and 200 mmol/kg or higher than 300 mmol/kg) helps programming the volume and concentration of the concentrated culture medium that has to be mixed with the sample. In order to reduce the required time for the process, the osmolality of the feed solution needs to be adjusted to a value higher than 50% of the final designated value (e.g. for the final value of 330 mmol/kg, we chose an osmolality of 220 mmol/kg for the feed solution). In the presented example the osmolality of the applied sample was close to zero, and as a result the osmolality of the feed solution was known. Thus, measuring the volume of the feed solution upon the FO process provides the possibility of estimating the osmolality of the feed solution. Therefore, in order to decrease the process duration, a solution with a high osmotic pressure could be used as the draw solution (e.g. MgCl2, 1 M). Overall, the presented system has a high flexibility to be programmed for pretreatment of samples with various initial osmolalities. In addition to adjusting the osmolality of the samples, due to the presence of the buffering substances in the concentrated solution, the pH is also adjusted to the required value. Furthermore, by changing some parameters, the operation of the osmosisbased system is flexible to be adapted for other applications as well. For example, by pumping the standard cell-culture medium as the feed solution and MgCl2 as the draw solution, we used the same system for concentrating the cell-culture medium. For two times concentration, the microprocessor was programmed to stop the circulation of the feed solution in the osmotic chamber as soon as the CLS detected the liquid level corresponding to the half volume of the initial feed solution. A similar process can be applied for pre-concentration of different samples. To avoid the loss of any ingredients of the feed solution due to the unwanted diffusion, changing the formulation of the draw solution can be helpful. In cases that the concentration of the ingredient is known, by having the same or smaller concentration of the ingredient in the draw solution, the diffusion of that specific substance will be avoided or reduced.
6. Conclusions In this paper we introduced a portable low-cost system for adjusting the osmolality and pH of an aqueous sample, on-line and automatically. In this system forward osmosis was exploited as a
well-established process to manipulate only the water content of the sample without denaturizing the ingredients after adding all the necessary substances. We demonstrated the application of this system for adjusting the osmolality and pH of Swiss environmental water samples that are to be evaluated by a cell-based biosensor. Controlling the osmolality and pH of such samples is imperative before applying them to a cell-culture. We studied the influence of flow-rate and temperature on the water flux that ultimately changes the process duration. The maximum water flux was provided by applying the feed and draw solutions at 400 ml/ min in opposite directions. By increasing the temperature from 25 1C to 37 1C, the water flux was increased by 2.9% per degree Celsius while the pH was stable. Using this system the turnaround time for pretreatment of 1 ml sample was 7.5 min and 5.5 min at 25 1C and 37 1C, respectively. At the higher temperature, a decline in the reflection coefficient of the membrane was observed. However, although the quality of the osmotic membrane is not perfect, the presented osmosis-based system prevents the sample dilution significantly compared to the conventional sample pretreatment techniques.
Acknowledgements Special thanks to Martial Geiser from HES-SO Valais, Sion Switzerland, Jules Hajj from the University of Saint-Joseph, Beirut Lebanon and Edward G. Beaudry from the Hydration Technology Innovations, Oregon, USA for their valuable discussions. The osmotic membranes were kindly provided by the Hydration Technology Innovations. The authors gratefully thank the staff at the Microsystems Technology Division of the CSEM for their technical assistance. This work was supported by the NanoTera “Livesense” project funded by the “Swiss National Science Foundation” (SNSF).
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