Preparation and characterization of nanosized P(NIPAM-MBA) hydrogel particles and adsorption of bovine serum albumin on their surface
© Zhu et al.; licensee Springer. 2012
Received: 27 August 2012
Accepted: 16 September 2012
Published: 24 September 2012
Thermosensitive polymer hydrogel particles with size varying from 480 to 620 nm were prepared through precipitation copolymerization of N-isopropylacrylamide with N,N′-methylenebisacrylamide (MBA) in water with ammonium persulfate as the initiator. Only polymer hydrogels without any coagula were obtained when MBA concentration in the monomer mixture was kept between 2.5 and 10.0 wt%; with increased MBA concentration, the monomer conversion was enhanced, the size of the hydrogels was increased, and their shrinking was lessened when heated from 25°C to 40°C. Bovine serum albumin adsorption on the surface of the hydrogels of different MBA content was measured at different pH levels and under different temperatures. The results demonstrated that the adsorption of the protein on the hydrogels could be controlled by adjusting the pH, the temperature of adsorption, and the crosslinking in the hydrogels. The results were interpreted, and the mechanisms of the polymerization were proposed.
KeywordsN-isopropylacrylamide Precipitation polymerization Hydrogel nanoparticles Bovine serum albumin
Poly(N-isopropylacrylamide), PNIPAM, has been known as a thermosensitive material. It exhibits a volume phase transition temperature (VPTT) around 32°C. Heskins et al.  were probably the first to report a study on its solution behaviors and ascribed the thermodynamic properties to an entropy effect. Since then, numerous studies on PNIPAM and its related polymers have been reported [2–14], and these materials have been widely used in different areas of high technology, particularly in biomedical areas. Since the report by Pelton et al. , the latex particles of PNIPAM and its copolymers have been used as supports for various biomolecules including proteins , enzymes , granulocyte , oligodeoxyribo-nucleotide  and antibodies . They are also used to the controlled drug delivery systems . Magnetic latex particles of these materials and their application in biomedical field have been also reported .
Nano- or microgels of PNIPAM and of its copolymers can be prepared by heterophase polymerizations with different crosslinkers in aqueous phase. The possible processes include precipitation polymerization, water-in-oil emulsion polymerization , and micro-emulsion polymerization . Among these, precipitation polymerization is probably the most frequently used technique [14–17]. Nagaoka et al. synthesized PNIPAM hydrogels by radiation polymerization . Kawaguchi et al.  prepared monodisperse PNIPAM hydrogel nanoparticles and used them as adsorbent for human gamma globulin. Through copolymerization of NIPAM with ionic monomers, pH and temperature sensitive hydrogels were also obtained, and this way the VPTT [19, 20] of the resultant materials was often shifted to a higher temperature. Kratz et al. prepared anionic hydrogels through copolymerization of NIPAM with acrylic acid . Elaissari et al. prepared cationic hydrogels by copolymerization of NIPAM with aminoethyl methacrylate hydrochloride in presence of a cationic initiator .
In a previous work, we have prepared monodisperse particles of poly(trihydroxy-methyl propane triacrylate-styrene), i.e., P(TMPTA-St), in ethanol and ethanol/water mixture [23, 24]. In this article, precipitation polymerization of NIPAM in water with N,N′-methylenebisacrylamide as the crosslinker and ammonium persulfate as the initiator was conducted. Thermosensitive hydrogel particles with sizes of 480 nm or larger were prepared, and adsorption of bovine serum albumin (BSA) on their surface was also studied.
Before use, N-isopropylacrylamide (NIPAM, CP grade, from TCI Development Co., Ltd., Shanghai, China) was recrystallized in hexane (AR, Tianjin Fuyu Fine Chemicals Co., Ltd., Tianjin, China), N,N′-methylenebisacrylamide (MBA, AR, Tianjin Kemiou Chemical Reagent Co., Ltd., China) recrystallized in methanol (AR, Tianjin Fuyu Chemicals), and ammonium persulfate (APS, AR, Tianjin Fuyu Chemicals) in water, respectively. Bovine serum albumin (AR, Shanghai Lanji Science and Technology Development Co., Ltd., China; M w = 67 kDa; isoelectric point, IEP 4.7) was stored in a refrigerator. Coomassie Brilliant Blue (G-250, AR, Shanghai Yixin Chemical Co., Ltd., Beijing, China) was used as received.
Preparation of hydrogel particles based on NIPAM
Hydrogel particles of P(NIPAM-MBA) were prepared by free radical precipitation polymerization. In a typical run, 1.8 g of NIPAM, 0.2 g of MBA, the crosslinker monomer, and 90.0 mL of deionized water were charged into a glass bottle of 120-mL capacity, which was located into a thermostat shaker at room temperature and shaken until the complete dissolution of the monomers, followed by N2 purge of the reaction system for 5 min and addition of 0.04 g of APS dissolved in 8 mL of water. The temperature of the thermostat shaker was then rapidly risen to 70°C within 1 h while keeping the shaking at 120 osc·min−1. The reaction was allowed to run for 6 h. The resultant polymers were separated out by repeated centrifugation at room temperature and washing with deionized water three times. Hydrogel particles with different monomer composition were prepared by varying the crosslinker amount.
Adsorption and desorption of BSA
To test the adsorption of BSA on the hydrogel particles at different pH levels, 1.5 g of dried particles was added into 150 mL of BSA aqueous solution of 0.5 g·L−1 concentration. The mixture was shaken up and evenly divided into three portions. Every portion was put in a bottle of 50 mL capacity with their pH adjusted respectively to 4, 7, and 11. The bottles were located into a thermostat shaker with temperature set at 40°C. BSA adsorption was processed for 2 h at a shaking speed of 120 osc·min−1. Of the mixture, 20 mL was taken out from the bottle and centrifuged for 5 min at 12,000 rpm to separate the particles out from the mixture. Of the supernatant, 400 μL was added into a 4-mL aqueous solution of Coomassie Brilliant Blue G-250 of known concentration. The amount of BSA remaining in the supernatant was determined by ultraviolet spectrophotometer (UV-2450, Shimadzu Corporation, Japan), and the amount of BSA adsorbed was thus obtained. BSA adsorption at 20°C was conducted by lowering the temperature of the mixture at the end of the adsorption at 40°C to 20°C, a temperature below the VPTT of the hydrogels, where they became hydrophilic and were highly hydrated. The bottles were kept shaken for another 2 h. The amount of BSA desorbed was determined the same way as described above.
At end of the polymerization, polymers were present under two forms: polymer particles and that remaining soluble in water. The yield of the polymer particles was determined gravimetrically by weighing the dried polymer powder collected upon centrifugation, and that of the soluble polymers was obtained by drying the supernatant. The sum of the two yields was considered as the total monomer conversion. Morphology of the polymer particles was examined using scanning electron microscope (SEM, QUANTA FEG-250, FEI Company, USA), and their number average size (Dn) was calculated by counting about 200 particles on the SEM pictures. Their size and size distribution (PDI) were also measured using a dynamic light scattering instrument (Nano-ZS, Malvern Instruments Ltd., UK). Variation of light transmittance of the samples at different temperature was followed using a photometer (Mod-662, Metrohm Ltd., Switzerland) at 565-nm wavelength.
Results and discussion
Effect of MBA content on particle formation and morphology
Precipitation polymerization of NIPAM-MBA at 70°C with different MBA amount
Particle yield (%)
Soluble polymer (%)
Monomer conversion (%)
It was found that stable latex was obtained when 2.5 wt% of MBA was used, and the resultant latex remained stable even after centrifugation at 12,000 rpm for 1 h. With MBA content increased to 5.0 wt%, the polymer precipitated out from the reaction mixture; phase separation occurred immediately upon the bottle shaking was stopped. Coagulation appeared when the MBA amount further increased to 12.5 wt%. It is commonly agreed that particle nucleation in this precipitation polymerization is done by oligomers grown up to a critical length, where they become insoluble and precipitate out to form the primary particles. This nucleation proceeds quite fast, and abundant primary particles can be formed in a short period. Once this period of nucleation is completed, particle growth is continued by an entropy mechanism as proposed by Stöver et al. , by which the monomers and the oligomers, prior to reaching their critical length, polymerize with the residual double bonds on the surface of the particles, instead of by adsorption of oligomers after reaching their critical length (i.e., by enthalpy mechanism).
As described in the ‘Methods’ section, the size of the particles was determined through dynamic light scattering at 25°C (Table 1, D1) and 40°C (D2), two different temperatures across the VPTT (about 32°C) of the hydrogels. It is obvious that the particles at 25°C were fully swelled, owing to strong interaction of their polymer chains with water; and at 40°C, the particles were deswelled to squeeze water molecules out because they were at the temperature above their VPTT. The particle size determined at 25°C (D1) was, therefore, significantly larger than that at 40°C (D2). Table 1 listed also the particle size obtained from SEM (Dn). The result demonstrated that Dn was slightly smaller than the hydrodynamic size of the particles at deswelled state (D2) regardless of MBA amount used. This has been often observed and attributed to the fact that these particles possess hydrophilic polymer segments or end chain groups on their surface. It is commonly known that light scattering in determination of latex particles does not make distinction between the veritable polymer particles and the hydrated layer on their surface; the size of the particles, thus, determined is therefore larger than their size at dry state obtained from TEM or SEM [26, 27]. Obviously, the size value determined at 40°C (D2) in this work reflected the real size of the particles, i.e., the hydrogel particle size from SEM (Dn). The size of the particles was of nano- or submicron scale.
Results in Table 1 demonstrated that, independent of the methods used for their determination, the size of the nanospheres was increasing with MBA content in the monomers, in agreement with the reported observation . This increase in particle size can be also interpreted based on the mechanisms of particle nucleation and growth. It is believed that the use of a crosslinker monomer, MBA in the present case, promoted significantly the increase in molecular weight of the oligomers, and therefore accelerated the formation of the primary particles. However, coalescence or combination of the primary particles might well occur when a too large number of primary particles was formed in a short period of time . This coalescence would result in particles with larger size, reducing the number of particles in the polymerization system, accompanied by formation of no spherical particles, particularly if a high concentration of crosslinker monomer was used and this coalescence occurred at high monomer conversion, where the subsequent polymerization of the remaining monomers was not enough to smooth the coalesced primary particles. Based on this consideration, the increase in particle size with increase in MBA is well understood so is the decrease in the number of particles (Np).
Data in Table 1 reveal that lower particle yields were observed with lower MBA content. This yield was slightly enhanced with increased MBA up to 7.5 wt% and remained practically unchanged afterwards. It is known that the use of crosslinker is a must in the precipitation polymerization, aiming at fabrication of uniform particles [23, 24, 29]. Crosslinker monomers greatly boost the molecular weight of the oligomers and promote the formation of the primary particles at beginning of the polymerization. Without the use of a crosslinker monomer, oligomers remain soluble in the polymerization medium for a longer time, and their accumulation leads often to coagula formation with progress of polymerization. With 2.5 wt% of MBA, the lowest in all the runs given in Table 1, a lowest particle yield of 87.14% was obtained along with a highest yield of soluble polymers of 12.35%. This was in good agreement with the above mechanisms of particle nucleation and growth. The decrease in particle yields was compensated by an increase in soluble polymers, and by consequence, high monomer conversion was achieved for all runs regardless of the MBA amount used.
It is also worth to note that abundant interlinkage was present between the nanospheres (Figure 1, inserted photos in particular), a distinct difference from the polymer particles prepared through emulsion polymerization. This observation was believed to be caused by the relatively heavy crosslinking in the particle polymer, and therefore attributable to the use of MBA in the polymerization. It is obvious that PNIPAM alone cannot be crosslinked. Polymer chains extending to water phase without crosslinking, if any, must be closely packed on the surface when drying up. It was reported that MBA polymer was more hydrophilic than NIPAM [14, 30], which may result in the particles with higher crosslinking at their surface layer rather than in their core. This MBA enrichment on the surface of the particles contributed to the formation of the interlinkage between the particles.
Thermo-sensitive properties of P(NIPAM-MBA) particles
It is seen that, below 10°C, the light transmittance in all the samples was 100%, regardless of the MBA content in the copolymers, indicating that all samples were transparent liquid and that the particles were fully swelled by water. However, when the temperature was risen to 15°C and above, the transmittance of the samples started to decline at rates dependent on the particle composition. PNIPAM homopolymer is known to have a VPTT at 32°C [2, 14]. Figure 2 demonstrated that incorporation of MBA rendered a broadened VPTT and raised its average value in the cross-linked copolymers.
For thermosensitive hydrogels, increasing the temperature from a point below its VPTT, where the particles are fully swelled with water, is a process in which the water molecules contained in the particles are expelled out due to the disruption of hydrogen bonding between water molecules and the hydrophilic amide groups, causing at the same time the packing up of the hydrophobic isopropyl groups in the polymer chains. This process makes the particles shrink to a smaller size. In Table 1, the diameters of the particles at 25°C (D1) and 40°C (D2) were determined for each samples of different MBA, which indeed confirmed the particle volume shrinking. The shrinking ratio, D1/D2, was rapidly decreasing with the increase in MBA, obviously owing to the increased crosslinking in the particles.
The influence of MBA amount was also clearly seen in Figure 2. While the sample with 2.5 wt% of MBA, the lowest among the four samples, was characterized by a most rapid decline in light transmittance reached a lowest value in a shortest time, the one with 10.0 wt% of MBA, the highest MBA amount, was characterized by a slowest decline with a highest final value of light transmittance reached in a longest period of time. The corresponding curves for the other samples lay in between these two samples, in the order of their MBA content. As aforementioned, the observed decline of the light transmittance was a reflection of the dehydration in the water-swollen hydrogel particles, owing to disruption of hydrogen bonding between water molecules and amide groups on polymer chains. This dehydration proceeded the way likely to squeeze water out from the fully hydrated particles by packing up more tightly the polymer chains. It is easy to conceive that the water squeezing was easier and more complete in a particle with lessened crosslinking, and it became harder when the particles were crosslinked at a higher degree; and there was also less water available at start of the experiment (at 5°C) due to the high crosslinking. This was exactly the case shown in Figure 2.
Evolutions of particle and soluble polymer yields during polymerization
Influence of initiator concentration
Precipitation polymerization of NIPAM-MBA with varied APS levels
Particle yield (%)
Soluble polymer (%)
Monomer conversion (%)
Adsorption and desorption of BSA on poly(NIPAM-MBA) hydrogel particles
All these results demonstrate that the adsorption of BSA molecules could be controlled by adjusting the crosslinking of the polymers in the particles, the pH, and the temperature of the experiments.
Nano and micro-sized hydrogel particles based on P(NIPAM-MBA) were prepared by precipitation polymerization in water at 70°C. Results revealed that the monomers polymerized very fast, the conversion reached about 95% within 10 min, and the proportion of the soluble polymers dramatically dropped down to about 12% at 1 h of polymerization time. The yields of the particles and their size were increased with MBA increase up to 7.5 wt%. Incorporation of MBA in the thermosensitive material caused a broadened VPTT and raised its average value in the cross-linked copolymers. Size determinations of the particles at temperatures above and below their VPTT confirmed a volume shrinking for the particles above their VPTT and the shrinking was rapidly weakened with increase in MBA, owing to the obviously increased crosslinking in the particles. As to the influence of the initiator APS on the polymerization, an obvious remark was that monomer conversion and the soluble polymers were both gradually increasing in accordance with increase in APS. Tests on BSA adsorption revealed that the amount of BSA adsorbed on the particles above its VPTT was always higher than that below. BSA adsorption was also enhanced at pH below the IEP of BSA. However, BSA adsorption decreased with increased crosslinking in the particles regardless of the pH. While the decrease was not important when MBA increased from 5.0 to 7.5 wt%, it was significant with MBA increased from 7.5 to 10.0 wt%.
XZ received her BSc degree in Chemistry in 1996 and MSc degree in Polymer Chemistry and Physics from Shandong University in 1999. After being employed as a lecturer in Shandong Institute of Light Industries for 3 years, she continued her study in Shandong University and obtained her PhD degree in Materials Science in 2005. She is currently an associate professor at the University of Jinan. Her main research interests focus mainly on preparations of functional polymer microspheres with different shapes through emulsion polymerization and precipitation polymerization, and aqueous dispersions of polyurethanes modified with polysiloxanes and/or polyacrylates. She has published 60 papers and holds four Chinese invention patents.
This work is financially supported by the Natural Science Foundation of China (NSFC; grant nos.: 20904016 and 21274054) and the Science Foundation of Shandong Province, China (grant no.: Z2008B07).
- Heskins M, Guillet JE: Solution properties of poly(N-isopropylacrylamide). J Macromol Sci 1968, 2: 1441–1455. 10.1080/10601326808051910View Article
- Hirokawa Y, Tanaka T: Volume phase transition in a nonionic gel. J Chem Phys 1984, 81: 6379–6380. 10.1063/1.447548View Article
- Pelton RH, Chibante P: Preparation of aqueous lattices with N-isopropylacrylamide. Colloid Surface 1986, 20: 247–256. 10.1016/0166-6622(86)80274-8View Article
- Kawaguchi H, Fujimoto K, Mizuhara Y: Hydrogel microspheres III. Temperature dependent adsorption of proteins on poly-N-isopropylacrylamide hydrogel microspheres. Colloid Polym Sci 1992, 270: 53–57. 10.1007/BF00656929View Article
- Okubo M, Ahmad H: Adsorption of enzymes onto submicron-sized temperature-sensitive composite polymer particles and its activity. Colloid Surface A 1999, 153: 429–433. 10.1016/S0927-7757(98)00464-6View Article
- Achiha K, Ojima R, Kasuya Y, Fujimoto K, Kawaguchi H: Interactions between temperature-sensitive hydrogel microspheres and granulocytes. Polym Adv Technol 1995, 6: 534–540. 10.1002/pat.1995.220060715View Article
- Delair T, Meunier F, Elaissari A, Charles MH, Pichot C: Amino-containing cationic latex-oligodeoxyribonucleotide conjugates: application to diagnostic test sensitivity enhancement. Colloid Surface A 1999, 153: 341–353. 10.1016/S0927-7757(98)00456-7View Article
- Kondo A, Kaneko T, Higashitani K: Development and application of thermo-sensitive immunomicrospheres for antibody purification. Biotech Bioeng 1994, 44: 1–6. 10.1002/bit.260440102View Article
- Qiu Y, Park K: Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001, 53: 321–339. 10.1016/S0169-409X(01)00203-4View Article
- Guo J, Yang W, Wang CC, He J, Chen J: Poly(N-isopropylacrylamide)-coated luminescent/magnetic silica microspheres: preparation, characterization, and biomedical applications. Chem Mater 2006, 18: 5554–5562. 10.1021/cm060976wView Article
- Sun Q, Deng Y: In situ synthesis of temperature-sensitive hollow microspheres via interfacial polymerization. J Am Chem Soc 2005, 127: 8274–8275. 10.1021/ja051487kView Article
- Braun O, Selb J, Candau F: Synthesis in microemulsion and characterization of stimuli-responsive polyelectrolytes and polyampholytes based on N-isopropylacrylamide. Polymer 2001, 42: 8499–8510. 10.1016/S0032-3861(01)00445-1View Article
- Chen Z, Xu L, Liang Y, Zhao M: pH-sensitive water-soluble nanospheric imprinted hydrogels prepared as horseradish peroxidase mimetic enzymes. Adv Mater 2010, 22: 1488–1492.View Article
- Pich A, Richtering W: Microgels by precipitation polymerization: synthesis, characterization, and functionalization. Adv Polym Sci 2010, 234: 1–37. 10.1007/12_2010_70View Article
- Jańczewski D, Tomczak N, Han MY, Vancso GJ: Introduction of quantum dots into PNIPAM microspheres by precipitation polymerization above LCST. Eur Polym J 2009, 45: 1912–1917. 10.1016/j.eurpolymj.2009.04.005View Article
- Meng ZY, Smith MH, Lyon LA: Temperature-programmed synthesis of micron-sized multi-responsive microgels. Colloid Polym Sci 2009, 287: 277–285. 10.1007/s00396-008-1986-8View Article
- Teng D, Hou J, Zhang X, Wang X, Wang Z, Li C: Glucosamine-carrying temperature- and pH-sensitive microgels: preparation, characterization, and in vitro drug release studies. J Colloid Interface Sci 2008, 322: 333–341. 10.1016/j.jcis.2008.03.014View Article
- Nagaoka N, Safranj A, Yoshida M, Omichi H, Kubota H, Katakai R: Synthesis of poly (N-isopropylacrylamide) hydrogels by radiation polymerization and cross-linking. Macromolecules 1993, 26: 7386–7388. 10.1021/ma00078a046View Article
- Pelton R: Temperature-sensitive aqueous microgels. Adv Colloid Interface Sci 2000, 85: 1–33. 10.1016/S0001-8686(99)00023-8View Article
- Karg M, Pastoriza-Santos I, Rodriguez-Gonzalez B, Klitzing RV, Wellert S, Hellweg T: Temperature, pH, and ionic strength induced changes of the swelling behavior of PNIPAM-poly(allylacetic acid) copolymer microgels. Langmuir 2008, 24: 6300–6306. 10.1021/la702996pView Article
- Kratz K, Hellweg T, Eimer W: Influence of charge density on the swelling of colloidal poly(N-isopropylacrylamide-co-acrylic acid) microgels. Colloid Surface A 2000, 170: 137–149. 10.1016/S0927-7757(00)00490-8View Article
- López-León T, Ortega-Vinuesa JL, Bastos-González D, Elaissari A: Cationic and anionic poly(N-isopropylacrylamide) based submicron gel particles: electrokinetic properties and colloidal stability. J Phys Chem B 2006, 110: 4629–4636.View Article
- Kong XZ, Gu XL, Zhu XL, Zhang LN: Precipitation polymerization in ethanol and ethanol/water to prepare uniform microspheres of poly(TMPTA-styrene). Macromol Rapid Commun 2009, 30: 909–914. 10.1002/marc.200800772View Article
- Gu XL, Zhu XL, Kong XZ, Zhang LN, Tan YB, Lu Y: Preparation of polymer uniform microspheres via precipitation polymerization in ethanol or ethanol-water mixture as new solvent. Acta Chim Sinica 2009, 67: 2486–2494.
- Downey JS, Frank RS, Li WH, Stöver HDH: Growth mechanism of poly(divinyl-benzene) microspheres in precipitation polymerization. Macromolecules 1999, 32: 2838–2844. 10.1021/ma9812027View Article
- Zhu X, Wu L, Kong XZ: Preparation and characterization of PMMA/CA core-shell nanoparticles. Acta Polym Sin 2010, 4: 427–434.
- Zou D, Li X, Zhu X, Kong XZ: Preparation of cationic latexes of poly(styrene-co-butyl acrylate) and their properties evolution in latex dilution. Chinese J Polym Sci 2012, 30: 278–286. 10.1007/s10118-012-1113-7View Article
- Meunier F, Pichot C, Elaissari A: Effect of thiol-containing monomer on the preparation of temperature-sensitive hydrogel microspheres. Colloid Polym Sci 2006, 284: 1287–1292. 10.1007/s00396-006-1514-7View Article
- Zhang Z, Gu XL, Zhu XL, Kong XZ: Preparation and formation mechanism of uniform polymer microspheres in precipitation polymerization of pentaerythritol triacrylate and styrene. Acta Phys-Chim Sin 2012, 28: 892–896.
- Huo D, Li Y, Qian Q, Kobayashi T: Temperature-pH sensitivity of bovine serum albumin protein-microgels based on cross-linked poly(N-isopropylacrylamide-co-acrylic acid). Colloid Surface B 2006, 50: 36–42. 10.1016/j.colsurfb.2006.03.020View Article
- Elaissari A: Thermally sensitive latex particles: preparation, characterization, and application in the biomedical field. In Handbook of Surface and Colloid Chemistry. 3rd edition. Edited by: Birdi KS. CRC Press, Boca Raton; 2009:539–566.
- Shamim N, Hong L, Hidajat K, Uddin MS: Thermosensitive-polymer-coated magnetic nanoparticles: adsorption and desorption of bovine serum albumin. J Colloid Interface Sci 2006, 304: 1–8. 10.1016/j.jcis.2006.08.047View Article
- Cheng X, Canavan HE, Graham DJ, Castner DG, Ratner BD: Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide. Biointerphases 2006, 1: 61–72. 10.1116/1.2187980View Article
- Duracher D, Elaissari A, Mallet F, Pichot C: Adsorption of modified HIV-1 capsid p24 protein onto thermosensitive and cationic core-shell poly(styrene)-poly(N-isopropylacryl-amide) particles. Langmuir 2000, 16: 9002–9008. 10.1021/la0004045View Article
- Ang WS, Elimelech M: Protein (BSA) fouling of reverse osmosis membranes: implications for wastewater reclamation. J Membrane Sci 2007, 296: 83–92. 10.1016/j.memsci.2007.03.018View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.