626 IEEE TRANSACTIONS ON NANOTECHNOLOGY, VOL. 10, NO. 3, MAY 2011

Functionalized Silica Nanotubes As AffinityMatrices for Bilirubin Removal

Cunfeng Song, Aifeng Zhang, Wei Shi, Hairong Jiang, and Dongtao Ge

Abstract—Arginine-immobilized silica nanotubes as affinity ma-trices were fabricated for bilirubin removal. The silica nanotubeswere prepared by depositing silica within the alumina membranethrough sol-gel method. Ligand arginine was immobilized on theinner and outer surfaces of silica nanotubes by activating withglutaraldehyde. The content of arginine of affinity matrices was∼20.1 mg/g nanotubes. Such arginine-immobilized affinity matri-ces were used to adsorb bilirubin from the bilirubin-phosphatesolution and bilirubin-albumin solution. The effects of tempera-ture, ionic strength, albumin concentration, and the adsorptionmechanism were investigated by batch experiments. The resultsshowed arginine-immobilized silica nanotubes achieved excellentadsorption capacity for bilirubin (∼63.6 mg/g nanotubes).

Index Terms—Affinity matrices, arginine, bilirubin, silicananotubes.

I. INTRODUCTION

B LOOD purification is therapeutic for various diseases thatare caused by exogenous or endogenous intoxications [1].

Usually, the toxins are divided into hydrophilic toxins andlipophilic toxins. There are many methods of blood purifica-tion involving removal toxins directly from plasma, includinghemodialysis, hemoultrafiltration, plasmapheresis [2], [3], butmany disadvantages appear in all these therapeutics. Dialy-sis and ultrafiltration membranes only allow hydrophilic smallmolecules to pass through, and they are often not so effective forsmall protein-bound molecules and middle molecules like themiddle chain fatty acids, aromatic amino acids, free phenols,and bilirubin. Plasmapheresis applies extracorporeal, nonspe-cific exchange of plasma with albumin or saline solutions. Thismethod removes most of the blood fluid phase and thereforecan only be used for a limited period of time and in specificclinical situations. Therefore, how to remove toxins, especiallylipophilic toxins, has become an important issue.

Affinity chromatography techniques present an efficientmethod of protein purification due to their ability to separate

Manuscript received May 7, 2010; revised July 6, 2010; accepted July 8,2010. Date of publication July 15, 2010; date of current version May 11, 2011.This work was supported in part by the National Nature Science Foundation ofChina under Grant 30870648, Grant 30870617, and Grant 30500127 and in partby the Natural Science Foundation of Fujian Province under Grant C0510005.The review of this paper was arranged by Associate Editor J. Li.

C. Song, A. Zhang, W. Shi, and D. Ge are with the Biomedical En-gineering Research Center/Department of Biomaterials, College of Materi-als, Xiamen University, Xiamen 361005, China (e-mail: mysongcf@163.com;zaf.8686@163.com; shiwei@xmu.edu.cn; gedt@xmu.edu.cn).

H. Jiang is with the College of Chemistry and Chemical Engineering, XiamenUniversity, Xiamen 361005, China (e-mail: jianghairong@xmu.edu.cn).

Digital Object Identifier 10.1109/TNANO.2010.2059037

almost any biomolecules on the basis of specific interactions be-tween the ligand on the matrices and the ligate (target proteins).At present, affinity chromatography has become a promisingtechnique for blood detoxification. Denizli et al. prepared al-kali blue 6B attached microbeads for the removal of phenol andnitrophenols [4]. Yi et al. synthesized a new chitosan immobi-lized with β-cyclodextrins and described its adsorption ability ofbilirubin [5]. Nevertheless, the existing matrices used for affin-ity chromatography often exhibit a low adsorption capacity;moreover, the pores in the matrices are tortuous, which greatlyenhance the diffuse resistance.

The deficiency of existing affinity matrices has stimulatedinterest to use nanomaterials [6], [7]. Nanotubes prepared bytemplate synthesis have a number of attributes that make thempotential candidates as affinity matrices: 1) nanotubes possess astraight pore structure that can effectively decrease the diffusionresistance of sample molecules in the interior of the matrix; 2)ligands can be immobilized on the inner and outer surfaces bychemical functionalization; 3) nanotubes have a large surfacearea, which creates the possibility of obtaining a high adsorp-tion capacity [7]. The use of nanotubes for blood purification,however, to the best of our knowledge, has not been reported sofar.

In this paper, we utilized functionalized silica nanotubes asaffinity matrices to remove lipophilic toxins, bilirubin, for thefirst time. The silica nanotubes were prepared by depositingsilica within the alumina membrane through sol-gel method.Ligand arginine (Arg) was immobilized on the inner and outersurfaces of silica nanotubes to obtain functionalized affinitymatrices. The preparation method, characteristics, and appli-cability of Arg-immobilized affinity nanotubes were presentedand discussed in detail.

II. EXPERIMENTAL METHODS

Alumina membranes (60-μm thickness, 100-nm poresize, and 47-mm diameter) were purchased from What-man. Tetraethyl orthosilicate (TEOS) was obtained fromShangtou Xilong Chemical Company (China). Bilirubin waspurchased from Shanghai Zhijie Biotechnology Company(China). Bovine serum albumin (BSA) was obtained fromSigma. Ninhydrin H2O, L-Arg and glutaraldehyde were theproducts of Sinopharm Chemical Reagent Company (China).All other chemicals were of analytical grade and used withoutany additional purification. All solutions were prepared usingdeionized Milli-Q water (Millipore).

Refrigerated centrifuge (Beckman Avanti J-25, USA) wasused for the collection of nanotubes. The morphologies of thenanotubes were visualized with the field-emission scanning

1536-125X/$26.00 © 2010 IEEE

SONG et al.: FUNCTIONALIZED SILICA NANOTUBES AS AFFINITY MATRICES FOR BILIRUBIN REMOVAL 627

electron microscopy (FESEM) (LEO, Germany) and Tecnai F30transmission electron microscopy (TEM) (FEI, Eindhoven, TheNetherlands). Fourier-transform infrared spectroscopy (FTIR)(Avatar360, Thermo Nicolet, USA) and X-ray photoelectronspectroscopy (XPS) (PHI Quantum 2000, USA) were used tocharacterize the surface properties of silica nanotubes after eachfunctionalization process. The concentrations of bilirubin andArg were determined using a Beckman coulter DU 800 Nucleicacid/protein analyzer (Beckmancoulter, Inc., USA).

A. Preparation of Silica Nanotubes

Silica nanotubes were synthesized within the pores of aluminamembranes through sol-gel method. The sol-gel silica precursorwas prepared by mixing absolute ethanol, TEOS, and 1 M HCl(50:5:1 by volume) [8]–[10]. This solution was allowed to hy-drolyze for 12 h. The alumina membranes were then immersedinto the above solution 10 min in vacuum to remove air fromthe pores of the membranes. The sol-impregnated membraneswere oven-cured overnight at 120 ◦C. Free-standing nanotubeswere obtained by etching the alumina membranes in 5 M HCl.After thorough washing with ethanol and deionized water, sil-ica nanotubes were centrifuged at 9000 g for 12 min, and thecollected solid was vacuum-heated at 100 ◦C for 3 h.

B. Immobilization of Arg

Silica nanotubes (5 mg) were shaken in 10 mL of 25 wt%glutaraldehyde aqueous solution for 5 h at room temperature.The activated nanotubes were washed by 10-mM phosphate-buffered saline (PBS) and deionized water to removed non-covalently bound glutaraldehyde. Subsequently, the activatednanotubes were incubated in 5 mL of 0.5-M Arg solution in10 mM PBS at pH 7.4 for 8 h. Arg-immobilized silica nan-otubes were recovered by centrifugation at 9000 g for 12 min.Unreacted Arg was removed by washing the nanotubes with 1M NaCl and deionized water extensively.

The amount of Arg immobilized on the silica nanotubes wasassayed by the ninhydrin method. Briefly, the ninhydrin reagentwas prepared by dissolving 50 mg SnCl2 and 400 mg ninhydrininto a mixture of 10 mL dimethyl sulfoxide and 2 mL of 2 Msodium acetate. A sample of 1-mL Arg solution was incubatedfor 15 min in a boiling water bath with 1-mL ninhydrin reagent,and then cooled to room temperature. An ethanol solution[8.0 mL, 50% (v/v) in pure water] was added to the mixture.The optical density of the resulting solution was determined at570 nm wavelength.

C. Adsorption Experiments

The Arg-immobilized nanotubes were tested for the adsorp-tion of bilirubin in 10 mM PBS (pH 7.4) and in bilirubin–BSAsolution by batch experiment. Because bilirubin is easily de-stroyed by exposure to direct sunlight or any other source ofultraviolet light, including fluorescent lighting, all adsorptionexperiments were carried out in a dark room. The amounts of

Fig. 1. Morphology of silica nanotubes. (a) SEM of nanotubes. (b) TEMimage of nanotubes.

bilirubin adsorbed were determined by the following equation:

q =(c0 − ct)v

m(1)

where q is the amount of bilirubin adsorbed onto unit mass ofthe nanotubes (mg/g); c0 and ct are the concentrations of thebilirubin in the initial and in the aqueous phase after adsorption,respectively (mg/L); v is the volume of the bilirubin solution (L);and m is the mass of the nanotubes (g). The concentration of thesolution of the free bilirubin was detected by spectrophotometryat the wavelength of 438 nm and the ones containing bilirubin–BSA complex at 460 nm.

D. Desorption and Regeneration

The bilirubin-adsorbed afnity nanotubes were regeneratedwith a solution containing 0.1 M NaOH and 1.0 M NaCl. Thebilirubin-adsorbed afnity nanotubes were placed in the desorp-tion medium and stirred for 4 h at room temperature at a stirringrate of 100 r/m. After desorption, the nanotubes were washedwith large volume of PBS (pH 7.4) and distilled water. Theregenerated affinity nanotubes were then reused for bilirubinadsorption.

III. RESULTS AND DISCUSSION

A. Characteristics of the Nanotubes

The SEM and the TEM images in Fig. 1 clearly demonstratedthe formation of hollow silica nanotubes within the membranepores. To obtain these images, the silica nanotubes samples weredissolved in ethanol and sonicated to have uniform dispersion.Then the dispersed samples were placed on the copper holderand dried in a vacuum overnight. Fig. 1 showed the silica nan-otubes have uniform diameter (∼100 nm), smooth surfaces, andultrathin wall thickness (∼3 nm). Nanotubes with such a size arepreferred for the application in affinity adsorption of bilirubin,because a high surface area to bind affinity ligands is providedand diffusion resistance of the bilirubin in the nanotubes de-creases effectively, based upon the straight pore structure andappropriate inner diameter.

The sequential steps of the functionalization were proved byFTIR spectroscopy (see Fig. 2). The Si-OH and Si-O-Si asym-metric stretching vibration at 946 and 1088 cm−1 , respectively,the Si-O-Si symmetric stretching vibration at 797 cm−1 , and theO-Si-O bending stretching vibration at 467 cm−1 , correspondingto silica were clearly evident in the spectra [see Fig. 2(a)]. The

628 IEEE TRANSACTIONS ON NANOTECHNOLOGY, VOL. 10, NO. 3, MAY 2011

Fig. 2. FTIR spectroscopy of the nanotubes. (a) Silica nanotubes. (b) Arg-immobilized silica nanotubes.

Fig. 3. XPS C 1 s spectra of Arg-immobilized silica nanotubes.

result confirmed that silica nanotubes were synthesized withinthe pores of alumina membranes by a sol-gel method. FTIR re-sults [see Fig. 2(b)] of the Arg-immobilized affinity nanotubesexhibited the peaks at 1645 cm−1 (C=N stretching vibration)and 1411 cm−1 (−COO stretching vibration), which indicatedthe release of the aldehyde group upon reacting with the aminogroup of Arg.

The surface of Arg-immobilized nanotubes was also ana-lyzed by using XPS (see Fig. 3). C 1 s core-level spectrum ofnanotubes surface exhibited four main components. The mainpeak centered at 284.9 eV was attributed to methylene carbons(−CH2−). The peak appearing around 286.0 eV was the char-acteristic of carbons bound to nitrogen (C−N). The two othercomponents appearing around 287.8 and 288.9 eV were char-acteristic of carbons bound to oxygen. The first one originatedfrom −CHO species (aldehyde group) and the second one from−COOH species (carboxylic acid group). Arg immobilized onthe activated silica nanotubes was further confirmed.

The amount of Arg immobilized on silica nanotubes can bedetermined spectrophotometrically at 570 nm, and the Arg con-tent of the nanotubes was ∼20.1 mg/g. The high Arg content of

Fig. 4. Adsorption rates of bilirubin on nanotubes. Initial bilirubin concentra-tion: 200 mg/L; temperature: 37 ◦C; medium: PBS (pH 7.4, 10 mM).

silica nanotubes can be ascribed to the high surface area of nan-otubes. In order to confirm that Arg was immobilized on boththe inner and outer surfaces of the prepared silica nanotubes,we carried out an experiment that allowed us to attach Arg toonly the inner surfaces of the nanotubes. While silica was stillwithin the pores of the template membrane, the inner surfaceswere activated by glutaraldehyde. The template membrane wasthen dissolved, and the glutaraldehyde on the inner surfaces wasused to immobilize the Arg ligand. The Arg content of these nan-otubes was about 9.6 mg/g nanotubes, which was almost halfthat of nanotubes with Arg immobilized on both the inner andouter surfaces.

B. Adsorption Rate

Albumin is the natural carrier of bilirubin in the blood. Biliru-bin is bound reversibly to albumin in two classes of bind-ing sites, lysine and Arg. Arg residues are closely associatedwith the strong hydrophobic anion-binding sites of serum albu-min [11], [12]. Therefore, we chose Arg as ligand for bilirubinadsorption. Fig. 4 showed the nonspecific and specific adsorp-tion rate curves of bilirubin onto the unmodified silica nan-otubes and Arg-immobilized silica nanotubes, respectively. Asseen here, there were relatively high adsorption rates observedat the beginning, and then adsorption equilibrium was reachedgradually for about 2 h.

The amount of bilirubin adsorption on the unmodified silicananotubes was quite low, only about 1.2 mg/g nanotubes. Whilemuch higher adsorption values up to 63.6 mg bilirubin /g wereobtained in the case of the Arg-immobilized nanotubes. Notethat a wide range of adsorption capacities was reported in lit-erature for bilirubin removal. Denizli and Kocakulak reachedadsorption capacities of 11.7 mg bilirubin /g polymer withCongo Red-modied poly (EGDMA–HEMA) microbeads [13].Sideman et al. proved biliribin adsorption capacity between2–24 mg/g with a macroreticular resin [14]. In our previouswork, we have developed lysine and polylysine immobilizedaffinity membrane chromatography and obtained the adsorption

SONG et al.: FUNCTIONALIZED SILICA NANOTUBES AS AFFINITY MATRICES FOR BILIRUBIN REMOVAL 629

Fig. 5. Effect of temperature on adsorption capacity. Initial bilirubin concen-tration: 200 mg/L; medium: PBS (pH 7.4, 10 mM).

capacities of bilirubin were 17.6–40.7 mg/g [15]–[17]. Compar-ison of these results, Arg-immobilized nanotubes exhibit a highaffinity adsorption toward bilirubin molecules.

C. Influence of the Adsorption Conditions on the AdsorptionCapacity

The effect of temperature on bilirubin adsorption was ob-tained under various temperatures, i.e., 25 and 37 ◦C, as shown inFig. 5. In general, adsorption decreases as temperature increases,because adsorption is an exothermic process. However, in biliru-bin case, it was different as also reported by others [18]–[20].One hypothesis to explain this unexpected behavior is that con-formational changes takes place in the bilirubin molecule. Thebilirubin molecule would change from a cis configuration to atrans configuration with increasing temperature, which in turncauses an increase in the adsorption capacity [21]. Data frombatch experiments also showed an equilibrium time of approxi-mate 2 h.

Fig. 6 presented the effect of the ionic strength on bilirubin ad-sorption, which showed that the adsorption capacity decreasedwith increasing NaCl concentration in the bilirubin solution.The binding of bilirubin to Arg is primarily achieved by elec-trostatic interactions between the positively charged functionalgroups of the positively charged functional groups of the con-stituent amino acids, and the negatively charged carboxyl groupson the bilirubin molecule [22]. When the NaCl concentrationchanged from 0.1 to 0.4 M, the adsorption of bilirubin decreasedby 63.2%. The decrease in the adsorption capacity as the ionicstrength increased can be attributed to weakened electrostatic in-teraction between the Arg-immobilized nanotubes and bilirubinmolecules.

It is generally accepted that bilirubin exists in serum in twodifferent forms: direct and indirect. The direct reacting type isthought to be bilirubin conjugated with glucuronic acid, render-ing it water soluble, while the indirect type is bound to blood pro-tein, albumin. Albumin is composed of 584 amino acid residueswith a molecular weight of approximately 66 000. Each albumin

Fig. 6. Effect of NaCl concentration on adsorption capacity. Initial bilirubinconcentration: 200 mg/L; temperature: 37 ◦C; medium: PBS (pH 7.4, 10 mM).

Fig. 7. Effect of BSA concentration on adsorption capacities. Initial bilirubinconcentration: 200 mg/L; temperature: 37 ◦C; adsorption time: 4 h.

molecule may have as many as 12 binding sites for bilirubin, butonly two sites bind bilirubin molecules tightly [23], [24]. It is re-ported that some sorbents, such as activated carbon, can removebilirubin only from the free or soluble phase, and the removal islimited by the tight binding of bilirubin to albumin [25], [26].Therefore, for successful use in the removal of bilirubin, a lig-and molecule should at least be capable of competing with theweak binding sites on albumin for the unconjugated bilirubin.We studied the effect of concentration of albumin on the adsorp-tion. The result was revealed in Fig. 7. It indicated that existenceof BSA obviously resulted in decrease in adsorption capacity ofthe Arg-immobilized nanotubes for bilirubin, and the adsorptioncapacity for bilirubin decreases with an increase in BSA con-centration. When BSA concentration is greater than 24 g/L, theamount of adsorbed bilirubin becomes insensitive to the albu-min concentration. Thus, Arg-immobilized nanotubes can wellcompete for those bilirubin molecules, which are weakly boundto albumin.

630 IEEE TRANSACTIONS ON NANOTECHNOLOGY, VOL. 10, NO. 3, MAY 2011

Fig. 8. Langmuir adsorption isotherm of Arg-immobilized afnity nanotubesfor bilirubin. Temperature: 37 ◦C; medium: PBS (pH 7.4, 10 mM); initialbilirubin concentration: 25, 50, 75, 100, 150, 200, 300 mg/L, respectively;adsorption time: 4 h.

D. Analysis of the Adsorption Mechanism

Langmuir adsorption isotherms were applied for the de-scription of the adsorption mechanism for bilirubin on Arg-immobilized nanotubes. Langmuir model is based on the as-sumption of adsorption homogeneity, such as equally availableadsorption sites, monolayer surface coverage, and no interactionbetween adsorbed species. The isotherms can be described asfollows:

qe =qmaxbce

1 + bce(2)

where ce (mg/L) is the equilibrium concentration of bilirubin insolution; qe (mg/g) is the adsorption capacity; qmax is the max-imum adsorption capacity (mg/g); and b is a constant. Equation(2) can be transformed to a linear form as follows:

1qe

=1

bqmaxce+

1qmax

. (3)

The data of the adsorption isotherm are presented in Fig. 8. Itcan be seen that the corresponding reciprocal plots (1/ce versus1/qe ) of the experimental data gave a linear plot (R2 = 0.998)for the Arg-immobilized nanotubes. In other words, a linear plotindicates the adsorption homogeneity and the data fit Langmuirmodel very well.

E. Regeneration of the Affinity Nanotubes

Regeneration is a crucial step in all affinity chromatographytechniques [7]. Desorption of the adsorbed bilirubin from theaffinity matrices was studied in a batch system. The adsorbedbilirubin on the nanotubes was removed when 0.1-M NaOHand 1.0-M NaCl solution was used as desorption agent. In orderto show the reusability of the affinity matrices, the adsorption–desorption cycle of bilirubin was repeated five times by using thesame nanotubes. After five cycles, the bilirubin adsorption ca-pacity could attain 59.2 mg/g nanotubes, which only decreased7%. The bilirubin adsorption was still remaining a relatively

high level. Moreover, no obvious changes of the morphologyof the affinity nanotubes were found in the recycling process.These results demonstrated the stability of Arg-immobilizednanotubes as affinity matrices.

IV. CONCLUSION

Affinity matrix of chromatographic plays an essential rolein affinity separation. In this paper, we developed affinity sil-ica nanotubes as affinity matrices for blood purification, whichwere prepared by depositing silica within the alumina mem-brane through sol-gel method. The affinity silica nanotubes havesmooth surfaces and high ligand content, which were benefi-cial to bilirubin adsorption. Batch experiments revealed affinitysilica nanotubes had good reusable performance and excellentadsorption capacity. Optimal adsorption could be achieved in anappropriate temperature, ionic strength, and albumin concentra-tion. The adsorption process presented a Langmuir adsorptionisotherm at the given range of the bilirubin concentration. Thesefeatures make the Arg-immobilized affinity nanotubes very goodcandidate for use in removal of bilirubin.

REFERENCES

[1] J. Stange, W. Ramlow, S. Mitzner, R. Schmidt, and H. Klinkmann, “Dial-ysis against a recycled albumin solution enables the removal of albumin-bound toxins artificial organs,” Artif. Org., vol. 17, no. 9, pp. 809–813,Sep. 1993.

[2] L. Yoo, D. Matalon, R. S. Hoffman, and D. S. Goldfarb, “Treatmentof pregabalin toxicity by hemodialysis in a patient with kidney failure,”Amer. J. Kidney Dis., vol. 54, no. 6, pp. 1127–1130, Dec. 2009.

[3] P. Toft, R. Schmidt, A. C. Broechner, B. U. Nielsen, P. Bollen, and K.E. Olsen, “Effect of plasmapheresis on the immune system in endotoxin-induced sepsis,” Blood Purif., vol. 26, no. 2, pp. 145–150, 2008.

[4] A. Denizli, G. Okan, and M. Ucar, “Dye-affinity microbeads for removalof phenols and nitrophenols from aquatic systems,” J. Appl. Polym. Sci.,vol. 83, no. 11, pp. 2411–2418, Mar. 2002.

[5] Y. Yi, Y. T. Wang, and W. Zhang, “Synthesis of a new chitosan immobilizedwith beta-cyclodextrins and its adsorption properties for bilirubin,” J.Appl. Polym. Sci., vol. 99, no. 3, pp. 1264–1268, Feb. 2006.

[6] L. Yu, C. M. Li, Q. Zhou, Y. Gan, and Q. L. Bao, “Functionalized multi-walled carbon nanotubes as affinity ligands,” Nanotechnology, vol. 18,no. 11, pp. 115614–115620, Feb. 2007.

[7] W. Shi, H. H. Cao, Y. Q. Shen, C. F. Song, D. H. Li, Y. Zhang, and D. T. Ge,“Chemically modified PPyCOOH microtubes as affinity matrix for proteinpurification,” Macromol. Chem. Phys., vol. 210, no. 17, pp. 1379–1386,Sep. 2009.

[8] M. Kang, L. Trofin, M. O. Mota, and C. R. Martin, “Protein capture insilica nanotube membrane 3-D microwell arrays,” Anal. Chem., vol. 77,no. 19, pp. 6243–6249, Oct. 2005.

[9] B. B. Lakshmi, C. J. Patrissi, and C. R. Martin, “Sol-gel template synthesisof semiconductor oxide micro- and nanostructures,” Chem. Mater., vol. 9,no. 11, pp. 2544–2550, Nov. 1997.

[10] C. J. Brinker and G. W. Scherer, Sol-Gel Science. New York: Academic,1990.

[11] A. Jonas and G. Weber, “Presence of arginine residues at the strong,hydrophobic anion binding sites of bovine serum albumin,” Biochemistry,vol. 10, no. 8, pp. 1335–1339, Apr. 1971.

[12] C. Jacobsen, “Chemical modification of the high-affinity bilirubin-bindingsite of human-serum albumin,” Eur. J. Biochem., vol. 27, no. 3, pp. 513–519, Jun. 1972.

[13] A. Denizli, M. Kocakulak, and E. Piskin, “Specific sorbents for bilirubinremoval from human plasma: Congored-modified poly(EGDMA/HEMA)microbeads,” J. Appl. Polym. Sci., vol. 68, no. 3, pp. 373–380, Apr. 1998.

[14] S. Sideman, L. Mor, D. Mordohovich, M. Mihich, O. Zinder, and J.M. Brandes, “In vivo hemoperfusion studies of unconjugated bilirubinremoval by ion exchange resin,” Trans. Amer. Soc. Artif. Intern. Org.,vol. 27, pp. 434–438, 1981.

SONG et al.: FUNCTIONALIZED SILICA NANOTUBES AS AFFINITY MATRICES FOR BILIRUBIN REMOVAL 631

[15] W. Shi, Y. Q. Shen, H. R. Jiang, C. F. Song, Y. Y. Ma, J. Mu, B. Y. Yang, andD. T. Ge, “Lysine-attached anodic aluminum oxide (AAO)-silica affinitymembrane for bilirubin removal,” J. Membr. Sci., vol. 349, no. 1–2,pp. 333–340, Mar. 2010.

[16] W. Shi, H. H. Cao, C. F. Song, H. R. Jiang, J. Wang, S. H. Jiang, J. Tu, andD. T. Ge, “Poly(pyrrole-3-carboxylic acid)-alumina composite membranefor affinity adsorption of bilirubin,” J. Membr. Sci., vol. 353, no. 1–2,pp. 151–158, May 2010.

[17] W. Shi, F. B. Zhang, and G. L. Zhang, “Adsorption of bilirubin with polyly-sine carrying chitosan-coated nylon affinity membranes,” J. Chromatogr.B, vol. 819, no. 2, pp. 301–306, May 2005.

[18] W. Shi, F. B. Zhang, G. L. Zhang, D. T. Ge, and Q. Q. Zhang, “Adsorptionof bilirubin on poly-L-lysine-containing nylon membranes: Applicationsin affinity chromatography,” Polym. Int., vol. 54, no. 5, pp. 790–795, May2005.

[19] S. Senel, F. Denizli, H. Yavuz, and A. Denizli, “Bilirubin removal fromhuman plasma by dye affinity microporous hollow fibers,” SeparationSci. Technol., vol. 37, no. 8, pp. 1989–2006, 2002.

[20] A. Lavin, C. Sung, A. M. Klibanov, and R. Langer, “Enzymatic removalof bilirubin from blood: A potential treatment for neonatal jaundice,”Science, vol. 230, no. 4725, pp. 543–545, Nov. 1985.

[21] R. A. Willson, A. F. Hofmann, and G. G. Kuster, “Toward an artificialliver. II. Removal of cholephilic anions from dogs with biliary obstruction,by hemoperfusion through charged and uncharged resins,” Gastroenterol-ogy, vol. 66, no. 1, pp. 95–107, Jan. 1974.

[22] W. Shi, F. B. Zhang, G. L. Zhang, L. Q. Jiang, Y. J. Zhao, and S. L. Wang,“Polylysine-immobilized affinity nylon membrane used for bilirubin ad-sorption,” Mol. Simul., vol. 29, no. 12, pp. 787–790, Dec. 2003.

[23] R. Brodersen, “Bilirubin solubility and interaction with albumin and phos-pholipid,” J. Biol., Chem., vol. 254, no. 7, pp. 2364–2369, Apr. 1979.

[24] M. Branca, A. Gamba, P. Manitto, D. Monti, and G. Speranza, “Thebinding of bilirubin to albumin: A study using spin-labelled bilirubin,”Biochim. Biophys. Acta, vol. 742, no. 2, pp. 341–351, Jan. 1983.

[25] A. Denizli, M. Kocakulak, and E. Piskin, “Bilirubin removal from humanplasma in a packed-bed column system with dye-affinity microbeads,” J.Chromatogr. B, vol. 707, no. 1–2, pp. 25–31, Apr. 1998.

[26] M. Kocakulak, A. Denizli, A. Y. Rad, and E. Piskin, “New sobent forbilirubin removal from human plasma: Cibacron blue F3 GA-immobilizedpoly(EGDMA-HEMA) microbeads,” J. Chromatogr. B, vol. 693, no. 2,pp. 271–276, Jun. 1997.

Authors’ photographs and biographies not available at the time of publication.