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ual immobilization of biomolecule on the glass surface usingysteine as a bifunctional linker
ata Sheo Bachan Upadhyay ∗, Nishant Verma1
epartment of Biotechnology, National Institute of Technology, Raipur, Chhattisgarh 492001, India
r t i c l e i n f o
rticle history:eceived 3 February 2014eceived in revised form 21 March 2014ccepted 4 April 2014vailable online xxx
eywords:ysteine
mmobilization
a b s t r a c t
A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cys-teine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with(3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate acysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase onboth groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. Thecysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm asdetermined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fouriertransform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual
ilver Nanoparticleslkaline Phosphataselass
immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% withamino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and theenzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine providesa new approach for enhanced immobilization and its integration into different industrial processes and
biosensor technology.
. Introduction
For different bioanalytical and biomedical processes, devel-pment of an immobilized enzyme system is a prerequisite. Iturther facilitates its broader biotechnological application in differ-nt fields. For an effective immobilization, a wide range of methodsave been developed and are still in progress. However, the over-ll objective is common, i.e. to enhance enzymatic properties suchs stability, activity and specificity [1]. These properties do notolely depend on the type of immobilization but also on the choicef support matrix. Immobilization on a glass surface is a cheap-st way of enzyme fixation besides having some advantages ofeing stable, nontoxic, easy handling and can easily be cleaned fore-use. These features can be further enhanced by different mod-fications including sol–gel precursor such as (3-mercaptopropyl)rimethoxysilane, which is a bifunctional molecule containing bothhiol and silane group and thus, has been immobilized on different
Please cite this article in press as: Upadhyay LSB, Verma N. Dual immobifunctional linker. Process Biochem (2014), http://dx.doi.org/10.1016
ubstrates including glass [2]. Among the various nanostructures,ilver nanoparticles are gaining importance as they provide a stablend a very large surface area for enzyme immobilization [3,4].
Cysteine is considered as a suitable agent for nanoparticles cap-ping due to the presence of a thiol group ( SH) which have highaffinity for binding to the nanoparticle surface, and other freefunctional groups ( NH2 and COOH) that can be used for theimmobilization of biomolecules. Covalent coupling strategies usingthiol groups of cysteine are widely used to conjugate biomoleculesto the solid supports [5–8]. Usually, it makes use of the reac-tions which involve heterodisulfide linkage formation and thus,allows site-specific reactions. But these thiol linkages are proneto self-oxidation and gives disulfides quite easily (at pH 7–9)in the presence of oxygen [9,10]. In this study, we described adual covalent immobilization technique on cysteine capped silvernanoparticles without involving any disulfide bonds for enzymeimmobilization. Here, cysteine capped silver nanoparticles (CSNPs)act as a linker between the glass surface of test tube and enzyme,alkaline phosphatase. Both functional groups of the cysteine wereactivated in a sequential manner using carbodiimide (for COOH)and glutaraldehyde (for NH2), and its immobilization efficiencyfor the enzyme was then determined.
2. Materials and methods
bilization of biomolecule on the glass surface using cysteine as a/j.procbio.2014.04.003
2.1. Chemicals
Silver nitrate (99% purity), 4-nitrophenyl phosphate diso-dium salt (99% purity) and glutaraldehyde (25% purity) were
btained from Loba Chemie, while l-cysteine, 1-(3-dimethylaminoropyl)-3-ethylcarbodimide hydrochloride (EDC) and N-ydroxysuccinimide (NHS, pure) were purchased from Himedia.3-Mercaptopropyl) trimethoxysilane (MPTS, 95% purity) was pur-hased from Sigma–Aldrich. Deionized (DI) water from Milliporeas used for the reagent preparation and throughout the process.
.2. Preparation of cysteine capped silver nanoparticles
For the synthesis of cysteine capped silver nanoparticles, potatoxtract was used as a reducing agent which was prepared by boil-ng fresh peeled off potato slices (30 g) in 100 mL of DI wateror 15 min, and filtered through whatmann filter paper No. 1. In00 mL aqueous solution of 1 mM silver nitrate, 1 mL of l-cysteine1 mM) was added, and incubated for 10 min at room tempera-ure (RT) with constant stirring on a magnetic stirrer. Next, 5 mLf potato extract was added to it, and pH of the solution wasaintained at 11 using sodium hydroxide (1 M). The solution was
hen heated at 60 ◦C for 30 min to develop a dark brown color,hich indicates the formation of cysteine capped silver nanopar-
icles (CSNPs). The nanoparticles were initially characterized byV–visible spectrophotometer (NanoDrop 1000, v3.8.1, Thermo-
cientific), by studying their surface plasmon resonance (SPR) inhe wavelength ranges from 220 to 750 nm. The size of synthe-ized CSNPs was analyzed by Particle size analyzer (PSA) (Zetasizerano ZS, Malvern Instruments, U.K,) using 1.5 mL solution at 25 ◦Cith 66.1 kcps count rate. The particles were separated by cen-
rifugation at 12,000 rpm for 30 min and dried at 37 ◦C overnighto obtain in powder form. The surface capping by cysteine wasonfirmed by analyzing KBr disks of powdered samples throughourier transform infrared (FTIR) spectroscopy (Thermo Nicolet,vatar 370, FT-IR spectrometer, Germany) in the range from 500
o 4000 cm−1 with 4 cm−1 resolution. To investigate the crys-alline structure, the XRD spectra of CSNPs powder was obtainedrom X-ray diffractometer (XPertPro Diffractometer, PANalytical,ermany) using CuK� radiation (� −1.540 A) in the 2� range of5◦–80◦ at 0.026◦/min scan rate.
.3. Enzyme immobilization on glass surface
The overall procedure is schematically illustrated in Fig. 1. Formmobilizing alkaline phosphatase on the surface of glass test tube,ollowing steps were performed:
.3.1. Glass surface silanizationBefore silanization, the glass surface must be free from any
mpurities. For cleaning, the test tubes (Borosil, 18 mm × 150 mm)ere thoroughly washed with detergent (Labolene, neutral pH),
ollowed by cleaning with methanol/HCl solution (1:1 volumetricatio) for 30 min at RT. After that, the test tubes were treated with
freshly prepared piranha solution (3:1 mixture of 98% H2SO4 and0% H2O2) at 70 ◦C in water bath for 30 min. The test tubes werehoroughly rinsed with DI water, and silanization was then doney incubating the test tubes with 10% MPTS methanolic solution1 mL) overnight at RT.
.3.2. Multi-deposition of cysteine capped silver nanoparticlesThe silanized test tubes were thoroughly washed with methanol
nd dried at RT. Then, the test tubes were given overnight incu-ation with freshly prepared CSNPs (1 mL) at RT. Next day, theest tubes were washed with DI water 2 times and again treated
Please cite this article in press as: Upadhyay LSB, Verma N. Dual immobifunctional linker. Process Biochem (2014), http://dx.doi.org/10.1016
or 20 min with 1 mL MPTS sol–gel, which was prepared accordingo the procedure as reported [11]. After attaching to first layer ofSNPs, this MPTS sol–gel leaves free thiol groups for the attach-ent of other CSNPs, thus increasing its deposition on the surface
PRESSochemistry xxx (2014) xxx–xxx
[12]. Finally, the sol was discarded and test tubes were again incu-bated with fresh CSNPs for 1 h. The tubes were then washed with DIwater twice and dried at RT. The surface characteristic of the MPTSmodified glass surface (Glass/MPTS/CSNP/MPTS-solgel/CSNP) wasthen analyzed by Scanning electron microscope (Jeol, JSM-6480 LV,Japan) at an accelerating voltage in the range of 10–20 kV.
2.3.3. Enzyme immobilizationA two-step approach was carried out for covalent enzyme
immobilization. In the first step, carboxyl group of cysteine wasemployed for immobilization using carbodiimide procedure. Theglass test tubes were treated with freshly prepared aqueous 30 mMNHS and 15 mM EDC solution (1:1 volumetric ratio, 1 mL) for 1.5 hto activate the carboxyl group [13]. After activation, the test tubeswere washed with DI water and incubated with alkaline phos-phatase (10.03 U/mg) for 24 h. In second step of immobilizationwhich utilized amino group of cysteine, the latter test tubes werewashed twice with Tris–HCl buffer (0.5 M, pH 9.0) and treatedwith 1% glutaraldehyde (GLT) solution for 2 h at 4 ◦C [14]. Thetubes were then washed two times with DI water and incubatedovernight with fresh alkaline phosphatase solution (1 mL) at 4 ◦C.Finally, the tubes were washed with Tris–HCl buffer (0.5 M, pH9.0), and enzyme assay was done in it using 1 mL final reactionvolume containing, 300 �L 4-nitrophenyl phosphate (5 mM, pH9) as a substrate and 700 �L Tris–HCl buffer (0.5 M, pH 9). Theyellow colored p-nitrophenol produced after 30 min of incuba-tion at 37 ◦C, was measured spectrophotometrically at 405 nm.One unit of activity is defined as the amount of enzyme whichliberated 1 �mol of 4-nitrophenol per minute under the assay con-ditions. A standard curve was generated by Lowry method, usingan aqueous solution of bovine serum albumin (BSA) protein andthe amount of immobilized enzyme was then calculated as thedifference between the amount of protein used for immobiliza-tion and the amount of protein left after immobilization. Eachactivation step of dual immobilization was performed separatelyin different tubes in order to compare the immobilization effi-ciency.
2.4. Reusability
The operational stability of immobilized enzyme was studiedby assaying the activity of immobilized alkaline phosphatase in thesame test tube for continuous 8 days with a time interval of 24 hbetween the assays. After each assay, the tubes were stored at 4 ◦Cin two different conditions. One set of glass tubes was stored inthe dried condition, while the other set was filled with Tris–HClbuffer (0.5 M, pH 9), and then stored. The experiment was carriedout in triplicates (n = 3), and a mean with standard deviation wascalculated and reported using Origin Pro (Version 8).
3. Results and discussion
3.1. Synthesis of cysteine capped silver nanoparticles
The CSNPs were synthesized by using potato extract as areducing agent. Before the reduction process, silver nitrate andcysteine were incubated to form a complex by thiolate bond-ing [15]. This complex was further reduced by potato extractat alkaline pH, to form cysteine capped silver nanoparticles.Four different techniques were used for the characterization ofcapped nanoparticles (Fig. 2). Initial characterization was done byUV–visible spectrophotometer in which a single sharp SPR peak
bilization of biomolecule on the glass surface using cysteine as a/j.procbio.2014.04.003
at 405 nm (�max) was observed (Fig. 2a), which is a characteristicproperty of nanoparticles owing to which they strongly absorbelectromagnetic waves in the visible region. The particle size in thesolution was then determined by PSA, and it showed a very narrow
L.S.B. Upadhyay, N. Verma / Process Biochemistry xxx (2014) xxx–xxx 3
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ig. 1. Schematic representation of the dual immobilization approach for immobiliilanized with MPTS (10%) and functionalized with CSNP to generate cysteine layelutaraldehyde (1%) in the aqueous phase to activate its carboxyl and amino group
ange size distribution with an average size of 61 nm (Fig. 2b). Theurface capping was confirmed by FTIR analysis of cysteine andysteine capped silver nanoparticles (Fig. 2c). The characteristicibration peaks of thiol group observed in cysteine (CYS) at550 (stretching) and 942 cm−1(bending) were disappeared inhe synthesized CSNPs. It indicates that the cysteine thiol groupttached to the synthesized silver nanoparticle surface via thiolateinkage to form cysteine capped silver nanoparticles [16–18].isappearance of similar thiol characteristic peak of cysteine,
or silver and gold nanoparticles capping has also been reported15,19]. Moreover, two other bands at 1590 cm−1 and 3350 cm−1
n cysteine, which corresponds to the asymmetric stretching ofOO−1 and NH3+ stretch were also observed in CSNP spectra with a
Please cite this article in press as: Upadhyay LSB, Verma N. Dual immobifunctional linker. Process Biochem (2014), http://dx.doi.org/10.1016
lightly shifted position, which further confirmed the attachmentf cysteine on nanoparticle surface. The shift in the band positionsn CSNP may be due to change in the dipole moment after bind-ng of cysteine on metal surface with high electron density. The
ig. 2. Characterization of cysteine capped silver nanoparticles by (a) UV–visible spectrophich showed the average particle size of 61 nm, (c) Fourier transform infrared spectrom
ysteine (CYS), and (d) X-ray diffractometer which confirmed the crystallinity of the synt
kaline phosphatase (AP) on the internal surface of a glass test tube. The tubes werech was sequentially treated with carbodiimide (30 mM NHS and 15 mM EDC) andtively, for the enzyme immobilization.
cysteine binding was further confirmed by FTIR spectra ofnon-cysteine modified glass surface in which no such bands, cor-responding to cysteine amino or carboxyl group were observed,except for Si-O-Si bond (at 1100 cm−1) which were formed dur-ing the silanization (Fig. S1, Supporting Information). Fig. 2d showsthe X-ray diffraction pattern for CSNPs which showed three distinctdiffraction peaks at 38.28, 64.53 and 77.52, indexed to (1 1 1), (2 2 0)and (3 1 1) planes of the face-centered cubic (FCC) silver. No otherpeaks correspond to silver oxide or any impurities were observed inXRD analysis, which indicate that the as-synthesized CSNPs were ofhigh purity and crystalline in nature (Fig. 2d). To further investigatethe oxidation state of synthesized nanoparticles, photoelectronspectra were recorded from V.G. Microtech Multilab ESCA 3000
bilization of biomolecule on the glass surface using cysteine as a/j.procbio.2014.04.003
spectrometer, equipped with AlK� X-ray source (h� = 1486.6 eV),with a step size of 0.1 eV. Higher resolution spectra of the Ag 3dregion showed two bands at 368.2 eV and 374.4 eV, correspondingto the binding energies of Ag 3d5/2 and Ag 3d3/2 respectively, which
hotometer showing a single and sharp SPR peak at 405 nm, (b) particle size analyzereter which confirmed the surface modification of silver nanoparticles (CSNP) by
a Recovery of the enzyme is calculated by assuming the specific activity of the soluble enzyme (10.03 U/mg) to be 100%.
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iggtctbiggs[f(tsoswbaTaswo
Fig. 4. Variation in the activity of glass immobilized alkaline phosphatase aftermultiple cycles of reuse, when stored at two different conditions. The glass tubes
highly depends on the storage conditions. Glass tubes stored inTris–HCl buffer, showed higher reusability than the tubes stored indried condition, at the same temperature. The immobilized enzyme
ig. 3. SEM micrographs of glass surface with (A) MPTS silanization and (B) noilanization (reference slide). After the silanization process, the surface of the glassas coated by a rough and continuous layer of MPTS sol–gel network.
onfirmed the existence of silver nanoparticles in non-oxidizedtate [20] (Fig. S2, Supporting Information).
.2. Glass surface immobilization
In the present work, we employed cysteine to enhance themmobilization efficiency for alkaline phosphatase on the internallass surface of test tube. Here, CSNPs acted as bridges betweenlass and enzyme. The advantage of using CSNPs is that, it enhanceshe initial enzyme loading capacity on single nanoparticle, as manyysteine groups are attached to it which can further be used forhe immobilization. Before the attachment, glass surface needs toe pre-processed to generate some type of functional groups on
ts surface to which nanoparticles can bind. In the present work,lass was silanized using (3-mercaptopropyl) trimethoxysilane toenerate thiol groups on its surface. The as-formed silanized glassurface was then treated with CSNPs to form a nanoparticles layer21]. To further increase the nanoparticles loading on glass sur-ace, MPTS sol–gel was prepared and used. The silanized glassGlass/MPTS/CSNP/MPTS-solgel/CSNP) was then analyzed by SEMo examine its surface characteristic. SEM analysis revealed thatilanization caused the formation of a rough but continuous layerf MPTS sol–gel network, when compared with untreated glassurface (Fig. 3). Now the glass-CSNP surface with bound cysteineas given two types of sequential treatments for enzyme immo-
ilization, carbodiimide treatment to activate its carboxyl groupnd glutaraldehyde treatment for the activation of amino group.he carboxyl activation was done prior to the amino group to avoid
Please cite this article in press as: Upadhyay LSB, Verma N. Dual immobifunctional linker. Process Biochem (2014), http://dx.doi.org/10.1016
ny aggregation caused by glutaraldehyde immobilized enzyme. Aimilar experiment was also carried out in another set of tubes, butith single group activation only. So, for complete immobilization
f enzyme on glass surface, two types of interaction occurred, one is
stored in Tris–HCl buffer (0.5 M, pH 9.0) filled condition, showed higher reusabil-ity of immobilized enzyme as compared to the tubes stored at dry condition. Eachobservation is an average of three repeats (n = 3) with standard deviation.
the covalent bond, which is formed during glass silanization (glass-MPTS) and enzyme immobilization (CSNP-enzyme), and the otheris the dative bond, which is formed between CSNP and the silanizedglass. Table 1 shows the efficiency comparison for dual and singletype of enzyme immobilization. It was observed that the enzymewas immobilized successfully with an increment in its activity, aswe moved from single group immobilization to dual immobiliza-tion. Here, we obtained a 73% activity for dual treatment, whichis 22% and 33% more than the amino group and carboxyl groupimmobilization, respectively. This activity is in favorable compar-ison with the efficiency of alkaline phosphatase immobilized onglass surface using the in vacuo cross-linking process [22]. Thisincrement in the enzymatic activity may be contributed by higherprotein loading2 as about 56% (1.8 mg out of 3.2 mg) of the enzymewas loaded with dual technique, while 37% (1.24 mg) and 35%(1.13 mg) loadings were obtained with amino group and carboxylgroup immobilization respectively. In order to check the stabilityof substrate (4-nitrophenyl phosphate) in the glass tubes, a controlexperiment was also performed in CSNP coated glass tubes, butwithout enzyme immobilization. No spontaneous hydrolysis of thesubstrate (4-nitrophenyl phosphate) was observed up to 6 h, whichclearly indicate that substrate hydrolysis occurs at the active site ofenzyme in enzyme immobilized glass tubes. The reusability exper-iment showed that the activity of the immobilized preparations
bilization of biomolecule on the glass surface using cysteine as a/j.procbio.2014.04.003
ept in wet condition (i.e. in presence of buffer) retains 85% ofts activity in reusability assay, while 40% activity was lost after
times of reutilization for dried storage immobilized enzyme sys-em (Fig. 4). It reveals that, in addition to improved immobilization,his dual approach may also provide stability to the enzyme, whichs a primary concern during any type of immobilization technique.
. Conclusion
In this work, we described a novel method for dual immo-ilization of alkaline phosphatase on cysteine modified glassest tube. The glass surface was thiol functionalized using (3-
ercaptopropyl) trimethoxysilane, and formed an interconnectingayer with CSNPs. Both functional groups were then activatedequentially to immobilize alkaline phosphatase. With the dualmmobilization technique, about 73% enzyme activity was recov-red which was higher than carboxyl/amino group immobilization.he immobilization method also provides stability to the enzymend it showed 85% retention of enzymatic activity after 8 times ofeuse. From the data, we can conclude that the enzyme was effi-iently immobilized with durability, and this dual approach coulde exploited for immobilizing other enzymes for the developmentf various enzymes related processes and technologies.
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.procbio.2014.04.003.
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