Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

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<ul><li><p>Tandem conjugation of enzyme and antibofor enzyme immunoassay</p><p>inory</p><p>ELISA</p><p>nzylinkiond onuri</p><p>much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface</p><p>ssay (s, pathsy maer, onrelativnces. T</p><p>on their surface. Thus,when they are used for preparation of enzymeand antibody conjugates, the resulting conjugates could vary signif-icantly in the enzyme-to-antibody ratios, resulting in poorreproducibility.</p><p>Itwould be advantageous to develop a conjugation approach thatcan provide controlled enzyme-to-antibody ratios. In the currentstudy,wedevelopeda layer-by-layer labelingstrategybyusing silicananoparticles as the conjugation scaffold. We rst immobilized theenzyme horseradish peroxidase (HRP) to the surface of preformedaminated silica nanoparticles. We then modied the HRP-labeled</p><p>Fujian 361005, China. Fax: +86 592 2187363.E-mail address: qgli@xmu.edu.cn (Q. Li).</p><p>1 These authors contributed equally to this work.2 Present address: Department of Genetics and Pathology, Rudbeck Laboratory,</p><p>Uppsala University, SE-751 85 Uppsala, Sweden.3 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; HRP, horserad-</p><p>ish peroxidase; HBsAg, hepatitis B surface antigen; NaBH3CN, sodium cyanoborohy-dride; TEOS, tetraethyl orthosilicate; APTMS, 3-aminopropyltrimethoxysilane;AEAPTMS, 3-(2-aminoethylamino)propyltrimethoxysilane; AEAEAPTMS, 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane; BSA, bovine serum albumin;IgG, immunoglobulin G; TMB, 3,30 ,5,50-tetramethylbenzidine; H2O2, hydrogen per-oxide; TEM, transmission electron microscope; UVVis, ultravioletvisible; S/N ratio,</p><p>Analytical Biochemistry 406 (2010) 813</p><p>Contents lists availab</p><p>Analytical Bio</p><p>journal homepage: www.esignal-to-noise ratio; CV, coefcient of variance.sitivity, uorogenic or chemiluminescent substrates are used insteadof chromogenic substrates [58]. Unfortunately, the readout for uo-rescence or chemiluminescence requires expensive instrumentationthat increases the overall cost of the assay. An alternative way to in-crease the sensitivity was achieved by amplication of the signalsgenerated from the enzymeantibody conjugate with a highenzyme-to-antibody ratio [9]. Such conjugates were assembled</p><p>Thepolymers, suchasdextranandpolylysinechains, usually con-tainmultiple functional groups that couldbe covalently linked toen-zyme and antibody molecules to form clustered enzymeantibodyconjugates [1013]. It was reported that by using polymer-basedconjugates, sensitivity could be improved signicantly. Micro-spheres, such as liposome [14,15], polystyrene microparticles[13,16,17], and silica nanoparticles [1821], have been used to pre-pare uorescent labels for immunoassays. Because they can be ex-ibly introduced with active functional groups onto their surface,thesemicrospheres are potential scaffolds for enzyme and antibodyconjugation [13,22,23]. The disadvantage of these microspheres,however, is that they often have the same type of functional groups</p><p>* Corresponding author at:. Molecular Diagnostics Laboratory, Department ofBiomedical Sciences and Key Laboratory of Ministry of Education for Cell Biologyand Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen,Silica nanoparticlesTandem conjugationHepatitis B surface antigen</p><p>Enzyme-linked immunosorbent atial role in the detection of biomarkerals due to its cost effectiveness, eareadout interpretation [14]. Howevtional absorption-based ELISA is itslimits its use to only abundant substa0003-2697/$ - see front matter 2010 Elsevier Inc. Adoi:10.1016/j.ab.2010.06.039antigen (HBsAg), thedetection limitwas three times lower than that of the commercially available ELISAkit. 2010 Elsevier Inc. All rights reserved.</p><p>ELISA)3 plays an essen-ogens, and drug residu-nipulation, and simplee drawback of conven-ely low sensitivity thato achieve improved sen-</p><p>through special scaffolds, such as polymer and microsphere, to forma cluster of enzyme and antibody molecules. Therefore, when oneantibody molecule of this type of conjugate binds to one antigen,tens or hundreds of enzyme molecules can bind to a single antigen.Subsequently, all of the enzymes will contribute to the substratecatalysis reaction, consequently leading to signal amplication.Keywords:the conventional antibodyenzyme conjugate used in ELISA,which often has one or two enzymemoleculesper antibody, the new type of conjugate contained more enzyme molecules per antibody and provided aRongqin Ke a,1,2, Wei Yang a,1, Xiaohu Xia a, Ye Xu a, QaMolecular Diagnostics Laboratory, Department of Biomedical Sciences and Key LaboratSchool of Life Sciences, Xiamen University, Xiamen, Fujian 361005, ChinabKey Laboratory of Chemical Biology of Fujian, Xiamen, Fujian 361005, China</p><p>a r t i c l e i n f o</p><p>Article history:Received 21 February 2010Received in revised form 17 June 2010Accepted 28 June 2010Available online 1 July 2010</p><p>a b s t r a c t</p><p>Wepresent a new type of ethe sensitivity of enzyme-layer-by-layer immobilizatof enzyme was immobilizeconjugate could be easily pll rights reserved.dy on silica nanoparticle</p><p>gge Li a,b,*</p><p>of Ministry of Education for Cell Biology and Tumor Cell Engineering,</p><p>meantibody conjugate that simplies the labeling procedure and increasesed immunosorbent assay (ELISA). The conjugates were prepared throughof enzyme and antibody on a silica nanoparticle scaffold. Amaximal amountthe nanoparticle, followed by antibody linkage through Dextran 500. Theed from unreacted reagents by simple centrifugations. In comparison with</p><p>le at ScienceDirect</p><p>chemistry</p><p>lsevier .com/locate /yabio</p></li><li><p>Preparation of aminated silica nanoparticles</p><p>y / RSilicananoparticleswere synthesizedusinga reversemicroemul-sionmethod [24,25]. Briey, 10 mlof cyclohexane, TritonX-100, andn-hexanol in a ratio of 3:1:1 (v/v) was stirred at room temperature,followed by the addition of 450 ll of water, 100 ll of TEOS, and70 ll of NH4OH (2830%), respectively. After reaction at room tem-perature for 24 h, the silica nanoparticles formed were isolated bycentrifugation (5900 RCF for 20 min) after adding 10 ml of acetone.The nanoparticles were rinsed with ethanol and water three times,silica nanoparticleswith ahydrophilic layer of oxidizeddextran, andthe antibody was conjugated to the oxidized dextran through Schiffreaction. The excess amount of reagents could be easily removed bybrief centrifugation. When used in an ELISA for the detection of thehepatitisB surfaceantigen (HBsAg), our conjugatesobtainedadetec-tion limit threefold lower than that of the commercial ELISA kit,whereas our results from 280 clinical serum samples agreed com-pletely with ELISA. In this article, we term our new type of en-zymeantibody conjugate as bifunctional silica nanoparticle.</p><p>Materials and methods</p><p>Chemicals</p><p>Triton X-100, sodium cyanoborohydride (NaBH3CN), tetraethylorthosilicate (TEOS), 3-aminopropyltrimethoxysilane (APTMS),3-(2-aminoethylamino)propyltrimethoxysilane (AEAPTMS), and 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEAEAPTMS), bovine serum albumin (BSA), and ProClinwere purchasedfrom SigmaAldrich (St. Louis, MO, USA). Dextran 500 (MW =500,000) was purchased from Amersham Biosciences (Uppsala,Sweden). HRPwas purchased fromBienzyme Laboratories (San Die-go, CA, USA). Monoclonal anti-HBsAg immunoglobulin G (IgG) anti-body (S04, B20), ELISAkits forHBsAg, colorimetric substrate forHRP,3,30,5,50-tetramethylbenzidine (TMB), hydrogen peroxide (H2O2),and human serum samples all were kindly provided by InTec Prod-ucts (Xiamen, China). All of the reagents were used without furtherpurication. Distilled water was used throughout the study.</p><p>Buffers</p><p>Dilution buffer consisted of 0.01 M TrisHCl containing 2 g/LBSA, 0.1% ProClin, and 9 g/L NaCl (pH 7.8). Washing buffer con-sisted of 0.01 M phosphate-buffered saline containing 0.5 g/LTween 20 (pH 7.4). Blocking buffer consisted of 0.01 M TrisHClcontaining 2% BSA, 4% sucrose, and 1% glycine (pH 7.8). Carbonatebuffer 1 consisted of 0.05 M Na2CO3NaHCO3 (pH 9.5), and carbon-ate buffer 2 consisted of 0.025 M Na2CO3NaHCO3 (pH 9.5).</p><p>Instruments</p><p>The colorimetric ELISAwasmeasuredbyamicrotiter plate reader(Thermo Labsystems Multiskan MK3 plate reader, Marietta, OH,USA). Transmission electron microscope (TEM) images of silicananoparticles were obtained from a JEOL JEM-2100 TEM (Tokyo, Ja-pan). Ultravioletvisible (UVVis) spectrawere recorded on a PGen-eral TU-1901 UVVis spectrophotometer (Beijing Purkinje GeneralInstrument,Beijing,China). All of thenanoparticleswere centrifugedon an Eppendorf 5415 D centrifuge (Hamburg, Germany) and soni-cated with a Scientz SB5200D ultrasonic cleaner (Ningbo, China).Microtitration plates were kindly provided by InTec Products.</p><p>Tandem conjugation of enzyme and antibodrespectively, to remove surfactant and unpolymerized materials.Silica nanoparticles (20 mg) were suspended in 1 ml of ethanol.</p><p>Then 2 ll of amino silane was added to the suspension to introduceamino groups onto the surface of silica nanoparticles (Fig. 1). Reac-tion was carried out at room temperature for 2 h with stirring, fol-lowed by incubation in a 65 C water bath for 5 min. Unreactedreagents were removed by washing with ethanol and water,respectively. The nanoparticles were resuspended in 1 ml of etha-nol. To conrm the existence of the amino groups on the surface ofsilica nanoparticles, one drop of salicylaldehyde was added to0.5 ml of nanoparticle suspension to observe color change [26].</p><p>Immobilization of HRP on surface of aminated silica nanoparticles</p><p>HRP was covalently linked to the aminated silica nanoparticlesusing the periodate oxidation method, as shown in Fig. 1. Here100 ll of HRP (4 mg/ml) was dialyzed against acetate buffer (pH5.2, 0.01 M) at 4 C and then mixed with 2 ll of 0.5 M NaIO4. Afterreaction for 25 min at room temperature in the dark with gentlestirring, the excess periodate was neutralized by adding 1 ll ofglycerol with stirring for 10 min. Unreacted reagents were re-moved by dialysis against acetate buffer (pH 5.2, 0.01 M). The solu-tion of oxidized HRP was adjusted to pH 9.5 with 0.1 M Na2CO3 andthen was added to 100 ll of aminated silica nanoparticle suspen-sion in carbonate buffer 1 (20 mg/ml). Aggregation occurred imme-diately on the addition of HRP, and the aggregated nanoparticleswere dispersed by ultrasonic treatment in an icewater bath. Afterreaction at 4 C for 6 h, NaBH3CN was added to a nal concentra-tion of 5 mM, followed by 12 h of incubation at 4 C. Finally, theunreacted reagents were removed by centrifugation and the pre-cipitated silica nanoparticles were washed with carbonate buffer1. After coupling, the color of nanoparticle suspension turned tolight brown from white.</p><p>Conjugation of antibody to surface of HRP-linked silica nanoparticles</p><p>Anti-HBsAg antibody (S04) was covalently linked to the HRP-immobilized silica nanoparticles through the oxidized dextran(Fig. 1), which was prepared by oxidation of Dextran 500 accordingto our previous work [26]. For this purpose, 100 ll (33 mg/ml inwater solution) of the oxidized Dextran 500 was added to 100 ll(20 mg/ml) ofHRP-conjugated silica nanoparticle suspension in car-bonate buffer 1 and reacted at 4 Cwith stirring for 3 h. The obtainedDextran 500-bridged, HRP-immobilized silica nanoparticles werewashed three times with carbonate buffer 2 to remove the excessoxidized Dextran 500. The anti-HBsAg antibody (100 ll, 3 mg/ml)wasdialyzedagainst carbonatebuffer2 for6 handadded to theDex-tran 500-bridged, HRP-immobilized silica nanoparticles. The mix-ture was allowed to react at 4 C for 6 h, and then NaBH3CN wasadded to a nal concentration of 0.005 M, followed by incubationat 4 C overnight.</p><p>An equal volume of blocking buffer was added to the mixtureand incubated overnight at 4 C. Then the nanoparticles were cen-trifuged and rinsed with 0.01 M TrisHCl buffer (pH 7.8) threetimes and nally suspended in the dilution buffer. The preparedimmunoconjugates were stored at 4 C before use.</p><p>Detection of HBsAg by ELISA</p><p>Bifunctional silica nanoparticles were diluted 1000-fold in thedilution buffer before use. Detection of HBsAg followed a standardELISA procedure. Briey, the monoclonal anti-HBsAg antibody B20(5 lg/ml in 0.02 M TrisHCl buffer, pH 7.4) was physically coatedonto the microtitration wells (100 ll/well). After incubation at 4 Covernight, themicrotitrationwells werewashed oncewithwashingbuffer andblockedwithblockingbuffer (200 ll/well) for12 hat4 C.</p><p>. Ke et al. / Anal. Biochem. 406 (2010) 813 9Then the blocking bufferwas thrown away, and 50 ll of HBsAg stan-dard solutionsor serumsampleswasadded toeachwell, followedbythe addition of 50 ll of diluted bifunctional silica nanoparticles or</p></li><li><p>y / R10 Tandem conjugation of enzyme and antibodHRP-labeled anti-HBsAg antibody. The plate was incubated at 37 Cfor 1 h and then washed ve times with washing buffer. SubstrateTMB + H2O2 (100 ll/well) was added, and the plate was incubatedat 37 C for 15 min. The reaction was then terminated by adding50 ll of 2 M H2SO4, and the absorbance at 450 nm with 630 nm asreference was recorded on a ThermoMK3 plate reader.</p><p>Results and discussion</p><p>Preparation of bifunctional silica nanoparticles</p><p>In the current work, silica nanoparticles were employed as thecarrier for enzyme and antibody immobilization. Thus, monodi-spersed silica nanoparticles with uniform morphology were vitalfor loading biomolecules on eachnanosphere, and this in turnwouldinuence the sensitivity, reproducibility, and analytical perfor-mance of the resulting immunoassay. Monodispersed spherical sil-ica nanoparticles prepared by a reverse microemulsion methodwere uniform (50 5 nm in diameter [mean standard deviation]),as observed with TEM (Fig. 2).</p><p>To introduce amino groups onto the surface, the nanoparticleswere modied with the amino silane under mild conditions. Theexistence of amino groups on the surface was conrmed by salicyl-aldehyde-mediated yellow color change.</p><p>Instead of direct encapsulation of HRP into the core of silicananoparticles, we immobilized HRP onto the surface of silica nano-particles. This method obviated the direct contact of HRP with or-ganic reagents that may cause a decrease or loss of the enzyme</p><p>Fig. 1. Schematic illustration of preparation procedure of the bifunctional silica nanoparwith HRP and antibody. Bare silica nanoparticles were rst aminated using amino silanperiodate oxidation method. The antibody was covalently linked to the HRP-conjugananoparticle-based ELISA. The immobilized capture antibody and the detection antibodsandwich with the antigen to be detected.. Ke et al. / Anal. Biochem. 406 (2010) 813activity. The large surface area of silica nanoparticle carriers al-lowed us to immobilize the maximal amount of HRP onto eachnanoparticle. Before antibody conjugation, we added a layer of</p><p>ticle and its use in immunoassay. (A) Preparation of silica nanoparticles conjugatedes. HRP was then covalently linked to the aminated silica nanoparticles using theted silica nanoparticles through oxidized Dextran...</p></li></ul>

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