Analytical Biochemistry 406 (2010) 8–13

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Analytical Biochemistry

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Tandem conjugation of enzyme and antibody on silica nanoparticlefor enzyme immunoassay

Rongqin Ke a,1,2, Wei Yang a,1, Xiaohu Xia a, Ye Xu a, Qingge Li a,b,*

a Molecular Diagnostics Laboratory, Department of Biomedical Sciences and Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering,School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, Chinab Key Laboratory of Chemical Biology of Fujian, Xiamen, Fujian 361005, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 February 2010Received in revised form 17 June 2010Accepted 28 June 2010Available online 1 July 2010

Keywords:ELISASilica nanoparticlesTandem conjugationHepatitis B surface antigen

0003-2697/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ab.2010.06.039

* Corresponding author at:. Molecular DiagnosticBiomedical Sciences and Key Laboratory of Ministryand Tumor Cell Engineering, School of Life Sciences,Fujian 361005, China. Fax: +86 592 2187363.

E-mail address: qgli@xmu.edu.cn (Q. Li).1 These authors contributed equally to this work.2 Present address: Department of Genetics and Pa

Uppsala University, SE-751 85 Uppsala, Sweden.3 Abbreviations used: ELISA, enzyme-linked immuno

ish peroxidase; HBsAg, hepatitis B surface antigen; Nadride; TEOS, tetraethyl orthosilicate; APTMS, 3-amAEAPTMS, 3-(2-aminoethylamino)propyltrimethoxysaminoethylamino)ethylamino]propyltrimethoxysilane;IgG, immunoglobulin G; TMB, 3,30 ,5,50-tetramethylbeoxide; TEM, transmission electron microscope; UV–Vissignal-to-noise ratio; CV, coefficient of variance.

We present a new type of enzyme–antibody conjugate that simplifies the labeling procedure and increasesthe sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared throughlayer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amountof enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. Theconjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison withthe conventional antibody–enzyme conjugate used in ELISA, which often has one or two enzyme moleculesper antibody, the new type of conjugate contained more enzyme molecules per antibody and provided amuch higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surfaceantigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.

� 2010 Elsevier Inc. All rights reserved.

Enzyme-linked immunosorbent assay (ELISA)3 plays an essen-tial role in the detection of biomarkers, pathogens, and drug residu-als due to its cost effectiveness, easy manipulation, and simplereadout interpretation [1–4]. However, one drawback of conven-tional absorption-based ELISA is its relatively low sensitivity thatlimits its use to only abundant substances. To achieve improved sen-sitivity, fluorogenic or chemiluminescent substrates are used insteadof chromogenic substrates [5–8]. Unfortunately, the readout for fluo-rescence or chemiluminescence requires expensive instrumentationthat increases the overall cost of the assay. An alternative way to in-crease the sensitivity was achieved by amplification of the signalsgenerated from the enzyme–antibody conjugate with a highenzyme-to-antibody ratio [9]. Such conjugates were assembled

ll rights reserved.

s Laboratory, Department ofof Education for Cell BiologyXiamen University, Xiamen,

thology, Rudbeck Laboratory,

sorbent assay; HRP, horserad-BH3CN, sodium cyanoborohy-

inopropyltrimethoxysilane;ilane; AEAEAPTMS, 3-[2-(2-BSA, bovine serum albumin;

nzidine; H2O2, hydrogen per-, ultraviolet–visible; S/N ratio,

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 amplification.

The polymers, such as dextran and polylysine chains, usually con-tain multiple functional groups that could be covalently linked to en-zyme and antibody molecules to form clustered enzyme–antibodyconjugates [10–13]. It was reported that by using polymer-basedconjugates, sensitivity could be improved significantly. Micro-spheres, such as liposome [14,15], polystyrene microparticles[13,16,17], and silica nanoparticles [18–21], have been used to pre-pare fluorescent labels for immunoassays. Because they can be flex-ibly introduced with active functional groups onto their surface,these microspheres 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 groupson 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.

It would be advantageous to develop a conjugation approach thatcan provide controlled enzyme-to-antibody ratios. In the currentstudy, we developed a layer-by-layer labeling strategy by using silicananoparticles as the conjugation scaffold. We first immobilized theenzyme horseradish peroxidase (HRP) to the surface of preformedaminated silica nanoparticles. We then modified the HRP-labeled

Tandem conjugation of enzyme and antibody / R. Ke et al. / Anal. Biochem. 406 (2010) 8–13 9

silica nanoparticles with a hydrophilic layer of oxidized dextran, 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 thehepatitis B surface antigen (HBsAg), our conjugates obtained a detec-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-zyme–antibody conjugate as bifunctional silica nanoparticle.

Materials and methods

Chemicals

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 ProClin were purchasedfrom Sigma–Aldrich (St. Louis, MO, USA). Dextran 500 (MW =500,000) was purchased from Amersham Biosciences (Uppsala,Sweden). HRP was purchased from Bienzyme Laboratories (San Die-go, CA, USA). Monoclonal anti-HBsAg immunoglobulin G (IgG) anti-body (S04, B20), ELISA kits for HBsAg, colorimetric substrate for HRP,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 furtherpurification. Distilled water was used throughout the study.

Buffers

Dilution buffer consisted of 0.01 M Tris–HCl 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 Tris–HClcontaining 2% BSA, 4% sucrose, and 1% glycine (pH 7.8). Carbonatebuffer 1 consisted of 0.05 M Na2CO3–NaHCO3 (pH 9.5), and carbon-ate buffer 2 consisted of 0.025 M Na2CO3–NaHCO3 (pH 9.5).

Instruments

The colorimetric ELISA was measured by a microtiter 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). Ultraviolet–visible (UV–Vis) spectra were recorded on a PGen-eral TU-1901 UV–Vis spectrophotometer (Beijing Purkinje GeneralInstrument, Beijing, China). All of the nanoparticles were 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.

Preparation of aminated silica nanoparticles

Silica nanoparticles were synthesized using a reverse microemul-sion method [24,25]. Briefly, 10 ml of cyclohexane, Triton X-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 (28–30%), 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,respectively, to remove surfactant and unpolymerized materials.

Silica nanoparticles (20 mg) were suspended in 1 ml of ethanol.Then 2 ll of amino silane was added to the suspension to introduce

amino 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 confirm 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].

Immobilization of HRP on surface of aminated silica nanoparticles

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 final 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.

Conjugation of antibody to surface of HRP-linked silica nanoparticles

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) of HRP-conjugated silica nanoparticle suspension in car-bonate buffer 1 and reacted at 4 �C with 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)was dialyzed against carbonate buffer 2 for 6 h and added to the Dex-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 final concentration of 0.005 M, followed by incubationat 4 �C overnight.

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 Tris–HCl buffer (pH 7.8) threetimes and finally suspended in the dilution buffer. The preparedimmunoconjugates were stored at 4 �C before use.

Detection of HBsAg by ELISA

Bifunctional silica nanoparticles were diluted 1000-fold in thedilution buffer before use. Detection of HBsAg followed a standardELISA procedure. Briefly, the monoclonal anti-HBsAg antibody B20(5 lg/ml in 0.02 M Tris–HCl buffer, pH 7.4) was physically coatedonto the microtitration wells (100 ll/well). After incubation at 4 �Covernight, the microtitration wells were washed once with washingbuffer and blocked with blocking buffer (200 ll/well) for 12 h at 4 �C.Then the blocking buffer was thrown away, and 50 ll of HBsAg stan-dard solutions or serum samples was added to each well, followed bythe addition of 50 ll of diluted bifunctional silica nanoparticles or

Fig. 1. Schematic illustration of preparation procedure of the bifunctional silica nanoparticle and its use in immunoassay. (A) Preparation of silica nanoparticles conjugatedwith HRP and antibody. Bare silica nanoparticles were first aminated using amino silanes. HRP was then covalently linked to the aminated silica nanoparticles using theperiodate oxidation method. The antibody was covalently linked to the HRP-conjugated silica nanoparticles through oxidized Dextran 500. (B) Working principle ofnanoparticle-based ELISA. The immobilized capture antibody and the detection antibody, which was conjugated onto the silica nanoparticle-harboring enzyme, formed asandwich with the antigen to be detected.

10 Tandem conjugation of enzyme and antibody / R. Ke et al. / Anal. Biochem. 406 (2010) 8–13

HRP-labeled anti-HBsAg antibody. The plate was incubated at 37 �Cfor 1 h and then washed five 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 Thermo MK3 plate reader.

Fig. 2. TEM image of the prepared silica nanoparticles.

Results and discussion

Preparation of bifunctional silica nanoparticles

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 each nanosphere, and this in turn wouldinfluence 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).

To introduce amino groups onto the surface, the nanoparticleswere modified with the amino silane under mild conditions. Theexistence of amino groups on the surface was confirmed by salicyl-aldehyde-mediated yellow color change.

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

activity. 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

Tandem conjugation of enzyme and antibody / R. Ke et al. / Anal. Biochem. 406 (2010) 8–13 11

Dextran 500 to the surface of the nanoparticles. One use of thislayer is to offer active groups for antibody conjugation, and anotheruse is to add hydrophilicity of the silica nanoparticle, rendering itmore easily dispersed in aqueous solution. Compared with a shortcross-linker such as glutaraldehyde, the use of Dextran 500 polyal-dehyde for enzyme conjugation resulted in minimal loss of enzymeactivity [10,11,27]. The enzyme conjugate could be kept at 4 �C forat least 1 year without obvious loss of activity.

The simple purification of our HRP and antibody conjugates is an-other advantage of our labeling strategy. Purification of the conven-tional antibody–enzyme conjugates often demands gel filtrationchromatography, which is time-consuming and laborious and oftenresults in loss of overall yield of the conjugate. In contrast, ourbifunctional silica nanoparticles could be purified by simple low-speed centrifugation. Moreover, the centrifugation procedure couldrecover all of the enzyme conjugates, which could be concentrated toany desired volume for later use. Therefore, our method is more con-venient and effective than the conventional preparations.

Effect of different lengths of spacer arms

Three different amino silanes were used to generate aminogroups on the surface of silica nanoparticles. These amino silanesare APTMS, AEAPTMS, and AEAEAPTMS, and they will generateamino groups that are 4, 7, and 10 atoms away from the surfaceof nanoparticles. The effect of different lengths of the spacer armson the conjugation of HRP with aminated silica nanoparticles wasinvestigated. Experimental results indicated that the amount ofunconjugated HRP that remained in the reaction mixture withthe 10-atom spacer arm was the smallest, whereas that with the4-atom spacer arm was the largest (Fig. 3A). We calculated thenumber of HRP molecules per silica particle based on the densityof SiO2 (1.96 g/cm3) [28], and the data are summarized in Table 1.This result showed that with an increase in the length of the spacerarm, more HRP molecules could be immobilized on the surface ofthe silica nanoparticles. The increased conjugation efficiency withlonger spacer arms could be attributed to the reduced steric hin-drance of HRP in access to the active sites on the surface of thenanoparticles [29].

The above results were also confirmed indirectly by using thesebifunctional nanoparticles in enzyme immunoassay. These bifunc-tional silica nanoparticles were diluted and used in the enzymeimmunoassay for detecting HBsAg standards of 1 ng/ml. The average

Fig. 3. Effects of different lengths of spacer arms. (A) Absorption spectrum of unconjugatspacer arms on the enzyme immunoassay. The bifunctional silica nanoparticles had thdilution buffer. The concentration of HBsAg standard measured here was 1 ng/ml.

signal-to-noise (S/N) ratio was calculated from four replicas. The re-sult turned out to be that the longer the spacer arm, the greater the S/N value (Fig. 3B). Although the silica nanoparticles modified with 7-and 10-atom spacer arms could immobilize nearly the same amountof HRP molecules, the latter tended to generate higher S/N ratios inthe enzyme immunoassay, probably due to the increased antibodymolecules immobilized, which might also increase the overall affin-ity of the antibody. However, we observed that although the nano-particle conjugates with the 10-atom spacer arm provided thehighest S/N ratio, they turned to aggregate during long-term storage.We attributed this phenomenon to the increased hydrophobicity ofthe nanoparticles with the long-chain alkanes. For this reason, thenanoparticles with the 7-atom spacer arm were used in the follow-ing experiments.

Effect of amount of HRP and antibody used for conjugation

The effect of the amount of HRP used for conjugation was alsoexamined. When 0.2 mg of HRP was added to 2 mg of aminated silicananoparticles, nearly all HRP molecules were immobilized on thesurface of the particles. This result indicated that 0.2 mg of HRPwas nonsaturated for conjugation of 2 mg of nanoparticles. In con-trast, when 0.4 mg of HRP was added, excess HRP remained uncon-jugated, indicating that 0.4 mg of HRP had already been saturatedfor conjugation of such an amount of nanoparticles. A larger amountof HRP was not examined after that.

We then compared these two conjugates in their performance forimmunoassay. To each 2.0 mg of HRP-modified silica nanoparticleswith different dilution ratios, 0.3 mg of anti-HBsAg antibodies wasadded. The resulting conjugates were used to detect 1 ng/ml HBsAgstandard solution. When compared with the HRP-nonsaturatedbifunctional silica nanoparticles, at all dilution ratios, the HRP-satu-rated nanoparticles always generated higher S/N ratios in the detec-tion of 1 ng/ml HBsAg standards (Fig. 4A). The result indicated that,to generate maximal S/N ratios in silica nanoparticle-based enzymeimmunoassay, the HRP used for conjugation should be saturated.Fortunately, saturated HRP conjugation could be easily achievedby using excess HRP for immobilization, and a simple centrifugationcould eliminate all unconjugated HRP.

To study the effect of the amount of antibodies used for conjuga-tion, the HRP-saturated silica nanoparticles were used to conjugatewith a varied amount of anti-HBsAg antibody. The resulting conju-gates were evaluated under different dilution ratios by detecting

ed HRP in the supernatant of the reaction mixture. (B) Effects of different lengths ofe same original concentration and were diluted to different concentrations by the

Table 1Effects of different lengths of spacer arms on conjugation of HRP to silicananoparticles.

Amino silane Spacer arm length (atoms) Number of HRP moleculesper SiO2 nanoparticle

APTMS 4 77AEAPTMS 7 310AEAEAPTMS 10 329

Table 2Intra- and interassay precision of SiO2 nanoparticle-based HBsAg immunoassay.

Concentration (ng/ml) 5.0 2.0 1.0

Intraassay CV (%) (n = 8) 6.10 5.40 5.50Interassay CV (%) (n = 6) 8.93 9.96 11.08

Fig. 5. Correlation analysis between ELISA and bifunctional silica nanoparticle-based ELISA for quantification of HBsAg in 30 positive serum samples (r = 0.992,P < 0.0001, n = 30).

12 Tandem conjugation of enzyme and antibody / R. Ke et al. / Anal. Biochem. 406 (2010) 8–13

1 ng/ml HBsAg standards. Interestingly, the results showed that theS/N ratio was not in direct proportion to the amount of antibodiesconjugated onto the nanoparticles, although nanoparticles harbor-ing more antibody molecules largely gave greater S/N ratios. Thenanoparticles carrying the largest number of antibodies behavedpoorly except at the lowest dilution (Fig. 4B). These data revealedthat the number of antibody molecules on the surface needs to bebalanced to achieve high affinity for best sensitivity.

Taken together, the above results demonstrated that using silicananoparticles as a carrier for conjugation between antibody andenzyme allowed more controllable and adjustable labeling. In com-parison, it is hard for current homobifunctional or heterobifunc-tional cross-linkers to control the amounts of the enzyme andantibody molecules in the conjugates.

Determination of HBsAg by sandwich ELISA

The linear range of the silica nanoparticle-based ELISA was 0.15–12.5 ng/ml with a linear regression value r2 = 0.998. The linear rangewas identical with conventional ELISA. The coefficients of variance(CVs) along the entire concentration range were less than 10%(n = 8). The detection limit, calculated as the response at the zero cal-ibrator plus 3 standard deviations, was 0.06 ng/ml, which was threetimes lower than a conventional ELISA described previously [26].The precision study of the silica nanoparticle-based ELISA usingthree different concentrations of HBsAg standards showed that theCV values of the intra- and interassays were between 5.4% and6.1% and between 8.93% and 11.08%, respectively (Table 2).

A total of 280 serum samples (220 HBsAg-negative and 60 HBsAg-positive) were measured by both conventional ELISA and nanoparti-cle-based ELISA. When the cutoff value was defined as two timesbackground signal, the two methods gave completely consistent re-

Fig. 4. Effects of the amount of HRP (A) and antibody (B) used for preparation of bifuncmodified 2-mg aminated silica nanoparticles. (B) Here 0.15 mg (1), 0.3 mg (2), and 0.45 mfor conjugation. The bifunctional silica nanoparticles used had the same original concentHBsAg standard measured here is 1.0 ng/ml, and the immunoassay followed a standard

sults for all of the samples. To study the correlation between conven-tional ELISA and our method, 30 positive serum samples weresubjected to quantitative detection by both methods. Because theconcentration of HBsAg in serum samples may be high enough to ex-ceed the upper limit of the linear range of both methods, theseHBsAg-positive serum samples were diluted with dilution bufferto make sure that the concentration of HBsAg was located in the lin-ear range. The original concentrations of HBsAg in serum sampleswere subsequently calculated according to the dilution ratios. Theobtained correlation coefficient, r = 0.992 (P < 0.0001, n = 30), dem-onstrated excellent correlation between the two methods (Fig. 5).

tional silica nanoparticles. (A) Here 0.4 mg (a) and 0.2 mg (b) of HRP were used forg (3) of antibody were used for coupling 2 mg of HRP-modified silica nanoparticles

ration, and all were diluted to different folds by dilution buffer. The concentration ofELISA.

Tandem conjugation of enzyme and antibody / R. Ke et al. / Anal. Biochem. 406 (2010) 8–13 13

Conclusions

In this work, we have developed a model system for preparationof antibody–enzyme conjugates on silica nanoparticle scaffold usingHRP and anti-HBsAg. The generated conjugates were used in ELISAfor the detection of HBsAg. Their easy preparation, low cost, and flex-ibility make silica nanoparticles an ideal scaffold for the preparationof antibody–enzyme conjugates. It can be conceived that a similarstrategy should be applicable to other enzyme labels as well as tosmall molecule labels used in immunoassays.

The beneficial nature of using silica nanoparticles as a scaffold isthat it converts a liquid phase-based, single-step reaction into a solidliquid-based, layer-by-layer reaction for enzyme conjugation. Thus,the one-step conjugation is divided into several controllable steps,and in each step the reactant ratio can be adjustable while the prod-ucts can be simply isolated through centrifugations. Because the ex-act labeling ratio is flexibly adjustable rather than strictly restrictedwith the number of active groups of the conjugated molecules, thewhole conjugation process can be optimized to achieve the best per-formance with high reproducibility. Therefore, not only is the purifi-cation of the conjugate greatly simplified, but also the sensitivity isimproved. We expect that this novel labeling strategy could findwide applications in various immunoassay formats.

Acknowledgments

We thank InTec Products (Xiamen, China) for providing humanserum samples and materials used in conventional ELISA. We alsothank Jieli Zhang and Ping Chen for technical assistance. The studywas financially supported by the National Natural Science Founda-tion of China (30500454) and the Natural Science Foundation ofFujian Province of China (2007J0112).

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