Ultrasensitive ELISA Using Enzyme-Loaded Nanospherical Brushes

Letter pubs.acs.org/ac

Ultrasensitive ELISA Using Enzyme-Loaded Nanospherical Brushes as Labels Zhenyuan Qu,† Hong Xu,*,† Ping Xu,† Kaimin Chen,§ Rong Mu,† Jianping Fu,‡ and Hongchen Gu*,† †

State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, P. R. China § Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China ‡ Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Improving the detection sensitivity of enzymelinked immunosorbent assay (ELISA) is of utmost importance for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient “chemical conjugation after electrostatic entrapment” (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boosted the detection limit of ELISA to 0.012 mIU mL−1, a 267-fold improvement as compared to conventional ELISA systems.

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silica nanoparticles,12 and mesoporous silica nanoparticles18,19 have been reported as good candidates for this purpose. However, the current particle systems share several common limitations including a relatively low enzyme loading capacity14 and a significant loss of enzyme activity during the immobilization process.20 To address these issues, herein we report the development of an ultrasensitive immunoassay system by using spherical poly(acrylic acid) (PAA) brushes (SPAABs) as labels. SPAABs have many superior properties over conventional particles as enzyme carriers: the three-dimensional, flexible, and soft PAA brushes can serve as an ideal scaffold for enzyme loading while preserving their biological activities.21−23 However, use of enzyme-loaded SPAABs as labels in ELISA still presents a significant challenge due to enzyme binding stability in SPAABs in a biological medium condition. In the present work, we leveraged a “chemical conjugation after electrostatic entrapment” (CCEE) method24 for covalent immobilization of proteins in SPAABs to demonstrate for the first time that enzyme-loaded SPAABs can be used as ultrasensitive and chemically stable labels in ELISA. Horseradish peroxidase (HRP), the most frequently used enzyme in ELISA, was employed. Human chorionic gonadotrophin (hCG), a bio-

nzyme-linked immunosorbent assay (ELISA) has become the gold standard for laboratorial and clinical analysis owing to its simplicity, low cost, and easy operation and instrumentation.1−3 However, one limitation of conventional ELISA using enzyme-antibody conjugates as labels is its relatively low sensitivity, which has become a bottleneck for meeting the ever-growing demand of early disease diagnosis based on low-abundant biomarkers. To solve this problem, efforts have been made in developing more sensitive substrates,4 introducing biotin−streptavidin systems,5 and performing ELISA in conjuction with polymerase chain reaction (immuno-PCR),6 etc. In recent years, the advent of a variety of innovated immunoassay techniques has promoted the frontier of detection sensitivity to an unprecedented level (∼fM or even lower).7−10 Representative techniques include biobar-code assay,7 Erenna immunoassay,8 Digital ELISA,9 and plasmonic ELISA,10 to name a few. Among various strategies to achieve higher sensitivity of ELISA, use of enzyme-loaded particles as labels presents a simple and promising method, where multiple enzymes are immobilized on the surface of a single particle to improve ELISA detection signal and thus enhance sensitivity.11,12 Currently, the most common way to prepare particle labels is through covalent immobilization of enzyme on particles via a variety of well-established conjugation methods.13 Many types of particles including liposome,14 gold nanoparticles,15 polymeric particles,16 micrometer-sized magnetic particles,17 © XXXX American Chemical Society

Received: July 9, 2014 Accepted: September 5, 2014

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uniformity.24,25 The CCEE process for covalent immobilization of HRP (Scheme 1a) consisted of two sequential steps: tandem electrostatic entrapment (step 1) and chemical conjugation (step 2). While step 1 leveraged the unique “Donnan effect”26 for SPAABs to achieve a heightened enzyme binding capacity, step 2 effectively turned labile electrostatic interaction into stable covalent binding, allowing resuspension of SPAAB-HRP into the biological medium or buffer used in immunoassays. The pH of buffer in step 1 and the EDC dosage in step 2 was optimized to achieve the high binding capacity of HRP (Figure S1 in the Supporting Information). Figure 1a shows a TEM image of SPAAB-HRP, displaying a clear core−shell structure with a uniform size distribution. The 80 nm silica core was surrounded by the PAA corona with an average dry thickness of 44 nm. Successful immobilization of HRP into SPAABs could be intuitively visualized by eye (Figure 1b). After immobilization, SPAABs possessed a characteristic brownish color of HRP. Immobilized HRP can be separated by centrifugation, leaving the supernatant colorless. For further verification, the SPAABHRP complex was subjected to UV−visible spectrometry. As shown in Figure 1c, the characteristic absorption of HRP was clearly seen in SPAAB-HRP, demonstrating their successful immobilization in SPAABs. The binding capacity was estimated to be about 677 μg mg−1 by subtracting the background absorption of SPAABs (which was fitted by an exponential function27) and comparing with the absorption of free HRP at 403 nm. Furthermore, no leaking of HRP was observed after SPAAB-HRP was redispersed in PBS (as judged from A403 in the supernatant). Noteworthily, excellent disparity of SPAABs was maintained after immobilization of HRP, as SPAAB-HRP could be stored in a dispersion state for weeks without notable precipitation. This property of HRP-loaded SPAABs is extremely important for their practical applications as labels in immunoassays to obtain reproducible results. The strong electrostatic repulsion and steric stabilization effect of PAA brushes and the simple CCEE immobilization process might have both contributed to this superb dispersity of SPAAB-HRP. For comparison, conventional carboxylated silica nanoparticles (SiO2−COOH) with a similar size (90 nm) were synthesized and conjugated with HRP via the NHS/EDC process at optimal conditions. The binding capacity of SiO2−COOH was estimated to be 14 μg mg−1 by the depletion method, much less than achieved using SPAABs. The catalytic property of SPAAB-HRP was measured using TMB/H2O2 as a substrate. As shown in Figure 2a, activities of single immobilized HRP relative to free HRP was estimated from the initial slope of catalysis kinetics. This activity could also be determined by the end-point method that was adopted in the subsequent immunoassay system (Figure 2b, see the Supporting Information for experimental conditions), where a linear relationship was observed between A450 and HRP concentration and activities of HRP were calculated from slopes of linear regression fitting. Both methods led to the same conclusion that relative activities of immobilized HRP as compared to free HRP (As) were ∼67% and 5.0% for SPAABHRP and SiO2−COOH-HRP, respectively. The remarkable enhancement in enzyme activity clearly demonstrated the advantage of SPAABs as efficient enzyme carriers. The retained high activity of immobilized HRP was consistent with previous reports,21 where enzymes are immobilized in SPAABs via electrostatic adsorption. Together, our data supported that the additional EDC conjugation step in the CCEE method did not

marker for early pregnancy and trophoblastic tumor, was used as the model analyte. The SPAAB-amplified ELISA immunoassay procedure was illustrated in Scheme 1: (i) SPAAB-HRP complex was prepared Scheme 1. (a) Covalent Immobilization of HRP Into SPAABs by CCEE Process, (b) Conjugation of Antibody onto SPAAB-HRP via NHS/EDC Process, and (c) SPAABs Amplified Sandwich ELISAa

a

Immunocomplex forms between SPAAB-HRP-Ab and immunomagnetic beads in the presence of analytes and can be separated by an external magnetic field. The signal is generated from the yellow product of TMB/H2O2 substrate under the catalysis of immobilized HRP.

by covalent immobilization of HRP in SPAABs via the CCEE process (Scheme 1a); (ii) SPAAB-HRP was decorated with a corona of antibody (anti-β-hCG antibody) via the Nhydroxysuccinimide/N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimidehydrochloride (NHS/EDC) process to obtain SPAAB-HRP-Ab with dual functionalities of recognizing antigen and generating signals (Scheme 1b); (iii) ELISA detection of hCG was achieved by a sandwich assay using antibody (anti-α-hCG antibody) functionalized magnetic beads as solid-phase substrates and SPAAB-HRP-Ab as labels (Scheme 1c). In the presence of analyte, an immunocomplex would form and could be further separated by an external magnetic field. The concentration of analyte was quantified using the signal amplified by HRP immobilized in SPAABs, which catalyzed its 3,3′,5,5′-tetramethylbenzidine (TMB)/ H2O2 substrate to produce yellow products with the maximum absorbance at 450 nm. The SPAABs used in this work was composed of an 80 nm silica core surrounded by densely grafted, long-stretching PAA chains synthesized via surface-initiated RAFT polymerization (SI-RAFT)25 (see Table S1 in the Supporting Information for characterization information). The SI-RAFT process allows a precise tailoring of PAA grafting density and lengths with high B

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Figure 1. (a) TEM image of SPAAB-HRP, the enlarged image clearly shows the core−shell structure of SPAAB-HRP. (b) States of SPAABs before and after immobilization of HRP. The color change of particles and their response to centrifugation indicate the success of HRP immobilization. Free HRP cannot be separated by centrifugation. (c) UV−visible spectra of free HRP, SPAABs, and SPAAB-HRP.

Figure 2. Activity of immobilized HRP relative to free HRP measured by (a) catalysis kinetics and (b) end-point method using TMB/H2O2 as substrates. The results were normalized to concentration of HRP for three forms of HRP.

Table 1. Comparison of SPAABs and SiO2−COOH as Carrier for HRP Immobilization particles SPAABs-HRP SiO2−COOH-HRP

immobilization method

Mparticlea

σ (μg mg−1)

Nb

As (%)

Neff

CCEE NHS/EDC

6.9 × 10 5.4 × 108a

677 14

10 600 172

67.3 5.0

7100 8.6

8a

a

See the Supporting Information for the calculation method of particle molecular weight. bN was calculated by eq 2 using 44 kDa as the molecular weight of HRP.

where As described the enzymatic activity of a single immobilized HRP relative to a free HRP, N was the number of HRP immobilized on a single particle, σ (mg HRP per mg particle) was the enzyme binding capacity, and Mparticle and MHRP were molecular weights of particle and HRP (44kD), respectively. Compared with conventional particles, SPAABs achieved an improvement of 48-fold in binding capacity for HRP and 13.4-fold in maintaining their enzyme activity, which together resulted in an almost 3-order of magnitude enhancement in catalysis efficiency (Table 1). After loading with HRP, the carboxyl groups remaining on SPAABs could be utilized for antibody conjugation via the NHS/EDC process (Scheme 1b). The influence of antibody

significantly affect activities of enzymes immobilized in SPAABs. The activity of immobilized enzymes in SPAABs could remain unchanged over a long period of time and kept the same in a biological sample (Figure S2 in the Supporting Information). The catalytic efficiency of SPAAB-HRP was characterized by the number of effective HRP on a single particle, which was calculated using the following equation:

Neff = A sN N=σ

(1)

M particle MHRP

(2) C

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tion of signal is visually evident, see Figure S5 in the Supporting Information). On the other hand, the background of SPAABamplified system (A450 at hCG = 0) was slightly higher than the conventional system, which was reflected by the intercepts of two linear regression equations. As a result, the limit of detection (LOD), defined by the signal at zero analyte concentration plus 3 standard deviations, was 0.012 mIU mL−1 (corresponding to 1.3 pg mL−1) for SPAAB-amplified system and 3.2 mIU mL−1 (corresponding to 0.35 ng mL−1) for conventional assay. By virtue of the ultrasensitive SPAAB-HRPAb labels, the detection sensitivity of ELISA was improved by 267-fold, much greater than similar immunoassay systems reported in the literature using conventional silica nanoparticles of similar sizes.29−31The LOD of the present SPAAB-amplified immunoassay system, which was in the low pg mL−1 range, was more sensitive or comparable to other similar particle-amplified immunoassay systems.17−19,32−34 Compared with even more sensitive immunoassay systems developed so far,7−10 the present technique improved the sensitivity in a simple way with no additional operation or specialized equipment needed. The SPAAB-amplified system could also be used for the detection of biological samples (e.g., hCG in fetal bovine serum) with the sensitivity improved by about 200-fold (Figure S6 in the Supporting Information), exhibiting potential applicability in detecting clinical samples. The drastic improvement of LOD of the SPAAB-amplified system clearly resulted from the efficient loading of HRP in SPAABs with their high activity properly preserved. In addition, the high density of antibody (134 μg per mg SPAABs, corresponding to ∼357 ng cm−2) immobilized on SPAAB-HRP also contributed to an enhanced sensitivity. All these improvements were attributable to the unique 3D architecture of SPAABs and our rational design of protein immobilization scheme: the massive inner space binding sites were reserved for high-capacity HRP immobilization whose substrates were small molecules that could diffuse rather freely, while the periphery region was decorated with an antibody corona to effectively capture analytes of interest (as we show in Scheme 1). Such a spatial covalent protein immobilization scheme in SPAABs has been previously studied by us,24 enabling a rational design and convenient control of protein distribution in SPAABs with tandem CCEE and NHS/EDC processes. In contrast, in preparation of conventional particles labels, competition of antibody and enzyme molecule for binding sites on the particle is a common issue, which inevitably affects detection sensitivity by either sacrificing the efficiency of capturing analytes or lowering the amplification factor.30 In summary, we have developed an ultrasensitive immunoassay system using HRP-loaded SPAABs as labels. The high capacity and high activity for covalent immobilization of HRP in SPAABs endows SPAAB-HRP with remarkable signal amplification capability. We envision that the ultrasensitive SPAAB-HRP can be used as universal amplification labels in biosensing and molecular diagnostics.

binding capacity and dosage of SPAAB-HRP-Ab on biosensing signal detection was examined (Figure S3a in the Supporting Information). A higher signal-to-background ratio (S/B) was achieved by SPAAB-HRP-Ab with high antibody binding capacity (SPAAB-HRP-AbH). This observation is consistent with the recent report that particles with a higher antibody coverage possess a greater association constant with antigens28 due to multiple attachment. A higher antibody concentration in solution for SPAAB-HRP-Ab might also contribute to the enhanced signal by accelerating the immunocomplex formation. The optimal dosage of SPAAB-HRP-Ab was set as 1 μg, where the highest S/B was reached (Figure S3a in the Supporting Information). In the same way, the concentration of anti-βhCG-Ab-HRP was optimized to be 1 μg mg−1 (Figure S3b in the Supporting Information). After conjugation of antibody, HRP-loaded SPAABs retained 60% of their catalysis activity (Figure S4 in the Supporting Information). Thus, one SPAABHRP-AbH carried ∼600 anti-β-hCG antibodies and 4200 effective HRPs (see the Supporting Information for calculation methods). ELISA detection of hCG using SPAAB-HRP-AbH as labels was performed and compared with conventional assays using anti-β-hCG-Ab-HRP as labels. Both systems were done at their respective optimal conditions. As shown in Figure 3, both systems exhibited excellent linearity between A450 and hCG concentration for the analyte concentration ranges tested. Judging from the slopes of linear regression fitting for the two systems, we conclude that optical signal from SPAAB-HRP-AbH was 400-fold improved as compared to conventional assays using anti-β-hCG-Ab-HRP as labels (The dramatic amplifica-

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ASSOCIATED CONTENT

S Supporting Information *

Figure 3. ELISA detection of hCG using TMB/H2O2 as substrates: (a) conventional assay using β-hCG-HRP as labels and (b) amplified system using SPAAB-HRP-Ab as labels (n = 3). The insets show the magnified results of low hCG concentration and have the same coordinate with the original graphs.

Experimental and additional figures (Figure S1−S6) and information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. D

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support for this work from the UMSJTU Collaboration on Biomedical Technologies (to J.F. and H.G., Grant No. 12X120010007), 863 High Tech Program (Grants 2013AA032203, 2012AA020103), SJTU funding (Grant YG2013MS29), Shmec Project (Grant 14ZZ023), and the U.S. National Science Foundation (Grant ECCS 1231826 and Grant CBET 1263889 to J.F.).

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