J. Phys. Chem. C 2008, 112, 17357–17361
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Magnetite Nanoparticle-Linked Immunosorbent Assay Lizeng Gao, Jiamin Wu, Sarah Lyle, Keith Zehr, Liangliang Cao, and Di Gao* Department of Chemical and Petroleum Engineering, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15261 ReceiVed: July 7, 2008; ReVised Manuscript ReceiVed: September 4, 2008
Using chitosan-modified magnetite nanoparticles (CS-MNPs) as a replacement of enzymes in conventional ELISA configurations, a magnetic nanoparticle-linked immunosorbent assay is developed. The CS-MNPs are synthesized by using a one-step solvothermal process, where chitosan in the reaction system is used both as a ligand and as a surface modification agent. The as-synthesized CS-MNPs have amine groups on their surface which provide good dispersibility in aqueous solutions and convenient sites for covalent linking of antibodies with the MNPs. They also possess catalytic properties that are able to catalyze color reactions in immunoassays and magnetic properties that can be used to capture, separate, and enrich antigens prior to the assay procedure. By employing both the catalytic and magnetic properties of the CS-MNPs, a capture-detection immunoassay is developed, where antigens can be captured, separated, and enriched prior to the assay procedure. Introduction Enzyme-linked immunosorbent assay (ELISA) is a versatile biochemical technique commonly used as a diagnostic tool and/ or a quality control check for detection of antigens in medicine, plant pathology, and various industries.1-6 A typical procedure of ELISA involves three steps: (i) immobilizing the antigen on a solid surface (such as a microtiter plate) either specifically or nonspecifically, (ii) binding the antigen with an antibody that is linked with an enzyme either by itself or via a secondary antibody, and (iii) adding a substrate of the enzyme and color agents that are able to produce a detectable chromogenic or fluorogenic signal via an enzymatic reaction indicating the quantity of the antigen in the sample. Most commonly used enzymes in ELISA include horseradish peroxidase (HRP) and alkaline phosphatase (AP).2-6 Although these enzymes are very effective in converting the substrate to elicit a chromogenic or fluorogenic signal, they lose their enzymatic activities gradually accompanying long-term storage, which limits the assay’s performance. In addition, a significant fraction of the cost of the ELISA technology is involved with production and purification of the enzyme-antibody conjugation. Recently, it has been found that magnetite (Fe3O4) nanoparticles (MNPs) possess an intrinsic peroxidase-like activity, which can be used similarly to the use of HRP to catalyze the color reaction of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2).7 Such a catalytic property of MNPs may be employed to replace the enzyme used in ELISA. The replacement of enzymes by MNPs in immunoassay may have the following advantages:8-11 (i) the catalytic property of MNPs is significantly more stable than enzymes such as HRP, which is favorable for long-term storage; (ii) MNPs are able to catalyze the color reaction in a much broader pH range than typical enzymes; and (iii) MNPs can be used to capture and separate the antigen from the sample with a simple magnetic field, which integrates the sample preparation with assay and, therefore, facilitates the overall analysis procedure. In order to be used in immunoassays, the MNPs need to have the following material properties. First, they need to be easily * To whom correspondence should be addressed. Tel: (412) 624-8488. Fax: (412) 624-9639. E-mail:
[email protected].
dispersed in aqueous solutions at a physiological pH.12 Second, they need to have functional groups on their surface which can be used for convenient linking of antibodies.13,14 Third, the MNPs need to have a large enough saturation magnetization per particle (which typically increases as the size of the particles increases) in order to be effectively separated from solution by using a moderate magnetic field, which puts a lower limit (typically around 100 nm) on the size of the MNPs that can be used in immunoassay. Fourth, after the MNPs aggregate upon applying a magnetic field, the aggregation should be easily redispersed in solution upon removal of the magnetic field, which puts an upper limit of about 30 nm on the crystalline domain size of the MNPs because further increasing the crystal size of the MNPs induces a superparamagnetic-ferromagnetic transition.15 Obviously, the third and the fourth requirements exclude the use of single crystalline MNPs as a good candidate for immunoassay applications, because there is no overlap on the size requirements. A recent report15 demonstrates that clusters of Fe3O4 nanocrystals may possess both a high magnetization and a superparamagnetic property, which is promising for the immunoassay application because it meets both the third and the fourth requirements. In this paper, we report a one-step method for synthesis of chitosan-modified MNPs (CS-MNPs) and the application of such CS-MNPs in an MNP-linked immunoassay. The as-synthesized CS-MNPs possess properties that meet all the abovementioned four requirements for applications in immunoassays: (i) they have amine groups on their surfaces that can be conveniently used for covalent linking of antibodies to the MNPs; (ii) the chitosan prevents aggregation of MNPs and promotes dispersion of the MNPs in aqueous solutions; (iii) they have a large enough magnetization and can be easily separated by a magnet from solution; and (iv) the aggregation of MNPs can be easily redispersed in solution upon removal of the magnetic field, which enables the integration of MNP-assisted sample preparation with the assay process. By employing both the magnetic and the catalytic properties of the synthesized CS-MNP, a capture-detection immunosorbent assay is developed, where the magnetic property is used to capture, separate, and enrich the antigen in the sample prior to the assay process, and the catalytic
10.1021/jp805994h CCC: $40.75 2008 American Chemical Society Published on Web 10/14/2008
17358 J. Phys. Chem. C, Vol. 112, No. 44, 2008 property is used to catalyze the color reaction for producing detection signals. Experimental Methods Chemicals. Sodium acetate (NaAc), ethanol and glutaraldehyde were purchased from Fisher Scientific. Ethylene glycol was purchased from J.T. Baker. Iron(III) chloride (FeCl3), sodium bicarbonate (NaHCO3), tween-20, chitosan (low molecular weight, viscosity 20-200 cps), phosphate-buffered saline (PBS), bovine serum albumin (BSA), hydrogen peroxide (H2O2) (30% stocking), iron(II, III) oxide nanopowder ( 1609). Representative SEM and TEM images of the as-synthesized MNPs are shown in panels b and c of Figure 1, respectively. It is observed that the diameter of the spherical MNPs is about 200 nm. The TEM image reveals that each particle is an assembly of even smaller Fe3O4 crystals with diameters varying from 10 to 20 nm. The as-synthesized CS-MNPs were easily dispersed in water by sonication and the dispersion remained stable for more than 1 h before precipitation was observed. In contrast, the MNP without chitosan modification aggregated rapidly and precipitated almost completely in less than 20 min. Figure 1d shows the optical images of the two types of MNPs taken 20 min after they were dispersed in water by sonication. Compared to the unmodified MNPs, CS-MNPs possess much higher dispersibility in water.
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Figure 3. FTIR spectra of chitosan and chitosan-modified MNPs.
Figure 1. Characterization of chitosan-modified MNPs (CS-MNPs). (a) XRD pattern. (b) SEM image. (c) TEM image and electron diffraction pattern (the inset). (d) Comparison of the dispersibility in water between CS-MNPs and unmodified MNPs: the unmodified MNPs aggregated rapidly and precipitated almost completely in less than 20 min; while the CS-MNP were easily dispersed in water by sonication and the dispersion remained stable for more than 1 h. The optical image was taken 20 min after the two types of particles were dispersed in water by sonication.
Figure 2. Demonstration of the superparamagnetic properties of the as-synthesized CS-MNPs. (a) Both the as-synthesized CS-MNPs and commercial iron magnetic nanoparticles were dispersed in water. (b) Both types of particles aggregated upon application of a magnetic field by a magnet. (c) Upon removal of the magnetic field, the CS-MNPs were easily redispersed in water with slightly shaking; however, the commercial iron magnetic nanoparticles stayed aggregated.
Each as-synthesized CS-MNP is a cluster of many Fe3O4 crystals with diameters varying from 10 to 20 nm rather than one single crystal. Fe3O4 particles with such a microstructure are likely to exhibit a superparamagnetic property.15 A significant advantage of MNPs with the superparamagnetic property for bioassay application is that after aggregated by a magnetic field, the aggregation of MNPs can be easily redispersed in solution upon removal of the magnetic field. We observed such a phenomenon using our as-synthesized CS-MNPs. As shown in Figure 2, after dispersing the as-synthesized CS-MNPs and commercial iron magnetic nanoparticles in water in two vials (Figure 2a), a magnet was used to induce aggregation of both types of particles (Figure 2b). Upon removal of the magnetic field, the CS-MNPs were easily redispersed in water with slightly shaking; however, the commercial iron magnetic nanoparticles stayed aggregated (Figure 2c). Figure 3 presents the FTIR spectra of pure chitosan and chitosan modified MNPs. The characteristic bands of pure chitosan are observed at 3420 (O-H stretching and N-H stretching vibrations), 1645 and 1548 (amide I, amide II and amino group), and 1076 cm-1 (C-O-C stretching vibration), which are consistent with previous publications.19,20 All these characteristic bands can be also seen in the spectrum of CSMNPs, in addition to a band appears at 604 cm-1, which can be related to Fe-O vibration.15 These results show the presence of chitosan on the surface of the as-synthesized nanoparticles even after extensive washing.
Figure 4. Color reaction of TMB catalyzed by the CS-MNPs in the presence of H2O2. (a) Absorption spectra: the black line is absorption spectrum of the reaction solution without the presence of CS-MNPs; the blue and the red lines are absorption spectra of the reaction solution mixed with CS-MNPs before and after addition of H2SO4 (2 N), respectively. Insets are optical images of the reaction solutions corresponding to the absorption spectra. (b) optical images of reaction mixtures comparing cases that (i) the color reaction is induced only by homogeneous catalysis due to iron ions leached from CS-MNPs and (ii) the color reaction is induced by heterogeneous catalysis with the presence of CS-MNPs.
The amino groups in chitosan provide convenient sites for covalent linkage of biomolecules such as antibodies to the MNP through linking their amino groups with the amino groups on the MNP using amino-cross-linkers such as glutaraldehyde. As a demonstration, we functionalized the CS-MNPs with mouse IgG. By monitoring the concentration of the antibodies in the solution before and after the immobilization step, we estimate that approximately 80% of the antibodies were immobilized on MNPs by using the current experimental procedure, which corresponds to about 100 mg mouse IgG per gram particles. The color reaction of TMB in the presence of H2O2 was used to demonstrate the catalytic activity of the CS-MNPs. As shown in Figure 4a, after mixing the MNPs with TMB and H2O2, the mixture turned from clear to blue (with a peak at 652 nm in the absorbance spectra). The color development was stopped by the addition of H2SO4 (2 N), changing the color to yellow (with a peak at 450 nm in the absorbance spectra. The color change induced by this reaction is similar to the color reaction of TMB catalyzed by HRP,3 which indicates that the synthesized CSMNPs have catalytic activities similar to HRP and may be used to catalyze the color reaction in ELISA. To investigate whether the color reaction was induced by homogeneous catalysis due to iron ions leached from CS-MNPs or by heterogeneous catalysis on the surface of CS-MNPs, CSMNPs were dispersed in 500 µL NaAc solution (0.1 M, pH 3.5) for 20 min and the mixture was separated to supernatant (solution with few or no CS-MNPs) and CS-MNPs precipitate. The supernatant was collected and mixed with 0.3% H2O2, 100 µg TMB; while the precipitated CS-MNPs were redispersed in 500 µL reaction buffer. Figure 4 shows the optical images of
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Figure 5. Schematics of the two configurations used in the MNP-linked immunosorbent assay. (a) The antigen is directly adsorbed on the plate through nonspecific interactions. (b) A sandwich format is used, where a capture antibody is first immobilized on the plate, and a detection antibody is linked to the MNP.
both mixtures taken after 10 min. Without the presence of CSMNPs, the supernatant showed little color changes in H2O2/ TMB system. Therefore, the observed color reaction is likely induced by heterogeneous catalysis on the surface of CS-MNPs. Stimulated by its peroxidase-like catalytic properties, we used the synthesized CS-MNP as a replacement for HRP in two immunoassay configurations (Figure 5) to detect mouse IgG and CEA tumor marker. In the first configuration, (Figure 5a), the mouse IgG was directly immobilized on the microtiter plate wells through nonspecific interaction, and the CS-MNP was functionalized with antimouse IgG. The assay procedure was very similar to a conventional ELISA as described in the following three steps: (i) the antimouse IgG functionalized CSMNPs were added to the mouse IgG-coated plate; (ii) TMB was used as a colorimetric substrate in the presence of H2O2, and (iii) the optical density (OD) at 652 nm was measured. In the second configuration (Figure 5b), a sandwich immunoassay format was used to detect CEA, which was constructed by using a CEA-specific monoclonal antibody (M111146) coated onto microtiter plate wells as a capture antibody and another CEAspecific monoclonal antibody (M111147) linked to the MNPs as a detection antibody. Typical results for these two assays are shown in panels a and b of Figure 6, respectively. There is a clear correlation between the concentration of the antigens and the detected optical signal. Because the assay format of these two configurations have been widely used in ELISA and the assay procedure is similar to ELISA, we name these assays “MNP-linked immunosorbent assay” to indicate that MNPs are used instead of an enzyme to catalyze the color reaction. The MNP-linked immunoassays shown in Figure 5 only employ the catalytic properties of the MNPs. By employing the superparamagnetic properties of the MNPs, a capture-detection assay can be constructed where antigens can be captured, separated from the sample, and enriched prior to the assay procedure. In such an assay, the as-synthesized CS-MNPs serve as both a magnetic antigen-enrichment agent and a catalyst in the color reaction due to their intrinsic dual functionalities. This is demonstrated using the experiment schematically shown in Figure 7. Briefly, the CS-MNPs were first functionalized with a capture antibody (anti-CEA M111147). Then, the functionalized MNPs were mixed with a series of samples containing CEA in different concentrations in PBS solution. After the CEA was captured by the MNPs, a magnetic field was applied to separate the MNPs from the rest of the sample. Another monoclonal CEA antibody (M111146) specific for CEA was coated onto microtiter plate
Figure 6. Typical results of MNP-linked immunosorbent assay for detection of (a) mouse IgG and (b) CEA using the configurations presented in panels a and b of Figure 5, respectively.
wells and used as a solid support in the sandwich format. Upon adding the MNPs-enriched sample to the wells, the MNPs that had captured antigens were immobilized on the plate through binding of the antigen with the antibody used as the solid support in the wells, while other MNPs that had not captured antigens were rinsed away. The MNPs immobilized on the surface of the wells catalyzed the color reaction upon addition of TMB and H2O2 to the wells. The amount of CEA in the sample was then quantified by measuring the optical density at 652 nm. Figure 8 shows a typical result of the assay that correlates the CEA concentration varying from 1-8 ng/mL to the optical density detected. The curve is compared to signals detected from negative controls where BSA solutions in the same concentration range were used. This capture-detection immunoassay based on CS-MNPs has a detection limit of about 1 ng/ml for CEA. In summary, the use of chitosan in the solvothermal process can effectively functionalize the surface of as-synthesized MNPs with amine groups which can be used for convenient covalent linking of the MNPs with antibodies. The presence of chitosan in the reaction system also produces sub-micrometer-sized MNPs as an assembly of nanometer-sized crystals. Such a structure is able to provide the MNPs with a large enough
Nanoparticle-Linked Immunosorbent Assay
J. Phys. Chem. C, Vol. 112, No. 44, 2008 17361 to replace the enzymes in conventional ELISA. By employing both the catalytic and the magnetic properties of the MNPs, we have demonstrated a capture-detection immunoassay, where the antigens are captured, separated, and enriched prior to the assay procedure. Acknowledgment. This work was supported by the ACS Petroleum Research Fund and the University of Pittsburgh Mascaro Sustainability Initiative. References and Notes
Figure 7. Schematics of the MNP-linked immunoassay that directly incorporates the antigen capture, separation, and enrichment with the assay by employing both the catalytic and superparamagnetic properties of the MNPs.
Figure 8. Typical result of the capture-detection assay that correlates the CEA concentration varying from 1 to 8 ng/mL to the optical density. The curve is compared to signals detected from negative controls where BSA solutions in the same concentration range were used.
saturation magnetization that can be used to capture, separate, and enrich antigens, while simultaneously enables the redispersion of the MNP aggregation in solution upon removal of the magnetic field. The as-synthesized MNPs are able to catalyze color reactions in the immunoassay and, therefore, can be used
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