Preparation of Highly Stable Oligo(ethylene glycol ...

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Copyright © 2007 American Scientiﬁc Publishers All rights reserved Printed in the United States of America

Journal of Nanoscience and Nanotechnology Vol. 7, 3754–3757, 2007

Preparation of Highly Stable Oligo(ethylene glycol) Derivatives-Functionalized Gold Nanoparticles and Their Application in LSPR-Based Detection of PSA/ACT Complex RESEARCH ARTICLE

Cuong Cao and Sang Jun Sim∗ Department of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746, Korea A sandwich immunoassay for PSA/ACT complex detection based on gold nanoparticle aggregation Delivered by Ingenta using two probes was developed. The functionalized colloidalto: gold nanoparticles (AuNPs) showed DTV - Technical Centerbut of also Denmark highly stable not only in the presence ofKnowledge high ionic strength in a wide pH range. The functionalized AuNPs were tagged with complex monoclonal antibody and goat PSA IP PSA/ACT : 192.38.67.112 polyclonal antibody and served as the probes induce13:39:16 aggregation of the colloidal particles. As a Wed, 14 Janto 2009 result, PSA/ACT complex was detected at concentrations as low as 1 ng/ml. This is the ﬁrst time that a new aggregation sandwich-immunoassay technique using two gold probes has been used, and the results are generally applicable to other LSPR-based immunoassays.

Keywords: Localized Surface Plasmon Resonance, Particle Aggregation, Prostate Cancer, Oligo(ethylene glycol).

1. INTRODUCTION Metallic nanoparticles have attracted much attention due to their unique optical properties. When the nanometer-sized metallic structures are excited by light, the nanoparticles will exhibit a strong UV-Vis absorption band that is not present in the spectrum of the bulk metal.1 This absorption band occurs when an incident photon frequency is resonant with the collective oscillation of the conduction electrons of the nanoparticles which leads to so-called localized surface plasmon resonance (LSPR).1 3 To model the LSPR of spherical nanoparticles, Mie theory is the simplest calculation available for estimating the extinction of a metallic nanoparticle. The wavelength shifts in the extinction maximum of nanoparticles can be used to detect moleculeinduced changes surrounding the nanoparticles.2 Nanoparticle aggregation induced by speciﬁc biomolecular interaction is one of the nanoparticle-based sensing mechanisms that enable the transduction of biological/chemical-binding events into optical signals based on the wavelength shifts in LSPR max .3 The sensing phenomenon induces a color change of the colloidal solution from red (free particles) to blue (aggregated particles), and it has been developed as simple colorimetric ∗

Author to whom correspondence should be addressed.

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assays for the detection of DNA hybridization,4 as well as having potential applications in immunoassays.5 Our study shows the ﬁrst time that using two probes for a sandwich immunoassay can enhance the wavelength shifts in LSPR max and thus the sensitivity of the assay. When a target antigen is bound to its gold-tagged primary antibody, the binding event will lead to the wavelength shift to the red area due to changing in dielectric constant of the medium surrounding environment. Moreover, if a gold-tagged secondary antibody is subsequently added, a considerable aggregation will be induced because of the fact that the polyclonal antibodies contain the entire antigen-speciﬁc antibody population, one antigen molecule can form a complex with several antibody molecules. Prostate speciﬁc antigen-1 -antichymotrypsin (PSA/ACT complex, MW 90 kDa) is chosen as a target analyte because of its important role in prostate cancer diagnosis.6 7 Beneﬁts of oligo(ethylene glycol) (OEG) are exploited to be used as linker for the chemical conjugation of PSA capture antibodies. OEG has been recognized for its ability to prevent non-speciﬁc adsorption of proteins.8 We functionalized the bare surface of AuNPs using various OEG mixtures of HS(CH2 11 (OCH2 CH2 6 OCH2 COOH and HS(CH2 11 (OCH2 CH2 3 OH to optimize the surface coverage of protein and to reduce the level of a nonspeciﬁc binding. Stability of the functionalized AuNPs has 1533-4880/2007/7/3754/004

doi:10.1166/jnn.2007.009

Cao and Sim

Preparation of Highly Stable OEG Derivatives-Functionalized Gold Nanoparticles and Their Application

also been examined under strong ionic strength and in wide range of pH.

(a)

2. EXPERIMENTAL DETAILS

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RESEARCH ARTICLE

PSA/ACT complex, PSA/ACT complex monoclonal antibody (PSA/ACT mAb) and goat PSA polyclonal antibody (PSA pAb) were supplied by BiosPaciﬁc, Inc. HAuCl4 , Na3 C6 H5 O7 , N -hydroxysuccinimide (NHS), N -ethyl-N (3-diethylaminopropyl) carbodiimide (EDC), 0.1 M phosphate buffered saline pH 7.2 (PBS) were purchased from Sigma-Aldrich. HS(CH2 11 (OCH2 CH2 6 OCH2 COOH and HS(CH2 11 (OCH2 CH2 3 OH (HS-OEG6 -COOH) (HS-OEG3 -OH) were purchased from Cos Biotech (Korea). (b) AuNPs were prepared by sodium citrate reduction of aqueous HAuCl4 solution as described by Turkevich et al.9 Before use, the pH was adjusted to 7.2 by adding 0.5 N NaOH, and then the solution was and stored at 4 C. This method yields spherical, ruby red particles withDelivered an average by Ingenta to: - Technical Center of Denmark diameter of about 21 nm, and DTV the surface plasmonKnowledge reso: 192.38.67.112 nance occurred at 526 nm. Then, the AuNPs wereIPcapped Wed, 143 -OH Jan 2009 13:39:16 with 1:10 molar ratio of HS-OEG6 -COOH/HS-OEG by mixing 9 ml of the gold solution and 1 ml of ethanolic solution of HS-OEG6 -COOH/HS-OEG3 -OH to yield an effective HS-OEG6 -COOH/HS-OEG3 -OH capping concentration of 0.5 mM. To prepare Au-PSA/ACT mAb and Au-PSA pAb probes, 1 ml of the functionalized AuNPs (A520 nm = 2) was mixed with 10 l of 1 M EDC/NHS. Fig. 1. (a) Size distribution and morphology (the inset) of AuNPs; (b) UV-Vis absorption spectra of AuNPs and AuNPs-mAb. After 3 min, 500 ml of the antibodies (1 mg/ml) was added to the activated AuNPs. The solution was mixed well, and then it was allowed to react at least 2 hrs at ambient temmaximum absorption peak at max = 526 nm, assigned to perature with a gentle mixing. Finally, the Au probes were the characteristic feature of gold plasmon resonance.10 On rinsed, collected, and stored in 0.1 M PBS buffer (pH 7.2) capping the AuNPs with 0.5 mM of 1:10 mixed solution at 4 C for up to 2 weeks. between HS-OEG6 -COOH and HS-OEG3 -OH, a damping The UV-Vis absorption spectra were recorded using an of surface plasmon band was happened as indicated by a Optizen 2120 UV plus spectrophotometer. Average diamsmall red shift (data not shown). These changes in the optieter of AuNPs was measured by dynamic light scatcal properties of the colloidal gold as being modiﬁed were tering (DLS) analysis (Brookhaven Instruments, USA). consistent with other previous studies,11 and indicated a Fourier transform infrared spectroscopy (FT-IR) spectra complete chemisorption of the OEG-thiol molecules on the were recorded by a Bruker IFS-66/S spectrometer. High gold surface. A higher red shift to 530 nm observed when resolution transmission electron microscopy (HR-TEM) the functionalized AuNPs were conjugated with PSA/ACT measurements were performed on a JEOL model JEMmAb indicated the formation of the protein layer on the 2100F instrument operated at an accelerating voltage of surface of the AuNPs. The same red shift of the wavelength 200 kV. was also observed when the secondary probe was prepared by conjugating it with PSA/ACT pAb (data not shown). The usefulness of the functionalized nanoparticles in bio3. RESULTS AND DISCUSSION logical applications depends on their stability,12 especially for those based on nanoparticle aggregation. Therefore, it 3.1. Synthesis of the Carboxyl-Stabilized AuNPs should be assured that the particle aggregation is caused Figure 1(a) shows that spherical, quite homogenous, ruby by the immune reactions, not due to pH or high ionic red AuNPs with Dmean = 227 nm ± 3.51 nm were synstrength of environment. In the study, when different conthesized by reduction of HAuCl4 using sodium citrate as centrations of NaCl ranging from 0% to 35% were added a reducing agent. In Figure 1(b), the UV-Vis spectrum of to the –COOH/–OH-stabilized AuNPs and rapidly mixed, the ruby red solution produced after chemical reduction no precipitation was observed, whereas a black precipitate with sodium citrate indicates formation of AuNPs with a was immediately formed in the citrate-passivated AuNP

RESEARCH ARTICLE

Preparation of Highly Stable OEG Derivatives-Functionalized Gold Nanoparticles and Their Application

Cao and Sim

Delivered by Ingenta to: DTV - Technical Knowledge Center of Denmark IP : 192.38.67.112 Wed, 14 Jan 2009 13:39:16

Fig. 2. UV-Vis absorption spectra of –OH/–COOH OEG thiolsstabilized AuNPs in high ionic strength (a), and in wide range of pH (b).

sample (data not shown). Figure 2(a) shows the normalized absorbance spectra of AuNPs peak at 526 nm for the 0% sample of NaCl. These spectra are quite indistinguishable even if the NaCl concentrations are increased up to 35%. Similar results are also observed for the nanoparticle’s stability as a function of pH (Fig. 2(b)). It is evident that the steric repulsion of the OEG-thiols-capped chains on the colloid surface improved the dispersion stability not only in the presence of the very high salt concentration conditions but also in a wide range of pH. In Figure 3, the transmittant spectrum of OEG thiolsstabilized AuNPs is similar to that of pure OEG thiols, and the main bands are clearly observed and identiﬁed as follows. The broad bands centered at about 3660 cm−1 are due to O-H vibrations of alcohol and from those of trapped water molecules. The bands observed at 2985 and 2990 cm−1 correspond to asymmetric and symmetric stretching of the alkyl chain (-CH2 -) which is in good agreement with IR spectrum of dodecanthiols.13 The strong peaks at 1064 cm−1 represent for C O C bending bands of OEGs. The C O bands of the carboxyl groups are observed in the spectra at 1747 cm−1 for the free –COOH OEG thiol, and at 1666 cm−1 for the –OH/–COOH OEG thiols-stabilized AuNPs. The shift in wavenumber is likely due to a change in their dipole moment when the free OEG thiols bind onto the gold surface with high 3756

Fig. 3. FT-IR spectra of free –OH OEG thiol (black spectrum), free –COOH OEG thiol (red spectrum) and –OH/–COOH OEG thiolsstabilized AuNPs (blue spectrum) at 4000–600 cm−1 .

electron density. Obviously, the C O bands of the carboxyl groups do not exist in the spectrum of the free –OH OEG thiol. However, the very weak peak at 2522 cm−1 , which attributes to the SH stretching vibration mode,13 disappears when the HS-OEG6 -COOH and HS-OEG3 -OH molecules adsorb on the surface of AuNPs, thus it gives a strong evidence that the S H bond is disrupted and HS-OEG6 -COOH/HS-OEG3 -OH anchors on the surface of gold through the S atom in the mercapto group. 3.2. Detection of PSA/ACT Complex by Nanoparticle Aggregation Induced by Double Probes (Sandwich) Method The nanoparticle aggregation was induced by adding 200 l of various concentrations of PSA/ACT complex ranging from 0 to 1000 ng/ml to 300 l of the PSA mAbgold probe (normalized at 530 = 035 − 04). After incubating at ambient room temperature for 1 h, 300 l of the normalized PSA pAb-gold probe was consequently added to the reaction, then, the UV-Vis spectra were recorded after 30 min. As shown in the Figure 4(a), PSA/ACT complex is determined for the whole range of investigated J. Nanosci. Nanotechnol. 7, 3754–3757, 2007

Cao and Sim

Preparation of Highly Stable OEG Derivatives-Functionalized Gold Nanoparticles and Their Application

probe is used (Fig. 4(b)). The results are much lower than those using two AuNPs probes, correlatively, due to the fact that binding reaction between PSA/ACT complex and PSA mAb likely induces the nanoenvironment’s dielectric constant (m surrounding the primary gold probe.

4. CONCLUSION

Fig. 4. (a) UV-Vis absorption spectra describing the detection of PSA/ACT complex using two AuNPs probes to increase interparticlecoupling aggregation; (b) UV-Vis absorption spectra describing the detection of PSA/ACT complex using one AuNPs probe.

concentrations. At 0 ng/ml of the analyte, the maximum absorption peak is centered around 531 nm. Up to a PSA/ACT complex concentration of 1 ng/ml, the max shift is observed for about 2 nm (peak at 533 nm). Moreover, the spectra are greatly shifted to red area if the analyte concentrations are increased. Therefore, the maximum wavelength shift is 31 nm for the 1000 ng/ml of PSA/ACT complex. The absorbance feature shows the onset of particle aggregation and is a result of plasmon coupling between aggregated nanoparticles.5 12 The results indicate that the PSA/ACT complex concentration could be determined by the sandwich-induced aggregation over a wide range, and limit of detection of the assay in the samples was 1 ng/ml of PSA/ACT complex, which is comparable with other immunological methods such as ELISA or SPR-based detection.7 14 It is because polyclonal antibodies contain an entire antigen-speciﬁc antibody population, where one antigen molecule can form a complex with several antibody molecules which leads to enhancement of aggregation-sensing assay. In fact, the max shifts are 2 nm and 5 nm for the 100 and 1000 ng/ml concentrations of PSA/ACT complex, respectively, if only the primary

Acknowledgments: This research was performed for the Core Environmental Technology Development Project for Next Generation funded by the Ministry of Environment of Korea.

References and Notes 1. C. L. Hayes and R. P. Van Duyne, J. Phys. Chem. B 105, 5599 (2001). 2. U. Kreibig and M. Vollmer, Cluster Material, Springer, Berlin, Heidelberg, New York (1995). 3. D. A. Stuart, A. J. Haes, C. R. Yonzon, E. M. Hicks, and R. P. Van Duyne, IEEE Proc.-Nanobiotechnol. 152, 13 (2005). 4. R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger, and C. A. Mirkin, Science 277, 1078 (1997). 5. N. T. K. Thanh and Zeev Rosenzweig, Anal. Chem. 74, 1624 (2002). 6. T. M. Chu, J. Clin. Lab. Anal. 8, 323 (1994). 7. C. Cao, J. P. Kim, B. W. Kim, H. Chae, H. C. Yoon, S. S. Yang, and S. J. Sim, Biosens. Bioelectron. 21, 2106 (2006). 8. R. G. Chapman, E. Ostuni, L. Yan, and G. M. Whitesides, Langmuir 16, 6927 (2000). 9. J. Turkevich, P. C. Stevenson, and J. Hillier, Discuss. Faraday Soc. 11, 55 (1951). 10. H. Otsuka, Y. Akiyama, Y. Nagasaki, and K. Kataoka, J. Am. Chem. Soc. 123, 8226 (2001). 11. N. Lala, A. G. Chittiboyina, S. P. Chavan, and M. Sastry, Colloid Surface A 205, 15 (2002). 12. R. C. Doty, T. R. Tshikhudo, M. Brust, and D. G. Fernig, Chem. Mater. 17, 4630 (2005). 13. A. Manna, T. Imae, T. Yogo, K. Aoi, and M. Okazaki, J. Colloid Interf. Sci. 256, 297 (2002). 14. D. A. Armbruster, Clin. Chem. 39, 181 (1993).

Received: 16 April 2007. Revised/Accepted: 23 May 2007. J. Nanosci. Nanotechnol. 7, 3754–3757, 2007

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The gold-protein conjugates were successfully synthesized and applied in LSPR-based detection of PSA/ACT complex. By functionalizing the AuNPs with –OH/-COOH OEG thiols, the particles showed their high stability not only in the presence of high ionic strength but also in a wide pH range. The functionalized AuNPs were tagged with PSA/ACT complex mAb and goat PSA pAb and served as the probes to induce aggregation of the colloidal particles. As a result, PSA/ACT complex was detected at concentrations as low as 1 ng/ml. This is the ﬁrst time that a new aggregation Delivered by Ingenta to: sandwich-immunoassay technique using two gold probes developed, and the results showed that the Technical Knowledge Center was of Denmark detection strategy could provide a useful tool for detection IP : 192.38.67.112 of the PSA-ACT complex, and it is generally applicable to Wed, 14 Jan 2009 13:39:16 other LSPR-based immunoassays.