Low-Level Detection of Poly(amidoamine) PAMAM Dendrimers Using Immunoimaging Scanning Probe Microscopy

Hindawi Publishing Corporation International Journal of Analytical Chemistry Volume 2012, Article ID 341260, 8 pages doi:10.1155/2012/341260 Research Article Low-Level Detection of Poly(amidoamine) PAMAM Dendrimers Using Immunoimaging Scanning Probe Microscopy Chevelle A. Cason,1 Thomas A. Fabré,1 Andrew Buhrlage,1 Kristi L. Haik,2 and Heather A. Bullen1 1 Department of Chemistry, Northern Kentucky University, Highland Heights, KY 41099, USA 2 Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY 41099, USA Correspondence should be addressed to Heather A. Bullen, Received 29 September 2011; Accepted 3 November 2011 Academic Editor: Charles L. Wilkins Copyright © 2012 Chevelle A. Cason et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Immunoimaging scanning probe microscopy was utilized for the low-level detection and quantification of biotinylated G4 poly(amidoamine) PAMAM dendrimers. Results were compared to those of high-performance liquid chromatography (HPLC) and found to provide a vastly improved analytical method for the low-level detection of dendrimers, improving the limit of detection by a factor of 1000 (LOD = 2.5 × 10−13 moles). The biorecognition method is reproducible and shows high specificity and good accuracy. In addition, the capture assay platform shows a promising approach to patterning dendrimers for nanotechnology applications. 1. Introduction Dendrimers are at the forefront of research in nanoscience due to the many interesting properties of these macromolecular systems including their precise architecture, highly reproducible shape, high uniformity and purity, low immunogenicity and toxicity, high loading capacity, and high shear resistance [1–5]. They have shown a great deal of versatility with applications in numerous areas such as drug delivery [6, 7], gene therapy [8, 9], chemotherapy [10], electrochemistry [11, 12], metal recovery [13], catalysis [14, 15], and sensors [16–18]. Development of new low-level detection and quantification methods is needed with the utilization of these nanomaterials. Currently, high-performance liquid chromatography (HPLC) is the predominate approach reported for dendrimer quantification [19, 20]. However, the primary focus of HPLC, along with capillary electrophoresis, has been to evaluate dendrimer purity and degree of conjugation [21–27]. Little has been reported within the literature with regard to the advancement of new quantification methods for dendrimers. This work introduces a biorecognition readout technique that has the potential to provide low-level detection of dendrimers. Biotinylated poly(amidoamine) PAMAM dendrimers were chosen as a model target. PAMAM dendrimers, which are highly water soluble, represent the most widely studied class of dendrimers. Functionalization of PAMAM dendrimer surfaces has proven useful in their utilization for various applications including drug delivery and chemical sensing [5, 6, 16]. Biotin-labeled dendrimers have been utilized in tumor [28] and antibody [29] targeting studies and biosensor design [30]. Biotinylated PAMAM dendrimers may also have the potential for delivering therapeutic drugs to the brain [31, 32]. We report here a readout method using an immunoassay platform and scanning probe microscopy (SPM) for lowlevel quantification of biotinylated G4 PAMAM dendrimers. The assay takes advantage of the documented specificity of biotin-avidin. Results are correlated with HPLC analysis. In addition, we briefly highlight the potential of this capture assay platform to selectively pattern PAMAM dendrimers onto a surface. Patterning of nanoparticles is relevant to a wide variety of applications in the fields of sensing, drug delivery, or development of nanodevices [33–35]. Dendritic architectures show promise in designing and developing sensor platforms with high sensitivity and stability [16]. 2 International Journal of Analytical Chemistry 2. Experimental ODT 2.1. Reagents. Poly(amidoamine) PAMAM dendrimers [core: ethylene diamine] (G = 4) dendri-PAMAM-(NH2 )32 were obtained from Dendritic Nanotechnologies, Inc. (Mt. Pleasant, MI). Biotinylated PAMAM dendrimers were prepared using sulfo-NHS-LC-biotin (Pierce EZ-Link Kit) as described previously [36]. Briefly, a 3 : 1 molar ratio of biotin/PAMAM dendrimers in 0.1 M phosphate buffer saline (PBS) was allowed to react for 2 h on an orbital shaker. Excess, unreacted biotin was then removed using Microcon filters (Millipore. Bedford, MA, USA). The biotinylation of dendrimers was evaluated using NMR spectroscopy. Biotinylated dendrimers were resuspended (1.0 mg/mL) in 1.0 M PBS until used. Octadecanethiol (ODT), 3,3 dithio-bis(propionic acid N-hydroxysuccinimide ester) (DSP), bovine serum albumin (BSA), Triton X-100, and avidin >98% were obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Avidin conjugated to Alexa Fluor 488 was purchased from Invitrogen (Invitrogen, Carlsbad, CA). Poly(dimethyl siloxane) (PDMS) was obtained from Dow Corning (Midland, MI). All organic solvents used were analytical, HPLC grade, from Sigma (Sigma-Aldrich, St. Louis, MO). DI water was obtained using a Milli-Q plus water purification system (Millipore, Bedford, MA). PBS and Borate buffers were prepared from Pierce buffer packs (Pierce Protein Research Products, Rockford, IL). 2.2. Capture Substrate Preparation. A modified approach was used for preparation of the capture substrate [37–39] utilizing template-stripped gold (TSG) for SPM imaging, as shown in Figure 1. TSG was prepared by evaporating gold onto p-type silicon wafers (University Wafer) with a resistive evaporator and affixing 1 × 1 cm glass pieces (ultrasonically cleaned 30 min each in diluted Contrad 70, DI water, and methanol) using two-part epoxy (Epoxy Technology) followed by curing at 150◦ C for 2 h. The glass pieces were gently detached from the silicon wafer revealing a smooth gold surface atop the glass chip. The TSG substrates were exposed for ∼30 s to an ODT soaked PDMS stamp (with a 3 mm diameter hole cut in the center), rinsed with ethanol, and dried under highpurity nitrogen. The substrates were then placed in a 0.1 mM solution of DSP in ethanol overnight. The capture platform was then rinsed with ethanol and dried under N2 . This formed the DSP-based adlayer in the areas on the substrate not covered by ODT. The hydrophobic ODT localized reagents in a confined sample area (3 mm ODT spot size) for the capture assay platform. To form the capture avidin surface, a 20 μL aliquot of avidin solution (500 μg/mL diluted in 50 mM borate buffer) was placed on top of the sample area and allowed to incubate for 6 h at room temperature in a humidity chamber. Substrates were then rinsed with 5 mL of 10 mM PBS (with 0.1% Triton X-100), and the surface area was incubated with a 2 (...truncated)