Elliot Chaikof Emory University/Georgia Institute of Technology
Ashutosh Chilkoti Duke University
Jennifer Elisseeff Whitaker Biomedical Engineering Institute
Joerg Lahann University of Michigan
CC1: Biomolecules at Interfaces
Tuesday PM, March 25, 2008
Room 3024 (Moscone West)
9:30 AM - **CC1.1
Delivering Drugs Layers At a Time - Nanoscale Assembly Approaches for Tunable Responsive Thin Films.
Paula Hammond 1 Show Abstract
1 Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
10:00 AM - CC1.2
Microscale Multiple Biomolecules Printing in One Step Using a PDMS Macrostamp.
Helene Lalo 1 , Jean-Christophe Cau 1 , Christophe Thibault 1 , Jean-Pierre Peyrade 1 , Christophe Vieu 1 , Veronique Le Berre 2 , Emmanuelle Trevisiol 2 , Jean-Marie Francois 2 Show Abstract
1 NanoBioSystem, LAAS-CNRS, université de Toulouse, Toulouse France, 2 , LISBP, CNRS-INRA, Université de Toulouse, Toulouse France
10:15 AM - CC1.3
Click Chemistry Surface Concentraion Gradients for Screening Cell Adhesion and Response.
Nathan Gallant 1 , Eric Amis 1 , Matthew Becker 1 Show Abstract
1 Polymers Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States
Biomimetic surfaces are engineered to present ligands for specific receptors, thereby controlling cell-material interactions to elicit a desired response. The presentation of these bioactive ligands strongly influences the cell response, and threshold concentrations are often necessary to support adhesion or trigger signals that encourage tissue formation. Extensive work has focused on optimizing the various aspects of ligand immobilization to enhance material directed cell function. In order to facilitate research on biomimetic and tissue engineered medical products, we have developed a novel and versatile method for fabricating continuously variable concentration gradients of surface conjugated biomolecules. This technology utilizes graded ultraviolet oxidation of self-assembled monolayers and upon further derivatization, converts the resulting carboxylate gradient into an increasing density of alkyne functionality appropriate for click chemistry surface conjugation of biomolecules. Thus, any appropriately engineered bioactive ligand (e.g., protein, peptide) possessing an accessible azide group can be immobilized onto the surface concentration gradient.Cell adhesion to adsorbed proteins or adhesive motifs engineered on surfaces is critical to biomaterials, tissue engineering, and biotechnological applications. One use of this versatile technology is as a testing platform for investigating cell adhesion to surfaces functionalized with adhesive motifs derived from the native extracellular matrix. This technology was used to fabricate a gradient of a glycine-arginine-glycine-aspartate-serine (GRGDS) linear adhesion peptide spanning a relevant range of the immobilized ligand. We used x-ray photoelectron spectroscopy to measure elemental surface concentrations along the gradient to calculate the immobilized peptide concentration. Automated microscopy was used to assess cell adhesion on RGD gradients. The number of adherent cells increased as a function of surface concentration before reaching saturation (≈100 pmol/cm2).We showed that versatile surface concentration gradients can be easily fabricated using changes in surface energy and click chemistry. Density gradients of an adhesive peptide controlled cell attachment as a function of surface concentration. This work demonstrates the ability to modulate a cell response with gradient substrates functionalized with a bioactive peptide, and is an example of the broad utility of this technology for high-throughput biomaterials research. Current work focuses on gradients of orthogonal chemistries for multiple biomolecule immobilizations to control cell adhesion and phenotype.This abstract, an official contribution of NIST, is not subject to copyright in the United States.
10:30 AM - CC1.4
Micropatterned Biorecognition Surfaces on Nonbiofouling Polymer by Living Radical Photopolymerization for High Sensitivity Biosensing.
Madoka Takai 1 , James Sibarani 1 , Kazuhiko Ishihara 1 Show Abstract
1 Department of Materials Engineering, School of Engineering and Center for NanoBio Integration, The University of Tokyo, Tokyo Japan
To create useful biomaterials for many biotechnology applications, interfaces are required that have both enhanced specific binding and reduced non specific binding. Thus, in applications such as biosensing, the tailoring of biointerface chemistry and the use of micro or nanofabrication technique becomes an important avenue for the production of surface with specific binding properties and minimal background interference. The aim of this study is to prepare micropatterned biorecognition layer on nonbiofouling surface bearing highly biocompatible poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) brushes for enhancing high signal/noise (S/N) ratio in biosensing using living radical photopolymerization based on diethyldithiocarbamate as photoiniferter (initiator, transfer, and terminator). The macrophotoiniferters comprised of 2-ethylhexyl methacrylate(EHMA) and 4-vinylbenzyl N,N-diethyldithiocarbamate (VBDC) (PEV) were synthesized with variation of VBDC content of 10% - 40% (PEV10, 20, 30, 40). Properly cleaned poly(ethylene terephthalate) (PET) was coated by dipping in 0.25 wt% of the macrophotoiniferter solutions in toluene. The photoiniferter-coated plates in aqueous solution of 0.3 M MPC monomer were irradiated with UV lamp (360 nm) at room temperature. The micropatterned biorecognition layers (methacrylpolyethylene glycol conjugated with N-succinimidyl carbonate, (PEG-NSC)) were also constructed by using UV irradiation(1hr) through photomask with 100 μm square in size and evaluated by contacting with FITC-albumin in PBS solution (pH 7.4). The density and the length of the poly(MPC) chains were governed via composition of VBDC in the macrophotoiniferter and UV irradiation time, respectively. The AFM observation showed that morphology and roughness of the poly(MPC)-modified surfaces varied depending on VBDC composition and UV irradiation time. The small RMS roughness (6.2-6.8) brushed like surfaces were formed by using the conditions of the PEV20, 30 and UV irradiation time 1 hr. The poly(MPC)-brush surfaces with the small RMS significantly reduce nonspesific protein adsorption although highly roughness surfaces still prone to lead inevitable nonspecific protein adsorption. Furthermore, the micropatterned biorecognition layer on the poly(MPC)-brush surface was achieved by evaluating of FITC-albumin conjugation. The strong intensity of fluorescence was observed only from the patterning area of FITC-albumin immobilized on bioactive layer. Living radical photopolymerization using PEV as macrophotoiniferter is a generic method for preparation of nonbiofouling surface bearing poly(MPC)-brush and for construction of micropatterned biorecognition layer on nonbiofouling surface. The micropattered biointerfaces having biorecognition and nonbiofouling prepared by photochemistry are applicable for high sensitivity biosensing and cell engineering.
10:45 AM - CC1.5
A Novel Class of Fibrils Based on poly-N-substituted Glycines (peptoids).
Modi Wetzler 1 , Ronald Zuckermann 2 , Annelise Barron 1 Show Abstract
1 Bioengineering, Stanford University, Stanford, California, United States, 2 Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States
11:30 AM - CC1.6
CAD/CAM Laser Microfabrication of Biointerfaces for Tissue Engineering.
Anand Doraiswamy 1 , Roger Narayan 1 , Douglas Chrisey 2 Show Abstract
1 Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States, 2 Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States
We have demonstrated the microfabrication of vascular tissue-like networks using a novel computer-aided design/computer-aided manufacturing (CAD/CAM) laser micromachining technology. Excimer laser (ArF, 193 nm, Energy: 0.2 to 2 J/cm2 and pulse-rate: 1-20) was integrated into a three-axis micropositioning system with variable feed rate. Various materials including Silicon (111) and agarose gel (1%) was used to study the laser ablation and subsequent cellular growth. Results on the extent of ablation (depth and width) in each material were systematically studied with increasing laser energy, aperture, and repetition rates. These results were used to develop CAD/CAM networks in agarose gels containing matrigel® (extra-cellular proteins), human elastin, vascular endothelial growth factor (VEGF: 1, 10, 100, 1000 ng/ml) and heparin (1, 10, 100, 1000 units/ml) for the controlled growth and proliferation of human aortic endothelial cells and human aortic smooth muscle cells. MTT analysis of cells showed VEGF and heparin induced proliferation of human aortic cells and inhibition of human aortic smooth muscle cells. The cells were also studied in co-culture within the patterns to develop vascular tissue-like networks, which formed at 72 hours and delaminated into a free-standing network. Live/dead® analysis of the cellular networks showed (>99%) viability at 72 hours without any loss in structural integrity. Results from previous developments of cellular networks of myoblasts, neuroblasts, and epithelials have also been highlighted. The CAD/CAM laser micromachining setup provides a direct-write approach to microfabrication of unique geometries that may be used to develop cellular and tissue networks for next-generation patient-specific care.
11:45 AM - CC1.7
Effect of Immobilization Strategies on Biological Function: Ephrin-A1 Signaling.
Khalid Salaita 1 2 , Pradeep Nair 1 , Jay Groves 1 2 Show Abstract
1 Chemistry, University of California, Berkeley, Berkeley, California, United States, 2 Physical Biosciences, Lawrence Berkeley National Laboratories, Berkeley, California, United States
Activation of the EphA2 receptor tyrosine kinase with its natively membrane-bound ligand, ephrin-A1, plays an important role in breast cancer biology. This talk examines the biological activity of the ephrin-A1 ligand as a function of how it is presented on a surface. Realtime fluorescence imaging, gene expression profiling, immunostaining, and western blots all show that ephrin-A1 is active when tethered to a fluid supported lipid membrane (SLB), and biological function is altered when presented in solution, tethered to a SLB in the gel-phase, or covalently immobilized onto a surface. Importantly, the chemical composition of the surface is identical in all cases, but its two-dimensional mobility drastically alters biological function.
12:00 PM - CC1.8
Recombinant Multimeric Integrin-Specific Ligands to Enhance Adhesive Activities.
Timothy Petrie 1 2 , Andres Garcia 1 2 Show Abstract
1 Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States, 2 Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
12:15 PM - CC1.9
Microcontact Printing for Controlled Patterned Ligand Tethering and Density.
Brandon Stanley 1 , Timothy Petrie 1 , Andres Garcia 1 Show Abstract
1 , Georgia Institute of Technology, Atlanta, Georgia, United States
12:30 PM - CC1.10
Fabrication of Biofunctional Surfaces displaying Multiple Peptide Ligands using Supported Bilayers.
Badriprasad Ananthanarayanan 1 3 , Matthew Black 1 3 , Dimitris Missirlis 1 3 , Matthew Tirrell 1 2 3 Show Abstract
1 Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California, United States, 3 Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, California, United States, 2 Materials, University of California, Santa Barbara, Santa Barbara, California, United States
There is great interest in decorating biomaterial interfaces with peptides. A wide variety of bioactive sequences, ranging from adhesion-promoting RGD motifs to antimicrobial peptides, can be used to confer appropriate functionality to a surface. It is very important to control the presentation of the peptide so that it is accessible to its receptors and therefore active. Another challenge is to develop ways to create multi-component surfaces that have complementary peptide functionalities.We use lipid-conjugated peptides ('peptide amphiphiles') and exploit their self-assembly to create biofunctional aggregates in solution and on surfaces. In particular, lipid-like peptide amphiphiles are incorporated in the bilayer membrane of lipid vesicles, and supported bilayers created by vesicle fusion are used as a platform to display peptide ligands. Supported bilayers provide an inherently low-background surface for interactions with cells and proteins, and can easily be patterned to produce complex displays.In this contribution we will discuss the fabrication of supported bilayers that display cell-adhesive RGD peptides and their characterization using fluorescence microscopy, FRAP and QCM. The accessibility of the peptide - as modified by including PEG spacer groups in the amphiphile structure - was found to play a significant role in determining the extent of cell attachment to these surfaces. Further, multiple-peptide displays were created by using GRGDSP along with its synergy site PHSRN in varying ratios to optimize the integrin-binding properties of the surface. We show that supported-bilayer based surface display is an effective way to create multi-component peptide surfaces and screen them for biological function.
12:45 PM - CC1.11
Role of Cationic Polymer and Surface Chemistry on Substrate-mediated Gene Delivery.
Fritz Simeon 1 2 , Too Phon 3 2 , Alan Hatton 1 2 Show Abstract
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States, 2 Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance (SMA), Singapore Singapore, 3 Biochemistry, National University of Singapore, Singapore Singapore
CC2: Designer Particles
Tuesday PM, March 25, 2008
Room 3024 (Moscone West)
2:30 PM - **CC2.1
Engineering Shape of Polymeric Micro and Nanoparticles for Drug Delivery.
Julie Champion 1 , Samir Mitragotri 1 Show Abstract
1 Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California, United States
Polymeric micro- and nanoparticles are routinely used for applications in drug delivery. Encapsulation of drugs in polymeric carriers protects them from enzymatic degradation and provides sustained release over prolonged periods. Further, encapsulation also allows drug targeting via cell and tissue-specific ligands. The performance characteristics of polymeric particles in the body, for example circulation times, macrophage clearance, targeting and drug release rates, depend on several particle parameters including size, shape, surface chemistry, and mechanical strength. We particularly focus on engineering particle shape, a design parameter that has received little attention in the past. We have devised methods to generate particles of several distinct shapes. Our studies show that particle shape makes a profound impact on important steps in drug delivery. Particle shape provides a new dimension in engineering of polymeric carriers and opens up new opportunities in drug delivery.
3:00 PM - **CC2.2
The Role of Nano and Sub-nano Scale Domains on Biointerface Perfomances.
Francesco Stellacci 1 Show Abstract
1 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States
3:30 PM - **CC2.3
Nanoparticle Surface Engineering for Tissue Targeting in Drug and Antigen Delivery.
Jeffrey Hubbell 1 2 , Melody Swartz 1 2 , Sai Reddy 1 , Andre van der Vlies 1 , Eleonora Simeoni 1 , Dominique Rothenfluh 1 Show Abstract
1 Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne Switzerland, 2 Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne Switzerland
4:30 PM - CC2.4
Systematic Investigation of Preparing Biocompatible, Single, and Small ZnS-capped CdSe Quantum Dots with Amphiphilic Polymers.
Robin Anderson 1 , Warrren Chan 1 Show Abstract
1 Institute for biomaterials and biomedical engineering, University of Toronto, Toronto, Ontario, Canada
The successful transfer of nanoparticles between solvents is critical for many applications. We evaluated the impact of amphiphillic polymer composition on the size, transfer efficiency, and biocompatibility of tri-n-octylphosphine oxide-stabilized semiconductor ZnS-capped CdSe and CdS-capped CdTeSe quantum dots (QDs). We also investigated the adsorption of various proteins onto the surface of these QDs and studied the effect of surface chemistry on non-specific protein binding. The results from these studies will have implications in the design of QDs and other nanoparticles for biological and biomedical applications.
4:45 PM - CC2.5
Initiated Chemical Vapor Deposition of Patterned Thin Films of pH-Responsive Hydrogels with Attached Quantum Dots.
Wyatt Tenhaeff 1 , Chia-Hua Lee 2 , Karen Gleason 1 Show Abstract
1 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Thin functional films of ionic hydrogels have been grafted to silicon and glass substrates by initiated chemical vapor deposition (iCVD). Prior to iCVD, the substrates were primed with 3-aminopropyldimethylethoxysilane, imparting the surface with a free amine group. Three monomers vapors were introduced into the reactor along with the initiator: 1.4 sccm of maleic anhydride (MA), 20 sccm of N,N-dimethylacrylamide (DMAA), and 0.4 sccm of di(ethylene glycol) divinyl ether (DEVE). At a filament temperature of 235 °C and an initiator flow of 0.4 sccm, the polymer film deposited at a rate of 16 nm/min. The DEVE crosslinker created a network by linking all of the polymer chains together, while maleic anhydride groups at the interface between the substrate and hydrogel film formed a covalent bond with the amine to tether the network to the substrate. Covalent linkages prevented the film from delaminating upon exposure to aqueous solutions; the films undergo 14-fold swelling in pH 7 buffer. The carboxylic acids that formed upon hydrolysis of maleic anhydride imparted anionic character to the film. In pH 1 buffer, the swelling ratio was 7. The composition of the film was 76% DMAA, 14% MA and 10% DEVE, as determined by X-ray photoelectron spectroscopy (XPS). Furthermore, for applications requiring hybrid nanocomposites, the reactivity of the intact anhydride functional groups was utilized. Anhydride groups were reacted with 2-aminoethanethiol (0.05 M solution in IPA) to form free sulfhydryl groups at the surface, determined by XPS and FTIR. High resolution XPS scans of the C1s region indicated that 94% of the anhydride groups at the surface were functionalized after 30 minutes in the 2-aminethanethiol solution at 70 °C. The functionalized films were soaked in a dispersion of CdSe/ZnS core-shell quantum dots in tetrahydrofuran, and stable linkages formed between sulfhydryls and the quantum dot surfaces. Cadmium, selenide, zinc, and sulfur signals in the XPS survey scans confirmed the presence of quantum dots on the surface of the hydrogels before and after ultrasonication in tetrahydrofuran for 30 seconds. The quantum dots have been linked to 7 μm square patterns that were synthesized by the direct deposition of the hydrogel through 2000 mesh TEM grids via iCVD. The pattern pitch was 12 μm. Fluorescent microscopy revealed that fluorescence was localized to the hydrogel regions. The surface properties of hydrogels are critical in determining biocompatibility and cell adhesion properties. Hydrogels incorporated into a biomedical microdevice or diagnostic microarray must form a stable interface between the substrate and liquid medium, while retaining surface functionalities. This platform for the vapor deposition of the hydrogel films shows great promise for such applications because the functionality of the deposited hydrogel is retained, the films are firmly attached to the substrate, and patterning is simple.
5:00 PM - CC2.6
Simultaneous Measurement of Glucose and Cholesterol by using Enzyme-Conjugated Nanocrystals.
Ki-Eun Kim 1 , Min-Kyu Oh 2 , Yun-Mo Sung 1 Show Abstract
1 Materials Science & Engineering, Korea University, Seoul Korea (the Republic of), 2 Chemical & Biological Engineering, Korea University, Seoul Korea (the Republic of)
Glucose and cholesterol biosensor market is growing very rapidly worldwide and also there is high need in simultaneous measurement of cholesterol and glucose to minimize the amount of blood drawing. Recently, many research groups have studied electrochemical biosensors using nanostructures giving many advantages such as high sensitivity and high accuracy. However, they show some critical limitations especially in designing of dual biosensors because they need complicate structures consisting of multi-chambers and electrodes. In order to achieve portable biosensor devices, small and simple structure optical biosensors are more desirable. In this study, one-single chamber optical dual biosensors were developed for the detection of both glucose and cholesterol using ZnSe and ZnO nanocrystals, respectively. ZnSe and ZnO nanocrystals have received considerable attention because they are stable in typical biomolecular sensing environment and can be synthesized easily through various methods. Also, they have novel optical and electrical properties related to quantum confinement. MUA (mercaptoundecanoic acid) capped ZnO and MAA (mercaptoacetic acid) treated ZnSe were synthesized first. It is ascertained through XPD, PL and UV-visible spectrum that synthesized ZnO and ZnSe nanocrystal quantum dots have not only good crystallinity though having a very small size but also superior optical property. Glucose oxidase (GOx) and cholesterol oxidase (GOx) were immobilized to the ZnO and ZnSe nanocrystals, respectively. The successful bioconjugation between nanoparticels and enzymes was confirmed using Fourier transform infrared spectroscopy (FTIR). Photoluminescence (PL) spectroscopy was used to detect glucose and cholesterol at the same time. PL peaks of GOx-ZnO and COx-ZnSe bioconjugates were positioned at different wavelength regions, 370 nm and 450 nm, respectively due to the energy band gap difference between ZnO and ZnSe. As concentration of the glucose and cholesterol in the solution increases, the intensity of the PL peaks from the GOx-ZnO and COx-ZnSe bioconjugates shows linear increase. Free electrons produced during the enzymatic oxidation reactions were effectively transferred to each nanocrystal through the MUA and MAA, and caused increase in the PL intensity. From the linear relationship and sensitive response of the PL intensity to the glucose and cholesterol concentration, the potential of the bioconjugates to be used for simultaneous glucose and cholesterol sensing was confirmed.
5:15 PM - CC2.7
Anisotropic Biphasic Nanoparticles for Cell Targeting:Surface Modification and Selective Biomolecule Immobilization.
Mutsumi Yoshida 1 , Kyung-Ho Roh 2 , Joerg Lahann 1 2 Show Abstract
1 Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 2 Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, Michigan, United States
Using an electrified co-jetting process, we have prepared anisotropic biphasic nanocolloids with two distinct phases.1,2 In this process, two parallel polymer solutions from a side-by-side capillary system are simultaneously jetted under an electric field to create the unique biphasic geometry. Owing to this setup, the two phases may be independently loaded as well as be surface-modified. As such, these particles can concurrently provide multi-functionality including targeting moieties on the surface, complex drug release profiles due to differing polymer degradation rates, or ‘smart’ imaging competence through stimuli-responsiveness.3 In this study, we report further modification of these particles, specifically designed for cell-targeting. Biotin- and acetylene-modified poly(acrylamide-co-acrylic acid) were prepared and chemistry confirmed by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Fluorescence-conjugated dextrans were added to the solutions of these polymers, which were used in the electrified co-jetting. Surface modifications were performed after fabrication of the particles presenting acetylene and/or biotin on the surface. Poly(ethylene glycol) (PEG) and was introduced to the particle surface via “click chemistry” to the acetylene group incubation to minimize non-specific interaction with cells. In addition, particles were incubated with streptavidin to add bio-functionality. Particles showed no significant interfacial diffusion or mixing, and the biphasic geometry maintained after thermal imidization and subsequent chemical modifications, as observed by confocal laser scanning microscopy. Preliminary results of biocompatibility assays suggest low cytotoxicity, with little effect of presence of the particles on cell morphology, proliferation, and release of cytosolic enzyme from damaged cells. Initial studies of surface modification revealed non-specific binding of non-modified particles to human umbilical vein endothelial cells (HUVECs), which was drastically reduced upon introduction of PEG. Addition of streptavidin to the surfaces of one hemisphere significantly increased binding to HUVECs, only when the cells were stained with biotinylated antibody against CD31. With the added ability to selectively target cells with directionality and controlled orientation, the biphasic nanocolloids may serve as a versatile platform requiring complex, multi-functionality, providing unique advantage over their conventional, isotropic counterparts. References: 1. Roh, K.-H., Martin, D.C. and Lahann, J. Biphasic Janus particles with nanoscale anisotropy. Nat Mater, 4, 759, 2005.2. Roh, K.-H., Yoshida, M. and Lahann, J. Water-Stable Biphasic Nanocolloids with Potential Use as Anisotropic Imaging Probes. Langmuir, 23, 5683, 2007.3. Yoshida, M., Roh, K.-H. and Lahann, J. Short-term biocompatibility of biphasic nanocolloids with potential use as anisotropic imaging probes. Biomaterials, 28, 2446, 2007.
5:30 PM - CC2.8
Biocompatible and Functional Quantum Dots via Heterogeneous Compact Ligands.
Bing Mei 1 2 , Kimihiro Susumu 1 , Igor Medintz 3 , Hedi Mattoussi 1 Show Abstract
1 Division of Optical Sciences, Code 5611, U.S. Naval Research Laboratory, Washington, District of Columbia, United States, 2 Department of Chemical Engineering, University of Massachusetts, Amherst, Massachusetts, United States, 3 Center for Bio/Molecular Science and Engineering, Code 6910, U.S. Naval Research Laboratory, Washington, District of Columbia, United States
Semiconductor quantum dots (QDs) have distinct advantages over traditional organic dyes, including tunable emission and a pronounced resistance to photo and chemical degradation. Their successful integration in biotechnology necessitates the preparation of water-soluble QDs that are highly luminescent, stable over a broad range of biological conditions and compatible with simple conjugation techniques. We have previously used PEG-terminated dihydrolipoic acid (DHLA-PEG) capping substrates to render the QDs water soluble and stable over a wide pH range. Recently, we further developed this scheme to prepare an array of DHLA-PEG ligands having a variety of functional end groups, including biotin, carboxyl, and amino groups. DHLA-PEG-COOH, for example, was prepared by coupling thioctic acid and succinic anhydride to the ends of amine-functionalized poly(ethylene glycol) followed by reduction of the 1,2-dithiolane group. All ligands are modular and each has a multidentate terminal group for strong anchoring on the QD surface, a PEG segment to promote hydrophilicity and a terminal group for biological linkage. Using combinations of these ligands (e.g., hydroxy-, amino-, or carboxyl-PEG ligands) during cap exchange, we prepared nanocrystals that have controllable fraction of targeted surface functions. For example, using mixed surface ligands of DHLA-PEG600 and DHLA-PEG600-COOH or DHLA-PEG600-NH2, QDs having up to 100% of carboxyl or amine terminal groups have been prepared. Surface charge of QDs can be tailored by simply tuning the molar ratio of ligands used. We then coupled DHLA–PEG–COOH functionalized QDs with amine functionalized dyes by EDC coupling and DHLA-PEG-NH2 functionalized QDs with isothiocyanate functionalized dyes. Absorption measurements confirmed QD-dye complex formation, with absorption spectra exhibiting features characteristic of the dye as well as the QD in the sample. In addition, fluorescence measurements showed a pronounced QD quenching, due to energy transfer from the QD to the proximal dye. These measurements were further used to extract the number of available binding sites (or number of surface ligands) per nanoparticle. Along with the ligand counting extracted from absorption and fluorescence measurements, we will also discuss synthesis and analytical characterization of the prepared ligands.