NANO LETTERS
Biological Assembly of Nanocircuit Prototypes from Protein-Modified CdTe Nanowires
2005 Vol. 5, No. 2 243-248
Ying Wang,† Zhiyong Tang,‡ Susheng Tan,§ and Nicholas A. Kotov*,†,‡ Department of Chemical Engineering and Departments of Biomedical Engineering and Materials Science and Engineering, 2300 Hayward, H. H. Dow Bldg, UniVersity of Michigan, Ann Arbor, Michigan 48109, and Department of Chemistry, Oklahoma State UniVersity, Stillwater, Oklahoma 74078 Received October 21, 2004; Revised Manuscript Received December 10, 2004
ABSTRACT CdTe nanowires made by self-organization of CdTe nanoparticles in aqueous media were separately conjugated with complementary biological connectors, such as antigen−antibody and biotin−streptavidin. Transmission electron microscopy images and Fo1 rster resonance energy transfer measurements in nanowire superstructures with different diameters indicate that biological affinity of the attached proteins results in the formation of crossbar and end-to-side connections between the nanowires. A prototype of a logical circuit made from a triangular arrangement of the nanowires spontaneously assembled on a Si substrate was examined by conducting atomic force microscopy. While diode-like behavior was observed in the sides of the triangle, the nanowire junction points were found to be nonconductive. It was attributed to high tunneling barrier created by protein molecules wedged between the nanowires. Suggestions are made how to reduce it or use the insulating gap between the nanowires as a framework for single-electron devices.
Introduction. One-dimensional nanomaterials, such as nanorods and nanowires (NWs), are promising building blocks for the miniaturization of electronic and photonic devices and biological sensors.1-7 To take full advantage of the material properties of NWs they need to be pre-aligned or otherwise ordered. Several physical or chemical strategies have been tested for the hierarchical assembly of NWs and other nanoscale building blocks. They include alignment of NWs in fluid flow,8 self-assembly with surface forces,9 and orientation induced by electrical/magnetic fields.10,11 Several problems have been identified for chemical or physical manipulation of NWs, such as high error frequencies and slow assembly speed, which should be addressed before their practical application. This can be done by developing novel techniques of preparation of NW arrays12 as well as methods to circumvent problems associated with the high probability of errors at the application level, such as computing algorithms.13 Very recently, an alternative strategy, biological assembly of nanocircuits, attracted the interest of many scientists. Biospecific recognition of building blocks made from different nanocolloids can result in new nanoscale * Corresponding author. E-mail:
[email protected]. † Department of Materials Science and Engineering, University of Michigan. ‡ Departments of Chemical Engineering and Biomedical Engineering, University of Michigan. § Department of Chemistry, Oklahoma State University. 10.1021/nl0482682 CCC: $30.25 Published on Web 12/29/2004
© 2005 American Chemical Society
superstructures14-17 or lead to the assembly of extended nanoparticle (NP) networks, which can grow to the size of macroscopic materials.18-21 Considerable progress has already been made regarding the functionalization of carbon nanotubes with proteins through bioconjugation, and the conjugated nanotubes have been used as biosensors.22-26 In principle, similar bioconjugates can be utilized to produce spatially and/or topologically organized NW superstructures with fairly high level of complexity and organization. Nevertheless, very limited efforts have been made to assemble NWs. This is partially due to the difficulties with preparation of NW dispersions in aqueous solutions, which is the natural media for biological assembly, from NW suspensions in organic solvents or from gas-phase-produced powders. Very recently, Caswell et al. and Salem et al. reported making the connection between metal gold nanorods or NWs by biotin-avidin connectors.18,27 Here, we demonstrate for the first time the biologically driven assembly of long semiconductor CdTe NWs in a crossbar and side-toend mode. We also investigate the electrical characteristics of this connection by conducting atomic force microscopy (AFM) for the fundamental assessment of the prospects of biologically coded circuit assembly. In the spontaneously assembled prototype of the NW circuit with a triangular geometry of a logic element, we demonstrate that biological ligands used for the connections prevent conductance from
Figure 1. Typical transmission electron microscopy (TEM) images of assembled building blocks of +NW-TGA-IgG (A-C) and NW-LCY-BSA + NW-TGA-IgD (D).
one NW to another. On the basis of these results, suggestions are made how to reduce the electron tunneling gap from one NW to another or to take advantage of it in the nanoscale circuits. Results and Discussion. CdTe NWs used here were prepared by the recently discovered process of spontaneous self-assembly of CdTe NPs.28 The reaction is simple, has a high yield, and is applicable to different materials including metals and semiconductors. It also results in NWs with uniform and controllable diameters. For this project, it is also essential that NWs are made directly in an aqueous environment with relatively high luminescence efficiency and variable surface chemistry.29 All of these features conveniently lend themselves to surface modification with biomolecules and thus, to the preparation of bioconjugates. We first chose complimentary antibody-antigen pair, specifically bovine serum albumin (BSA) and corresponding anti-BSA antibody (IgG), as the connectors to bridge NWs. The choice was based on the availability of the detailed structural information for BSA and its ability to form different molecular complexes.30 Thioglycolic-acid (TGA) stabilized CdTe NWs (photoluminescent peak 533 nm) and L-cystein (LCY) stabilized CdTe NWs (photoluminescent peak 670 nm) prepared through self-organization of nanoparticles28 were used to form the building blocks. NWconjugated BSA and anti-BSA IgG were prepared by the sulfo-NHS (N-hydroxysulfo-succinimide) and EDC (1-ethyl3(3-dimethylaminopropyl) carbodiimide hydrochloride) reaction.31 The carboxylic acid groups of TGA and LCY will form an amide bond with the primary amine groups of the protein. Typically a reaction mixture containing 0.05 mM 244
CdTe NW, 0.2 mg/mL antigen or antibody, 1 mM NHS, 0.02 M EDC in pH 7.0 sodium phosphate buffer was prepared and kept at room temperature for 4 h, then dialysis with Spectra/Por Membrane, MWCO 100000 (Spectrum Laboratories, Inc.), in pH 7.0 buffer to get rid of the redundant free proteins in the mixture. As a start, CdTe-TGA NWs were conjugated with BSA and IgG respectively to give NW-TGA-BSA and NW-TGA-IgG complexes. Then equal amounts of these two building blocks were mixed and the transmission electron microscopy (TEM) images of as-formed structures revealed that NWs prefer crossbar (Figure 1 A, B) and end-to-side connection (Figure 1C, D). To verify that the linkage between NWs were due to the antigen-antibody linkage rather that just a random overlap of NW in TEM images, two NWs with different morphology and emission wavelength, λfl, were made by taking advantage of the fine control over the NW emission afforded by the NPfNW transition.28 L-cystein-stabilized NWs were made quite long with aspect ratio over 500 and emitting in the red at λfl ) 670 nm. The NWs stabilized by TGA were made relatively short with aspect ratio of 20 ∼ 40 and emitting in the green, λfl ) 525 nm. NW- LCY was functionalized with BSA, while NW-TGA was conjugated to IgG. Similarly, crossbar and side-to-end binding predominated in the assembled complex (Figures 1D, 2A,B). As longer NWs were involved in bioconjugation, it was more difficult to remove the free protein from the mixture. As a result, some background due to the presence of free proteins was observed in TEM images (Figure 2 A, B). Control experiments without addition of BSA or IgG gave distinctly different results with well-dispersed NWs infrequently connecting to each other Nano Lett., Vol. 5, No. 2, 2005
Figure 2. (A,B) Transmission electron microscopy images of assembled NW-TGA-IgG with NW-LCY-BSA. (C) Control experiment: NW-TGA mixed with NW-LCY.
(Figure 2C). High negative charge of the NWs prevents the close approach of them to each other. Their aggregates and NW crossings are rare events without the self-assembling proteins. These results indicate that antigen-antibody interactions are essential for the formation of observed NW assemblies. Predominantly perpendicular arrangements of NWs in the bioconjugates (Figures 1, and 2 A,B) stem, most likely, from the electrostatic repulsion minimized for the crossbar geometry. Biconjugation techniques had been widely used to assemble gold nanoparticles or NWs into 3-D or 1-D structures.32-35 Most of the reported results were based on the morphological changes observed in TEM images. More convincing pieces of evidence concerning the effect of biomolecules are desirable. Compared to other colloids and high aspect ratio particles, CdTe NWs are prepared in aqueous phase with adjustable photoemission, which offer an unequivocal way to obtain evidence of conjugate formation by optical techniques. The direct evidence supporting the formation of superstructures from CdTe NWs due to biospecific interactions can be found from Fo¨rster resonance energy transfer (FRET) phenomena.14,36,50 NWs with green fluorescence (Green-NW, λmax ) 533 nm) and red, i.e., emission and red fluorescence (Red-NW, λmax ) 641 nm) are individually conjugated with IgG and BSA to form Green-NW-IgG and Red-NW-BSA complexes. When Green-NW-IgG is combined with Red-NW-BSA, the mutual affinity of the antigen and antibody brought the NWs close enough together to allow the resonance dipole-dipole coupling required for FRET to occur. As expected, a significant enhancement of the Red-NW-BSA red emission at 641 nm and the corresponding quenching of the green emission of Green-NW-IgG at 533 nm are observed after the self-assembly of the labeled biospecific ligands in the complex (Figure 3). The kinetics of the reaction in Figure 3 is very characteristic to the reactions of antigen-antibody recognition.14 Thus, the energy of the excitonic state in the green-emitting NW was transferred to the similar state of the red-emitting NW with lower exciton energy. These results demonstrated that proteins were covalently bonded to the Nano Lett., Vol. 5, No. 2, 2005
Figure 3. Fluorescence spectra recorded at different times after mixing Green-NW-IgG with green emission (533 nm) with RedNW-BSA with red emission (641 nm). The red and green profiles correspond to 1 and 230 min time periods passed after the point of mixing, respectively.
NW surface and the NW-BSA/NW-IgG complex indeed “linked” to each other to form an assembly. A different set of complementary bioconnectors, i.e., biotin-streptavidin (SA), was also used to assemble mini circuits from CdTe NWs. The biomolecular recognition of biotin by SA is characterized by the extraordinary high affinity constant of about 1014 dm3 mol-1, which makes it the strongest ligand-receptor interaction currently known.37 First, biotin hydrazide (BH) reacted with carboxylic acid groups on NWs stabilized by L-cystein to form hydrazone linkage or by carbodiimide reaction (NW-LCY-BH), while the streptavidin was connected to NW-TGA with much lower aspect ratio through conventional EDC/NHS reaction (NW-TGA-SA).14 The second set of conjugates was prepared by reacting BH with carboxylic acid groups of thioglycolic acid on NW-TGA to form NW-TGA-BH. The addition of NW-TGA-SA to NW-LCY-BH (Figure 4A, 4B) or addition of SA to NW-TGA-BH (Figure 4C) produced a high portion of crossbar and side-to-end assembly. The FRET was observed after mixing the NW-TGA-SA with green emission and NW-LCY-BH 245
Figure 4. Transmission electron microscopy images of assembled building blocks of (A, B) NW-LCY-BH + NW-TGA-SA. (C) NW-TGA-BH + SA.
with red emission (not shown here), which proved that such a system retains substantial bioactivity and can form supramolecular assembly. One of the main features of the building blocks made of NWs is their potential application as electronic devices. So, the electric properties of the assembled networks has attracted much attention.1;2 Conductive atomic force microscopy (C-AFM) is a powerful tool for exploring the electrical characteristics of nanoscale structures.38-40 Compared with scanning tunneling microscope, C-AFM can precisely locate the objects by using topographic features acquired simultaneously and independently from electrical properties. As we also reported recently, C-AFM in conjunction with CdTe NWs similar to the ones used here can be used as a nanolithography tool, removing a thin portion of NW under the tip in selected areas.41 Prototypes of resonance tunneling diodes with modulated band gap along the NW were made in this way.41 Here C-AFM will be used to establish conductivity through the biologically assembled NWs and their junctions. Arsenic-doped silicon wafers were used as substrates because they have high conductivity with saturation tunneling current of 10 nA at 0.2 V.42 We first deposited NW-TGA-BSA on the silicon wafer, then absorb a layer of NW-MSA-IgG (MSA: mercaptosuccinic acid). Due to antigen-antibody affinity they preferentially attached to NW-TGA-BSA in the manner presented in Figures 2 and 4. In this part of the project, we were particularly interested in the crossbar arrangements of the NWs. An archetypal triangular arrangement of the NWs of different diameters (two NWs 10∼12 nm and one NW ∼5 nm in diameter) were spontaneously assembled using BSA-IgG connectors on the surface of the wafer (Figure 5). Then the C-AFM probe was positioned directly on top of the NWs and bias was applied between the tip and the wafer. In this way, I-V curves were taken in several selected points of special significance, for instance the middle points of the sides of the triangle and the juncture points where one NW intersects the other one. As shown in Figure 5, diode-like 246
Figure 5. (A) AFM topography image of NW-TGA-BSA/ NW-MSA-IgG samples deposited on arsenic-doped silicon wafer. (B) Current vs bias dependences of the CdTe NW on the arsenicdoped silicon wafer. Nano Lett., Vol. 5, No. 2, 2005
curves with various magnitudes of turn-on voltage from 0.8 to 6 V were obtained for the middle points on the sides. The thicker wires, display substantially lower turn-on voltage (Figure 5 points B, C) than the thinnest wire (Figure 5 point A), as one would expect. However, we want to point out that it is difficult to unequivocally correlate the transverse conductivity with the diameters of NWs, because the magnitude of tunneling current is highly dependent on the contact between the sample and the substrate. What is probably more important is that there was no current going through the cross points (Figure 5, points D, E, F) with positive bias up to 10 V. This indicates that the NW junctions are nonconductive. The insulation effects should be attributed to the presence of protein molecules between the NWs. The diameter of the streptavidin-biotin complex is estimated to be 5 nm,43-45 which creates high tunneling barrier. The presence of the physical gap can also be seen in TEM images in Figure 1D. This problem of the insulating nature of the crossover points should be seriously considered when contemplating the prospects of biologically coded assembly of electronic circuits. The tunneling barrier can be reduced when DNA oligomers are used as NW connectors, resulting in a smaller effective separation between them as compared to proteins, which may be a better choice for this task. It can possibly be eliminated completely by annealing at high temperature in appropriate atmosphere, when biological coatings are either carbonized or completely removed. It is important to emphasize, however, that the 5 nm gap existing between the wires in the protein-driven assembly can also be viewed as a resource for the creation of hybrid NP-NW devices in these junctions, such as single-electron devices. For instance, metal NP bioconjugate with biotin can be specifically adsorbed on one of the four docking spots on the streptavidin between the top and the bottom NW of the junction. The recent data on the preparation of CdTe NW-Au NP superstructures46 demonstrate the first step toward such NP-NW hybrid devices. Se and Te NWs47,48 including those made from CdTe NPs precursors in aqueous media49 can also be a useful component of such circuits combining higher transversal conductivity and semiconducting material whose band gap can be varied. Conclusion. Aqueous CdTe NWs are assembled with complementary connectors, antigen-antibody and biotinstreptavidin. Crossbar and end-to-side connections are found to be the dominant assembly fashions. The formation of conjugate complex is confirmed by Fo¨rster resonance energy transfer between NWs with different emission wavelength. Studies performed with the help of conductive AFM demonstrate that the junction points where one NW crosses the other are nonconducting, thus impeding the charge transport between the wires. According to TEM results, the insulating gap between them is estimated to be several nanometers, which is sufficient for the assembly of a NP between the crossing NWs. One of the important current tasks in the area of bottom-up assembly is to understand the distribution of the protein molecules on the NW surface and Nano Lett., Vol. 5, No. 2, 2005
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Nano Lett., Vol. 5, No. 2, 2005
