Bio/Nano-Materials Subgroup


Back Row: Fengwei, GengFeng, Pinal, Jae-seung, Dave, Wes, Drew, Brandon, Matt M.

Front Row: Haley, Can, Savka, Abigail, Jill, Sarah, Dwight, Ade, Xiaoyang

Programmed Assembly of DNA-Functionalized Nanoparticles


          Our subgroup is using the unique recognition interactions of DNA to direct the assembly of nanometer-sized particles. This methodology allows for the fabrication of new materials with highly tunable properties and for the development of novel biomolecule sensing and separation technologies. Some of our work in this area is in collaboration with Professor George C. Schatz. This work is highly interdisciplinary, utilizing knowledge and techniques from many areas of science, including organic and inorganic chemistry, materials science, and biochemistry.

         We apply this strategy towards the synthesis of new nanostructured materials made from metals, semiconductors (e.g. "quantum dots"), and inorganic and polymer insulators. We not only can design the physical characteristics of these materials by changing the DNA linker and particle composition, control the construction and deconstruction of the materials by hybridizing and dehybridizing the linking DNA. We use a number of different spectroscopic and microscopic techniques to characterize these materials, including UV-visible spectroscopy, light scattering, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The outstanding materials characterization facilities at Northwestern make it possible for us to investigate the chemical and physical properties of these materials.

References: 

Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607.

Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. "DNA-Directed Synthesis of Binary Nanoparticle Network Materials," J. Am. Chem. Soc. 1998, 120, 12674-12675.

Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C.; “What Controls the Optical Properties of DNA-Linked Gold Nanoparticle Assemblies??J. Am Chem. Soc. 2000, 122, 4640-4650.

Jin, R. C.; Wu, G.S.; Li, Z.; Mirkin, C. A.; Schatz, G. C. What controls the melting properties of DNA-linked gold nanoparticle assemblies? J. Am Chem. Soc. 2003, 125, 1643-1654.

Reviews:

Mirkin, C. A. "Towards DNA Based Technology for Preparing Nanocluster Circuits and Arrays," MRS Bulletin, 2000, 25, 43-54.

Mirkin, C. A. “Programming the Assembly of Two- and Three-Dimensional Architectures with DNA and Nanoscale Inorganic Building Blocks?Inorg. Chem., 2000, 39, 2258-2272.

Storhoff, J. J.; Mirkin, C. A. "Programmed Materials Synthesis with DNA," Chem. Rev., 1999, 99, 1849-1862.

Nanoparticle-Based DNA Detection Methods

By studying the materials properties of nanoparticles functionalized with DNA, we have been able to identify many novel structures, which are proving useful in new biodetection schemes.

In 1997, we developed a highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes. We found that introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. The color changes associated with DNA detection were detected visually on a reverse-phase silica plate (Figure 2-1a), allowing for unoptimized detection of about 10 femtomoles of an oligonucleotide. In years since, we have made many advanced in understanding how nanomaterials can be used in highly sensitive and selective detection schemes for biomolecules.

Specifically, we have shown that the sharp melting profiles of DNA-nanoparticle aggregates allow for extremely selective detection of oligonucleotides in that very small changes in temperature permits discrimination of single base pair mismatches. We have exploited these properties in various chip-based detection methods that rely upon either light scattering (Figure 2-1b) or silver staining (Figure 2-1c) to detect nanoparticles captured onto a surface by target DNA that has been immobilized by surface-bound oligonucleotides (a so-called "sandwich assay"). RAMAN dye labeled oligonucleotide-gold probes used in conjunction with silver staining allows for multiplexed detection of DNA targets, because each target probe yields a distinct SERS signal that can be read via RAMAN spectroscopy (Figure 2-d).

We developed an electrical detection method for DNA (Figure 2-1e). In this method, target DNA is captured in the gap between two electrodes using a sandwich assay. Following capture, silver is plated onto the nanoparticles, bridging the gap between the two electrodes and thus allowing current flow. The current flow is translated into DNA detection. Using this method, we can detect target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of 100,000:1.

In a collaboration with Hupp group, we developed a real-time DNA detection method that utilizes single-strand-DNA-modified nanoparticle probes and micropatterned chemoresponsive diffraction gratings interrogated simultaneously at multiple laser wavelengths (Figure 2-1f). The surface-bound nanoparticle probe-based assay with the chemoresponsive diffraction grating signal transduction scheme results in an experimentally-simple DNA detection protocol, displaying attributes of both detection methodologies - the high sensitivity and selectivity afforded by nanoparticle probes and the experimental simplicity, wavelength-dependent resonant enhancement features, and miniaturization potential provided by the diffraction-based sensing technology.

References:

Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles, Science, 1997, 277, 1078-1080.

Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes, J. Am. Chem. Soc., 1998, 120, 1959-1964.

Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA Array Detection with Nanoparticle Probes Science, 2000, 289, 1757-1760.

Taton, T. A.; Lu, G.; Mirkin, C. A. Two-Color Labeling of Oligonucleotide Arrays via Size-Selective Scattering of Nanoparticle Probes, J. Am. Chem. Soc., 2001, 123, 5164-5165.

Park, S.-J.; Taton, T. A.; Mirkin, C. A. Array-Based Electrical Detection of DNA Using Nanoparticle Probes, Science, 2002, 295, 1503-1506.

Bailey, R. C.; Nam, J.-M.; Mirkin, C. A.; Hupp, J. T. Real-Time Multicolor DNA Detection with Chemoresponsive Diffraction Gratings and Nanoparticle Probes, J. Am. Chem. Soc. 2003, 125, 13541-13547.

Cao, Y.; Jin, R.; Mirkin, C. A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection, Science 2002, 297, 1536-1540.

Cao, Y.; Jin, R.; Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Raman-Dye-Labeled Nanoparticle Probes For Proteins, J. Am. Chem. Soc. 2003, accepted for publication.

 

Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins

Figure 3-1. The basic concept of bar-code DNA.

An ultrasensitive method for detecting protein analytes has been recently developed. The system is based on magnetic microparticle probes with antibodies that specifically bind a target of interest [prostate specific antigen (PSA), amyloid beta oligomer] and nanoparticle probes that are encoded with DNA (Figure 3-1) that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes (Figure 3-2). Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows one to determine the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe (Figure 3-2). Because the nanoparticle probe Figure 3-1. The basic concept of bar-code DNA. carries with it a large number of oligonucleotides per protein binding event, there is substantial amplification and one can detect PSA at 30 attomolar concentration (Figure 3-3 inset). Alternatively, one can do polymerase chain reaction on the oligonucleotide barcodes and boost the sensitivity to 3 attomolar (Figure 3-3). Comparable clinically accepted conventional assays for detecting the same target have sensitivity limits of ~ 3 pM, 6 orders of magnitude less sensitive than what is observed with this new method.

Figure 3-2. The bio-bar-code assay. (Left) Probe design and preparation.
(Right) Target protein detection and bar-code DNA amplification and identification

Figure 3-3. Scanometric detection of PSA-specific barcode DNA. PSA concentration (sample volume of 10 ml) was varied from 300 fM to 3 aM and a negative control sample where no PSA was added (control) is shown. For all seven samples, 2 ml of anti-dinitrophenyl (10 pM) and 2 ml of b-galactosidase (10 pM) were added as background proteins. Also shown is PCR-less detection of PSA (30 aM and control) with 30 nm NP probes (inset). Chips were imaged with the Verigene ID system.

References:

Nam, J.-M.; Park, S.-J.; Mirkin, C. A. Bio-barcodes based on oligonucleotide-modified nanoparticles, J. Am. Chem. Soc.2002, 124, 3820-3821.

Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins, Science 2003, 301, 1884-1886.

Nanoparticle Compositions and Shapes

Figure 5-1. Rayleigh light-scattering of nanocrystals: shape, size and composition matter

Size provides important control over many of the physical and chemical properties of nanoscale materials, including luminescence, conductivity, and catalytic activity.  Colloid chemists have achieved excellent control over particle size for several spherical metal and semiconductor compositions. 

An intriguing possibility for new nanoparticle compositions lies in combinations of materials, for example in a core-shell format.  Such materials are likely to have physical properties which are very different from particles of the individual components, and the potential for tunable properties by varying composition ratios.   We have devised a low-temperature method for generating one type of core-shell particle consisting of a core of Ag and a monolayer shell of Au.  The desirable optical properties of the silver core particles are preserved.  However, now the particles can be readily functionalized with thiol-modified oligonucleotides because of the thin gold shell.  We are currently extending this strategy to prepare other particles such as copper and platinum to create a series of core-shell particles with tailorable physical properties by virtue of the choice of core but the surface chemistry and stability of the native, and oligonucleotide-modified, pure gold nanoparticles.

Single Strand for Bio-Barcode Assay


         Although the original barcode assay provides several significant new analytical capabilities as compared to existing techniques, it is not yet in its optimal form. The current design, which typically employs gold nanoparticles as the barcode carrier, has several disadvantages. First, in the case of nucleic acid targets, three different oligonucleotides are required, which is synthetically demanding and costly. Second, it is difficult to achieve consistent and 100% barcode DNA loading on the support strands attached to the Au-NP surface, through hybridization. This inefficiency reduces the signal amplification possible and increases variability, limiting the quantitative capabilities of the assay. Third, changes in assay-specific buffer systems (e.g. salt concentrations, surfactants, buffering salts) can lead to changes in the hybridization efficiency of the barcode sequences to the Au-NP linked complements. Fourth, designing recognition sequences, barcode sequences, and barcode-support sequences that do not exhibit cross-reactivity, especially in the case of multiplexed analyte detection, becomes more difficult as the number of targets increases.

         As a means of addressing these drawbacks, we have designed a new bio-barcode assay that uses a single strand oligonucleotide barcode, which incorporates the recognition element and is terminated with an alkylthiol to generate Au-NP probes. This assay relies on a ligand-exchange process involving dithiothreitol (DTT), a common disulfide reducing agent to liberate the covalently attached barcode sequences from the Au-NP probes for assay readout. DTT has been demonstrated to efficiently remove thiolated oligonucleotides from gold surfaces. The major advantage of this novel DTT-induced barcode release system is that fewer oligonucleotides are required (from 3 to 1), to prepare the Au-NP probes which makes this method less synthetically challenging and particle probe preparation much simpler to perform.

         The utility of this new bio-barcode assay is demonstrated by detecting a mock mRNA target (3' poly-A single-stranded DNA) using both scanometric and fluorescent readouts. Furthermore, the ligand exchange method for releasing the barcodes increases the assay’s reliability, while maintaining its high sensitivity. Finally, the DTT-based assay provides quantitative information over the low attomolar to mid-femtomolar (fM) target concentration range when coupled with the scanometric assay readout method.

References: 

Thaxton, C.S., Hill, H.D., Georganopoulou, D.G., Stoeva, S.I., Mirkin, C.A. "A Bio-Barcode Assay Based Upon DTT-Induced Oligonucleotide Release" Anal. Chem. 2005, in press.

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