Shiping Song

Target-Responsive Structural Switching for Nucleic Acid-Based Sensors

Interest in the development of sensitive, selective, rapid, and cost-effective biosensors for biomedical analysis, environmental monitoring, and the detection of bioterrorism agents is rapidly increasing. A classic biosensor directly transduces ligand-target binding events into a measurable physical readout. More recently, researchers have proposed novel biosensing strategies that couple ligand-induced structural switching of biomolecules with advanced optical and electronic transducers. This approach has proven to be a highly general platform for the development of new biosensors. In this Account, we describe a series of electrochemical and optical nucleic acid sensors that use target-responsive DNA structures. By employing surface-confined DNA structures with appropriate redox labels, we can monitor target-induced structural switching of DNA or aptamer-specific small molecule probes by measuring electrochemical currents that are directly associated with the distance between the redox label and the electrode surface. We have also demonstrated significant improvements in sensing performance through optimization of the DNA self-assembly process at electrode surfaces or the introduction of nanomaterial-based signal amplification. Alternatively, gold nanoparticles interact differently with folded and unfolded DNA structures, which provides a visual method for detecting target-induced structural switching based on the plasmonic change of gold nanoparticles. This novel method using gold nanoparticles has proven particularly suitable for the detection of a range of small-molecule targets (e.g., cocaine) and environmentally toxic metal ions (e.g., Hg(2+)). Rational sequence design of DNA aptamers improves the sensitivity and increases the reaction kinetics. Recently, we have also designed microfluidic devices that allow rapid and portable mercury detection with the naked eye. This Account focuses on the use of bulk and nanoscale gold and DNA/aptamer molecules. We expect that researchers will further expand the analyte spectrum and improve the sensitivity and selectivity of nucleic acid sensors using functional biomolecules, such as DNAzymes, peptide aptamers and engineered proteins, and nanomaterials of different sizes, dimensions and compositions, such as carbon nanotubes, graphene, silicon nanowires, and metal nanoparticles or nanorods.

Functional nanoprobes for ultrasensitive detection of biomolecules

There has been great interest in developing new nucleic acid and protein detection methods for both clinical and numerous non-clinical applications. In a long-lasting effort to improve the detection ability of bioassays, functional nanomaterials have been actively explored to greatly enhance the sensitivity during the last two decades. This tutorial review focuses on recent progress in biosensor development by exploiting several unique optical, electronic and catalytic properties of a range of nanomaterials, such as gold nanoparticles, quantum dots, silicon nanowires, carbon nanotubes and graphene. In addition, a perspective on new opportunities offered by emerging technologies (e.g. DNA nanotechnology) is provided.

Visual Cocaine Detection with Gold Nanoparticles and Rationally Engineered Aptamer Structures

A novel bioassay strategy is designed to detect small-molecule targets such as cocaine, potassium, and adenosine, based on gold nanoparticles (AuNPs) and engineered DNA aptamers. In this design, an aptamer is engineered to be two pieces of random, coil-like single-stranded DNA, which reassembles into the intact aptamer tertiary structure in the presence of the specific target. AuNPs can effectively differentiate between these two states via their characteristic surface-plasmon resonance-based color change. Using this method, cocaine in the low-micromolar range is selectively detected within minutes. This strategy is also shown to be generic and applicable to the detection of several other small-molecule targets.

A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing.

A critical challenge in surface-based biomolecular detection is the reduced accessibility of target molecules to probes arranged on a heterogeneous surface compared to probe–target recognition in homogeneous solution.[1–5] To improve the recognition abilities of such heterogeneous surface probes, much effort has been devoted to control the surface chemistry, conformation, and packing density of the probe molecules as well as the size and geometry of the surface.[6–11] Here, we devise a new concept to achieve improved probe–target recognition properties by introducing a probe bearing a 3D DNA nanostructure-based chip platform. DNA nanotechnology has attracted intense interest because the unparalleled self-recognition properties of DNA offer flexibility and convenience for the ‘bottom-up’ construction of exquisite nanostructures with high controllability and precision.[12–20] Our strategy to design and construct 3D nanostructured recognition probes on a surface provides a significantly enhanced spatial positioning range and accessibility of the probes on a surface over previously reported linear or stem-loop probe structures.[2,7]

Ultrasensitive, Multiplexed Detection of Cancer Biomarkers Directly in Serum by Using a Quantum Dot-Based Microfluidic Protein Chip

Sensitive and selective detection for cancer biomarkers are critical in cancer clinical diagnostics. Here we developed a microfluidic protein chip for an ultrasensitive and multiplexed assay of cancer biomarkers. Aqueous-phase-synthesized CdTe/CdS quantum dots (aqQDs) were employed as fluorescent signal amplifiers to improve the detection sensitivity. Secondary antibodies (goat anti-mouse IgG) were conjugated to luminescent CdTe/CdS QDs to realize a versatile fluorescent probe that could be used for multiplexed detection in both sandwich and reverse phase immunoassays. We found that our microfluidic protein chip not only possessed ultrahigh femtomolar sensitivity for cancer biomarkers, but was selective enough to be directly used in serum. This protein chip thus combines the high-throughput capabilities of a microfluidic network with the high sensitivity and multicolor imaging ability offered by highly fluorescent QDs, which can become a promising diagnostic tool in clinical applications.

Aptamer-based multicolor fluorescent gold nanoprobes for multiplex detection in homogeneous solution.

The design of a novel multicolor fluorescent gold nanoprobe for homogeneous detection of small-molecule targets is reported, which combines the specific binding abilities of aptamers with the ultrahigh quenching ability of gold nanoparticles (AuNPs). Dye-tagged aptamers and their complementary sequence with thiol labels are co-assembled at the surface of AuNPs. As a proof of concept, it is demonstrated that such a multicolor fluorescent gold nanoprobe can simultaneously detect adenosine, potassium ion, and cocaine with high selectivity. This potentially generic strategy is shown to be promising for rapid screening of small molecular targets.

Graphene Oxide-Facilitated Electron Transfer of Metalloproteins at Electrode Surfaces

Graphene is a particularly useful nanomaterial that has shown great promise in nanoelectronics. Because of the ultrahigh electron mobility of graphene and its unique surface properties such as one-atom thickness and irreversible protein adsorption at surfaces, graphene-based materials might serve as an ideal platform for accommodating proteins and facilitating protein electron transfer. In this work, we demonstrate that graphene oxide (GO) supports the efficient electrical wiring the redox centers of several heme-containing metalloproteins (cytochrome c, myoglobin, and horseradish peroxidase) to the electrode. Importantly, proteins retain their structural intactness and biological activity upon forming mixtures with GO. These important features imply the promising applications of GO/protein complexes in the development of biosensors and biofuel cells.

Unmodified gold nanoparticles as a colorimetric probe for potassium DNA aptamers

Unmodified gold nanoparticles effectively differentiate unfolded and folded DNA, thus providing a novel approach to colorimetrically probe aptamer-based recognition processes.

DNA Nanostructure-Decorated Surfaces for Enhanced Aptamer-Target Binding and Electrochemical Cocaine Sensors

The sensitivity of aptamer-based electrochemical sensors is often limited by restricted target accessibility and surface-induced perturbation of the aptamer structure, which arise from imperfect packing of probes on the heterogeneous and locally crowded surface. In this study, we have developed an ultrasensitive and highly selective electrochemical aptamer-based cocaine sensor (EACS), based on a DNA nanotechnology-based sensing platform. We have found that the electrode surface decorated with an aptamer probe-pendant tetrahedral DNA nanostructure greatly facilitates cocaine-induced fusion of the split anticocaine aptamer. This novel design leads to a sensitive cocaine sensor with a remarkably low detection limit of 33 nM. It is also important that the tetrahedra-decorated surface is protein-resistant, which not only suits the enzyme-based signal amplification scheme employed in this work, but ensures high selectivity of this sensor when deployed in sera or other adulterated samples.

Graphene-Based High-Efficiency Surface-Enhanced Raman Scattering-Active Platform for Sensitive and Multiplex DNA Detection

We have developed a surface-enhanced Raman scattering (SERS)-active substrate based on gold nanoparticle-decorated chemical vapor deposition (CVD)-growth graphene and used it for multiplexing detection of DNA. Due to the combination of gold nanoparticles and graphene, the Raman signals of dye were dramatically enhanced by this novel substrate. With the gold nanoparticles, DNA capture probes could be easily assembled on the surface of graphene films which have a drawback to directly immobilize DNA. This platform exhibits extraordinarily high sensitivity and excellent specificity for DNA detection. A detection limit as low as 10 pM is obtained. Importantly, two different DNA targets could be detected simultaneously on the same substrate just using one light source.