Basri Gulbakan

A Dual Platform for Selective Analyte Enrichment and Ionization in Mass Spectrometry Using Aptamer-Conjugated Graphene Oxide

This study demonstrates the use of aptamer-conjugated graphene oxide as an affinity extraction and detection platform for analytes from complex biological media. We have shown that cocaine and adenosine can be selectively enriched from plasma samples and that direct mass spectrometric readouts can be obtained without a matrix and with greatly improved signal-to-noise ratios. Aptamer-conjugated graphene oxide has clear advantages in target enrichment and in generating highly efficient ionization of target molecules for mass spectrometry. These results demonstrate the utility of the approach for analysis of small molecules in real biological samples.

DNA-guided metal-nanoparticle formation on graphene oxide surface.

Over the last decade, DNA has been widely employed as a scaffold to form inorganic metallic nanoparticles (MNPs). The unique programmable structure provided by Watson-Crick base pairing and the tunable properties of DNA have been used for growth and positioning of nanoparticle structures. This has enabled new synthetic strategies, such as DNA-metallization,[1-8] DNA-mediated nanoparticle synthesis,[9-15] and DNA-controlled positioning of nanoparticles,[16-17] to create novel, efficient and useful miniaturized optical sensors, electronic devices, circuits and medical theranostic kits. The remarkable molecular recognition properties and self-assembly capabilities of DNA have been intensively utilized for several years. For example, DNA-directed inorganic nanowire particles have been studied for observing conformational changes of double-stranded DNA by addition of metal ions [1]. Moreover, the assembly of nanocrystals of semiconductor materials using DNA as a template has been examined to overcome insulation of DNA in electronic circuits.[18] Thus, the binding affinity of metal ions to oxygen-containing mono/di/tri-phosphate groups and N bonds of bases prevents excessive deposition on DNA with increases in concentration. [19] In addition to its ability to self-assemble, DNA has also been used to produce unique dispersed nanoparticles and to control the positions of these particles on programmable DNA scaffolds. As an example, DNA/RNA sequence, structure and composition have been used to fine tune the size, shape and physicochemical properties of nanoparticles and to generate biocompatible and easily functionalizable nanomaterials. [11-12]

A Logical Molecular Circuit for Programmable and Autonomous Regulation of Protein Activity Using DNA Aptamer-Protein Interactions

Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo. (1) As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here, we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy.

Aptamer Conjugated Multifunctional Nanoflowers as a Platform for Targeting, Capture and Detection in Laser Desorption Ionization Mass Spectrometry

Although many different nanomaterials have been tested as substrates for laser desorption and ionization mass spectrometry (LDI-MS), this emerging field still requires more efficient multifuncional nanomaterials for targeting, enrichment, and detection. Here, we report the use of gold manganese oxide (Au@MnO) hybrid nanoflowers as an efficient matrix for LDI-MS. The nanoflowers were also functionalized with two different aptamers to target cancer cells and capture adenosine triphosphate (ATP). These nanoflowers were successfully used for metabolite extraction from cancer cell lysates. Thus, in one system, our multifunctional nanoflowers can (1) act as an ionization substrate for mass spectrometry, (2) target cancer cells, and (3) detect and analyze metabolites from cancer cells.

Enrichment and detection of rare proteins with aptamer-conjugated gold nanorods.

Rare protein enrichment and sensitive detection hold great potential in biomedical studies and clinical practice. This work describes the use of aptamer-conjugated gold nanorods for the efficient enrichment of rare proteins from buffer solutions and human plasma. Gold nanorod (AuNR) surfaces were modified with a long PEG chain and a 15-mer thrombin aptamer for protein enrichment and detection. Studies of the effect of surface modification on enrichment efficiency of thrombin showed that a change of only one EG(6) linker unit, i.e., from 2EG(6) to 3EG(6), could increase thrombin protein capture efficiency by up to 47%. Furthermore, a 1 ppm sample of thrombin in buffer could be enriched with around 90% efficiency using a low concentration (0.19 nM) of gold nanorod probe modified with 3EG(6) spacer, and with the same probe, effective capture was achieved down to 10 ppb (1 ng) thrombin in plasma samples. In addition to α-thrombin enrichment, prothrombin was also efficiently captured from plasma samples via gold nanorods conjugated with 15-mer thrombin aptamer. Our work demonstrates efficient enrichment of rare proteins using aptamer-modified nanomaterials, which can be used in biomarker discovery studies.

Aptamers: turning the spotlight on cells

This article is a review of the development and application of aptamer probes for cell imaging. Aptamers selected against whole cells have been modified with different fluorescent dyes and nanomaterials, such as gold nanoparticles, quantum dots, and superparamagnetic iron oxide, for their use as imaging probes of live cells. These probes have been successfully used for cell imaging both in vitro and in vivo by optical imaging, magnetic resonance imaging (MRI), computed tomography (CT), and positron-emission tomography (PET). In this article, we discuss the development of different aptamer-based probes currently available for imaging of live cells and their applications in the biomedical field.

Post-Genomics Nanotechnology Is Gaining Momentum: Nanoproteomics and Applications in Life Sciences

The post-genomics era has brought about new Omics biotechnologies, such as proteomics and metabolomics, as well as their novel applications to personal genomics and the quantified self. These advances are now also catalyzing other and newer post-genomics innovations, leading to convergences between Omics and nanotechnology. In this work, we systematically contextualize and exemplify an emerging strand of post-genomics life sciences, namely, nanoproteomics and its applications in health and integrative biological systems. Nanotechnology has been utilized as a complementary component to revolutionize proteomics through different kinds of nanotechnology applications, including nanoporous structures, functionalized nanoparticles, quantum dots, and polymeric nanostructures. Those applications, though still in their infancy, have led to several highly sensitive diagnostics and new methods of drug delivery and targeted therapy for clinical use. The present article differs from previous analyses of nanoproteomics in that it offers an in-depth and comparative evaluation of the attendant biotechnology portfolio and their applications as seen through the lens of post-genomics life sciences and biomedicine. These include: (1) immunosensors for inflammatory, pathogenic, and autoimmune markers for infectious and autoimmune diseases, (2) amplified immunoassays for detection of cancer biomarkers, and (3) methods for targeted therapy and automatically adjusted drug delivery such as in experimental stroke and brain injury studies. As nanoproteomics becomes available both to the clinician at the bedside and the citizens who are increasingly interested in access to novel post-genomics diagnostics through initiatives such as the quantified self, we anticipate further breakthroughs in personalized and targeted medicine.