Aniruddh Solanki

GRAPHENE-ENCAPSULATED NANOPARTICLE-BASED BIOSENSOR FOR THE SELECTIVE DETECTION OF BIOMARKERS

Nanomaterials such as silicon nanowires (SiNWs), [ 1 , 2 ] carbon nanotubes (CNTs), [ 3–6 ] and graphene, [ 7,8 ] have gained much attention for use in electrical biosensors due to their nanoscopic and electrical properties. For instance, SiNWs and CNTs can be integrated into fi eld-effect transistors (FETs) to detect small amounts of target biomolecules with high sensitivity and selectivity by measuring electrical disturbances induced by the binding of these biomolecules to the surface of the nanostructure. [ 9,10 ] The detection of biomarker proteins with high sensitivity and selectivity is vital for the early diagnosis of many diseases including cancer and HIV. For this purpose, carbonbased nanomaterials such as CNTs and graphene have attracted signifi cant attention for fabricating highly sensitive FET-based biosensors. [ 6 , 8,9 , 11–15 ] In particular, the use of graphene in FETbased biosensors is becoming more and more appealing not only due to its unique properties, such as higher 2D electrical conductivity, superb mechanical fl exibility, large surface area, and high chemical and thermal stability, but also due to its ability to overcome the limitations of CNTs, such as variations in electrical properties of CNT-based devices and the limited surface area of CNTs. [ 16–24 ] Nevertheless, there have been only a few reports on the development of graphene FET-based biosensors, [ 14 , 25 ] and their potential as biosensors has not been fully explored. It is therefore critical to develop nanoscopic graphenebased biosensors that are simple in device structure, small in size, and allow label-free detection and real-time monitoring of biomarkers, all of which are essential criteria for biosensors. A key challenge in the above requirements is the achievement of both, well-organized 2D or 3D graphene structures, in microscopic and nanoscopic biosensing devices and well-defi ned bioconjugation chemistry on graphene.

Nanotechnology for regenerative medicine: nanomaterials for stem cell imaging

Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of microenvironmental signals and novel methods to track and guide transplanted stem cells. The application of nanotechnology to stem cell biology would be able to address those challenges. This review details the current challenges in regenerative medicine, the current applications of nanoparticles in stem cell biology and further potential of nanotechnology approaches towards regenerative medicine, focusing mainly on magnetic nanoparticle- and quantum dot-based applications in stem cell research.

Selective Inhibition of Human Brain Tumor Cells through Multifunctional Quantum‐Dot‐Based siRNA Delivery

One of the most promising new chemotherapeutic strategies is the RNA interference (RNAi)-based approach, wherein small double-stranded RNA molecules can sequence-specifically inhibit the expression of targeted oncogenes.[1] In principle, this method has high specificity and broad applicability for chemotherapy. For example, the small interfering RNA (siRNA) strategy enables manipulation of key oncogenes that modulate signaling pathways and thereby regulate the behavior of malignant tumor cells. To harness the full potential of this approach, the prime requirements are to deliver the siRNA molecules with high selectivity and efficiency into tumor cells and to monitor both siRNA delivery and the resulting knock-down effects at the single cell level. Although several approaches such as polymer- and nanomaterial-based methods[2] have been attempted, limited success has been achieved for delivering siRNA into the target tumor cells. Moreover, these types of approaches mainly focus on the enhancement of transfection efficiency, knock-down of non-oncogenes (e.g. green fluorescent protein (GFP)), and the use of different nanomaterials such as quantum dots (QDs), iron oxide nanoparticles, and gold nanoparticles.[3,4] Therefore, to narrow the gap between current nanomaterial-based siRNA delivery and chemotherapies, there is a clear need to develop methods for target-oriented delivery of siRNA [5], for further monitoring the effects of siRNA-mediated target gene silencing via molecular imaging probes[4], and for investigating the corresponding up/down regulation of signaling cascades.[6] Perhaps most importantly, to begin the development of the necessary treatment modalities, the nanomaterial-based siRNA delivery strategies must be demonstrated on oncogenes involved in cancer pathogenesis.

Axonal alignment and enhanced neuronal differentiation of neural stem cells on graphene-nanoparticle hybrid structures.

Human neural stem cells (hNSCs) cultured on graphene-nanoparticle hybrid structures show a unique behavior wherein the axons from the differentiating hNSCs show enhanced growth and alignment. We show that the axonal alignment is primarily due to the presence of graphene and the underlying nanoparticle monolayer causes enhanced neuronal differentiation of the hNSCs, thus having great implications of these hybrid-nanostructures for neuro-regenerative medicine.

ZnO thin film transistor immunosensor with high sensitivity and selectivity

A zinc oxide thin film transistor-based immunosensor (ZnO-bioTFT) is presented. The back-gate TFT has an on-off ratio of 108 and a threshold voltage of 4.25 V. The ZnO channel surface is biofunctionalized with primary monoclonal antibodies that selectively bind with epidermal growth factor receptor (EGFR). Detection of the antibody-antigen reaction is achieved through channel carrier modulation via pseudo double-gating field effect caused by the biochemical reaction. The sensitivity of 10 fM detection of pure EGFR proteins is achieved. The ZnO-bioTFT immunosensor also enables selectively detecting 10 fM of EGFR in a 5 mg/ml goat serum solution containing various other proteins.

Controlling Differentiation of Neural Stem Cells Using Extracellular Matrix Protein Patterns

The ability of stem cells to differentiate into specialized lineages within a specific microenvironment is vital for regenerative medicine. For harnessing the full potential of stem cells for regenerative therapies, it is important to investigate and understand the function of three types of micro-environmental cues—soluble signals, cell–cell interactions, and insoluble (physical) signals—that dynamically regulate stem cell differentiation.[1] Neural stem cells (NSCs) are multipotent and differentiate into neurons and glial cells,[2] which can provide essential sources of engraftable neural cells for devastating diseases such as Alzheimer’s disease,[3] Parkinson’s disease[4] and spinal cord injury.[5] One of the major challenges involved in the differentiation of NSCs is to identify and optimize factors which result in an increased proportion of NSCs differentiating into neurons as opposed to glial cells. To this end, soluble cues such as brain-derived neurotrophic factor (BDNF),[6] sonic hedgehog (Shh),[7] retinoic acid (RA),[6c] and neuropathiazol[8] have been shown to significantly increase neuronal differentiation of NSCs in vitro. However, the research toward studying the function of the other two microenvironmental cues (cell–cell interactions and insoluble cues) during the neurodifferentiation of NSCs is limited, mainly due to the lack of availability of methods for the investigation.[9] While various aspects such as cell–cell interactions,[10] combinations of extracellular matrix (ECM) proteins,[1a,11] and physical properties of substrates have been shown to play a vital role in determining the fate of other adult stem cells such as mesenchymal stem cells (MSCs),[12] cardiac stem cells,[13] and hematopoetic stem cells,[14] little is known about the influence of such factors on the neuronal differentiation of NSCs. Therefore, there is a pressing need to develop methods for investigating the role of cell–cell interactions and insoluble signals in selectively inducing the differentiation of NSCs into specific neural cell lineages.

Nanotopography-mediated Reverse Uptake for siRNA Delivery into Neural Stem Cells to Enhance Neuronal Differentiation

RNA interference (RNAi) for controlling gene expression levels using siRNA or miRNA is emerging as an important tool in stem cell biology. However, the conventional methods used to deliver siRNA into stem cells result in significant cytotoxicity and undesirable side-effects. To this end, we have developed a nanotopography-mediated reverse uptake (NanoRU) delivery platform to demonstrate a simple and efficient technique for delivering siRNA into neural stem cells (NSCs). NanoRU consists of a self-assembled silica nanoparticle monolayer coated with extracellular matrix proteins and the desired siRNA. We use this technique to efficiently deliver siRNA against the transcription factor SOX9, which acts as a switch between neuronal and glial fate of NSCs. The knockdown of SOX9 enhanced the neuronal differentiation and decreased the glial differentiation of the NSCs. Our NanoRU platform demonstrates a novel application and the importance of nanotopography-mediated siRNA delivery into stem cells as an effective method for genetic manipulation.

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is a single vehicle that not only solubilizes hydrophobic small molecules in physiological solutions but also forms complexes with siRNA molecules, making it an attractive delivery system for controlling stem cell differentiation. Herein, we report the synthesis and application of DexAM to simultaneously deliver hydrophobic small molecules and siRNA into neural stem cells to significantly enhance their neuronal differentiation.

Label-free polypeptide-based enzyme detection using a graphene-nanoparticle hybrid sensor.

A graphene-nanoparticle (NP) hybrid biosensor that utilizes an electrical hysteresis change to detect the enzymatic activity and concentration of Carboxypeptidase B was developed. The results indicate that the novel graphene-NP hybrid biosensor, utilizing electrical hysteresis, has the ability to detect concentrations of targeted enzyme on the micromolar scale. Furthermore, to the knowledge of the authors, this is the first demonstration of a graphene-based biosensor that utilizes a hysteresis change resulting from metallic NPs assembled on a graphene surface.

A Step Closer to Complete Chemical Reprogramming for Generating iPS Cells

The advent of induced pluripotent stem cells (iPSCs) can overcome some of the current limitations of hESC-based cell therapy. For instance, using genetic engineering, the Yamanaka group manipulated murine somatic cells through the expression of four transcription factors (TFs) Oct4, Klf4, Sox2, and cMyc, and reprogrammed the cells to a pluripotent state. [2] Similar genetic manipulations subsequently resulted in the generation of human iPSCs. [3] Efforts in many labs are now concentrated on applying the iPSC technology to tissue-replacement therapies as well as on modeling diseases in vitro. [4] Other advances include generating iPSCs from patients with different diseases in an effort to cause the in vitro differentiation of these iPSCs into the cell types affected by the disease. [5] The use of iPSCs also helps in gaining significant insights into understanding the mechanisms underlying pluripotency and differentiation. However, before iPSCs can be clinically relevant, several limitations, including the use of viral vectors and the slow kinetics and low efficiency of induction, need to be addressed. [6] One of the most critical issues is the presence of transgenes in the iPSCs. Typically, iPSCs are generated by transducing somatic cells with transgenes, which are integrated within the cell’s genome, by using retroviruses or lentiviruses. The integrated transgenes are silenced, and the endogenous genes encoding the TFs are activated. However, transgene reactivation (especially c-Myc) poses a significant threat as it can lead to tumorigenesis. [7] Furthermore, the erroneous expression of these