Potentio-tunable FET sensor having a redox-polarizable single electrode for the implementation of a wearable, continuous multi-analyte monitoring device.

The emerging field of wearable devices for monitoring bioanalytes calls for the miniaturization of biochemical sensors. The only commercially available electrochemical wearable monitoring medical devices for bioanalytes are the amperometric continuous glucose monitoring (CGM) systems. The use of such amperometric methods to monitor glucose levels requires a relatively large electrode surface area for sufficient redox species collection, allowing accurate measurements to be made. Consequently, miniaturization of such sensors bearing large electrodes is challenging. Furthermore, it is difficult to introduce and deploy more than one electrode-based sensor per device, thereby limiting the number of analytes that can be monitored in parallel. To address these limitations, we have employed a non-referenced, single polarizable electrode coupled to a fin-shaped field-effect transistor (Fin-FET). We have discovered that by passivating the FET area by a relatively thick oxide and/or polytetrafluoroethylene (PTFE) polymer, leaving only the polarizable working electrode (WE) exposed, we can monitor redox analytes at the micromolar to millimolar concentration range. We attribute this effect to the WE polarization by the solution redox species. We have exploited the superior sensitivity of the adjacent silicon-based Fin-FET to detect changes in sensor electrode potentials induced by the redox species. Furthermore, we demonstrated the correlation between a specific analyte and the biasing WE potential on the accumulation/depletion of the coupled Fin-FET channel as manifested by the transistor source-drain current. Moreover, we utilized the analyte-electrode potential interaction, which is analyte-specific, to tune the specificity of the sensor towards an analyte of choice. In addition, we demonstrated the use of a single-electrode potentiometric sweep to assist in identifying the accumulation/depletion as a result of analyte-WE state. Collectively, the tiny potentio-tunable electrochemical sensor (PTEchem sensor) area is ~50 × 50 µm, and dedicated wireless transducer facilitates the use of this sensor for wearable continuous, multi-metabolite monitoring.

Cellular Metabolomics by a Universal Redox-Reactive Nanosensors Array: From the Cell Level to Tumor-on-a-Chip Analysis.

Although biosensors based on nanowires-field effect transistor were proved extraordinarily efficient in fundamental applications, screening of charges due to the high-ionic strength of most physiological solutions imposes severe limitations in the design of smart, "real-time" sensors, as the biosample solution has to be previously desalted. This work describes the development of a novel nanowire biosensor that performs extracellular real-time multiplex sensing of small molecular metabolites, the true indicators of the body's chemistry machinery, without any preprocessing of the biosample. Unlike other nanoFET devices that follow direct binding of analytes to their surfaces, our nanodevice acts by sensing the oxidation state of redox-reactive chemical species bound to its surface. The device's surface array is chemically modified with a reversible redox molecular system that is sensitive to H2O2 down to 100 nM, coupled with a suite of corresponding oxidase enzymes that convert target metabolites to H2O2, enabling the direct prompt analysis of complex biosamples. This modality was successfully demonstrated for the real-time monitoring of cancer cell samples' metabolic activity and evaluating chemotherapeutic treatment options for cancer. This distinctive system displays ultrasensitive, selective, noninvasive, multiplex, real-time, label-free, and low-cost sensing of small molecular metabolites in ultrasmall volumes of complex biosamples, in the single-microliter scale, placing our technology at the forefront of this cutting-edge field.

Multicolor Spectral-Specific Silicon Nanodetectors based on Molecularly Embedded Nanowires.

Silicon-based photodetectors cannot distinguish between different wavelengths. Therefore, these detectors relay on color-specific filters to achieve color separation. Color filters add complexity to color sensitive device fabrication, and hinder miniaturization of such devices. Here, we report an ultrasmall (as small as ∼20 nm by 300 nm), red-green-blue-violet (RGBV) filter-free spectrally gated field effect transistor (SGFET) detectors. These photodetectors are based on organic-silicon nanowire hybrid FET devices, capable of detecting specific visible wavelength spectrum with full width at half-maxima (fwhm) under 100 nm. Each SGFET is controlled by a distinctive RGBV spectral range, according to its specific organic fluorophore functionalization. The spectral-specific RGBV detection is accomplished via covalent attachment of different fluorophores. The fluorophore molecules inject electrons into the nanowire structure as a result of light absorption at the appropriate RGBV spectral range. These photoinduced electrons modify the occupancies of the oxide's surface states, shifting the device threshold voltage, thus changing its conductivity, and functioning as a negative stress bias in a p-type SiNW FETs. A positive biasing can be achieved via UV light-induced ionization, which leads to detrapping and translocation of electrons at the oxide layer. Furthermore, a novel theoretical model on the mechanism of action of these devices was developed. Also, we show that suspended SGFETs can function as nonvolatile memory elements, which unlike fast-relaxing on-surface SGFETs, can store discrete "on" (RGBV illumination) and "off" (UV illumination) states for several days at ambient conditions. We also demonstrate a unique single-nanowire multicolor photodetector, enabling in principle a broad spectral detection over a single silicon nanowire element. These highly compact, spectral-controlled nanodevices have the potential to serve in various future novel optoelectric applications.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

IKAP/Elp1 involvement in cytoskeleton regulation and implication for familial dysautonomia.

Deficiency in the IKAP/Elp1 protein leads to the recessive sensory autosomal congenital neuropathy which is called familial dysautonomia (FD). This protein was originally identified as a role player in transcriptional elongation being a subunit of the RNAPII transcriptional Elongator multi-protein complex. Subsequently, IKAP/Elp1 was shown to play various functions in the cytoplasm. Here, we describe experiments performed with IKAP/Elp1 downregulated cell lines and FD-derived cells and tissues. Immunostaining of the cytoskeleton component α-tubulin in IKAP/Elp1 downregulated cells revealed disorganization of the microtubules (MTs) that was reflected in aberrant cell shape and process formation. In contrast to a recent report on the decrease in α-tubulin acetylation in IKAP/Elp1 downregulated cells, we were unable to observe any effect of IKAP/Elp1 deficiency on α-tubulin acetylation in the FD cerebrum and in a variety of IKAP/Elp1 downregulated cell lines. To explore possible candidates involved in the observed aberrations in MTs, we focused on superior cervical ganglion-10 protein (SCG10), also called STMN2, which is known to be an MT destabilizing protein. We have found that SCG10 is upregulated in the IKAP/Elp1-deficient FD cerebrum, FD fibroblasts and in IKAP/Elp1 downregulated neuroblastoma cell line. To better understand the effect of IKAP/Elp1 deficiency on SCG10 expression, we investigated the possible involvement of RE-1-silencing transcription factor (REST), a known repressor of the SCG10 gene. Indeed, REST was downregulated in the IKAP/Elp1-deficient FD cerebrum and IKAP/Elp1 downregulated neuroblastoma cell line. These results could shed light on a possible link between IKAP/Elp1 deficiency and cytoskeleton destabilization.

Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules.

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the biological effects of defined molecules on these human stem cells. We show that hMSC treated for five days with retinoic acid (RA) in the absence of serum undergo several transcriptional changes causing an inhibition of ERK related pathways. We found that RA induces the loss of hMSC properties such as differentiation potential to either osteoblasts or adipocytes. We also found that RA inhibits cell cycle progression in the presence of proliferating signals such as epidermal growth factor (EGF) combined with basic fibroblast growth factor (bFGF). In the same manner, RA showed to cause a reduction in cell adhesion and cell migration. In contrast to these results, the addition of EGF+bFGF to serum free cultures was enough to upregulate ERK activity and induce hMSC proliferation and cell migration. Furthermore, the addition of these factors to differentiation specific media instead of serum was enough to induce either osteogenesis or adipogenesis. Altogether, our results show that hMSC's ability to survive without serum enables the identification of signaling factors and pathways that are involved in their stem cell biological characteristics without possible serum interferences.

Enriched population of PNS neurons derived from human embryonic stem cells as a platform for studying peripheral neuropathies.

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.