Time-encoded electrical detection of trace RNA biomarker by integrating programmable molecular amplifier on chip.

One of the serious challenges facing modern point-of-care (PoC) molecular diagnostic platforms relate to reliable detection of low concentration biomarkers such as nucleic acids or proteins in biological samples. Non-specific analyte-receptor interactions due to competitive binding in the presence of abundant molecules, inefficient mass transport and very low number of analyte molecules in sample volume, in general pose critical hurdles for successful implementation of such PoC platforms for clinical use. Focusing on these specific challenges, this work reports a unique PoC biosensor that combines the advantages of nanoscale biologically-sensitive field-effect transistor arrays (BioFET-arrays) realized in a wafer-scale top-down nanofabrication as high sensitivity electrical transducers with that of sophisticated molecular programs (MPs) customized for selective recognition of analyte miRNAs and amplification resulting in an overall augmentation of signal transduction strategy. The MPs realize a programmable universal molecular amplifier (PUMA) in fluidic matrix on chip and provide a biomarker-triggered exponential release of small nucleic acid sequences easily detected by receptor-modified BioFETs. A common miRNA biomarker LET7a was selected for successful demonstration of this novel biosensor, achieving limit of detection (LoD) down to 10 fM and wide dynamic ranges (10 pM-10 nM) in complex physiological solutions. As the determination of biomarker concentration is implemented by following the electrical signal related to analyte-triggered PUMA in time-domain instead of measuring the threshold shifts of BioFETs, and circumvents direct hybridization of biomarkers at transducer surface, this new strategy also allows for multiple usage (>3 times) of the biosensor platform suggesting exceptional cost-effectiveness for practical use.

Treating cryptosporidiosis: A review on drug discovery strategies.

Despite several decades of research on therapeutics, cryptosporidiosis remains a major concern for human and animal health. Even though this field of research to assess antiparasitic drug activity is highly active and competitive, only one molecule is authorized to be used in humans. However, this molecule was not efficacious in immunocompromised people and the lack of animal therapeutics remains a cause of concern. Indeed, the therapeutic arsenal needs to be developed for both humans and animals. Our work aims to clarify research strategies that historically were diffuse and poorly directed. This paper reviews in vitro and in vivo methodologies to assess the activity of future therapeutic compounds by screening drug libraries or through drug repurposing. It focuses on High Throughput Screening methodologies (HTS) and discusses the lack of knowledge of target mechanisms. In addition, an overview of several specific metabolic pathways and enzymatic activities used as targets against Cryptosporidium is provided. These metabolic processes include glycolytic pathways, fatty acid production, kinase activities, tRNA elaboration, nucleotide synthesis, gene expression and mRNA maturation. As a conclusion, we highlight emerging future strategies for screening natural compounds and assessing drug resistance issues.

Spatially selective manipulation of cells with single-beam acoustical tweezers.

Acoustical tweezers open major prospects in microbiology for cells and microorganisms contactless manipulation, organization and mechanical properties testing since they are biocompatible, label-free and have the potential to exert forces several orders of magnitude larger than their optical counterpart at equivalent power. Yet, these perspectives have so far been hindered by the absence of spatial selectivity of existing acoustical tweezers - i.e., the ability to select and move objects individually - and/or their limited resolution restricting their use to large particle manipulation only and/or finally the limited forces that they could apply. Here, we report precise selective manipulation and positioning of individual human cells in a standard microscopy environment with trapping forces up to ~200 pN without altering their viability. These results are obtained with miniaturized acoustical tweezers combining holography with active materials to synthesize specific wavefields called focused acoustical vortices designed to produce stiff localized traps with reduced acoustic power.

Selective electrohydrodynamic concentration of waterborne parasites on a chip.

Concentrating diluted samples is a key step to improve detection capabilities. The wise use of scaling laws shows the advantages of working with sub-microliter-sized samples. Rapid progress in MEMS technologies has driven the design of integrated platforms performing many biochemical operations. Here we report a new concentrator device based on electro-hydrodynamic forces which can be easily integrated into electrowetting-on-dielectric (EWOD) platforms. This approach is label-free and applicable to a wide range of micro-objects. The detection and analysis of two common waterborne parasites, Cryptosporidium and Giardia, is a perfect test case due to their global health relevance. By fully controlling the interplay of the various forces acting on the micron-sized Cryptosporidium parvum and Cryptosporidium muris oocysts, we show that it is possible to concentrate them on the side of a 10 µL initial drop and then extract them efficiently from a droplet of a few hundred nanoliters. We performed a finite element modeling of the forces acting on the parasites' oocysts to optimize the electrodes' shapes. We obtained state-of-the-art concentration factors of 12 ± 0.4 times and 2 to 4 times in the sub-region of the drop and the extracted droplet, respectively, with an efficiency of 70 ± 6%. Furthermore, this device had the ability to selectively concentrate parasites of different species out of a mix. We demonstrated this by segregating C. parvum oocysts from either Giardia lamblia cysts or its related species, C. muris oocysts.

Spatiotemporal control of DNA-based chemical reaction network via electrochemical activation in microfluidics.

In recent years, DNA computing frameworks have been developed to create dynamical systems which can be used for information processing. These emerging synthetic biochemistry tools can be leveraged to gain a better understanding of fundamental biology but can also be implemented in biosensors and unconventional computing. Most of the efforts so far have focused on changing the topologies of DNA molecular networks or scaling them up. Several issues have thus received little attention and remain to be solved to turn them into real life technologies. In particular, the ability to easily interact in real-time with them is a key requirement. The previous attempts to achieve this aim have used microfluidic approaches, such as valves, which are cumbersome. We show that electrochemical triggering using DNA-grafted micro-fabricated gold electrodes can be used to give instructions to these molecular systems. We demonstrate how this approach can be used to release at specific times and locations DNA- based instructions. In particular, we trigger reaction-diffusion autocatalytic fronts in microfluidic channels. While limited by the stability of the Au-S bond, this easy to implement, versatile and scalable technique can be used in any biology laboratory to provide new ways to interact with any DNA-based computing framework.

Electrowetting on Immersed Conducting Hydrogel.

Conducting polymers demonstrate an interesting ability to change their wettability at ultralow voltage (<1 V). While the conducting hydrogel poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) is increasingly used as an interface with biology partly thanks to its mechanical properties, little is known about the electrical control of its wettability. We rely on the captive bubble technique to study this hydrogel property under relevant conditions (fully immerged). We here report that the wettability variations of PEDOT:PSS are driven by an electrowetting phenomenon in contrast to other conducting polymers which are thought to undergo wettability changes due to oxido-reduction reactions. In addition, we propose a modified electrowetting model to describe the wettability variations of PEDOT:PSS in aqueous solution under ultralow voltage and we show how these variations can be tuned in different ranges of contact angles (above or under 90°) by coating the PEDOT:PSS surface.

Ab initio infrared vibrational modes for neutral and charged small fullerenes (C20, C24, C26, C28, C30 and C60).

We calculate the infrared (IR) absorption spectra using DFT B3LYP(6-311G) for a range of small closed-cage fullerenes, Cn, n=20, 24, 26, 28, 30 and 60, in both neutral and multiple positive and negative charge states. The results are of use, notably, for direct comparison with observed IR absorption in the interstellar medium. Frequencies fall typically into two ranges, with C-C stretch modes around 1100-1500 cm(-1) (6.7-9.1 µm) and fullerene-specific radial motion associated with under-coordinated carbon at pentagonal sites in the range 600-800 cm(-1) (12.5-16.7 µm). Notably, negatively charged fullerenes show significantly stronger absorption intensities than neutral species. The results suggest that small cage fullerenes, and notably metallic endofullerenes, may be responsible for many of the unassigned interstellar IR spectral lines.This article is part of the themed issue 'Fullerenes: past, present and future, celebrating the 30th anniversary of Buckminster Fullerene'.

Electrical impedance sensor for quantitative monitoring of infection processes on HCT-8 cells by the waterborne parasite Cryptosporidium.

Cryptosporidium is the main origin of worldwide waterborne epidemic outbreaks caused by protozoan parasites. Its resilience to water chemical treatments and the absence of therapy led to consider it as a reference pathogen to assess water quality and as a possible bioterrorism agent. We here show that an electrical impedance-based device is able to get insights on Cryptosporidium development on a cell culture and to quantify sample infectivity. HCT-8 cells were grown to confluency on Interdigitated Microelectrode Arrays (IMA's) during 76h and then infected by Cryptosporidium parvum during 60h. The impedimetric response was measured at frequencies ranging from 100Hz to 1MHz and a 7min sampling period. As the infection progresses the impedance signal shows a reproducible distinct succession of peaks at 12h post infection (PI), 23h PI and 31h PI and local minima at 9h PI, 19h PI and 28h PI. An equivalent circuit modeling-based approach indicates that these features are mostly originated from paracellular pathway modifications due to host-parasite interactions. Furthermore, our data present for the first time a real-time monitoring of early parasitic stage development with alternating zoite and meront predominances, observed respectively at peaks and local minima in the impedimetric signal. Finally, by quantifying the magnitude of the impedimetric response, we demonstrate this device can also be used as an infectivity sensor as early as 12h PI thus being at least 6 times faster than other state of the art techniques.

Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces.

The paper reports on a surface plasmon resonance (SPR)-based approach for the sensitive and selective detection of lysozyme. The SPR sensor consists of a 50 nm gold film coated with a thin film of reduced graphene oxide (rGO) functionalized with anti-lysozyme DNA aptamer. The SPR chip coating with rGO matrix was achieved through electrophoretic deposition of graphene oxide (GO) at 150 V. Electrophoretic deposition resulted in partial reduction of GO to rGO with a thickness depending on the deposition time. For very short time pulses of 20 s, the resulting rGO film had a thickness of several nanometers and was appropriate for SPR sensing. The utility of the graphene-based SPR sensor for the selective and sensitive detection of proteins was demonstrated using lysozyme as model protein. Functionalization of rGO matrix with anti-lysozyme DNA aptamer through π-stacking interactions allowed selective SPR detection of lysozyme. The graphene-based SPR biosensor provides a means for the label-free, concentration-dependent and selective detection of lysozymes with a detection limit of 0.5 nM.

Controlling fullerene addition sequences, regioselectivity and magic numbers via metal encapsulation.

A systematic density functional study of chlorine addition to C(70) up to C(70)Cl(12) confirms experimental observations of regioselectivity and stability of C(70)Cl(10). We show that K@C(70) follows an alternative addition sequence with different isomers and magic numbers to C(70) such as KC(70)Cl(3). This prediction is important for controlling functionalisation behaviour via encapsulation and endofullerene purification.

Enzyme-free sugar sensing in microfluidic channels with an affinity-based single-wall carbon nanotube sensor.

We present a novel nonenzymatic carbon nanotube sensor integrated in a microfluidic channel for the detection of sugars. The sensor is assembled as a liquid-gated field-effect transistor, with the transistor channel composed of 1 to 10 nanotubes, which are controllably functionalized with boronic acid receptors. The devices show sensitivity to glucose in a concentration range of 5 to 30 mM. Furthermore, by controlling the type of nanotube-receptor coupling (as covalent or noncovalent) and by deploying a sensitive impedance-based detection technique, we corroborate in detail the transduction mechanism of our affinity-based sensor. In the case of covalent coupling, charge carrier scattering along the nanotubes is the dominant mechanism. While in the noncovalent case, surface charge effects dominate. The identification of the mechanism along with the tunability of the chemical coupling and the cost-effective integration in microchannels constitute a solid basis for the entry of nanotube-based sensors in lab-on-a-chip applications.

Site-specific self-assembled liquid-gated ZnO nanowire transistors for sensing applications.

A scalable bottom-up solution-based approach for the site-specific realization of ZnO nanowire (ZnO-NW)-based field-effect transistors for sensing applications in liquids is reported. The nanowires are grown across predefined electrodes patterned by photolithography. Site specificity is attained by the use of nanoparticles acting as seeds. Using integrated on-chip microchannels and microfabricated gate electrodes, electrochemically gated ZnO-NW network transistors functioning in liquids are demonstrated. The optimized devices are rendered sensitive to pH through chemical functionalization. The unique combination of the sensitivity, site specificity, scalability, and cost effectiveness of the technique opens up avenues for the routine realization of one-dimensional nanostructure-based chemical and biosensors for analytical and diagnostic applications.

Correlation of structural and electronic properties in a new low-dimensional form of mercury telluride.

Using high resolution electron microscopy and first principles quantum mechanical calculations we have explored the fundamental physics and chemistry of the semiconductor, HgTe grown inside single wall carbon nanotubes. This material forms a low-dimensional structure based on a repeating Hg2Te2 motif in which both atom species adopt new coordination geometries not seen in the bulk. Density-functional theory calculations confirm the stability of this structure and demonstrate conclusively that it arises solely as a consequence of constrained low dimensionality. This change is directly correlated with a modified electronic structure in which the low-dimensional form of HgTe is transformed from a bulk semimetal to a semiconductor.

Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.