Using Impedance Flow Cytometry for Rapid Viability Classification of Heat-Treated Bacteria.

In the future, rapid electrical characterization of cells with impedance flow cytometry promises to be a fast and accurate method for the evaluation of cell properties. In this paper, we investigate how the conductivity of the suspending medium along with the heat exposure time affects the viability classification of heat-treated E. coli. Using a theoretical model, we show that perforation of the bacteria membrane during heat exposure changes the impedance of the bacterial cell from effectively less conducting than the suspension medium to effectively more conducting. Consequently, this results in a shift in the differential argument of the complex electrical current that can be measured with impedance flow cytometry. We observe this shift experimentally through measurements on E. coli samples with varying medium conductivity and heat exposure times. We show that increased exposure time and lower medium conductivity results in improved classification between untreated and heat-treated bacteria. The best classification was achieved with a medium conductivity of 0.045 S/m after 30 min of heat exposure.

An enzymatic nucleic acid vertical flow assay.

Vertical flow assays have been developed in recent years addressing limitations of the lateral flow assays, including limited multiplexing capability, quantitation, and hook effect. In the present study, the first passive paper-based vertical flow assay is developed for the detection of the nucleic acid target. Horseradish peroxidase was used as an enzymatic tracer with a high potential for signal amplification. In order to achieve the best signal-to-noise ratio, different parameters of paper-based assays were optimized. The sample is heat denatured and hybridized with a specific probe to form a dual-labeled hybridization complex. A small volume of diluted sample, 12 µl, can be analyzed within 6 min on the assay in a sandwich format. Assay specificity was evaluated by testing different unrelated samples, and also, 1.7 nM was obtained as the limit of detection (LOD) using the 0 + 3SD method, which is equivalent to 8.5 fmol of double-stranded DNA in the 12 µl sample volume. The linear range of 3-194 nM with a 0.978 correlation coefficient was obtained according to the calibration curve. The developed assay was evaluated with 45 hepatitis B virus clinical plasma samples, and the result showed 100% consistency of the assay with the real-time PCR benchmark. In the present study, we sought to develop a mere detection system for nucleic acid targets, and to investigate the possibility of using enzyme reporter in a passive vertical flow assay.

Continuous Microfluidic Production of Citrem-Phosphatidylcholine Nano-Self-Assemblies for Thymoquinone Delivery.

Lamellar and non-lamellar liquid crystalline nanodispersions, including liposomes, cubosomes, and hexosomes are attractive platforms for drug delivery, bio-imaging, and related pharmaceutical applications. As compared to liposomes, there is a modest number of reports on the continuous production of cubosomes and hexosomes. Using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC), we describe the continuous production of nanocarriers for delivering thymoquinone (TQ, a substance with various therapeutic potentials) by employing a commercial microfluidic hydrodynamic flow-focusing chip. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to characterize TQ-free and TQ-loaded citrem/SPC nanodispersions. Microfluidic synthesis led to formation of TQ-free and TQ-loaded nanoparticles with mean sizes around 115 and 124 nm, and NTA findings indicated comparable nanoparticle size distributions in these nanodispersions. Despite the attractiveness of the microfluidic chip for continuous production of citrem/SPC nano-self-assemblies, it was not efficient as comparable mean nanoparticle sizes were obtained on employing a batch (discontinuous) method based on low-energy emulsification method. SAXS results indicated the formation of a biphasic feature of swollen lamellar (Lα) phase in coexistence with an inverse bicontinuous cubic Pn3m phase in all continuously produced TQ-free and TQ-loaded nanodispersions. Further, a set of SAXS experiments were conducted on samples prepared using the batch method for gaining further insight into the effects of ethanol and TQ concentration on the structural features of citrem/SPC nano-self-assemblies. We discuss these effects and comment on the need to introduce efficient microfluidic platforms for producing nanocarriers for delivering TQ and other therapeutic agents.

Investigating the Use of Impedance Flow Cytometry for Classifying the Viability State of E. coli.

Bacteria detection, counting and analysis is of great importance in several fields. When viability plays a major role in decision making, the counting of colony-forming units grown on agar plates remains the gold standard. However, because plate counts depend on the growth of the bacteria, it is a slow procedure and only works with culturable species. Impedance flow cytometry (IFC) is a promising technology for particle detection, counting and characterization. It relies on the perturbation of an electric field by particles flowing through a microfluidic channel. The perturbation is directly related to the electrical properties of the particles, and therefore provides information about their composition and structure. In this work we investigate whether IFC can be used to differentiate viable cells from inactivated cells. Our findings demonstrate that the specific viability state of the bacteria has to be considered, but that with proper characterization thresholds, IFC can be used to classify bacterial viability states. By using three different inactivation methods-ethanol, heat and autoclavation-we have been able to show that the impedance response of Escherichia coli depends on its viability state, but that the specific response depends on the inactivation method. With these findings we expect to be able to optimize IFC for more reliable bacteria detection and counting in the future.

A hydrodynamic flow focusing microfluidic device for the continuous production of hexosomes based on docosahexaenoic acid monoglyceride.

Cubosomes and hexosomes are emerging platforms for drug and nutraceutical delivery applications. In addition to common high- and low-energy batch emulsification methods for the preparation of these nano-self-assemblies, it is important to introduce suitable microfluidic devices with a precision control of the flow parameters for their continuous production. Microfluidics has several advantages including cost effectiveness, short-production time, and control of the nanoparticle size and size distribution. In the present study, a hydrodynamic flow focusing polyimide microfluidic device was employed for the continuous production of hexosomes based on docosahexaenoic acid monoglyceride (MAG-DHA), in the presence of the stabilizer Pluronic F127. The size, structural, morphological and size characterizations of the continuously produced MAG-DHA nanodispersions were investigated through an integrated approach involving synchrotron small angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy. We report on a simple process for the microfluidic synthesis of hexosomes with sizes ranging from 108 to 138 nm and relatively narrow size distributions as the polydispersity indices were in the range of 0.14-0.22. At the applied total volumetric flow rates (TFRs) ranging of 50-150 µL min-1 and flow rate ratios (FRRs) of 10-30, it was evident from SAXS findings that ethanol has only a slight effect on the lattice parameter of the internal inverse hexagonal (H2) phase of the produced hexosomes. In addition to hexosomes, cryo-TEM observations indicated the coexistence of vesicular structures and smaller nano-objects. The formation of these nano-objects that are most likely normal micelles was also confirmed by SAXS, particularly on increasing FRR from 10 to 20 or 30 at TFR of 150 µL min-1. Taking into account the reported positive health effects of MAG-DHA, which is a long-chain omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglyceride, in various disorders including cancer, the produced hexosomes are attractive for the delivery of ω-3 PUFAs, drugs, nutraceuticals, and their combinations.

Electrochemical sensing of biomarker for diagnostics of bacteria-specific infections.

AIM: Pseudomonas aeruginosa is a pathogen that is prevalent in serious infections in compromised patients worldwide. A unique virulence factor of this bacterium is the redox-active molecule pyocyanin, which is a potential biomarker for the identification of P. aeruginosa infections. Here we report a direct, selective and rapid detection technique of pyocyanin. MATERIALS & METHODS: Pyocyanin was detected by amperometry at a relatively high potential where the pyocyanin signal was unaffected by background contributions. RESULTS & CONCLUSION: Pyocyanin was detected at concentrations down to 125 nM in a 50 µM mixture of interfering compounds with a reproducibility of r(2) = 0.999 (n = 5) within 200 s. The results document a step toward a point-of-care technique for diagnosis of P. aeruginosa infections.

Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry.

This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electrical impedance spectroscopy in order to perform a theoretical study to clarify our results. This study focuses on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy.

Advanced microtechnologies for detection of chromosome abnormalities by fluorescent in situ hybridization.

Cytogenetic and molecular cytogenetic analyses, which aim to detect chromosome abnormalities, are routinely performed in cytogenetic laboratories all over the world. Traditional cytogenetic studies are performed by analyzing the banding pattern of chromosomes, and are complemented by molecular cytogenetic techniques such as fluorescent in situ hybridization (FISH). To improve FISH application in cytogenetic analysis the issues with long experimental time, high volumes of expensive reagents and requirement for trained technicians need to be addressed. The protocol has recently evolved towards on chip detection of chromosome abnormalities with the development of microsystems for FISH analysis. The challenges addressed by the developed microsystems are mainly the automation of the assay performance, reduction in probe volume, as well as reduction of assay time. The recent focus on the development of automated systems for performing FISH on chip is summarized in this review.

Electrostatic force microscopy of self-assembled peptide structures.

In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO(2) surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated.

High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris.

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200-300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

Dynamic in situ chromosome immobilisation and DNA extraction using localized poly(N-isopropylacrylamide) phase transition.

A method of in situ chromosome immobilisation and DNA extraction in a microfluidic polymer chip was presented. Light-induced local heating was used to induce poly(N-isopropylacrylamide) phase transition in order to create a hydrogel and embed a single chromosome such that it was immobilised. This was achieved with the use of a near-infrared laser focused on an absorption layer integrated in the polymer chip in close proximity to the microchannel. It was possible to proceed to DNA extraction while holding on the chromosome at an arbitrary location by introducing protease K into the microchannel.

Stability of diphenylalanine peptide nanotubes in solution.

Over the last couple of years, self-organizing nanotubes based on the dipeptide diphenylalanine have received much attention, mainly as possible building blocks for the next generation of biosensors and as drug delivery systems. One of the main reasons for this large interest is that these peptide nanotubes are believed to be very stable both thermally and chemically. Previously, the chemical and thermal stability of self-organizing structures has been investigated after the evaporation of the solvent. However, it was recently discovered that the stability of the structures differed significantly when the tubes were in solution. It has been shown that, in solution, the peptide nanotubes can easily be dissolved in several solvents including water. It is therefore of critical importance that the stability of the nanotubes in solution and not after solvent evaporation be investigated prior to applications in which the nanotube will be submerged in liquid. The present article reports results demonstrating the instability and suggests a possible approach to a stabilization procedure, which drastically improves the stability of the formed structures. The results presented herein provide new information regarding the stability of self-organizing diphenylalanine nanotubes in solution.

Superparamagnetic bead interactions with functionalized surfaces characterized by an immunomicroarray.

Magneto-resistive sensors capable of detecting superparamagnetic micro-/nano-sized beads are promising alternatives to standard diagnostic assays based on absorbance or fluorescence and streptavidin-functionalized beads are widely used as an integral part of these sensors. Here we have developed an immunomicroarray for systematic studies of the binding properties of 10 different micro-/nano-sized streptavidin-functionalized beads to a biotin substrate immobilized on SiO(2) with or without surface modification. SiO(2) surface cleaning, immobilized substrate concentration and surface blocking conditions were optimized. Polyethylene glycol-based surfaces with different end groups on the anchor molecule, 2,4,6-trichloro-1,3,5-triazine (TsT), were synthesized and compared with the standard (3-aminopropyl)triethoxysilane (APTS)/glutaraldehyde chemistry. APTS/glutaraldehyde, directly linked TsT and bare H(2)O(2)-activated SiO(2) performed better than polyethylene glycol-modified surfaces. Two beads, Masterbeads and M-280 beads, were found to give superior results compared with other bead types. Antibody/antigen interactions, illustrated by C-reactive protein, were best performed with Masterbeads. The results provide important information concerning the surface binding properties of streptavidin-functionalized beads and the immunomicroarray can be used when optimizing the performance of bead-based biosensors.

Manipulation of biological samples using micro and nano techniques.

The constant interest in handling, integrating and understanding biological systems of interest for the biomedical field, the pharmaceutical industry and the biomaterial researchers demand the use of techniques that allow the manipulation of biological samples causing minimal or no damage to their natural structure. Thanks to the advances in micro- and nanofabrication during the last decades several manipulation techniques offer us the possibility to image, characterize and manipulate biological material in a controlled way. Using these techniques the integration of biomaterials with remarkable properties with physical transducers has been possible, giving rise to new and highly sensitive biosensing devices. This article reviews the different techniques available to manipulate and integrate biological materials in a controlled manner either by sliding them along a surface (2-D manipulation), by grapping them and moving them to a new position (3-D manipulation), or by manipulating and relocating them applying external forces. The advantages and drawbacks are mentioned together with examples that reflect the state of the art of manipulation techniques for biological samples (171 references).

Qualitative mapping of structurally different dipeptide nanotubes.

Biological self-assembled structures are receiving increasing focus within micro- and nanotechnology, for example, as sensing devices, due to the fact that they are cheap to produce and easy to functionalize. Therefore, methods for the characterization of these structures are much needed. In this paper, electrostatic force microscopy (EFM) was used to distinguish between hollow nanotubes formed by self-assembly by a simple aromatic dipeptide, L-phenylalanine, silver-filled peptide-based nanotubes, and silver wires placed on prefabricated SiO2 surfaces with a backgate. The investigation shows that it is possible to distinguish between these three types of structures using this method. Further, an agreement between the detected signal and the structure of the hollow peptide was demonstrated; however only qualitative agreement with the mathematical expressing of the tubes is shown.

Scanning conductance microscopy investigations on fixed human chromosomes.

Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value for the dielectric constant of different human chromosomes.