Editorial: The European Symposium on Biochemical Engineering Sciences, Dublin 2016.

The European Symposium on Biochemical Engineering Sciences, Dublin 2016.

Evaluation and characterization of axial distribution in expanded bed: II. Liquid mixing and local effective axial dispersion.

Expanded bed adsorption (EBA) is a promising technology to capture proteins directly from unclarified feedstock. In order to better understand liquid mixing along the bed height in expanded beds, an in-bed sampling method was used to measure residence time distribution at different bed heights. A 2cm diameter nozzle column was tested with agarose raw beads (3% crosslinked agarose containing tungsten carbide). Two settled bed heights (11.5 and 23.1cm) with different expansion factors (1.4-2.6) were investigated and the number of theoretical plates (N), the height equivalent of theoretical plate (HETP) and the local effective axial dispersion coefficient (Dax) were calculated for each bed height-defined zone. The effects of expansion factor, settled bed height and mobile phase were evaluated. The results showed that N increased with the increase of expansion factors, but Dax was unaffected under fixed bed heights. Dax and HETP were found similar as a function of relative bed height for two settled bed heights tested. Higher mobile phase viscosity resulted in stronger axial dispersion. In addition, the local effective Dax under the expansion factor near 2.0 had a different profile which showed minimum values at 0.6-0.8 relative bed height, and the potential mechanism was discussed. These results would be useful for the characterization of axial dispersion and modeling protein adsorption in expanded beds under varying operation conditions.

Transmission line model analysis of transcription factors binding to oligoduplexes - differentiation of the effect of single nucleotide modifications.

Advanced impedance spectroscopy analysis based on the transmission line model (TLM) is explored as a novel QCM acoustic biosensing platform for the detection of the single point mutation effect on the binding of the transcription factors (TFs) to immobilized DNA oligoduplexes and the characterization of the protein-DNA mechanical properties.

An amorphous silicon photodiode microfluidic chip to detect nanomolar quantities of HIV-1 virion infectivity factor.

A hydrogenated amorphous silicon (a-Si:H) photosensor was explored for the quantitative detection of a HIV-1 virion infectivity factor (Vif) at a detection limit in the single nanomolar range. The a-Si:H photosensor was coupled with a microfluidic channel that was functionalized with a recombinant single chain variable fragment antibody. The biosensor selectively recognizes HIV-1 Vif from human cell extracts.

Kinetic characterization of the retinoic X receptor binding to specific and unspecific DNA oligoduplexes with a quartz crystal microbalance.

Quartz Crystal Microbalance (QCM) biosensor technology was used to study the interaction of the DNA-binding domain (DBD) of the transcription factor RXRα with immobilized specific (DR1) and unspecific (DR1neg) DNA oligoduplexes. We identify the QCM sensor frequency at the susceptance minimum (fBmin) as a better measuring parameter, and we show that fBmin is proportional to the mass adsorbed at the sensor surface and is not influenced by interferences coming from viscoelastic variations of the adsorbed layers or buffers. This parameter was used to study the binding of RXRα to DNA and to calculate the association and dissociation kinetic constants of RXRαDBD-DR1 interaction. We show that RXRαDBD binds to DNA both as a monomer and as a homodimer, and that the mechanism of binding is salt dependent and occurs in two steps. The QCM biosensor data reveal that a high ionic strength buffer prevents the unspecific interactions and at a lower ionic strength the dissociation of RXRαDBD-DR1 occurs in two phases.

Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model.

A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, G and G, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HRE(wt)) and another with a random sequence (HRE(neg)) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HRE(wt) and HRE(neg), respectively. Furthermore we show that the binding of Haa1DBD to HRE(wt) leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HRE(wt) oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein-DNA biophysics and kinetics.

Acoustic detection of cell adhesion to a coated quartz crystal microbalance - implications for studying the biocompatibility of polymers.

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D-lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.

Acoustic detection of cell adhesion on a quartz crystal microbalance.

An acoustic quartz crystal microbalance (QCM) was used to signal and follow the cell­adhesion process of epithelial cells [human embryonic kidney(HEK) 293T and cervical cancer (HeLa) and fibroblasts [African Green Monkey kidney cells (COS-7)] onto gold surfaces. Cells were applied on the sensor and grown under serum-free and serum-supplemented culture media. The sensor resonance frequency (Δf) and motional resistance (ΔR) variations were measured during cell growth to monitor cell adhesion processes. Fingerprints of the adhesion processes, generated using the QCM signal, were found to be specific for each cell type while enabling the identification of the phases of the adhesion process. Under serum-free conditions, the deposition of HEK 293T and HeLa cells was characterized by a decrease of Δf with constant ΔR, whereas for COS­7 cells, this initial deposition was signaled by variations of ΔR at constant Δf. Toward the end of the adhesion process, fingerprints were characterized by a continuous increase of ΔR consistent with the increase in viscoelasticity. The morphology of adherent cells was visualized by fluorescent microscopy, enabling the association of the cell morphology with QCM signals.

Acoustic wave biosensors: physical models and biological applications of quartz crystal microbalance.

Piezoelectric sensors are acoustic sensors that enable the selective and label-free detection of biological events in real time. These sensors generate acoustic waves and utilize measurements of the variation of the wave propagation properties as a signal for probing events at the sensor surface. Quartz crystal microbalance (QCM) devices, the most widespread acoustic resonators, allow the study of viscoelastic properties of matter, the adsorption of molecules, or the motility of living cells. In a tutorial-like approach, this review addresses the physical principles associated with the QCM, as well as the origin and effects of major interfering phenomena. Special attention is paid to the possibilities offered by QCM that go beyond microweighing, and important recent examples are presented.

Piezoelectric biosensors assisted with electroacoustic impedance spectroscopy: a tool for accurate quantitative molecular recognition analysis.

In this work, electroacoustic impedance analysis based on a modified Butterworth-Van Dyke (BVD) model is used to complement resonance frequency measurements of piezoelectric crystal sensors for the identification and removal of interfering signals. This approach enables the accurate use of the Sauerbrey correlation to establish a direct relationship between mass deposited at the sensor surface and measured frequency variations. Kinetic models can thus be evaluated and binding constants estimated directly from the measured data. We further demonstrate the usefulness of this approach by applying it to the study of the formation of 11-hydroxy-1-undecanothiol self-assembled monolayers (SAM) as well as to the binding of streptavidin to immobilized biotin. Kinetic and equilibrium parameters were estimated from transient analysis, adsorption isotherms, Scatchard and Hill plots obtained from the frequency data for both the alkanethiol and streptavidin films. This strategy based on electroacoustic impedance assisted quartz-crystal microbalance (QCM) biosensors is expected to be a major contribution for the use of these piezoelectric devices as a reliable and cheap detection system that can easily be integrated into analytical techniques.

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies.

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.

Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor.

In this paper recombinant single-chain fragments (scFv-4BL), and single domain antibodies (4BL-V(H)) and (4BL-V(H)D) generated against HIV1 virion infectivity factor (Vif) are used to develop piezoimmunosensors for HIV1 recognition. Mixed self assembled monolayers were generated at the surface of gold coated crystal sensors to which scFv-4BL, 4BL-V(H), or 4BL-V(H)D were immobilized. Impedance analysis was used to discriminate interfering signals from frequency variation data and to increase the sensor sensitivity. The elimination of interfering signals enabled the quantification of the amount of immobilized protein and gave some indication on the viscoelasticity of immobilized biofilms. All the modified sensors were able to specifically recognize HIV1 Vif in liquid samples. The results indicate that lower sensitivities are obtained with 4BL-V(H) single domain antibodies, possibly due to its higher hydrophobic character. The sensitivity obtained when using scFv-4BL was reestablished when using the more hydrophilic 4BL-V(H)D single domain. 4BL-V(H)D piezoimunosensors were effective in recognizing HIV1 Vif from protein mixtures and from cell extracts of human embryonic kidney cells expressing HIV1 Vif. The results presented in this paper demonstrate the potential applicability of the developed piezoimmunosensors to monitor HIV1 infection evolution.

Molecular construction of bionanoparticles: chimaeric SIV p17-HIV I p6 nanoparticles with minimal viral protein content.

VLPs (virus-like particles) are promising delivery vectors for molecular therapy, since they combine the major advantages of viral vectors with significantly fewer viral vector disadvantages. The present paper describes the molecular construction of chimaeric VLPs based on minimal SIV (simian immunodeficiency virus) and HIV1 components. A chimaeric protein was constructed by fusion of SIV matrix protein (p17) and HIV1 p6 protein, and we demonstrated that the chimaeric proteins assemble as 80 nm nanoparticles containing approximately 7700 chimaeric protein units. Chimaeric VLPs are released from HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40) and are fully encapsulated with lipid membrane. Chimaeric VLPs are produced at 3.7-fold higher levels when compared with SIV p17 VLPs owing to duplication of a PTAP (Pro-Thr-Ala-Pro) domain previously shown as essential for virus particle release. The chimaeric VLPs constructed in the present paper were efficiently pseudotyped with vesicular-stomatitis-virus glycoprotein, as shown by immunoprecipitation assays.

Influence of electrolytes in the QCM response: discrimination and quantification of the interference to correct microgravimetric data.

In this work we demonstrate that the presence of electrolytes in solution generates desorption-like transients when the resonance frequency is measured. Using impedance spectroscopy analysis and Butterworth-Van Dyke (BVD) equivalent electrical circuit modeling we demonstrate that non-Kanazawa responses are obtained in the presence of electrolytes mainly due to the formation of a diffuse electric double layer (DDL) at the sensor surface, which also causes a capacitor like signal. We extend the BVD equivalent circuit by including additional parallel capacitances in order to account for such capacitor like signal. Interfering signals from electrolytes and DDL perturbations were this way discriminated. We further quantified as 8.0+/-0.5 Hz pF-1 the influence of electrolytes to the sensor resonance frequency and we used this factor to correct the data obtained by frequency counting measurements. The applicability of this approach is demonstrated by the detection of oligonucleotide sequences. After applying the corrective factor to the frequency counting data, the mass contribution to the sensor signal yields identical values when estimated by impedance analysis and frequency counting.

Multiplex PCR-single-base extension genotyping of multiple glutathione S-transferase polymorphisms.

Identification of genetic polymorphisms has recently gained increased interest, since they can be used as markers to identify the genes that predispose to disease. This emerging role of genetic polymorphism in clinical association has created the need for high-throughput genotyping methodologies. The present study describes the development of an SBE (single-base extension) methodology for the parallel identification of genetic polymorphisms in glutathione S-transferase genes, a superfamily of phase II drug-metabolizing enzymes. Oligonucleotide PCR primers were designed for simultaneous amplification of GSTM1, GSTP1 and GSTT1 gene loci SBE primers were also designed to be specific for each loci and to stop one nucleotide 5'-upstream of the polymorphic location. A specific tag was associated with each SBE primer to guarantee further discrimination by length. After simultaneous amplification of the target gene loci from genomic DNA extracted from human blood samples, SBE reactions were performed with fluorescently labelled dideoxynucleotide triphosphates. Individual genotypes were identified after separation of each tag-SBE probe by PAGE. The multiplex/SBE methodology was validated with previously genotyped DNA samples extracted from 21 individuals and it was used in a blind assay to genotype additional 64 individuals. The results show that SBE leads to the same results as the current 'gold standard' restriction-fragment-length-polymorphism-based genotyping methodologies, since SBE is a robust and accurate genotyping methodology that enables the parallel identification of multiple polymorphisms in the same reaction.

Electrochemical DNA sensor for detection of single nucleotide polymorphisms.

In recent years there has been an increased interest in using biosensors for the recognition and monitoring of molecule interactions. DNA sensors and gene chips are particularly relevant for directly applying the information gathered from the genome projects. In this work electrochemical techniques are used to develop methodologies to detect DNA polymorphisms in human genes using cytochrome P450 3A4 (CYP3A4) as a model gene. CYP3A4*1B oligonucleotides were immobilized on the surface of a gold electrode and hybridized with fully complementary oligonucleotide sequences as well as with mismatched sequences corresponding to the CYP3A4*1A reference sequence. The methodology developed is based on double-stranded DNA's ability to transport charge along nucleotide stacking. The perturbation of the double helix pi-stack introduced by a mismatched nucleotide reduces electron flow and can be detected by measuring the attenuation of the charge transfer. The methodology developed could identify CYP3A4*1A homozygotes by the 5 microC charge attenuation observed when compared with DNA samples containing at least one CYP3A4*1B allele.