Guilherme N. M. Ferreira

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.

Electric-field-pulse-assisted covalent immobilization of DNA in the nanosecond time scale

A single square voltage pulse is used to achieve the selective covalent bonding of DNA probes to a functionalized thin-film surface (SiO2) on a plastic substrate. Significant covalent bonding occurs with only 100 ns of pulse duration, representing a decrease in immobilization time of more than ten orders of magnitude from the 2 h needed without electric field. A systematic study of the effect of the duration and rise/fall times of the voltage pulse on the immobilization of DNA probe molecules is presented. Successful hybridization of the DNA probes immobilized using this technique with a complementary target strand was achieved.

Thin Film Micro Arrays with Immobilized DNA for Hybridization Analysis

Abstract : In this work, a procedure to immobilize DNA probes on a microarray patterned on a flexible plastic substrate is developed. The method involves the chemical activation of a thin film surface, the introduction of amine functionality via a silanization step, the coupling of an adequate crosslinker and finally the immobilization of the DNA probe. The response of different thin-film materials and plastic substrates to the immobilization procedure is discussed. The DNA probes immobilized in the patterned pixels were then allowed to hybridize with complementary target DNA labeled with a fluorescent molecule. A prototype array of thin film pixels of SiO2 functionalized by silanization deposited over a polyimide substrate is demonstrated.

Purification of Bionanoparticles

Abstract The recent demand for nanoparticulate products such as viruses, plasmids, protein nanoparticles, and drug delivery systems have resulted in the requirement for predictable and controllable production processes. Protein nanoparticles are an attractive candidate for gene and molecular therapy due to their relatively easy production and manipulation. These particles combine the advantages of both viral and non‐viral vectors while minimizing the disadvantages. However, their successful application depends on the availability of selective and scalable methodologies for product recovery and purification. Downstream processing of nanoparticles depends on the production process, producer system, culture media and on the structural nature of the assembled nanoparticle, i.e., mainly size, shape and architecture. In this paper, the most common processes currently used for the purification of nanoparticles, are reviewed.

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.

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.

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 ∼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.

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.

Acoustic detection of cell adhesion on a quartz crystal microbalance

An acoustic quartz crystal microbalance (QCM) was used to signal and follow the cell‑adhesion 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 COS‑7 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.

Editorial: ECAB focus issue: Engineered catalysts, robust, cost-effective and integrated bioprocesses and high-throughput screening

How can technology and industrial biotechnology contribute to a more bio-based economy? At the 3rd European Congress of Applied Biotechnology (ECAB3) in Nice in 2015, relevant topics and technologies and their contribution to sustainability were presented and discussed. In this issue of Biotechnology Journal, five special articles are selected from this conference, highlighting processes and technologies envisaging the development of engineered catalysts, robust bioprocesses, as well as high-throughput screening methods for process development.