Electrochemical and piezoelectric DNA biosensors for hybridisation detection

DNA biosensors (or genosensors) are analytical devices that result from the integration of a sequence-specific probe and a signal transducer. Among other techniques, electrochemical and piezoelectric methods have recently emerged as the most attractive due to their simplicity, low instrumentation costs, possibility for real-time and label-free detection and generally high sensitivity.Focusing on the most recent activity of worldwide researchers, the aim of the present review is to give the readers a critical overview of some important aspects that contribute in creating successful genosensing devices. Advantages and disadvantages of different sensing materials, probe immobilisation chemistries, hybridisation conditions, transducing principles and amplification strategies will be discussed in detail. Dedicated sections will also address the issues of probe design and real samples pre-treatment. Special emphasis will be finally given to those protocols that, being implemented into an array format, are already penetrating the molecular diagnostics market.     

DNA biosensors for the detection of aflatoxin producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis>

Abstract  In this work, we report on the development of a DNA-based piezoelectric biosensor specific for the detection of an amplicon of the aflD gene of Aspergillus flavus and A. parasiticus. Expression of this gene is consistently correlated with a strain’s ability to produce aflatoxins that are considered very potent liver carcinogens in various animal species and humans. The DNA biosensor has been characterized with synthetic oligonucleotides and amplicons. Moreover, it has been applied to the analysis of real samples consisting of amplicons of DNA extracted from flours and feed contaminated with A. flavus and A. parasiticus. Graphical Abstract        

Signal enhancement of electrochemical DNA biosensors for the detection of trace heavy metals

Heavy-metal pollution has attracted intensive attention from the public because of the severe threats of heavy metals to the ecosystem and human health. Ultralow concentration of heavy metals in aquatic environment leads to the urgent needs of sensitive approaches for heavy-metal detection. Electrochemical DNA biosensors present outstanding superiority in convenience, selectivity, and sensitivity compared with conventional methods. To achieve the ultralow detection limit, efforts have been made to implement signal enhancement strategies to develop electrochemical DNA biosensors with enhanced sensing performance. This review focuses on the recent progress in signal enhancement strategies applied to electrochemical DNA biosensors for heavy-metal-ion detection including nicking enzyme–assisted amplification, the utilization of core–shell nanoparticles, and nanocomposites modification.     

Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells

Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP–MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker and the guanine moiety anodic oxidation and cyclic voltammetry with K₃[Fe(CN)₆] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 μg mL⁻¹. Figure Schematic illustration of preparation of the nanostructured films: (a) layer-to-layer coverage (DNA/nanomaterial/SPE); (b) mixed coverage (DNA-nanomaterial/SPE)     

Analysis of the evolution of the detection limits of electrochemical DNA biosensors

In this paper we critically review detection limits of electrochemical DNA biosensors enabling DNA detection without target labelling. The review includes transduction principles and latest breakthroughs. To compare the efficiency of each type of electrochemical DNA biosensor, a simple DNA biosensors classification is established on the basis of the nature of the bio-electrochemical transduction.     

Chemiluminescence biosensors for DNA detection using graphene oxide and a horseradish peroxidase-mimicking DNAzyme

In this communication, we demonstrate that graphene oxide (GO) greatly inhibits the peroxidatic activity of a horseradish peroxidase-mimicking DNAzyme. Combining this observation with the unique DNA/GO interactions, an ultrasensitive GO-based chemiluminescence DNA biosensing platform is developed.     

用于半胱氨酸检测的电化学生物传感器研究进展

半胱氨酸(Cys)作为人体重要的非必需氨基酸之一,对蛋白质合成、渗透调节、解毒过程、神经系统功能和抗氧化过程均发挥着重要作用,近年来广泛应用于医学临床、食品加工和生化研究领域。因此,发展快速、准确检测半胱氨酸浓度的方法具有重要意义。本文简要介绍了半胱氨酸的基本知识以及半胱氨酸的传统检测方法,对半胱氨酸定量检测的电化学传感器的研制与应用进行了重点阐述,并对其发展前景进行了展望。     

用于褪黑素检测的电化学生物传感器研究进展

褪黑素是人体松果体分泌的一种重要的神经递质,近年来其在控制昼夜节律和提供免疫抗炎特性等生理调节作用方面备受关注。因此,发展可靠、快速检测体内和体外样本中褪黑素浓度的方法,对于探索褪黑素的临床应用和生物学特性具有重要的意义。本文对褪黑素及其传统检测方法进行简要介绍,重点阐述了近几年报道的用于生物样本和药物样本中褪黑素定量检测的电化学传感器,并对褪黑素传感器的未来发展方向进行展望。     

Profiling Differential Expression of Cellulases and Metabolite Footprints in Aspergillus terreus

This study reports differential expression of endoglucanase (EG) and β-glucosidase (βG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G₁, G₃ and G₄ as well as putative positional isomers (G₁/G₂, G₂/G₃) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of βG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four βG isoforms.     

Aptamer-based biosensors for the detection of HIV-1 Tat protein

Two biosensors have been constructed using an RNA aptamer as biorecognition element. The aptamer, specific for HIV-1 Tat protein, has been immobilised on the gold surface of piezoelectric quartz crystals or surface plasmon resonance (SPR) chips to develop a quartz crystal microbalance (QCM)-based and an SPR-based biosensor, respectively. Both the biosensors were modified with the same immobilisation chemistry based on the binding of a biotinylated aptamer on a layer of streptavidin. The binding between the immobilised aptamer and its specific protein has been evaluated with the two biosensors in terms of sensitivity, reproducibility and selectivity. A protein very similar to Tat, Rev protein, has been used as negative control. The two biosensors both were very reproducible in the immobilisation and the binding steps. The selectivity was high in both cases.     

Nanomaterial-based biosensors for detection of prostate specific antigen

Screening serum for the presence of prostate specific antigen (PSA) belongs to the most common approach for the detection of prostate cancer. This review (with 156 refs.) addresses recent developments in PSA detection based on the use of various kinds of nanomaterials. It starts with an introduction into the field, the significance of testing for PSA, and on current limitations. A first main section treats electrochemical biosensors for PSA, with subsections on methods based on the use of gold electrodes, graphene or graphene-oxide, carbon nanotubes, hybrid nanoparticles, and other types of nanoparticles. It also covers electrochemical methods based on the enzyme-like activity of PSA, on DNA-, aptamer- and biofuel cell-based methods, and on the detection of PSA via its glycan part. The next main section covers optical biosensors, with subsections on methods making use of surface plasmon resonance (SPR), localized SPR and plasmonic ELISA-like schemes. This is followed by subsections on methods based on the use of fiber optics, fluorescence, chemiluminescence, Raman scattering and SERS, electrochemiluminescence and cantilever-based methods. The most sensitive biosensors are the electrochemical ones, with lowest limits of detection (down to attomolar concentrations), followed by mass cantilever sensing and electrochemilumenescent strategies. Optical biosensors show lower performance, but are still more sensitive compared to standard ELISA. The most commonly applied nanomaterials are metal and carbon-based ones and their hybrid composites used for different amplification strategies. The most attractive sensing schemes are summarized in a Table. The review ends with a section on conclusions and perspectives.

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Graphical abstract Schematic representation of nanostructure-based biosensors for detection of prostate specific antigen using various detection schemes and biorecognition elements such as antibodies (Abs), aptamers (APT), lectins (LEC), and molecularly imprinted polymers (MIP).

     

Optical leaky waveguide biosensors for the detection of organophosphorus pesticides

Organophosphorus (OP) pesticides can be rapidly detected by integrating organophosphorus hydrolase with an optical leaky waveguide biosensor. This enzyme catalyses the hydrolysis of a wide range of organophosphorus compounds causing an increase in the pH. Thus, the direct detection of OP is possible by monitoring of the pH changes associated with the enzyme's activity. This article describes the use of an optical, leaky waveguide clad with absorbing materials for the detection of OP pesticides by measuring changes in refractive index, absorbance and fluorescence. In the most effective configuration, a thick sensing layer was used to increase the amount of immobilized enzyme and to increase the light interaction with the sensing layer, resulting in a greatly enhanced sensitivity. The platforms developed in this work were successfully used to detect paraoxon and parathion down to 4 nM concentrations.     

Cellular impedance biosensors for drug screening and toxin detection

Cell-based impedance biosensing is an emerging technology that can be used to non-invasively and instantaneously detect and analyze cell responses to chemical and biological agents. This article highlights the fabrication and measurement technologies of cell impedance sensors, and their application in toxin detection and anti-cancer drug screening. We start with an introduction that describes the capability and advantages of cell-based sensors over conventional sensing technology, followed by a discussion of the influence of cell adhesion, spreading and viability during cell patterning on the subsequent impedance measurements and sensing applications. We then present an electronic circuit that models the cell-electrode system, by which the cellular changes can be detected in terms of impedance changes of the circuit. Finally, we discuss the current status on using cell impedance sensors for toxin detection and anti-cancer drug screening.     

Surface properties of Aspergillus oryzae spores investigated by atomic force microscopy

Spores of the filamentous fungus Aspergillus oryzae have a great biotechnological potential for the production of highly active proteins. To date, little is known about the molecular mechanisms of spore aggregation, a phenomenon observed during germination in liquid medium. Here, atomic force microscopy (AFM) imaging and force measurements were used to characterize, under aqueous conditions, the surface morphology and macromolecular interactions of A. oryzae spores in relation to their aggregation behavior. Dormant spores were covered with a discontinuous layer of about 35 nm thickness, as revealed by height images. High-resolution deflection images showed that this layer consisted of rodlets, 10±1 nm in diameter, that were assembled in parallel to form fascicles interlaced with different orientations. The germinating spore surface was much rougher and showed streaks oriented in the scanning direction, indicating that the probe was interacting with soft material. Retraction force curves were strikingly different depending on the spore physiological state: while dormant spores exhibited non-adhesive properties, germinating spores showed single or multiple attractive forces of 400±100 pN magnitude, along with characteristic elongation forces and rupture lengths ranging from 20 to 500 nm. These elongation forces are attributed to the stretching of long, flexible cell surface macromolecules and suggested to play a role in the aggregation process by promoting bridging interactions.     

A review of biosensors and biologically-inspired systems for explosives detection

This article provides a review of the published literature describing the use of biosensors and biologically-inspired systems for explosives detection. The review focusses on the use of antibodies, enzymes, biologically-inspired synthetic ligands and whole-cell biosensors, providing a flavour of the range of technology, formats and approaches that can be used to detect explosives using biological systems.     

Modification of Escherichia coli single-stranded DNA binding protein with gold nanoparticles for electrochemical detection of DNA hybridization

The unique binding event between Escherichia coli single-stranded DNA binding protein (SSB) and single-stranded oligonucleotides conjugated to gold (Au) nanoparticles is utilized for the electrochemical detection of DNA hybridization. SSB was attached onto a self-assembled monolayer (SAM) of single-stranded oligonucleotide modified Au nanoparticle, and the resulting Au-tagged SSB was used as the hybridization label. Changes in the Au oxidation signal was monitored upon binding of Au tagged SSB to probe and hybrid on the electrode surface. The amplified oxidation signal of Au nanoparticles provided a detection limit of 2.17 pM target DNA, which can be applied to genetic diagnosis applications. This work presented here has important implications with regard to combining a biological binding event between a protein and DNA with a solid transducer and metal nanoparticles.