logo ban ban logo

Účelem vzdělání není zaplnit mysl, ale otevřít ji. Čím více poznatků si osvojíme, tím víc si uvědomíme, co ještě neznáme.

mendel

Výzkum

Electrochemical Sensors and Biosensors for Influenza Detection – Literature Survey 2012-2013

Because of new development in the assays for “pathogen determination and quantification”, this review follows our previously published one focused on sensors and biosensors in the field of electrochemical detection of influenza virus [1]. Very fast development of this global research area leading to the application of new methods and materials in pathogen determination is obvious [2-4]. Therefore, the update of electrochemical detection approaches for influenza virus in the years 2012 and 2013 is summarized in this text.
Family of influenza viruses contains three genera: Influenza A, Influenza B and Influenza C. These three genera differ from each other in inside, species-specific nucleo-protein antigen, the number of gene segments, host specificity and clinical manifestations protein [5]. Because of the fact that the individual subtypes of influenza virus differ from each other by the variations in membrane virus constitution (membrane protein, ion channels, matrix proteins), various ways of detection based on individual parts of virus are investigated. Utilization of individual differences could help in detection of individual subtypes of influenza virus.
2. SENSORS
Our last review, which is updated by this text, was divided on two basic parts according to the way of virus determination. These two basic ways were called as sensors and biosensors. The difference in both approaches lies in the presence of bio-recognition element in the sensor construction. Developmentally, the usage of electrochemical sensors is an older way. Reasons for the course change from sensors to biosensors in the area of virus determination have been summarized [6,7]. Nevertheless, development in this area is still going on.

While the development in the application of mercury and amalgam electrodes is negligible, the usage of carbon and gold electrodes is widespread. Gold electrode has well defined surface and thus, these are very suitable for the electrode surface modification. This is probably the main reason why the latest investigation is focused to this electrode material. Particularly, modification of gold electrode surface through the chemical reagents [8,9] or magnetoimmunosensing entity [10,11] was published. Chemical modification is in this case presented by two approaches, where the first one is based on the functional architecture introducing receptor molecules as a sensing entity that mimics those found in the membrane of target cells of influenza A virus [8]. The artificial receptors are built by sequential assembly of 1-octanethiol/octyl-galactoside hybrid bilayer, followed by an enzyme-mediated functionalization of the terminal galactoside groups with sialic acid molecules. The detection mechanism relies hence on the specific affinity between the sialic acid-galactose receptor moieties anchored on the modified electrode surface and the hemagglutinin (HA) viral surface protein. In contrast to immunosensors based on antibodies as bioreceptor, the sialylated modified gold electrode is also able to distinguish among influenza phenotypes. The second way of chemical modification is represented by the diazonium salt as modifier of electrode surface [9]. More precisely, 4-carboxy phenyl groups located on the electrode surface were prepared for the indium tin oxide, gold and glassy carbon electrode. Modified glassy carbon electrode was tested for the functionality as influenza ODN hybridisation biosensor [9]. In addition, modification of gold electrode surface using magnetoimmunosensing entities was based on the connection of gold electrode with magnet and next usage of magnetic particles as catchers and carriers of influenza virus [10,11]. The electrochemical response of suggested system was obtained from realized bienzymatic strategy. The first enzyme functional as tracer was tagged on immunomagnetic beads, which could be accumulated on the magneto controlled gold electrode and the second enzyme was immobilized on the electrode by layer-by-layer technique. This construction allowed obtaining the catalytically reduced electrochemical signal of H2O2 after the immunoreaction.

While the development in the application of mercury and amalgam electrodes is negligible, the usage of carbon and gold electrodes is widespread. Gold electrode has well defined surface and thus, these are very suitable for the electrode surface modification. This is probably the main reason why the latest investigation is focused to this electrode material. Particularly, modification of gold electrode surface through the chemical reagents [8,9] or magnetoimmunosensing entity [10,11] was published. Chemical modification is in this case presented by two approaches, where the first one is based on the functional architecture introducing receptor molecules as a sensing entity that mimics those found in the membrane of target cells of influenza A virus [8]. The artificial receptors are built by sequential assembly of 1-octanethiol/octyl-galactoside hybrid bilayer, followed by an enzyme-mediated functionalization of the terminal galactoside groups with sialic acid molecules. The detection mechanism relies hence on the specific affinity between the sialic acid-galactose receptor moieties anchored on the modified electrode surface and the hemagglutinin (HA) viral surface protein. In contrast to immunosensors based on antibodies as bioreceptor, the sialylated modified gold electrode is also able to distinguish among influenza phenotypes. The second way of chemical modification is represented by the diazonium salt as modifier of electrode surface [9]. More precisely, 4-carboxy phenyl groups located on the electrode surface were prepared for the indium tin oxide, gold and glassy carbon electrode. Modified glassy carbon electrode was tested for the functionality as influenza ODN hybridisation biosensor [9]. In addition, modification of gold electrode surface using magnetoimmunosensing entities was based on the connection of gold electrode with magnet and next usage of magnetic particles as catchers and carriers of influenza virus [10,11]. The electrochemical response of suggested system was obtained from realized bienzymatic strategy. The first enzyme functional as tracer was tagged on immunomagnetic beads, which could be accumulated on the magneto controlled gold electrode and the second enzyme was immobilized on the electrode by layer-by-layer technique. This construction allowed obtaining the catalytically reduced electrochemical signal of H2O2 after the immunoreaction. immobilized on the surface of a gold electrode microsystem via standard thiol chemistry. Such architecture serves as sensor for DNA detection which is based on hybridization.

Figure 1. Schematic of the DNA detection system based on the stem-loop structured DNA probe. Probe was formed by the introduction of four ferrocene moieties at the 5' end of a stem-loop oligonucleotide and a C6-thiol modifier group at the 3' end. This 4Fc-DNA was

3. BIOSENSORS
3.1. Hybridisation on electrodes ODNs belong to the first choice targets for formation of biosensing platform for pathogens. The simplest determined step is obviously hybridisation of target sequence. This procedure could be easily recorded by electrochemical methods. Well written overview of electrochemical real-time nucleic acid amplification was published by Patterson et al. [12]. This review is aimed on general pathogens quantification including influenza virus and divides presented information into four parts according the way of nucleic acid amplification as follows: solid polymerase chain reaction (PCR), solution-phase PCR using electrochemical reporters of product formation, solution-phase qPCR using sequence specific reporters, and isothermal amplification [12].
The newest ways of influenza determination based on hybridization reaction are connected with the application of electrochemical labels [13-19]. Usage of ferrocene as a modifier of specific ODN sequence was reported by Chatelain et al. [13]. They used four-ferrocene modified oligonucleotide at the 5'-end and a C6-thiol modifier group at the 3'-end as a probe for DNA detection with a gold electrode microsystem (Fig. 1). The probe sequence had a stem-loop structure that fold efficiently on the electrode, and thus optimized electron transfer. Such architecture served as sensor for DNA detection based on hybridization.
Grabowska et al. constructed sensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode (via Au-S bond formation) [14]. This sensor was used for simultaneous determination of two different oligonucleotide targets. One of the probes, bound on its S'-end with ferrocene, was related to sequence encoding part of hemagglutinin from H5N1 virus. The second probe, bound on its S'-end with methylene blue, was related to the fragment of neuraminidase from the same virus. Such sensor is able to detect main markers of the influenza virus, hemagglutinin and neuraminidase.
Our group published magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene [17]. Paramagnetic particles covered with dT25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labelled with quantum dots (CdS, ZnS and/or PbS) were used. The obtained signals identified mutations present in the neuraminidase gene sequence.
3.2. Quartz crystal microbalance Quartz crystal microbalance (QCM) contributed to the influenza research in last two years by two various ways. The first way is represented by the detection of influenza virus [20,21] and the second by the study of influenza virus binding capabilities [22,23]. Application of QCM for influenza detection was done through two various parts of influenza virus. In the first case, the aptamer was used for the formation of switch on/off system based on the crosslinked polymeric hydrogel [21]. A selected aptamer with high affinity and specificity against H5N1 surface protein was used, and hybridization between the aptamer and ssDNA formed the crosslinker in the polymer hydrogel. The aptamer hydrogel was immobilized on the gold surface of QCM sensor using a self-assembled monolayer method. The hydrogel remained in the state of shrink if no H5N1 virus was present in the sample because of the crosslinking between the aptamer and ssDNA in the polymer network. When it exposed to the target virus, the binding reaction between the aptamer and H5N1 virus caused the dissolution of the linkage between the aptamer and ssDNA resulting in the abrupt swelling of the hydrogel. The second part of virus used for detection was hemagglutinin, its binding capabilities, respectively [20]. Diltemiz et al. used 4-aminophenyl boronic acid as a new ligand for binding of sialic acid (having an important role in binding of HA) via boronic acid sugar interaction. QCM sensor surface was modified with thiol groups and then 4-aminophenyl boronic acid and sialic acid were immobilized on sensor surfaces, respectively. Further studies of influenza binding capabilities were focused on hemagglutinin and its interaction. The first example of such study was the work of Takahashi et al. [22]. They studied the association of a sulphated galactosyl ceramide (sulphatide) with the viral envelope glycoprotein hemagglutinin. To determine whether the ectodomain of HA could bind to sulphatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was applied. sHA showed subtype-specific antigenicity and binding ability to both sulphatide and gangliosides. Kinetics of sHA binding to sulphatide was demonstrated by QCM analysis. The second example was the work of Wangchareansak et al. focused on behaviour of N-acetylglucosamine [23]. N-acetylglucosamine is a part of the oligosacharide ligand responsible for the first binding step of virus (ligand-virus interactions) to a host cell. For immobilization on the gold surface, N-acetylglucosamine was linked to p-nitrophenol, and the nitro group was reduced and then bound to cysteine via two-step synthesis.

Práce je spojená s projektem NanoBioMetalNet CZ.1.07/2.4.00/31.0023

Zemědělská 1/1665
613 00 Brno
Budova D		Tel.: +420 545 133 350
Fax.: +420 545 212 044