Special Issue "Live Cell-Based Sensors"
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A special issue of Sensors (ISSN 1424-8220). This special issue belongs to the section "Biosensors".
Deadline for manuscript submissions: closed (31 October 2012)
Special Issue Editor
Guest Editor
Prof. Dr. Akiyoshi Taniguchi
1 Advanced Medical Materials Group, Biomaterials Center, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba-shi Ibaraki, 305-004 Japan
2 Wased University-NIMS Joint Graduate Program, 1-1 Namiki, Tsukuba, Ibaraki, Japan
E-Mail: taniguchi.akiyoshi@nims.go.jp
Phone: +81-29-860-4505
Fax: +81-29-860-4714
Interests: cell- based biosensor; nanotoxicology; in vitro co-culture; microfluidics
Special Issue Information
Dear Colleagues,
Living cells maintain life functions by responding quickly and with great sensitivity to changes in the external environment. Consequently, sensors using live cells are thought to be able to perform analyses faster and with more sensitivity than previously possible. Cell-based sensors can be roughly divided into two types. The first uses microorganisms such as Escherichia coli or yeast as sensing elements (Microbial cells). The second type uses human and animal cells (Mammalian cells). The first type can be cultivated rather easily and has the advantages of being inexpensive and portable. The second type is more complex but has the advantage of potentially being used with human subjects. Most research in this area is concentrated on the first type, microbial sensors, but research on sensors that use mammalian cells has recently become more widespread. Live cell-based sensors may potentially be used as an evaluation technology in medical and pharmaceutical fields, as well as for cytotoxicity inspection of medical supplies, nanomaterials, biomaterials, environmental factors and other materials. The special issue of the journal Sensors will cover these different types of live cell-based sensors and applications for these different fields.
Prof. Dr. Akiyoshi Taniguchi
Guest Editor
Submission
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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Sensors is an international peer-reviewed Open Access monthly journal published by MDPI.
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Keywords
- microbial cells
- mammalian cells
- biotechnology
- biosensor
- cell culture
- cytotoxicity
Published Papers (19 papers)
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Received: 10 June 2011; in revised form: 8 July 2011 / Accepted: 12 July 2011 / Published: 18 July 2011
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Abstract: The increasing use of nanomaterials in consumer and industrial products has aroused concerns regarding their fate in biological systems. An effective detection method to evaluate the safety of bio-nanomaterials is therefore very important. Titanium dioxide (TiO2), which is manufactured worldwide in large quantities for use in a wide range of applications, including pigment and cosmetic manufacturing, was once thought to be an inert material, but recently, more and more studies have indicated that TiO2 nanoparticles (TiO2 NPs) can cause inflammation and be harmful to humans by causing lung and brain problems. In order to evaluate the safety of TiO2 NPs for the environment and for humans, sensor cells for inflammation detection were developed, and these were transfected with the Toll-like receptor 4 (TLR4) gene and Nuclear Factor Kappa B (NF-κB) reporter gene. NF-κB as a primary cause of inflammation has received a lot of attention, and it can be activated by a wide variety of external stimuli. Our data show that TiO2 NPs-induced inflammation can be detected by our sensor cells through NF-κB pathway activation. This may lead to our sensor cells being used for bio-nanomaterial safety evaluation.
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Received: 2 September 2011; in revised form: 22 September 2011 / Accepted: 29 September 2011 / Published: 12 October 2011
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Abstract: This study demonstrates a novel cell manipulation microdevice for cell docking, culturing, cell-cell contact and interaction by microfluidic manipulation of heterogeneous cell suspensions. Heterogeneous cell suspensions include disparate blood cells of natural killer cells and leukemia cancer cells for immune cell transplantation therapy. However, NK cell alloreactivity from different healthy donors present various recovery response levels. Little is still known about the interactions and cytotoxicity effects between donor NK cells and recipient cancer cells. The cell-based micro device first showed the capability of cell docking, movement, contact and cell-cell interaction with respect to cell cytotoxicity of NK cells against cancer cells. With various flow tests for live cell loading, flow rates of 10 μL/h were chosen for injection in the central and side flows such that both types of suspension cells could be gently docked at the gap structure in a reaction zone. The trapping number of particles and cells was linearly proportional to the gap length. Finally, the cytotoxicity of around 40% was found to be similar in the case of dilute cells and a large cell population. As a result, the cell manipulation microdevice has been validated for live suspensions of natural killer and cancer cells, and exhibited the capability to measure the cytotoxicity of dilute cell suspensions.
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Received: 29 November 2011; in revised form: 26 December 2011 / Accepted: 29 December 2011 / Published: 30 December 2011
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Abstract: Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-mm-diameter microwells (91.45%) was higher than that in 20-mm-diameter microwells (83.19%) at an injection flow rate of 2.8 mL/min. However, most of the occupied 20-mm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis.
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Received: 26 November 2011; in revised form: 30 December 2011 / Accepted: 9 January 2012 / Published: 11 January 2012
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Abstract: The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.
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Received: 21 December 2011; in revised form: 13 January 2012 / Accepted: 13 January 2012 / Published: 18 January 2012
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Abstract: Cell-based biosensing is a “smart” way to obtain efficacy-information on the effect of applied chemical on cellular biological cascade. We have proposed an engineered post-synapse model cell-based biosensors to investigate the effects of chemicals on ionotropic glutamate receptor (GluR), which is a focus of attention as a molecular target for clinical neural drug discovery. The engineered model cell has several advantages over native cells, including improved ease of handling and better reproducibility in the application of cell-based biosensors. However, in general, cell-based biosensors often have low signal-to-noise (S/N) ratios due to the low level of cellular responses. In order to obtain a higher S/N ratio in model cells, we have attempted to design a tactic model cell with elevated cellular response. We have revealed that the increase GluR expression level is not directly connected to the amplification of cellular responses because the saturation of surface expression of GluR, leading to a limit on the total ion influx. Furthermore, coexpression of GluR with a voltage-gated potassium channel increased Ca2+ ion influx beyond levels obtained with saturating amounts of GluR alone. The construction of model cells based on strategy of amplifying ion flux per individual receptors can be used to perform smart cell-based biosensing with an improved S/N ratio.
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Received: 1 December 2011; in revised form: 13 January 2012 / Accepted: 17 January 2012 / Published: 1 February 2012
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Abstract: Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events—in this case in choroid plexus functions, however with applicability to other cell types as well.
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Received: 29 December 2011; in revised form: 24 January 2012 / Accepted: 3 February 2012 / Published: 6 February 2012
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Abstract: Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.
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Received: 1 February 2012; in revised form: 28 February 2012 / Accepted: 5 March 2012 / Published: 8 March 2012
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Abstract: Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni2+ and Cu2+) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity.
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Received: 1 May 2012; in revised form: 24 May 2012 / Accepted: 24 May 2012 / Published: 30 May 2012
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Abstract: A series of studies aimed at developing methods and systems of analyzing epigenetic information in cells and in cell networks, as well as that of genetic information, was examined to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional DNA information-processing events. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes, population effects and community effects. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an “algebraic” system (emphasis on temporal aspects) and as a “geometric” system (emphasis on spatial aspects). Exploiting the combination of latest microfabrication technology and measurement technologies, which we call on-chip cellomics assay, we can control and re-construct the environments and interaction of cells from “algebraic” and “geometric” viewpoints. In this review, temporal viewpoint of epigenetic information, a part of the series of single-cell-based “algebraic” and “geometric” studies of celluler systems in our research groups, are summerized and reported. The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs.
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Received: 12 June 2012; in revised form: 29 June 2012 / Accepted: 4 July 2012 / Published: 9 July 2012
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Abstract: K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments) to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC) and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.
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Received: 8 June 2012; in revised form: 4 July 2012 / Accepted: 18 July 2012 / Published: 26 July 2012
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Abstract: Bioactive microcapsules containing Bacillus thuringiensis (BT) spores were generated by a combination of a hydro gel, microfluidic device and chemical polymerization method. As a proof-of-principle, we used BT spores displaying enhanced green fluorescent protein (EGFP) on the spore surface to spatially direct the EGFP-presenting spores within microcapsules. BT spore-encapsulated microdroplets of uniform size and shape are prepared through a flow-focusing method in a microfluidic device and converted into microcapsules through hydrogel polymerization. The size of microdroplets can be controlled by changing both the dispersion and continuous flow rate. Poly(N-isoproplyacrylamide) (PNIPAM), known as a hydrogel material, was employed as a biocompatible material for the encapsulation of BT spores and long-term storage and outstanding stability. Due to these unique properties of PNIPAM, the nutrients from Luria-Bertani complex medium diffused into the microcapsules and the microencapsulated spores germinated into vegetative cells under adequate environmental conditions. These results suggest that there is no limitation of transferring low-molecular-weight-substrates through the PNIPAM structures, and the viability of microencapsulated spores was confirmed by the culture of vegetative cells after the germinations. This microfluidic-based microencapsulation methodology provides a unique way of synthesizing bioactive microcapsules in a one-step process. This microfluidic-based strategy would be potentially suitable to produce microcapsules of various microbial spores for on-site biosensor analysis.
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Received: 17 July 2012; in revised form: 5 September 2012 / Accepted: 5 September 2012 / Published: 26 September 2012
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Abstract: In this study the mitochondrion is regarded as a target to reveal compounds that may be used to combat various diseases. Consequently, the sexual structures of yeasts (with high mitochondrial activity) were identified as sensors to screen for various anti-mitochondrial drugs that may be toxic to humans and that are directed, amongst others, against fungal diseases and cancer. Strikingly, these sensors indicated that chloroquine is a potent pro-mitochondrial drug which stimulated yeast sexual reproduction. In addition, these sensors also showed that some Non-Steroidal Anti-Inflammatory drugs (NSAIDs), anti-malarial drugs, antifungal and anticancer drugs are anti-mitochondrial. These yeast sensor bio-assays may fast track studies aimed at discovering new drugs as well as their mechanisms and should now be further evaluated for selectivity towards anti-/ pro-mitochondrials, fertility drugs and contraceptives, using in vitro, in vivo, in silico and omics research.
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Received: 27 September 2012; in revised form: 8 November 2012 / Accepted: 8 November 2012 / Published: 12 November 2012
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Abstract: Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10–9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.
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Received: 8 October 2012; in revised form: 12 November 2012 / Accepted: 12 November 2012 / Published: 22 November 2012
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Abstract: Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. We have developed a novel method termed enzyme-mediated activation of radical sources (EMARS) to identify coclustering molecules on the cell surface under living conditions, which features a radical formation from an aryl azide reagent by horseradish peroxidase (HRP). For identification of molecules labeled by the EMARS reaction, antibody array system and mass spectrometry-based proteomics approaches are available. Spatio- temporally-regulated interaction between b1 integrin and ErbB4 involved in fibronectin-dependent cell migration and therapeutic antibody-stimulated interaction between FGFR3 and CD20 were discovered using the EMARS method.
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Received: 17 September 2012; in revised form: 8 November 2012 / Accepted: 9 November 2012 / Published: 3 December 2012
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Abstract: Recent progress in patterned microelectrode manufacturing technology and microfluidics has opened the way to a large variety of cellular and molecular biosensor-based applications. In this extremely diverse and rapidly expanding landscape, silicon-based technologies occupy a special position, given their statute of mature, consolidated, and highly accessible areas of development. Within the present work we report microfabrication procedures and workflows for 3D patterned gold-plated microelectrode arrays (MEA) of different shapes (pyramidal, conical and high aspect ratio), and we provide a detailed characterization of their physical features during all the fabrication steps to have in the end a reliable technology. Moreover, the electrical performances of MEA silicon chips mounted on standardized connector boards via ultrasound wire-bonding have been tested using non-destructive electrochemical methods: linear sweep and cyclic voltammetry, impedance spectroscopy. Further, an experimental recording chamber package suitable for in vitro electrophysiology experiments has been realized using custom-design electronics for electrical stimulus delivery and local field potential recording, included in a complete electrophysiology setup, and the experimental structures have been tested on newborn rat hippocampal slices, yielding similar performance compared to commercially available MEA equipments.
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Received: 29 October 2012; in revised form: 30 November 2012 / Accepted: 4 December 2012 / Published: 6 December 2012
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Abstract: A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 µM, obtained with the strain C. reinhardtii after 40 minutes of exposure.
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Received: 1 November 2012; in revised form: 31 December 2012 / Accepted: 11 January 2013 / Published: 24 January 2013
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Abstract: This paper reviews the state of the art of artificial tactile sensing, with a particular focus on bio-hybrid and fully-biological approaches. To this aim, the study of physiology of the human sense of touch and of the coding mechanisms of tactile information is a significant starting point, which is briefly explored in this review. Then, the progress towards the development of an artificial sense of touch are investigated. Artificial tactile sensing is analysed with respect to the possible approaches to fabricate the outer interface layer: synthetic skin versus bio-artificial skin. With particular respect to the synthetic skin approach, a brief overview is provided on various technologies and transduction principles that can be integrated beneath the skin layer. Then, the main focus moves to approaches characterized by the use of bio-artificial skin as an outer layer of the artificial sensory system. Within this design solution for the skin, bio-hybrid and fully-biological tactile sensing systems are thoroughly presented: while significant results have been reported for the development of tissue engineered skins, the development of mechanotransduction units and their integration is a recent trend that is still lagging behind, therefore requiring research efforts and investments. In the last part of the paper, application domains and perspectives of the reviewed tactile sensing technologies are discussed.
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Received: 11 March 2013; in revised form: 18 April 2013 / Accepted: 3 May 2013 / Published: 6 May 2013
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Abstract: Whole-cell biosensors are a good alternative to enzyme-based biosensors since they offer the benefits of low cost and improved stability. In recent years, live cells have been employed as biosensors for a wide range of targets. In this review, we will focus on the use of microorganisms that are genetically modified with the desirable outputs in order to improve the biosensor performance. Different methodologies based on genetic/protein engineering and synthetic biology to construct microorganisms with the required signal outputs, sensitivity, and selectivity will be discussed.
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Received: 6 March 2013; in revised form: 15 April 2013 / Accepted: 18 April 2013 / Published: 14 May 2013
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Abstract: Biosensors are devices that are capable of detecting specific biological analytes and converting their presence or concentration into some electrical, thermal, optical or other signal that can be easily analysed. The first biosensor was designed by Clark and Lyons in 1962 as a means of measuring glucose. Since then, much progress has been made and the applications of biosensors are today potentially boundless. This review is limited to their clinical applications, particularly in the field of oncohematology. Biosensors have recently been developed in order to improve the diagnosis and treatment of patients affected by hematological malignancies, such as the biosensor for assessing the in vitro pre-treatment efficacy of cytarabine in acute myeloid leukemia, and the fluorescence resonance energy transfer-based biosensor for assessing the efficacy of imatinib in chronic myeloid leukemia. The review also considers the challenges and future perspectives of biosensors in clinical practice.
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Last update: 24 December 2012
