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1162 KiB

Open AccessArticle

by Søren Fjelstrup, Marie Bech Andersen, Jonas Thomsen, Jing Wang, Magnus Stougaard, Finn Skou Pedersen, Yi-Ping Ho, Marianne Smedegaard Hede and Birgitta Ruth Knudsen

Sensors 2017, 17(6), 1201; https://doi.org/10.3390/s17061201 - 24 May 2017

Cited by 15 | Viewed by 7286

Abstract

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to [...] Read more.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution. Full article

(This article belongs to the Special Issue Genosensing)

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1476 KiB

Open AccessArticle

Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

by Cheng Zhang, Yiping Chen, Xinmiao Liang, Guanhua Zhang, Hong Ma, Leng Nie and Yu Wang

Sensors 2017, 17(5), 961; https://doi.org/10.3390/s17050961 - 26 Apr 2017

Cited by 18 | Viewed by 7051

Abstract

Quantum dots (QDs) are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV) [...] Read more.

Quantum dots (QDs) are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV) gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB) patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 10³ IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation. Full article

(This article belongs to the Special Issue Genosensing)

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2150 KiB

Open AccessArticle

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops

by Suely Moura-Melo, Rebeca Miranda-Castro, Noemí De-los-Santos-Álvarez, Arturo J. Miranda-Ordieres, José Ribeiro Dos Santos Junior, Rosana A. Da Silva Fonseca and María Jesús Lobo-Castañón

Sensors 2017, 17(4), 881; https://doi.org/10.3390/s17040881 - 18 Apr 2017

Cited by 9 | Viewed by 5971

Abstract

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic [...] Read more.

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines. Full article

(This article belongs to the Special Issue Genosensing)

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2003 KiB

Open AccessArticle

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

by Changbei Ma, Haisheng Liu, Kefeng Wu, Mingjian Chen, Liyang Zheng and Jun Wang

Sensors 2017, 17(4), 760; https://doi.org/10.3390/s17040760 - 03 Apr 2017

Cited by 17 | Viewed by 4911

Abstract

MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical [...] Read more.

MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical method for cost-effective and sensitive detection of miRNA using a super 2-aminopurine (2-AP)-labeled hairpin probe (HP) and exonuclease I activity. Specifically, the fluorescence of 2-AP is strongly quenched when it is incorporated within DNA. In the presence of a target miRNA, HP attains an open conformation by hybridizing with the target miRNA to form a double-stranded structure with a protruding 3′-terminus. Next, the digestion of the protruding 3′-terminus is triggered by exonuclease I, during which 2-AP is released free in solution from the DNA, thereby increasing fluorescence. This method is highly sensitive, with a detection limit of 0.5 nM—10 times lower than a previously reported quencher-free fluorescence method. Furthermore, this method has potential applications in clinical diagnosis and biomedical research. Full article

(This article belongs to the Special Issue Genosensing)

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Review

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1454 KiB

Open AccessReview

Genotyping and Bio-Sensing Chemosensory Proteins in Insects

by Guoxia Liu, Philippe Arnaud, Bernard Offmann and Jean-François Picimbon

Sensors 2017, 17(8), 1801; https://doi.org/10.3390/s17081801 - 04 Aug 2017

Cited by 11 | Viewed by 5621

Abstract

Genotyping is the process of determining differences in the genetic make-up of an individual and comparing it to that of another individual. Focus on the family of chemosensory proteins (CSPs) in insects reveals differences at the genomic level across various strains and biotypes, [...] Read more.

Genotyping is the process of determining differences in the genetic make-up of an individual and comparing it to that of another individual. Focus on the family of chemosensory proteins (CSPs) in insects reveals differences at the genomic level across various strains and biotypes, but none at the level of individuals, which could be extremely useful in the biotyping of insect pest species necessary for the agricultural, medical and veterinary industries. Proposed methods of genotyping CSPs include not only restriction enzymatic cleavage and amplification of cleaved polymorphic sequences, but also detection of retroposons in some specific regions of the insect chromosome. Design of biosensors using CSPs addresses tissue-specific RNA mutations in a particular subtype of the protein, which could be used as a marker of specific physiological conditions. Additionally, we refer to the binding properties of CSP proteins tuned to lipids and xenobiotic insecticides for the development of a new generation of biosensor chips, monitoring lipid blood concentration and chemical environmental pollution. Full article

(This article belongs to the Special Issue Genosensing)

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2989 KiB

Open AccessReview

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

by Linda K. Medlin and Jahir Orozco

Sensors 2017, 17(5), 1184; https://doi.org/10.3390/s17051184 - 22 May 2017

Cited by 69 | Viewed by 9947

Abstract

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic [...] Read more.

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks. Full article

(This article belongs to the Special Issue Genosensing)

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4786 KiB

Open AccessReview

Screening Genotoxicity Chemistry with Microfluidic Electrochemiluminescent Arrays

by Itti Bist, Kiran Bano and James F. Rusling

Sensors 2017, 17(5), 1008; https://doi.org/10.3390/s17051008 - 03 May 2017

Cited by 7 | Viewed by 4644

Abstract

This review describes progress in the development of electrochemiluminescent (ECL) arrays aimed at sensing DNA damage to identify genotoxic chemistry related to reactive metabolites. Genotoxicity refers to chemical or photochemical processes that damage DNA with toxic consequences. Our arrays feature DNA/enzyme films that [...] Read more.

This review describes progress in the development of electrochemiluminescent (ECL) arrays aimed at sensing DNA damage to identify genotoxic chemistry related to reactive metabolites. Genotoxicity refers to chemical or photochemical processes that damage DNA with toxic consequences. Our arrays feature DNA/enzyme films that form reactive metabolites of test chemicals that can subsequently react with DNA, thus enabling prediction of genotoxic chemical reactions. These high-throughput ECL arrays incorporating representative cohorts of human metabolic enzymes provide a platform for determining chemical toxicity profiles of new drug and environmental chemical candidates. The arrays can be designed to identify enzymes and enzyme cascades that produce the reactive metabolites. We also describe ECL arrays that detect oxidative DNA damage caused by metabolite-mediated reactive oxygen species. These approaches provide valuable high-throughput tools to complement modern toxicity bioassays and provide a more complete toxicity prediction for drug and chemical product development. Full article

(This article belongs to the Special Issue Genosensing)

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2427 KiB

Open AccessReview

Electrochemical Genosensing of Circulating Biomarkers

by Susana Campuzano, Paloma Yáñez-Sedeño and José Manuel Pingarrón

Sensors 2017, 17(4), 866; https://doi.org/10.3390/s17040866 - 14 Apr 2017

Cited by 37 | Viewed by 7281

Abstract

Management and prognosis of diseases requires the measurement in non- or minimally invasively collected samples of specific circulating biomarkers, consisting of any measurable or observable factors in patients that indicate normal or disease-related biological processes or responses to therapy. Therefore, on-site, fast and [...] Read more.

Management and prognosis of diseases requires the measurement in non- or minimally invasively collected samples of specific circulating biomarkers, consisting of any measurable or observable factors in patients that indicate normal or disease-related biological processes or responses to therapy. Therefore, on-site, fast and accurate determination of these low abundance circulating biomarkers in scarcely treated body fluids is of great interest for health monitoring and biological applications. In this field, electrochemical DNA sensors (or genosensors) have demonstrated to be interesting alternatives to more complex conventional strategies. Currently, electrochemical genosensors are considered very promising analytical tools for this purpose due to their fast response, low cost, high sensitivity, compatibility with microfabrication technology and simple operation mode which makes them compatible with point-of-care (POC) testing. In this review, the relevance and current challenges of the determination of circulating biomarkers related to relevant diseases (cancer, bacterial and viral infections and neurodegenerative diseases) are briefly discussed. An overview of the electrochemical nucleic acid–based strategies developed in the last five years for this purpose is given to show to both familiar and non-expert readers the great potential of these methodologies for circulating biomarker determination. After highlighting the main features of the reported electrochemical genosensing strategies through the critical discussion of selected examples, a conclusions section points out the still existing challenges and future directions in this field. Full article

(This article belongs to the Special Issue Genosensing)

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9589 KiB

Open AccessReview

DNA Sequencing Sensors: An Overview

by Jose Antonio Garrido-Cardenas, Federico Garcia-Maroto, Jose Antonio Alvarez-Bermejo and Francisco Manzano-Agugliaro

Sensors 2017, 17(3), 588; https://doi.org/10.3390/s17030588 - 14 Mar 2017

Cited by 50 | Viewed by 18197

Abstract

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA [...] Read more.

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS) meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years. Full article

(This article belongs to the Special Issue Genosensing)

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