The authors have nothing to disclose.

1. processável PANI Síntese <ol><li> Dissolve-se anilina (1 ml, 11 mmol) completamente em 60 ml de clorofórmio num balão de 250 ml de fundo redondo. Agita-se a 600 rpm durante 5 min e arrefecer a 0-5 ° C com gelo. Isso normalmente leva 15-20 min (Figura 2A). </li><li> Adicionar sulfonato de sódio dodecil benzeno (NaDBS) (7,44 g, 21 mmol) para a solução de anilina em um balão de fundo redondo, enquanto se agitava a 600 rpm. </li><li> Dissolver persulfato de amónio (APS) (3,072 g, 13,5 mmol) em 20 ml de água e adicionar tudo isso gota a gota ao longo de 30 min para evitar o superaquecimento da reação. </li><li> Efectua-se a reacção a 0-5 ° C durante 24 h, e permitir-lhe atingir a temperatura ambiente durante mais 24 h. </li><li> Observe-se a mistura de reacção inicialmente transformar branca leitosa após 15 min, em seguida, castanho escuro após 2 horas, e, finalmente, para verde escuro após 24 h (Figura 2B-F). </li><li> Filtra-se a solução PANI-NaDBS com um funil de Buchner. Misturar com 80 ml de clorofórmio e 120 ml de água, em queeparation funil (Figura 2G). </li><li> Incubar a solução durante 24 horas à temperatura ambiente e recolher o PANI verde escuro a partir do funil de separação, deixando NaDBS que não reagiram e APS no sobrenadante aquoso. </li></ol> 2. A mistura PANI-sonda e irradiação UV <ol><li> Diluir solução PANI 10x com clorofórmio-água (1: 3 v / v) e misturar 200 ul de PANI diluída com 6,4 pmol de oligonucleótidos de DNA sonda por agitação suave durante 15 min num tubo de microcentrífuga. </li><li> Irradiar a solução de ADN com 1.200 PANI μJ / cm 2 de UV em um reticulador durante 2 min. É crítico que a exposição UV é limitado ao valor indicado. A exposição prolongada à radiação UV compromete a mudança de fluorescência no PANI, provavelmente devido à ligação covalente cruzada de PANI e ADN. </li><li> complexos por centrifugação a 17000 xg durante 6 minutos, e lava-se com solução salina tamponada com fosfato (PBS). Sedimentar novamente, e re-suspensão em PBS. </li></ol> 3. Hybridization de PANI-sonda <ol><li> Adicionar 8 uL de 100 uM oligonucleótidos de ADN complementares ou ácidos nucleicos alvo com 200 ul de complexos PANI-sonda. </li><li> Realizar hibridação por mistura de balanço solução durante 15 min a 40 ° C. </li><li> Agregar as complexos PANI por centrifugação a 17000 xg durante 6 min. Lavar com PBS e re-suspensão em água. </li></ol> 4. Emissão constante Medição Estado de fluorescência <ol><li> Adicionar PANI de diferentes tratamentos para uma microplaca de 96 poços e medir a emissão de fluorescência na gama de 270-850 nm por excitação a 250 nm. Um pico de emissão para PANI devem ser observadas cerca de 500 nm. </li></ol> 5. Medição da Microscopia de Fluorescência duplex hibridado <ol><li> Gota revestimento PANI sobre uma lamela de vidro de borosilicato e seco a 40 ° C durante 48 h. </li><li> Adicionar sonda (8 uL de 100 uM) num filme de PANI e secou-se irradiar com luz UV (1200 μJ / cm2 </suP&gt;) durante 2 min. </li><li> Lava-se a película PANI-sonda com PBS e seco a 40 ° C durante 48 h. </li><li> Realizar a hibridação durante 15 min pela adição de ácidos nucleicos alvo. Esta poderia ser uma amostra biológica, ou um oligonucleótido alvo de controlo (8 uL de 100 uM). Siga com uma lavagem PBS. </li><li> Obtenção das imagens fluorescentes em aumento de 40 vezes, com um filtro de passagem longa de 500 nm. </li></ol>

<ol>
	<li>Hahm, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+polymers+in+protein+detection+platforms:+optical,+electrochemical,+electrical,+mass-sensitive,+and+magnetic+biosensors.">Functional polymers in protein detection platforms: optical, electrochemical, electrical, mass-sensitive, and magnetic biosensors.</a> Sensors (Basel). 11 (3), 3327-3355 (2011).</li><li>Rahman, M. M., Li, X. B., Lopa, N. S., Ahn, S. J., Lee, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+DNA+hybridization+sensors+based+on+conducting+polymers.">Electrochemical DNA hybridization sensors based on conducting polymers.</a> Sensors (Basel). 15 (2), 3801-3829 (2015).</li><li>Sengupta, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+Intrinsic+Properties+of+Polyaniline+to+Detect+Nucleic+Acid+Hybridization+through+UV-Enhanced+Electrostatic+Interaction.">Utilizing Intrinsic Properties of Polyaniline to Detect Nucleic Acid Hybridization through UV-Enhanced Electrostatic Interaction.</a> Biomacromolecules. 16 (10), 3217-3225 (2015).</li><li>Song, E., Choi, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polyaniline+Nanowire+and+Its+Applications+in+Chemiresistive+Sensing.">Conducting Polyaniline Nanowire and Its Applications in Chemiresistive Sensing.</a> Nanomaterials. 3 (3), 498 (2013).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyaniline+nanofibres+for+fluorescent+nucleic+acid+detection.">Polyaniline nanofibres for fluorescent nucleic acid detection.</a> Nanoscale. 3 (3), 967-969 (2011).</li><li>Oliveira Brett, A. M., Chiorcea, A. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+of+DNA+Immobilized+onto+a+Highly+Oriented+Pyrolytic+Graphite+Electrode+Surface.">Atomic Force Microscopy of DNA Immobilized onto a Highly Oriented Pyrolytic Graphite Electrode Surface.</a> Langmuir. 19 (9), 3830-3839 (2003).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(m-Phenylenediamine)+Nanospheres+and+Nanorods:+Selective+Synthesis+and+Their+Application+for+Multiplex+Nucleic+Acid+Detection.">Poly(m-Phenylenediamine) Nanospheres and Nanorods: Selective Synthesis and Their Application for Multiplex Nucleic Acid Detection.</a> PLoS ONE. 6 (6), e20569 (2011).</li><li>Namgoong, H., Woo, D. J., Lee, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-chemical+structure+of+polyaniline+synthesized+by+self-stabilized+dispersion+polymerization.">Micro-chemical structure of polyaniline synthesized by self-stabilized dispersion polymerization.</a> Macromol Res. 15 (7), 633-639 (2007).</li><li>John, A., Palaniappan, S., Djurado, D., Pron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+preparation+of+solution+processable+conducting+polyaniline+by+inverted+emulsion+polymerization+using+didecyl+ester+of+4-sulfophthalic+acid+as+multifunctional+dopant.">One-step preparation of solution processable conducting polyaniline by inverted emulsion polymerization using didecyl ester of 4-sulfophthalic acid as multifunctional dopant.</a> J Polym Sci A: Polym Chem. 46 (3), 1051-1057 (2008).</li><li>El-Dib, F. I., Sayed, W. M., Ahmed, S. M., Elkodary, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+polyaniline+nanostructures+in+micellar+solutions.">Synthesis of polyaniline nanostructures in micellar solutions.</a> J Appl Polym Sci. 124 (4), 3200-3207 (2012).</li><li>Tsotcheva, D., Tsanov, T., Terlemezyan, L., Vassilev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Investigations+of+Polyaniline+Prepared+in+the+Presence+of+Dodecylbenzenesulfonic+Acid.">Structural Investigations of Polyaniline Prepared in the Presence of Dodecylbenzenesulfonic Acid.</a> J Therm Anal Calorim. 63 (1), 133-141 (2001).</li><li>Jia, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyaniline-DBSA/organophilic+clay+nanocomposites:+synthesis+and+characterization.">Polyaniline-DBSA/organophilic clay nanocomposites: synthesis and characterization.</a> Synthetic Met. 128 (1), 115-120 (2002).</li><li>Kim, B. -. J., Oh, S. -. G., Han, M. -. G., Im, S. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Polyaniline+Nanoparticles+in+Micellar+Solutions+as+Polymerization+Medium.">Preparation of Polyaniline Nanoparticles in Micellar Solutions as Polymerization Medium.</a> Langmuir. 16 (14), 5841-5845 (2000).</li><li>Scales, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corona-Stabilized+Interpolyelectrolyte+Complexes+of+siRNA+with+Nonimmunogenic,+Hydrophilic/Cationic+Block+Copolymers+Prepared+by+Aqueous+RAFT+Polymerization&#8224;.">Corona-Stabilized Interpolyelectrolyte Complexes of siRNA with Nonimmunogenic, Hydrophilic/Cationic Block Copolymers Prepared by Aqueous RAFT Polymerization&#8224;.</a> Macromolecules. 39 (20), 6871-6881 (2006).</li><li>Kadashchuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+trions+in+conjugated+polymers.">Localized trions in conjugated polymers.</a> Phys Rev B. 76 (23), 235205 (2007).</li><li>Chang, H., Yuan, Y., Shi, N., Guan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+DNA+Biosensor+Based+on+Conducting+Polyaniline+nanotube+Array.">Electrochemical DNA Biosensor Based on Conducting Polyaniline nanotube Array.</a> Anal. Chem. 79, 5111-5115 (2007).</li><li>Zhu, N., Chang, Z., He, P., Fang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemically+fabricated+polyaniline+nanowire-modified+electrode+for+voltammetric+detection+of+DNA+hybridization.">Electrochemically fabricated polyaniline nanowire-modified electrode for voltammetric detection of DNA hybridization.</a> Eletrochim. Acta. 51, 3758-3762 (2006).</li></ol>

The work was supported by the University of Southern Mississippi College of Science and Technology and Mississippi College of Science and technology and Mississippi INBRE program (Award Number P204M103476 from the National Institute of general Medical Science).

Um sensor baseado em ácidos nucleicos de PANI requer a solubilização do polímero em água, a fim de interagir com o DNA e RNA. A dispersão de PANI em água é realizada usando tensioactivos, formando micelas, como relatado previamente 8. Além dos NaDBS utilizados aqui outros agentes tensioactivos aniónicos, como éster de dodecilo de ácido 4-sulfophthalic, tensioactivos não iónicos tais como etoxilato de nonil-fenol, ou tensioactivos catiónicos como brometo de cetiltrimetil amónio também pode ser...

Conjugated polymers provide many options for molecular sensors. This includes fluorescence, electronic, and colorimetric responses1. There have been many efforts to incorporate conjugated polymers in nucleic acid sensors. However, most systems require secondary detection, limiting sensing options2. Recently, we reported a conjugated polymer-based sensor platform built on polyaniline (PANI) that exploits properties of this polymer, creating a label-free system3. PANI is an extensively conjugated electro-active polymer with properties such as fluorescence and resistance that are suitable for measuring biological systems4. The excitons within the structure are not localized leading to mobility of the positive charge between monomeric subunits. This provides a flexible scaffold of positive charges that can interact with the negatively charged backbone of DNA5,6. Importantly, electrostatically attached DNA is orientated such that nitrogenous bases can participate in base pairing. Association with DNA alters the electronic properties of PANI, an effect that can be enhanced by UV irradiation (Figure 1)3. Using this system, oligonucleotides complementary to target nucleic acids can be immobilized on PANI. Multiple studies have demonstrated that upon hybridization electrostatically adsorbed oligonucleotides dissociate from PANI or other cationic matrices due to conformational changes caused by the switch to a double-stranded DNA structure3,5,7.
In a sensor system where probe attachment modulates conjugated polymer properties, hybridization events can be transduced without labels or enzymatic modification of probes or target nucleic acids. Conjugated polymers offer great flexibility in detection methods, one of which is fluorescence. Through monitoring PANI fluorescence, concentrations of target nucleic acids as low as 10-11 M (10 pM) can be detected3. Detection is rapid, occurring within 15 minutes of hybridization, and specific where a single mismatch in a target molecule can be differentiated3. 
Fabrication of PANI-sensors is straightforward. High molecular weight PANI can be generated that is well-dispersed in water using standard synthesis procedures involving aniline monomer, surfactant, and controlled addition of an oxidant. Yield can be very high and unreacted oxidant removed by washing with water, ensuring no further PANI growth. PANI-probe association occurs spontaneously upon mixture, and complex formation is enhanced by mild UV exposure. Hybridization can be carried out immediately, and the changes in PANI fluorescence assayed following a short incubation. The simplicity of this technology makes it highly accessible to many laboratories.

<table><tbody><tr><td>Aniline&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>A7401-500&amp;nbsp;</td><td>ACS, liquid, refrigerated</td></tr><tr><td>Ammonium peroxydisulfate</td><td>Fisher Scientific&amp;nbsp;</td><td>A682-500&amp;nbsp;</td><td>ACS, crystalline</td></tr><tr><td>Sodium dodecylbenzene sulfonate</td><td>Pfaltz &amp;amp; Bauer&amp;nbsp;</td><td>D56340&amp;nbsp;</td><td>95% solid</td></tr><tr><td>Chloroform</td><td>Fisher Scientific&amp;nbsp;</td><td>MCX 10601&amp;nbsp;</td><td>Liquid</td></tr><tr><td>DNA primers</td><td>MWG operon</td><td>n/a</td><td>custom DNA sequence ~20&amp;nbsp;bps</td></tr><tr><td>Microplate&amp;nbsp;</td><td>USA Scientific&amp;nbsp;</td><td>1402-9800&amp;nbsp;</td><td>96 well, polypropylene as it is unreactive to chloroform</td></tr><tr><td>Microplate Adhesive Film</td><td>USA Scientific&amp;nbsp;</td><td>2920-0000&amp;nbsp;</td><td>Reduces well-to-well contamination, sample spillage and evaporation</td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific&amp;nbsp;</td><td>12-544-D&amp;nbsp;</td><td>PANI coated on UV irradiated cover glass</td></tr><tr><td>UV crosslinker&amp;nbsp;</td><td>UVP&amp;nbsp;</td><td>HL-2000&amp;nbsp;</td><td>Energy: X100 &amp;mu;J/cm&lt;sup&gt;2&lt;/sup&gt;; Time: 2 min</td></tr><tr><td>Hybridization Oven</td><td>VWR</td><td>01014705 T</td><td>Temperature: 400&amp;nbsp;&amp;#176;C; with rocking for 15 min</td></tr><tr><td>Glass Apparatus&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td>Three necked round bottom flask for reaction; dropping funnel, stoppers, condenser, separating funnel</td></tr><tr><td>Microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Leica IMC S80</td><td>Magnification 20X; Pseudo color 536 nm; Exposure 86 msec; Gain 1.0x; Gamma 1.6</td></tr><tr><td>Microplate Reader</td><td>Molecular Devices&amp;nbsp;</td><td>89429-536</td><td></td></tr></tbody></table>

a polyaniline-based sensor of nucleic acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Figura 2A capta a configuração de reacção no início do processo de polimerização, isto é, antes da adição APS. A formação de micelas é o passo inicial na reacção de síntese de processo de PANI ocorre na interface micelar. A Figura 2B mostra uma solução leitosa após 5 min. 30 min após a APS é adicionada a reacção se vira para uma cor ligeiramente castanha. A Figura 2C mostra a alteração de cor associada c...

Watch this Scientific Journal Video about A Polyaniline-based Sensor of Nucleic Acids at JoVE.com

A Polyaniline-based Sensor of Nucleic Acids

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Um sensor baseado em Polianilina de Ácidos Nucleicos

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

a-polyaniline-based-sensor-of-nucleic-acids

Research

JoVE Journal

Biochemistry

8.0K visualizações.  University of Southern Mississippi. Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Vídeo: A Polyaniline-based Sensor of Nucleic Acids

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricação de Eletroquímica-DNA Biossensores para Detecção Reagentless de ácidos nucléicos, proteínas e pequenas moléculas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Bioengenharia

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodologia sintética para Asymmetric Ferroceno Derivados Sistemas Bio-conjugadas via Metodologia base de resina em Fase Sólida

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Química

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Seleção de Aptamer de DNA de ligação éster de ácido ftálico, caracterização e aplicação de um Aptasensor eletroquímica

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

A Biochip eletroquímica microfluídica à base de Análise de hibridação DNA livre-Label

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Reconhecimento Molecular de engenharia com Polymers Bio-mimético em únicos Walled nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

O uso de um ensaio de β-lactamase-baseado titulometria Biosensor para detectar interações biomoleculares

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desenvolvimento de um Biosensor eletroquímico do DNA para detectar um patógeno de origem alimentar

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Detecção visual de vários ácidos nucleicos em uma matriz capilar

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

<meta charset="utf8"></head><body>Nanomáquinas DNAzyme de precisão para análise discriminativa de RNA e DNA

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>Autor em destaque: Avanços em nanossensores de DNA - Abordando os desafios de sensibilidade e seletividade na detecção molecular

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - Nanomáquinas baseadas para reconhecimento de ácidos nucleicos

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Um sensor baseado em Polianilina de Ácidos Nucleicos

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