The authors have nothing to disclose.

1. verarbeitbaren PANI Synthese <ol><li> Auflösen Anilin (1 ml, 11 mmol) vollständig in 60 ml Chloroform in einem 250 ml Rundkolben. Rühre bei 600 Umdrehungen pro Minute für 5 Minuten und Abkühlen auf 0-5 ° C mit Eis. Dies dauert in der Regel 15 bis 20 min (2A). </li><li> Hinzufügen Natriumdodecylbenzolsulfonat (NaDBS) (7,44 g, 21 mmol) zu der Anilin-Lösung in einem Rundkolben während bei 600 rpm gerührt wurde. </li><li> Man löst Ammoniumpersulfat (APS) (3,072 g, 13,5 mmol) in 20 ml Wasser und fügen Sie alle tropfenweise über 30 Minuten eine Überhitzung der Reaktion zu vermeiden. </li><li> Führen Sie die Reaktion bei 0-5 ° C für 24 Stunden, und lassen Sie es Raumtemperatur für weitere 24 Stunden zu erreichen. </li><li> Beachten Sie das Reaktionsgemisch zunächst drehen milchig weiß nach 15 min, dann dunkelbraun nach 2 h und schließlich zu dunkelgrün nach 24 h (2B-F). </li><li> Filtern Sie die PANI-NaDBS Lösung mit einem Büchner-Trichter. Mischen Sie mit 80 ml Chloroform und 120 ml Wasser in alseparation Trichter (2G). </li><li> Inkubiere die Lösung 24 h bei Raumtemperatur und sammle die dunkelgrüne PANI aus dem Scheidetrichter, so dass nicht-umgesetztes NaDBS und APS in dem wässrige Überstand. </li></ol> 2. PANI-Sonde Mischen und UV-Bestrahlung <ol><li> Verdünnen Sie PANI Lösung 10x mit Chloroform-Wasser (1: 3 v / v) und mischen Sie 200 ul verdünnt PANI mit 6,4 &amp; mgr; mol Sonden-DNA-Oligonukleotide durch sanftes für 15 min rocking in ein Mikrozentrifugenröhrchen. </li><li> Bestrahlen , um die PANI-DNA - Lösung mit 1200 &amp; mgr; J / cm 2 UV in einem Vernetzer für 2 min. Es ist kritisch, dass die UV-Belichtung mit der angegebenen Menge beschränkt ist. Verlängerte Exposition gegenüber UV kompromittiert die Fluoreszenzänderung in PANI, wahrscheinlich durch kovalente Vernetzung von PANI und DNA. </li><li> Pellet-Komplexe durch Zentrifugation bei 17.000 xg für 6 min und Waschen mit phosphatgepufferter Salzlösung (PBS). Pellet erneut und resuspendieren in PBS. </li></ol> 3. Hybridization von PANI-Sonde <ol><li> Aus 8 ul 100 uM komplementären DNA-Oligonucleotide oder Zielnukleinsäuren zu 200 ul PANI-Sonde-Komplexe. </li><li> Führen Hybridisierung durch Lösungsgemisch 15 min bei 40 rocking ° C. </li><li> Pellet die PANI-Komplexe durch Zentrifugation bei 17.000 xg für 6 min. Waschen mit PBS und Resuspendieren in Wasser. </li></ol> 4. Emissions Steady-State-Fluoreszenzmessung <ol><li> In PANI aus verschiedenen Behandlungen in einer 96-Well-Mikroplatte und messen Emissionsfluoreszenz im 270-850 nm-Bereich durch Anregung bei 250 nm. Ein Emissionsspitze für PANI sollte etwa 500 nm zu beobachten. </li></ol> 5. Fluoreszenzmikroskopie Messung der hybridisierten Duplex <ol><li> Drop-Mantel PANI auf einem Borosilikat-Deckglas und trocken bei 40 ° C für 48 Stunden. </li><li> In Sonde (8 ul 100 uM) auf einem getrockneten PANI Film und bestrahlen es mit UV - Licht (1200 &amp; mgr; J / cm 2 </sup&gt;) für 2 min. </li><li> Waschen Sie die PANI-Sonde Film mit PBS und trocken bei 40 ° C für 48 Stunden. </li><li> Führen Sie die Hybridisierung für 15 min von Ziel-Nukleinsäuren hinzufügen. Dies könnte eine biologische Probe oder eine Kontroll Zieloligonucleotid (8 ul 100 uM) sein. Folgen Sie mit einem PBS waschen. </li><li> Besorgen Sie sich die Fluoreszenzbilder bei 40-facher Vergrößerung, mit einem 500 nm Langpassfilter. </li></ol>

<ol>
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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).

A PANI-basierten Sensor von Nukleinsäuren erfordert die Solubilisierung des Polymers in Wasser, um mit DNA und RNA zu interagieren. Die Dispersion von PANI in Wasser Tenside erreicht Verwendung bilden Mizellen wie zuvor 8 berichtet. Neben den hier NaDBS andere anionische Tenside wie dodecylester von 4-Sulfophthalsäure, nicht - ionische Tenside wie Nonylphenolethoxylat oder kationische oberflächenaktive Mittel wie Cetyltrimethylammoniumbromid könnte auch für die Synthese von verarbeitbaren PANI 9,10 verwendet. Die Synthese hier beschrieben beginnt mit einem festen Verhältnis des anionischen Tensids zu dem Monomer NaDBS Anilin bei einem 2: 1-Molverhältnis. Das Tensid NaDBS hat einen hohen Hydrophilie-Lipophilie-Gleichgewicht (HLB) ermöglicht Polymerisation von Anilin-Monomer in Chloroform-Wasser emulgiert durch Gleichgewicht zwischen den mizellaren polymeren Öltröpfchen und Wasser herzustellen. Das Oxidationsmittel APS wird dann bei einer 1: 1,5-Molverhältnis von Mizellen gr zu erzeugen,aufgrund PANI, wobei der innere Kern als Keimbildner wirkt. Dies führt zu Schwanz Befestigung von Monomeren zu leiten, während auch PANI in einem dynamischen Gleichgewicht mit dem umgebenden Lösungsmittel 11,12 zu halten. Die Reaktion läuft in einer gesteuerten Weise , bis hochmolekulare Polymere sind 13 gebildet. Dieses Protokoll wird Partikel erzeugen , die von 50 nm bis 1500 nm 3 betragen. Critical eine Reaktion zu etablieren, wo PANI wächst langsam noch löslich bleibt ist die langsame Zugabe des Oxidationsmittels APS, niedrige Temperatur, und das Fehlen von starken Protonensäuren. Einige Optimierung von Reaktions Proportionen kann notwendig sein, gut verteilt PANI zu erzeugen. PANI durch dieses Verfahren synthetisiert ist stabil für mehr als ein Jahr als Emulsion. Übermäßige Verdünnung sollte der HLB-Gleichgewicht der Dispersion vermieden werden kann gestört werden, was PANI auszufällen. High-Speed-Mischung kann helfen, die PANI in Lösung zu halten. Perfekte Löslichkeit ist nicht wünschenswert, da es Pellet formati ausschließen würdeauf, was ein wichtiges Kriterium für Hybridisierungsverfahren in einer Emulsionsbasis Sensor ist. Nach dem Erzeugen PANI dispergiert, DNA-Sonde Oligos an die Polymermatrix durch elektrostatische Wechselwirkung gebunden. Wechselwirkung von PANI Imingruppen und die Phosphate des Rückgrats der DNA spontan auftreten. Unspezifische PANI-Nukleinsäure-Wechselwirkungen Analytsensorik in komplexen biologischen Proben 14 erschweren. Zu erreichen Assoziation zwischen PANI und Sonden-DNA, die aus spontanen Interaktionen unterschieden werden kann, wird der Komplex mit UV bestrahlt. Die Schaffung von polaren Gruppen durch erhöht UV-ausgelöste Ladungslokalisierung induzierte Benetzbarkeit der Polymeroberfläche 15. Optimale Belichtung beträgt ca. 2 min und kann bestätigt werden , wenn PANI Fluoreszenz bei 500 nm erhöht ist etwa 5X (Abbildung 3). Überexposition des Komplexes UV kann auf eine Vernetzung zwischen PANI und DNA führen, die im Gegensatz zu elektrostatischen Wechselwirkungen, does nicht eine Höhe von PANI Fluoreszenz führen. Die Prozesssensoren der Erzeugung ist einfach und schnell. Die Polymerisationsreaktion wird nach 48 Stunden beendet, indem 24 Stunden, gefolgt von nicht-umgesetztes Oxidationsmittel und NaDBS zu trennen. Synthese müssen als die Menge an Vorrat PANI durchgeführt durch das Protokoll in diesem Artikel hergestellt selten werden ist sehr groß im Verhältnis zur Menge in einem Sensor verwendet wird. PANI Emulsion bei hohen Konzentrationen stabil ist; jedoch jede Verdünnung, die während der Sondenbefestigung auftritt, kann zu Ausfällen von PANI führen, Sensorleistung zu beeinträchtigen. Die unmittelbare Verwendung von PANI-Sonde Komplexe Ziele zu erkennen, wird dieses Problem zu vermeiden. Gemeinsam können Schaffung von Komplexen und Hybridisierung in weniger als einer Stunde durchgeführt werden. PANI-Sonde Filme sind stabiler und können nach einer Langzeitlagerung verwendet werden. Eine Einschränkung dieser Sensortechnologie ist, dass es nicht sehr zugänglich Multiplexen. Jedes Detektionsereignis muss in einem separaten Röhrchen durchgeführt werden. Filme könneneine bessere Format zum Multiplexen als verschiedene Sonden auf unterschiedlichen Regionen eines Films beobachtet werden konnte. Eine Probe konnte über den Spot-Array angewendet werden, und die Fluoreszenz an jedem Ort und Stelle verwendet Ausdruck eines anderen Gens zu bewerten. Der Erfassungsmechanismus hier beschrieben wird, liefert mehrere Vorteile gegenüber anderen Systemen. Berichte von Nukleinsäuren durch eine Dissoziation Erfassungsmechanismus PANI Beteiligung stützen sich auf Fluorophore 5 Sonde DNA - Oligos angebracht. Andere Strategien nutzen , kovalente Bindung von Sonden, die 16-17 einzelne Basenpaar - Unterschiede in der Ziel - Nukleinsäuren zu unterscheiden scheitern. Das Weglassen der Markierung oder andere Nebenmoden Detektions in dieser Technologie schafft Möglichkeiten andere Eigenschaften von elektroaktiven PANI zu verwenden. Die Sensorantwort könnte somit durch die Befestigungs-Ablösung Dynamik des Sensors verändert die Messung der elektrochemischen Eigenschaften erweitert werden. Labs, die diese Technik annehmen wird, die s findenEnsor System werden zusätzlich zu schnell und unkompliziert kostengünstig.

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.

2A erfasst zu Beginn des Polymerisationsverfahrens des Reaktionsaufbau, also vor APS hinaus. Mizellenbildung ist der erste Schritt in dem Reaktionsverfahren-PANI - Synthese an der mizellaren Schnittstelle auftritt. 2B zeigt eine milchige Lösung nach 5 min. 30 min nach dem APS zugegeben wird die Reaktion dann auch in eine leicht braune Farbe. 2C zeigt die Farbänderung mit der Bildung von Oligomeren verbunden. 2D eine dunkelbraune Farbe nach 4 h zeigt, eine hohe Konzentration an kurzkettigen PANI angibt, im Einklang mit eine sehr langsame Reaktion. Endlich , nach 48 Stunden eine dunkelgrüne Lösung mit hoher PANI Molekulargewicht beobachtet werden kann, der in Chloroform-Wasser (Abbildung 2E). 2F zeigt eine gut dispergiert sind , homogene PANI Lösung im Scheidetrichter ohne Phasentrennung dispergiert wird. (Abbildung 2) oder auf der Oberfläche der Drop-beschichteten Folien werden können (Abbildung 3). Die basale Fluoreszenz von PANI Filme relativ gering ist (3A). Sonden-DNA wird über dem Film hinzugefügt, und man ließ bei Raumtemperatur trocknen. Wenn die erwarteten PANI DNA-Komplex bildet, kann eine intensive Zunahme der Fluoreszenz beobachtet (3B) werden. Electron Zustrom von der negativ geladenen DNA verursacht höhere Elektronendichte in PANI-Molekülen, die Fluoreszenzzunahme verursacht. Nach der Hybridisierung induzierte Dissoziation der Sonde-Ziel - Duplex, kehrt PANI Fluoreszenz zu basalen Intensität (3C). Die Dissoziation wird durch Veränderungen in der DNA-Rückgrat bei der Hybridisierung, dass ein Kompromiss PANI-Sonde Wechselwirkung verursacht. Erfassungs Nukleinsäuren mit dieser Technologie können auch direkt in Emulsion (3D) durchgeführt werden. Ein Vorteil der Emulsion der Verwendung<html> -basierten System ist die Kompatibilität mit Plattenleser Instrumenten. Dies ermöglicht eine hochgenaue Messung von PANI Fluoreszenz und vermeidet durch Unregelmäßigkeiten in Filmbeschichtung verursachten Schwierigkeiten. A PANI-basierten Sensor in Emulsions Format ist sehr spezifisch. Die Einführung einer einzelnen Fehlpaarung (mm) in der Ziel - Oligo ausfällt basal PANI Fluoreszenz (Figur 3D) wiederherzustellen. <img alt="Abbildung 1" src="/files/ftp_upload/54590/54590fig1.jpg" /> Abbildung 1. Befestigung der Sonden durch UV - Exposition vermittelt erhöht PANI Fluoreszenz. Die Hybridisierung von Target - Nukleinsäuren führt zu Ablösung von PANI, und Reversion des Polymers auf die basale Fluoreszenz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig1large.jpg" target="_blank">eine größere Ansicht dieser Figur zu sehen.</a> iles / ftp_upload / 54590 / 54590fig2.jpg &quot;/&gt; Abbildung 2. Synthese von verarbeitbaren PANI. (A) Beginn der Polymerisationsreaktion. (B) Nach 5 min bei APS wird langsam zugegeben. (C) 30 Minuten nach der APS - Zugabe. (D) Nach 1 Stunde. (E) Nach dem 4 Std. (F) Grünes Produkt nach 48 Stunden der Reaktion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54590/54590fig3.jpg" /> Abbildung 3. Verwendung PANI Fluoreszenz Nucleinsäurehybridisierung nachzuweisen. (A) PANI - Film erzeugt durch Tropfenbeschichtung auf Borosilicatglas und anschließend bei 40 ° C für 48 Stunden getrocknet. (B) PANI Fluoreszenz nach Sondenimmobilisierung. (C)Nach der Hybridisierung der Ziel-DNA mit PANI-immobilisierten Sonden. (D) Fluoreszenz bei 500 nm von PANI Emulsion vor und nach der Hybridisierung mit entweder komplementär oder einzelne Fehlpaarung (mm) Zieloligonukleotide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig3large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

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.

Ein Polyanilin basierenden Sensor von 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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.0K Aufrufe.  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.

Serie: 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.

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

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. ...

Forschung

Biotechnik

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.

Synthesemethoden für Asymmetric Ferrocen abgeleitete Bio-Konjugat-Systemen über Festphasen-Resin-basierte Methodik

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. ...

Chemie

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.

Phthalsäure Acid Ester-Bindung DNA-Aptamer Auswahl, Charakterisierung und Anwendung eines elektrochemischen 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 ...

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.

Einem mikrofluidischen Biochip-basierte Elektrochemische für Markierungsfreie DNA-Hybridisierungsanalyse

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.

Technik Molekulare Erkennung mit Bio-Mimetikum Polymers auf einwandigen Kohlenstoff-Nanoröhrchen

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.

Die Verwendung eines β-Lactamase-basierte Conductimetric Biosensor-Assays, biomolekulare Wechselwirkungen zu erkennen

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.

Entwicklung der eine elektrochemische DNA-Biosensor, einem Foodborne Krankheitserreger zu erkennen

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.

Visuelle Erkennung von mehreren Nukleinsäuren in einem Kapillar 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 ...

<meta charset="utf8"></head><body>Präzisions-DNAzyme-Nanomaschinen für die diskriminative RNA- und DNA-Analyse

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 im Rampenlicht: Fortschritte bei DNA-Nanosensoren – Bewältigung der Herausforderungen bei Empfindlichkeit und Selektivität beim molekularen Nachweis

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 - basierte Nanomaschinen zur Nukleinsäureerkennung

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

Ein Polyanilin basierenden Sensor von Nucleic Acids

Zusammenfassung

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