The authors have nothing to disclose.

1. Processable PANI Synthesis
<ol>
	<li>Dissolve aniline (1 ml, 11 mmol) completely in 60 ml of chloroform in a 250 ml round-bottom flask. Stir at 600 rpm for 5 min and cool to 0-5 &#176;C with ice. This usually takes 15-20 min (Figure 2A).</li>
	<li>Add sodium dodecyl benzene sulphonate (NaDBS) (7.44 g, 21 mmol) to the aniline solution in a round-bottomed flask while stirring at 600 rpm.</li>
	<li>Dissolve ammonium persulphate (APS) (3.072 g, 13.5 mmol) in 20 ml water and add all of it drop-wise over 30 min to avoid overheating the reaction.</li>
	<li>Carry out the reaction at 0-5 &#176;C for 24 hr, and allow it to reach room temperature for another 24 hr.</li>
	<li>Observe the reaction mixture initially turn milky white after 15 min, then dark brown after 2 hr, and finally to dark green after 24 hr (Figure 2B-F).</li>
	<li>Filter the PANI-NaDBS solution with a Buchner funnel. Mix with 80 ml chloroform and 120 ml water in a separation funnel (Figure 2G).</li>
	<li>Incubate the solution for 24 hr at room temperature and collect the dark green PANI from the separation funnel, leaving unreacted NaDBS and APS in the aqueous supernatant.</li>
</ol>
2. PANI-probe Mixing and UV Irradiation
<ol>
	<li>Dilute PANI solution 10x with chloroform-water (1:3 v/v) and mix 200 &#181;l of diluted PANI with 6.4 &#181;mol of probe DNA oligonucleotides by gentle rocking for 15 min in a microfuge tube.</li>
	<li>Irradiate the PANI-DNA solution with 1,200 &#181;J/cm2 of UV in a crosslinker for 2 min. It is critical that UV exposure is limited to the indicated amount. Extended exposure to UV compromises the fluorescence change in PANI, likely due to covalent cross-linking of PANI and DNA.</li>
	<li>Pellet complexes by centrifugation at 17,000 x g for 6 min, and wash with phosphate buffered saline (PBS). Pellet again, and re-suspend in PBS.</li>
</ol>
3. Hybridization of PANI-probe
<ol>
	<li>Add 8 &#181;l of 100 &#181;M complementary DNA oligonucleotides or target nucleic acids to 200 &#181;l of PANI-probe complexes.</li>
	<li>Perform hybridization by rocking solution mixture for 15 min at 40 &#176;C.</li>
	<li>Pellet the PANI complexes by centrifugation at 17,000 x g for 6 min. Wash with PBS and re-suspend in water.</li>
</ol>
4. Emission Steady State Fluorescence Measurement
<ol>
	<li>Add PANI from different treatments into a 96 well microplate and measure emission fluorescence in the 270-850 nm range by excitation at 250 nm. An emission peak for PANI should be observed around 500 nm.</li>
</ol>
5. Fluorescence Microscopy Measurement of Hybridized Duplex
<ol>
	<li>Drop coat PANI on a borosilicate glass coverslip and dry at 40 &#176;C for 48 hr.</li>
	<li>Add probe (8 &#181;l of 100 &#181;M) on a dried PANI film and irradiate it with UV light (1,200 &#181;J/cm2) for 2 min.</li>
	<li>Wash the PANI-probe film with PBS and dry at 40 &#176;C for 48 hr.</li>
	<li>Perform hybridization for 15 min by adding target nucleic acids. This could be a biological sample or a control target oligonucleotide (8 &#181;l of 100 &#181;M). Follow with a PBS wash.</li>
	<li>Obtain the fluorescent images at 40X magnification, with a 500 nm long pass filter.</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. -. 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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. 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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).

A PANI-based sensor of nucleic acids requires solubilization of the polymer in water in order to interact with DNA and RNA. The dispersion of PANI in water is accomplished using surfactants, forming micelles as previously reported8. In addition to the NaDBS used here other anionic surfactants like dodecyl ester of 4-sulfophthalic acid, nonionic surfactants like nonyl phenol ethoxylate, or cationic surfactants like cetyltrimethyl ammonium bromide could also be used for synthesis of processable PANI9,10. The synthesis described here begins with a fixed proportion of the anionic surfactant NaDBS added to the monomer aniline at a 2:1 molar ratio. The surfactant NaDBS has a high hydrophile-lipophile balance (HLB) allowing polymerization of aniline monomer emulsified in chloroform-water by establishing equilibrium between the micellar polymeric oil droplets and water. The oxidant APS is then added at a 1:1.5 molar ratio to generate micelles of growing PANI, where the inner core acts as a nucleating agent. This leads to head to tail attachment of monomers, while also keeping PANI in dynamic equilibrium with the surrounding solvent11,12. The reaction proceeds in a controlled manner until high molecular weight polymers are formed13. This protocol will generate particles that range from 50 nm to 1,500 nm3. Critical to establishing a reaction where PANI grows slowly yet remains soluble is the slow addition of the oxidant APS, low temperature, and the absence of strong protonic acids. Some optimization of reactant proportions may be necessary to generate well-dispersed PANI. PANI synthesized by this method is stable for more than one year as an emulsion. Excessive dilution should be avoided as the HLB balance of the dispersion may be disrupted, causing PANI to precipitate. High speed mixing can help to keep the PANI in solution. Perfect solubility is not desirable, as it would preclude pellet formation, which is an important criterion for hybridization procedures in an emulsion-based sensor.
After generating dispersed PANI, DNA probe oligos are attached to the polymer matrix through electrostatic interaction. Interaction of PANI imine groups and the phosphates of the backbone of DNA occur spontaneously. Non-specific PANI-nucleic acid interactions complicate analyte sensing in complex biological samples14. To achieve association between PANI and probe DNA that can be distinguished from spontaneous interactions, the complex is irradiated with UV. The creation of polar moieties induced by UV-triggered charge localization increases wettability of the polymer surface15. Optimal exposure is approximately 2 min, and can be confirmed if PANI fluorescence at 500 nm is elevated roughly 5X (Figure 3). Over-exposure of the complex to UV can lead to crosslinking between PANI and DNA, which unlike electrostatic interactions, does not cause an elevation of PANI fluorescence.
The process of generating sensors is easy and quick. The polymerization reaction is completed after 48 hours followed by 24 hours to separate from unreacted oxidant and NaDBS. Synthesis will need to be carried out infrequently as the quantity of stock PANI produced by the protocol in this article is very large relative to the amount used in a sensor. PANI emulsion at high concentrations is stable; however, any dilution that occurs during probe attachment can lead to precipitation of PANI, compromising sensor performance. The immediate use of PANI-probe complexes to detect targets will avoid this issue. Together, creation of complexes and hybridization can be done in under an hour. PANI-probe films are more stable and can be used after long-term storage. A limitation of this sensor technology is that it is not highly amenable to multiplexing. Each detection event must be carried out in a separate tube. Films may provide a better format for multiplexing as different probes could be spotted on different regions of a film. A sample could be applied over the spot array, and the fluorescence at each spot used to evaluate expression of a different gene.
The detection mechanism described here provides several advantages over other systems. Reports of detecting nucleic acids through a dissociation mechanism involving PANI rely on fluorophores attached to probe DNA oligos5. Other strategies use covalent attachment of probes, which fail to distinguish single base-pair differences in target nucleic acids16-17. The omission of labeling or other secondary modes of detection in this technology creates opportunities to use other properties of electro-active PANI. The sensor response thus could be extended to the measurement of electrochemical properties altered by the attachment-detachment dynamics of the sensor. Labs that adopt this technique will find the sensor system to be cost effective in addition to fast and straightforward.

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.

Figure 2A captures the reaction setup at the start of the polymerization process, i.e., before APS addition. Micelle formation is the initial step in the reaction process-PANI synthesis occurs at the micellar interface. Figure 2B shows a milky solution after 5 min. 30 min after APS is added the reaction turns to a slightly brown color. Figure 2C shows the color change associated with the formation of oligomers. Figure 2D shows a dark brown color after 4 hr, indicating a high concentration of short chain PANI, consistent with a very slow reaction. Finally after 48 hr a dark green solution of high molecular weight PANI can be observed that is dispersed in chloroform-water (Figure 2E). Figure 2F shows a well-dispersed, homogenous PANI solution in the separating funnel with no phase separation.
Sensing nucleic acids can be carried out in emulsion (Figure 2) or on the surface of drop-coated films (Figure 3). The basal fluorescence of PANI films is relatively low (Figure 3A). Probe DNA is added over the film, and allowed to dry at room temperature. When the expected DNA-PANI complex forms, an intense increase in fluorescence can be observed (Figure 3B). Electron influx from the negatively charged DNA causes higher electron density in PANI molecules, causing the fluorescence increase. After hybridization induced dissociation of the probe-target duplex, PANI fluorescence returns to basal intensity (Figure 3C). Dissociation is caused by changes in the DNA backbone upon hybridization that compromise PANI-probe interaction. Sensing nucleic acids with this technology can also be performed directly in emulsion (Figure 3D). A benefit of using the emulsion-based system is compatibility with plate reader instruments. This permits a highly precise measurement of PANI fluorescence, and avoids difficulties caused by irregularities in film coating. A PANI-based sensor in emulsion format is highly specific. The introduction of a single mismatch (mm) in the target oligo fails to restore basal PANI fluorescence (Figure 3D).
<img alt="Figure 1" src="/files/ftp_upload/54590/54590fig1.jpg" /> 
Figure 1. Attachment of probes mediated by UV exposure increases PANI fluorescence. Hybridization of target nucleic acids results in detachment from PANI, and reversion of the polymer to basal fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54590/54590fig2.jpg" /> 
Figure 2. Synthesis of processable PANI. (A) Start of polymerization reaction. (B) After 5 min when APS is slowly added. (C) 30 min after APS addition. (D) After 1 hr. (E) After 4 hr. (F) Green product after 48 hr of reaction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54590/54590fig3.jpg" /> 
Figure 3. Use of PANI Fluorescence to detect nucleic acid hybridization. (A) PANI film created through drop-coating on borosilicate glass, followed by drying for 48 hr at 40 &#176;C. (B) PANI fluorescence after probe immobilization. (C) Following hybridization of target DNA with PANI-immobilized probes. (D) Fluorescence at 500 nm of PANI emulsion before and after hybridization with either complementary or single mismatch (mm) target oligonucleotides. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</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.

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

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JoVE Journal

Biochemistry

8.0K Views.  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.

Video: 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.

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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.

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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.

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.

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.

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.

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.

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.

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

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

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.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

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

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

A Polyaniline-based Sensor of Nucleic Acids

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