Name: fabrication of electrochemical-dna biosensors for the reagentless detection of nucleic acids, proteins and small molecules
Uploaded: 2011-06-01T13:00:00
Duration: 13 min 15 s
Description: Watch this Scientific Journal Video about Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules at JoVE.com

This work was funded by a grant (OPP1015402) from the Bill and Melinda Gates Foundation through the Grand Challenges Explorations Initiative, and by the NIH through grants GM062958-01 and 2R01EB002046. This work was performed in part under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

1. Setting the Stage 
<ol>
	<li>Purchase the relevant, chemically modified probe DNA from a custom oligonucleotide synthesis company such as Biosearch Technologies (Novato, CA) or Midland Certified (Midland, TX). The probe is modified during synthesis by the addition of a C6 thiol at its 3&#x2019;-end and a redox-active methylene blue at its 5&#x2019;-end.</li>
	<li>Dissolve the probe DNA in phosphate buffered saline pH 7.4 to a concentration of 200 &mu;M and verify its concentration by measuring its absorbance at 260 nm using a spectrophotometer. Due to the methylene blue moiety on the DNA probe the solution should have a visible blue tint. </li>
	<li>Freshly prepare 1 mL of a 10 mM solution of tris(2-carboxyethyl)phosphine TCEP in distilled, deionized water (DI-water). In our experience, these TCEP solutions remain fresh for one week when stored in the dark at 4&deg;C.</li> 
	<li>To reduce any disulfide bonds that might be present in the probe DNA solution, combine 1 &mu;L of the probe DNA stock solution with 2 &mu;L of the TCEP solution and mix gently with a pipette. Incubate the mixture for one hour in a dark, refrigerated container. The initially blue solution should become clear as the TCEP reversibly reduces the methylene blue. If the solution does not become clear, repeat the procedure using a fresh TCEP solution or, possibly, at room temperature.</li> 
	<li>After one hour dilute the reduced DNA probe solution with 1 mL of buffer; this will dilute it to a concentration of 200nM. Later on, you will incubate a set of gold disk electrodes in 200 &mu;L portions of this diluted probe solution.</li>
	<li>Freshly prepare at least 2 mL of 2mM mercaptohexanol in phosphate buffered saline.</li> 
</ol>
2. Sensor Preparation 
<ol>
	<li>Combine 0.05 micron alumina powder with water on a fine polishing cloth. Polish a set of gold disk electrodes (CH Instruments, Austin, TX) by pressing the gold surface firmly into the wet cloth, and moving them in a figure eight pattern for approximately three minutes per electrode.</li>
	<li>Rinse the polished electrodes with DI-water and immerse them into Eppendorf tubes filled with the same. Sonicate for five minutes to remove any residual alumina powder.</li> 
	<li>Place the electrodes into a 0.5 M sulfuric acid solution, attach them to a potentiostat along with a platinum counter and silver/silver chloride reference electrode, and run a series of voltammograms to oxidize, reduce, and electrochemically clean their surfaces. Following this perform a second electrochemical cleaning in a solution of 0.01 M KCl in 0.1 M sulfuric acid. The fine details of these cleaning procedures can be found in our Nature Protocols paper1, and in the supplement to this video.</li>
	<li>Arrange a set of 2 mL Eppendorf tubes in a rack and fill each with 200 &mu;L of the probe DNA solution. The concentration of the probe DNA in this solution will define the density with which the probe DNAs pack on the sensor surface. Sensor performance is strongly dependent on probe density, with the optimal density varying from one sensor architecture to the next. The probe concentration used at this step should thus be optimized for each new type of sensor2. For the probe architectures we have investigated to date, the probe DNA concentrations we employ in this step range from 15 nM to 2 &mu;m, with 200 nM being a typical value.</li> 
	<li>Rinse the gold disk electrodes with DI-water, and then immerse them in the relevant probe DNA solution in an Eppendorf tube for one hour. At this point, the probe DNA will attach to the gold electrode surface via the formation of a thiol-on-gold self assembled monolayer.</li> 
	<li>Rinse the electrodes with DI-water, immerse them in 2mM mercaptohexanol in an Eppendorf tube and store them in a dark place for 3 hours to overnight at room temperature to ensure complete formation of the self assembled monolayer. This step incorporates the mercaptohexanol as part of a mixed monolayer to ensure the formation of a stable monolayer. To prevent evaporation you may want to seal the electrode into the Eppendorf tube with parafilm. Sensors can be stored in this solution for several days if necessary.</li>
	<li>When you are ready to use the sensor, rinse it with DI-water and then soak it in buffer for at least ten minutes. Sensors aimed at antibody detection must also be immersed in a 100 nM solution of the relevant recognition strand prior to use, followed by an extremely quick rinse with buffer.</li>
</ol>
3. Sensor Testing, DNA Detection 
<ol>
	<li>In this protocol, a 17 nucleotide probe strand is affixed to a gold electrode. It has a methylene blue redox reporter at its 3&#x2019;-end (Figure 1). When the probe molecule hybridizes with a capture strand, the current through the sensor circuit decreases.</li> 
	<li>Rinse a fresh sensor with DI-water and immerse it in a blank sample lacking the target in order to record the background signal it produces. Attach the sensor to the working electrode lead of a potentiostat. Place a platinum counter electrode and a silver/silver chloride reference electrode into the solution.</li> 
	<li>Run a square wave measurement from 0 to -0.6 V with an amplitude of 25 mV and a step size of 1mV. The optimal square wave frequency will depend on the details of the probe architecture2,3; for the probe architectures we have employed the optimal values are typically in the range 60 to 600 Hz. You should see a rounded peak at approximately -0.35 V, the redox potential of methylene blue (the peak potential may shift slightly depending on the precise pH of your test solution). The height from baseline current to this peak is proportional to the efficiency electron transfer between the methylene blue and the gold electrode (Figure2). Save this background measurement.</li>
	<li>Move the electrodes to a solution that contains the target DNA molecule of interest, equilibrate (5 to 120 min depending on the size, structure and concentration of the target4,5) and collect a second square wave voltammagram. The height of the peak at -0.35 V will change from the initial, background measurement. The magnitude of this change is related to the concentration of the analyte. It is the main output data of this sensor (Figure 3).</li> 
	<li>Measuring relative signal change, the percentage decrease or increase in signal relative to the background peak, is often more reproducible than measuring the absolute change in current, as this corrects for variations in the surface area of the electrode. To do this, the difference between the peak current and the background peak current are divided by the background peak current.</li>
</ol>
4. Sensor Regeneration 
<ol>
	<li>Once your measurements are complete, move the sensor into a container filled with DI-water for 30 s, or squirt it with a steady stream of DI-water for 30 s. Repeat this two more times with fresh deionized water. Note: some analytes are resistant to this approach; for them try aggressive rinsing in 6 M guanidine hydrochloride or 70% ethanol.</li>
	<li>Put the sensor back into a solution where measurements will be made. Within a minute, the peak height should have returned to its original value. It is worth noting that these sensors often exhibit a slightly larger signal change during their first test and regeneration cycle, and highly consistent results during the subsequent cycles6. </li>
</ol>
5. Sensor Testing, Antibody Detection 
<ol>
	<li>In this protocol, the methylene-blue and thiol-modified DNA probe used in these sensors serves as an "anchor" strand7. It is attached directly to the gold electrode. This is then hybridized with a second, "recognition" DNA strand that has been covalently conjugated to the relevant antigen (Figure 4). We have had good luck with commercial synthesis houses, such as Biosearch Technologies and Panagene, for the synthesis of the needed DNA-antigen chimeras. The hybridization step is carried out by transferring a pre-fabricated sensor into an Eppendorf tube containing 100 nM of the relevant recognition DNA strand in PBS for 1 hour.</li> 
	<li>Place the sensor in the relevant blank solution. Attach it to the working electrode lead of a potentiostat and place a platinum counter electrode and silver/silver chloride reference electrode into the solution.</li>
	<li>Perform square wave voltammetry as described above. For the particular probe architecture we have employed here the optimal square wave frequency is 60 Hz. You should see a rounded peak around -0.35 V. Save this background measurement. </li>
	<li>Transfer the electrodes to a solution containing the target analyte, incubate for 5 to 60 min, and collect a second square wave voltammagram. If the target antibody is present the peak at -0.35 V will decrease. The magnitude of this change is related to the antibody concentration.</li>
</ol>
6. Sensor Testing, Small Molecule Detection 
<ol>
	<li>In this case, the probe molecule on the sensor surface is an aptamer, a DNA or RNA molecule that has been selected in vitro to bind a specific molecular analyte, that changes its structure (folds) upon binding to its target analyte8,9 (Figure 5). Here we employ a cocaine-binding, DNA aptamer developed by the Stojanovic10,11 lab.</li> 
	<li>Rinse a fresh sensor with DI-water and immerse it in a blank sample lacking the target in order to record the background signal it produces. Attach the sensor to the working electrode lead of a potentiostat. Place a platinum counter and a silver/silver chloride reference into the solution.</li> 
	<li> Perform square wave voltammetry as described above. For the particular probe architecture we have employed here the optimal square wave frequency is 200Hz (But 60 Hz also works). You should see a rounded peak around -0.35 V. Save this background measurement.</li> 
	<li> Transfer the electrodes to a solution containing the target analyte, incubate for &tilde;5 min, and collect a second square wave voltammagram. The height of the peak at -0.35 V will change. The magnitude of this change is related to the concentration of the target analyte. If you cannot obtain a cocaine sample, procaine, the use of which is unregulated, can be used as a substitute. </li>
</ol>
7. Representative Results: 
When used to detect DNA using the first architecture, the signal should decrease by at least 60% when equilibrated at 200 nM target. 
After three brief rinses in deionized water, the signal should return very close (within 0.1-5%) to its original value. Antibody detection sensors should undergo 
a signal decrease of 40 to 80%. Aptamer-based sensors for the detection of cocaine exhibit a signal increase of up to 200% depending on the frequency and surface 
coverage at which they operate. For the cocaine sensor, a low surface coverage is best3.
<img src="/files/ftp_upload/2922/2922fig1.jpg" alt="Figure 1" /> 
Figure 1. Detection of DNA with an electrochemical DNA biosensor.
<img src="/files/ftp_upload/2922/2922fig2.jpg" alt="Figure 2" /> 
Figure 2. Screen shot showing the signal produced by an E-DNA biosensor during square wave voltammetry. 
<img src="/files/ftp_upload/2922/2922fig3.jpg" alt="Figure 3" /> 
Figure 3. Screen shot showing the signals produced by an E-DNA biosensor during square wave voltammetry, before 
and after hybridization with an analyte.
<img src="/files/ftp_upload/2922/2922fig4.jpg" alt="Figure 4" /> 
Figure 4. Detection of antibodies with a scaffold biosensor.
<img src="/files/ftp_upload/2922/2922fig5.jpg" alt="Figure 5" /> 
Figure 5. Detection of cocaine or procaine with an electrochemical aptamer biosensor. 
<table border="1">
	<tbody>
		<tr>
			<td>Custom Oligo</td>
			<td>Sequence</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Linear Probe DNA (LP17)</td>
			<td>5&#x2032;-HS&#x2212;(CH2)6&#x2212;TGGATCGGCGTTTTATT&#x2212;(CH2)7&#x2212;NH&#x2212;MB-3&#x2032;</td>
			<td>HPLC Purified, can be ordered with S-S</td>
		</tr>
		<tr>
			<td>Target Analyte DNA </td>
			<td>AATAAAACGCCGATCCA</td>
			<td>Unmodified </td>
		</tr>
		<tr>
			<td>Recognition Strand</td>
			<td>5'-Antigen-TEG- CAGTGGCGTTTTATTCTTGTTACTG-3'</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scaffold Anchor </td>
			<td>5'-HS-(CH2)6-GCAGTAACAAGAATAAAACGC CACTGC-(CH2)7-MB</td>
			<td>HPLC Purified, can be ordered with S-S</td>
		</tr>
		<tr>
			<td>A4 Cocaine Aptamer</td>
			<td>5'-HS-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-MethyleneBlue-3'</td>
			<td>HPLC Purified, can be ordered with S-S</td>
		</tr>
	</tbody>
</table>
Table 1. Probe and Target DNA Sequences.

<ol>
	<li>Xiao, Y., Lai, R. Y., Plaxco, K. W. <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+electrode-immobilized,+redox-modified+oligonucleotides+for+electrochemical+DNA+and+aptamer-based+sensing.">Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing.</a> Nat. Protocols. 2, 2875-2880 (2007).</li><li>Ricci, F., Lai, R. Y., Heeger, A. J., Plaxco, K. W., Sumner, 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=Effect+of+Molecular+Crowding+on+the+Response+of+an+Electrochemical+DNA+Sensor.">Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor.</a> Langmuir. 23, 6827-6834 (2007).</li><li>White, R. J., Phares, N., Lubin, A. A., Xiao, Y., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Electrochemical+Aptamer-Based+Sensors+via+Optimization+of+Probe+Packing+Density+and+Surface+Chemistry.">Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry.</a> Langmuir. 24, 10513-10518 (2008).</li><li>Lubin, A. A., Vander Stoep Hunt, B., White, R. J., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Probe+Length,+Probe+Geometry,+and+Redox-Tag+Placement+on+the+Performance+of+the+Electrochemical+E-DNA+Sensor.">Effects of Probe Length, Probe Geometry, and Redox-Tag Placement on the Performance of the Electrochemical E-DNA Sensor.</a> Analytical Chemistry. 81, 2150-2158 (2009).</li><li>Lubin, A. A., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding-Based+Electrochemical+Biosensors:+The+Case+for+Responsive+Nucleic+Acid+Architectures.">Folding-Based Electrochemical Biosensors: The Case for Responsive Nucleic Acid Architectures.</a> Accounts of Chemical Research. 43, 496-505 (2010).</li><li>Lubin, A. A., Lai, R. Y., Baker, B. R., Heeger, A. J., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-Specific,+Electronic+Detection+of+Oligonucleotides+in+Blood,+Soil,+and+Foodstuffs+with+the+Reagentless,+Reusable+E-DNA+Sensor.">Sequence-Specific, Electronic Detection of Oligonucleotides in Blood, Soil, and Foodstuffs with the Reagentless, Reusable E-DNA Sensor.</a> Analytical Chemistry. 78, 5671-5677 (2006).</li><li>Cash, K. J., Ricci, F., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrochemical+Sensor+for+the+Detection+of+Protein?Small+Molecule+Interactions+Directly+in+Serum+and+Other+Complex+Matrices.">An Electrochemical Sensor for the Detection of Protein?Small Molecule Interactions Directly in Serum and Other Complex Matrices.</a> Journal of the American Chemical Society. 131, 6955-6957 (2009).</li><li>Baker, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electronic,+Aptamer-Based+Small-Molecule+Sensor+for+the+Rapid,+Label-Free+Detection+of+Cocaine+in+Adulterated+Samples+and+Biological+Fluids.">An Electronic, Aptamer-Based Small-Molecule Sensor for the Rapid, Label-Free Detection of Cocaine in Adulterated Samples and Biological Fluids.</a> Journal of the American Chemical Society. 128, 3138-3139 (2006).</li><li>Ferapontova, E. E., Olsen, E. M., Gothelf, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+Aptamer-Based+Electrochemical+Biosensor+for+Detection+of+Theophylline+in+Serum.">An RNA Aptamer-Based Electrochemical Biosensor for Detection of Theophylline in Serum.</a> Journal of the American Chemical Society. 130, 4256-4258 (2008).</li><li>Stojanovic, M. N., Landry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-Based+Colorimetric+Probe+for+Cocaine.">Aptamer-Based Colorimetric Probe for Cocaine.</a> Journal of the American Chemical Society. 124, 9678-9679 (2002).</li><li>Stojanovic, M. N., Prada, P. d. e., Landry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-Based+Folding+Fluorescent+Sensor+for+Cocaine.">Aptamer-Based Folding Fluorescent Sensor for Cocaine.</a> Journal of the American Chemical Society. 123, 4928-4931 (2001).</li><li>Cash, K. J., Heeger, A. J., Plaxco, K. W., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+Reusable,+DNA+Pseudoknot-Based+Electrochemical+Sensor+for+Sequence-Specific+DNA+Detection+in+Blood+Serum.">Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum.</a> Analytical Chemistry. 81, 656-661 (2009).</li><li>Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrochemical+Sensor+for+Single+Nucleotide+Polymorphism+Detection+in+Serum+Based+on+a+Triple-Stem+DNA+Probe.">An Electrochemical Sensor for Single Nucleotide Polymorphism Detection in Serum Based on a Triple-Stem DNA Probe.</a> Journal of the American Chemical Society. 131, 15311-15316 (2009).</li><li>Zuo, X., Xiao, Y., Plaxco, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Specificity,+Electrochemical+Sandwich+Assays+Based+on+Single+Aptamer+Sequences+and+Suitable+for+the+Direct+Detection+of+Small-Molecule+Targets+in+Blood+and+Other+Complex+Matrices.">High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices.</a> Journal of the American Chemical Society. 131, 6944-6945 (2009).</li><li>Xia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrochemical+Supersandwich+Assay+for+Sensitive+and+Selective+DNA+Detection+in+Complex+Matrices.">An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices.</a> Journal of the American Chemical Society. 132, 14346-14348 (2010).</li><li>Patterson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Triplex-Forming+Oligonucleotide+Probes+for+the+Reagentless,+Electrochemical+Detection+of+Double-Stranded+DNA.">Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA.</a> Analytical Chemistry. 82, 9109-9115 (2010).</li><li>Ferguson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Microfluidic+Electrochemical+DNA+Sensor.">Integrated Microfluidic Electrochemical DNA Sensor.</a> Analytical Chemistry. 81, 6503-6508 (2009).</li><li>Swensen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous,+Real-Time+Monitoring+of+Cocaine+in+Undiluted+Blood+Serum+via+a+Microfluidic,+Electrochemical+Aptamer-Based+Sensor.">Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor.</a> Journal of the American Chemical Society. 131, 4262-4266 (2009).</li></ol>

There is nothing to disclose.

An important note is that none of the experiments described above will work properly unless the electrodes have been properly cleaned. Here is a guide to our electrochemical cleaning procedure. When working with CH Instruments potentiostats, we run these cleaning steps using a set of three macro programs.
Phase Zero (E-clean O) 
Immerse the electrodes in 0.5M H2SO4 and connect them to the working electrodes of a potentiostat. Also attach and immerse an Ag/AgCl reference and platinum counter electrode. Start with an oxidation step (2 V for 5 s) and then a reduction step (0.35 V for 10 s).
Phase One (E-clean 1) 
Initiate oxidation and reduction scans under the same acidic conditions (0.5M H2SO4) from 0.35 to 1.5 V (20 scans at a scan rate of 4 V/s and a sample interval of 0.01 V, followed by four scans at a scan rate of 0.1 V/s and a sample interval of 0.01 V).
Phase Two (E-clean 2) 
Conduct another set of electrochemical oxidation and reduction scans under acidic conditions (0.01 M KCl/0.1 MH2SO4) covering four different potential ranges (all performed for 10 segments at a scan rate of 0.1 V s 1 and a sample interval of 0.01 V): (i) potential range from 0.2 to 0.75 V; (ii) potential range from 0.2 to 1.0 V; (iii) potential range from 0.2 to 1.25 V; (iv) potential range from 0.2 to 1.5 V.
Many types of gold electrodes can be used to conduct these experiments. In addition to gold disk electrodes such as those employed here, we have had success with microfabricated gold surfaces, gold wire, and gold on printed circuit boards.
Along with the sensors described in this paper, many other electrochemical DNA biosensor architectures have been reported. This includes sensors with a pseudoknot12, triple strand13, sandwich14, super sandwich15, or triplex16 architecture. 
In the future, we expect that these sensors will be used in point of care medical diagnostics. They have been successfully integrated into several microfluidic devices17,18, and offer many advantages over optical analyte detection systems. In particular, these sensors can function in turbid, optically dense and highly auto-fluorescent samples.

<table border="1">	
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Gold Disk Electrodes</td>
			<td>CH Instruments</td>
			<td>CHI101</td>
			<td>Can be re-used</td>
		</tr>
		<tr>
			<td>Synthetic Probe DNA</td>
			<td>Biosearch Technologies</td>
			<td>Custom</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Synthetic Target DNA</td>
			<td>Sigma Genosys </td>
			<td>Custom</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mercaptohexanol</td>
			<td>Sigma Aldrich</td>
			<td>725226-1G</td>
			<td>Store in cool dark place</td>
		</tr>
		<tr>
			<td>Platinum Electrode</td>
			<td>BASi</td>
			<td>MW-1032</td>
			<td>Can be re-used</td>
		</tr>
		<tr>
			<td>Ag/AgCl Reference</td>
			<td>BASi</td>
			<td>MF-2052</td>
			<td>Can be re-used</td>
		</tr>
		<tr>
			<td>Polishing Cloth</td>
			<td>Buehler </td>
			<td>40-7212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alumina Polish</td>
			<td>Buehler</td>
			<td>40-6325-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline Buffer, pH 7.4</td>
			<td>Sigma Aldrich</td>
			<td>P7059-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CH Instruments 605A</td>
			<td>CH Instruments</td>
			<td>605A</td>
			<td>Use any potentiostat</td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Sigma Aldrich</td>
			<td>N4637-500ML</td>
			<td>Stored frozen</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Fisher Scientific</td>
			<td>ND-2000</td>
			<td>Use any UV-Vis </td>
		</tr>
		<tr>
			<td>PCR Mix </td>
			<td>Bio-Rad</td>
			<td>170-8862</td>
			<td>Stored frozen</td>
		</tr>
		<tr>
			<td>Cocaine</td>
			<td>Sigma Aldrich</td>
			<td>C5776</td>
			<td>DEA License Required</td>
		</tr>
		<tr>
			<td>Procaine</td>
			<td>Sigma Aldrich</td>
			<td>P9879</td>
			<td>Substitute for Cocaine</td>
		</tr>
		<tr>
			<td>Anti-Flag Antibody</td>
			<td>Sigma Aldrich</td>
			<td>F1804-1mg</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules at JoVE.com

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

"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.

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. ...

fabrication-electrochemical-dna-biosensors-for-reagentless-detection

Research

JoVE Journal

Bioengineering

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

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 ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...