“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. 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.
1. Setting the Stage
2. Sensor Preparation
3. Sensor Testing, DNA Detection
4. Sensor Regeneration
5. Sensor Testing, Antibody Detection
6. Sensor Testing, Small Molecule Detection
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.
Figure 1. Detection of DNA with an electrochemical DNA biosensor.
Figure 2. Screen shot showing the signal produced by an E-DNA biosensor during square wave voltammetry.
Figure 3. Screen shot showing the signals produced by an E-DNA biosensor during square wave voltammetry, before
and after hybridization with an analyte.
Figure 4. Detection of antibodies with a scaffold biosensor.
Figure 5. Detection of cocaine or procaine with an electrochemical aptamer biosensor.
Custom Oligo | Sequence | Comments |
Linear Probe DNA (LP17) | 5′-HS−(CH2)6−TGGATCGGCGTTTTATT−(CH2)7−NH−MB-3′ | HPLC Purified, can be ordered with S-S |
Target Analyte DNA | AATAAAACGCCGATCCA | Unmodified |
Recognition Strand | 5′-Antigen-TEG- CAGTGGCGTTTTATTCTTGTTACTG-3′ | |
Scaffold Anchor | 5′-HS-(CH2)6-GCAGTAACAAGAATAAAACGC CACTGC-(CH2)7-MB | HPLC Purified, can be ordered with S-S |
A4 Cocaine Aptamer | 5′-HS-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-MethyleneBlue-3′ | HPLC Purified, can be ordered with S-S |
Table 1. Probe and Target DNA Sequences.
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.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
Gold Disk Electrodes | CH Instruments | CHI101 | Can be re-used |
Synthetic Probe DNA | Biosearch Technologies | Custom | |
Synthetic Target DNA | Sigma Genosys | Custom | |
Mercaptohexanol | Sigma Aldrich | 725226-1G | Store in cool dark place |
Platinum Electrode | BASi | MW-1032 | Can be re-used |
Ag/AgCl Reference | BASi | MF-2052 | Can be re-used |
Polishing Cloth | Buehler | 40-7212 | |
Alumina Polish | Buehler | 40-6325-016 | |
Phosphate buffered saline Buffer, pH 7.4 | Sigma Aldrich | P7059-1L | |
CH Instruments 605A | CH Instruments | 605A | Use any potentiostat |
Newborn Calf Serum | Sigma Aldrich | N4637-500ML | Stored frozen |
NanoDrop | Fisher Scientific | ND-2000 | Use any UV-Vis |
PCR Mix | Bio-Rad | 170-8862 | Stored frozen |
Cocaine | Sigma Aldrich | C5776 | DEA License Required |
Procaine | Sigma Aldrich | P9879 | Substitute for Cocaine |
Anti-Flag Antibody | Sigma Aldrich | F1804-1mg |