In the Laboratory
Label-Free Detection of DNA Hybridization by Cyclic Voltammetry
W
An Advanced Undergraduate Analytical Chemistry Laboratory Experiment Temitope Aiyejorun, Janusz Kowalik, Jiri Janata, and Mira Josowicz* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332; *
[email protected] The use of electropolymerized conducting polymers for the label-free electrochemical detection of DNA hybridization (1) is effective in demonstrating their practical application in analytical chemistry. Here we show how the different aspects of analytical chemistry, biochemistry, and electrochemical detection are juxtaposed. This laboratory puts undergraduates in touch with current research significant to finding faster and more convenient ways for DNA screening. Electrochemical “Label-Free” DNA Hybridization Detector The basic concept of a DNA biosensor relies on the immobilization of single-stranded (ss) oligonucleotides (probe) on a transducer to create an ssDNA recognition detector. The DNA detector recognizes its complementary oligonucleotides (target) and forms a hybrid with it. The DNA duplex between the complementary DNA strands yields detectable and analytically useful signals. It has paved the way to detecting the DNA hybridization by means of different recognition principles, which can be electrochemical (2), optical (3), gravimetrical (4), or surface plasmon resonance-based (5) in nature. The electrochemical transducers deliver a fast, simple, and low-cost methodology leading to successful discrimination between the complementary and the noncomplementary oligonucleotides. In a typical electrochemical DNA hybridization detector, the ssDNA is immobilized on a conjugated polymer film, such as polypyrrole. Polypyrrole (PPy) is formed by simultaneous electrooxidation of pyrrole and polymerization on a platinum electrode immersed in a solution of an electrolyte containing anion X−, for example, perchlorate or chloride (Scheme I). During the electrochemical oxidation of PPy the removal of electrons from the polypyrrole π electron orbitals takes place. The “liberated” electron travels to the positively charged Pt electrode on which the PPy is deposited. In chemical terms, the electron-deficient species in PPy is called a cation-radical, while materials scientists prefer to call it a polaron.
m+
n
N H
+ mX
Eox
− + (X )m + 2nH + 2ne−
N H
n
Scheme I. Electropolymerization of pyrrole. X− is the dopant anion that is inserted from electrolyte and m is number of dopant anions.
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Obviously, the existing π system of the PPy that is interrupted by this “defect”, still maintains its properties as a vehicle for electrical charge transport. The positive “defect” is redistributed within the π system much like a Gaussian distribution, and is not stationary. In summary, the PPy becomes positively charged and needs to be compensated by a negatively charged entity in order for the system to maintain charge neutrality (6–8). An important property of this material is that it can be reversibly reduced and reoxidized by controlling the potential applied to the polymer-modified platinum electrode. A convenient method for repetitive sweeping the electrode potential, E, from the positive to negative direction while monitoring current, i, is cyclic voltammetry. When a positive potential is applied to the PPy-modified electrode, the electrons leave the electrode. Simultaneously, the chloride anions from the electrolyte penetrate the bulk of the polymer to compensate for the electrochemically generated charge imbalance at the electrode (9, 10). During the reduction of PPy, the polymer gains electrons that are being used by the polymer “to fill” the positive “defects” (polarons) and the dopant anions, X−, are expelled from the PPy back to the electrolyte. Again, that process helps to preserve the charge neutrality in the polymer. Furthermore, the cyclic voltammetry traces the quantity of electrons that are transferred to the electrode or from the electrode when the potential E is applied to the modified electrode (working electrode). Therefore, it is possible to determine the quantity of charge that is accepted and removed from the polymer during the potential cycling from the area under the recorded i–E curve. Consequently, the repetitive cycling of the PPy film should result in identical cyclic voltammograms (CV). It is important to realize that the use of this electrochemically controlled anion-exchange process is the basis for the understanding of the operation of the anticipated development of DNA hybridization detector. Therefore we need to conduct one more thought experiment. Suppose, we create a barrier at the PPy–solution interface that carries negative charge. Will the electrostatic barrier affect the quantity of the anions penetrating the bulk of the PPy film? The answer is no, because the electrostatic barrier will affect the kinetics of the exchange of the anions but not the quantity. Therefore, the slower kinetics of negative ions penetrating bulk of the PPy layer affects the shape of the recorded CV. DNA hybridization can be diagnosed from the changes of the CV shape. A proper layout of the modified electrode is needed to create an electrostatic barrier for chloride ions on top of the
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Outline of Experiments The electrically conducting PPy is electropolymerized on the Pt-electrode first. This layer is then modified by another polymer bearing phosphonate groups by electropolymerization of TPTC3PO3H2, as shown in Figure 1. In contrast to the mobile anions coming from the electrolyte that dope the PPy layer, the ionized phosphonate groups, PO32−, of the pTPTC3PO3H2 layer are immobile, as they are covalently bound to the polymer. The polymers are deposited in two different thicknesses; the PPy layer that controls the electrochemically driven anion-exchange is always thicker than the pTPTC3PO3H2 layer, which only links the phosphonate groups at the surface of the modified electrode to the divalent magnesium cation. The magnesium ion as a linker forms a mixed salt (complex) between the phosponate group of the bilayer and the phosphate group of the DNA. It is known that metal ions such as Mg2+ or Ca2+ stabilize biological membranes by charge neutralization after binding to the carboxylated and phosphorylated head groups of lipids. Furthermore, these cations are also used to stabilize nucleic acids by alleviating electrostatic repulsion between the negatively charged phosphates (11). Divalent Mg2+ and Ca2+ are both “hard” ions and prewww.JCE.DivCHED.org
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Figure 1. Schematic of the fabricated label-free DNA hybridization detector. The layers are arranged in the sequence of their deposition on the Pt-electrode.
eⴚ
Cl
ⴚ
*
red
n
PPy layer. In this context, it is useful to think of a DNA molecule as a polyanion. In DNA, the building blocks consisting of nucleic base and deoxyribose are linked to each other as esters via a phosphoryl moiety. This arrangement leaves the remaining acid hydroxy group unbound and negatively charged under appropriate pH conditions. Such a site can be bound to any other negatively charged entity via multivalent ions, like Mg2+. The single strand, ssDNA, is capable of forming a double helix with complementary strand by establishing hydrogen bonds between complementary nucleic bases. The shape of the macromolecule becomes cylinder-like; the inner core consists of the electrically neutral nucleic bases, while the external coat contains ionizable phosphate groups. The hybridization process places the negative charges on the outer part of the double helix. Consequently, the presence of the negative charges on the outer part of the double helix defines the type of the electrostatic barrier for the PPy layer. To link that electrostatic barrier to the PPy modified electrode we need to introduce another polymer on top of the PPy layer. It is required that this polymer be of limited thickness and contain a functional group that is able to link the phosphate groups of the DNA probe using a complex bond formation. We chose a grafted thin layer of 2,5-bis(2-thienyl)N-(3-phosphorylpropyl)pyrrole, pTPTC3PO3H2. The presence of the phosphonic acid groups on the pTPTC3PO3H2 allows the link with the phosphate groups of the ssDNA probe through the divalent magnesium ions. The assembled electrochemical label-free DNA hybridization detector is then exposed to a solution of a target DNA (noncomplementary or complementary ssDNA) and the CV response is tested. The analytical signal of the detector, i = f(E ), is recorded before and after exposure to the target DNA. If the target DNA strand is complementary, the barrier height increases, hence, the chloride ion exchange between the PPy and the solution becomes kinetically hindered resulting in a shape change of the CV.
2ⴙ
Mg
S
N
O Oⴚ P OH
OR ⴚ O P O O
DNA probe
OR ⴚ O P O O
DNA target
S
ox Pt
pTPTC3-PO3H2
PPy
eⴚ
Cl
ⴚ
DNA hybridization detector
Figure 2. Principle of the electrochemical detection of DNA hybridization with the label-free DNA hybridization detector.
fer to bind to “hard” oxygen-containing ligands to form bidentate complexes (Figure 2). The significance of the labelfree DNA hybridization detector is its ability to form a duplex with a target DNA as shown in Figure 2. Once the hybridization of the DNA probe with the complementary target oligonucleotide (ODN) has occurred, the phosphate groups remain on the outside of the duplex. The attached target ODN present an electrostatic barrier at the surface of the label-free DNA hybridization detector. The hybridization event slows the electrochemically driven chloride anion exchange at the PPy-electrode–electrolyte interface. To test the reliability of the electrochemical label-free DNA hybridization detector, we conducted experiments with ODN of different lengths (27–38 bases), sequences, and concentrations in the range of 0.1–10 µM. Cyclic voltammetry was used to detect the hybridization event. The change in the CV area (shape and magnitude) indicates that hybridization has taken place.
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Materials An ODN of 27 base pairs with a complementary and noncomplementary ODN is suitable for the experiment, but it is good for the application to choose a sequence that is readily found in nature. The TPTC3PO3H2 is not commercially available; however, it can be obtained from us1 in small quantities or synthesized by the research advisor as outlined in the Supplemental Material.W All other chemicals such as pyrrole, TRIS buffer, salts for the experiments are commercially available. They are all listed in the Supplemental Material along with the Material Safety Data Sheet (MSDS) information.W Instrumentation To control the potential of the working electrode with respect to the reference electrode, any commercial potentistat– galvanostat can be used. We have used the potentiostat–galvanostat model CH660, CH Instruments. For electrochemical polymerization of PPy and TPTC3PO3H2 in acetonitrile, the silver兾silver ion (Ag兾0.1M AgNO3 in acetonitrile) is used as a reference electrode. For the experiments conducted in aqueous TRIS兾HCl buffer solution the silver兾silver chloride (Ag兾AgCl in 3 M KCl) is used. The platinum disc electrode from Bioanalytical Systems, with the disc diameter of 1.5 mm (A = 0.018 cm2) is used as the working electrode. However, any other smaller size working electrode can be also used. Platinum foil serves as the auxiliary electrode. A standard UV–vis spectrophotometer is used to determine concentrations of DNA solutions. Hazards Pyrrole and acetonitrile are flammable liquids, therefore, keep away from sparks, hot plates, and open flame. Acetonitrile is considered a human carcinogen. Avoid inhalation of pyrrole and acetonitrile vapors since they may cause irritation of the digestive and the respiratory tract and irritation of the nervous system. Handle them in adequately ventilated space (fume hood). Pyrrole, acetonitrile, tris(hydroxymethyl)aminomethane, tetra-n-butyl-ammonium perchlorate may lead to eye and skin irritation. Silver nitrate is a strong oxidizer, therefore, contact with other reducing agents may cause fire. It causes skin inflammation and discoloration owing to exposure. Results and Discussion The electropolymerization of pyrrole has been described in numerous publications; therefore, the polymerization procedure is given only in the Supplemental Material.W After complexing Mg2+ with the phosphonic acid groups of the pTPTC3PO3H2 layer, as shown in Figure 1, the chloride ion exchange of the PPy bilayer is characterized by voltammograms recorded in aqueous TRIS buffer solution containing Cl− ions (pH = 7.3). Usually, at least five voltammograms are recorded. However, to evaluate the response, only the last CV is used (Figure 3). The CV demonstrates the ability of the modified electrode to accumulate and release anions from the PPy layer as 1210
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the potential of the working electrode at the electrode–electrolyte interface is changed. When the target DNA is complementary to the DNA probe, a hybrid duplex of the oligonucleotides is formed at the pTPTC3PO3H2 surface. The response of the detector to a noncomplementary and complementary DNA target is shown as curve B and C, respectively, in Figure 3. It is useful to describe the changes in the CV experiments recorded in Figure 3 as a difference between the currents as a function of potential. Figure 4 is the result of the subtraction of curve B from curve A shown as curve E and
Figure 3. Voltammograms of the detector response recorded in 0.1 M TRIS/HCl buffer solution at the sweep rate of 20 mV/s vs Ag/ AgCl reference electrode; (A) CV of the detector with immobilized DNA probe only. (B) CV of the same detector as in curve A but in response to exposure to a noncomplementary target DNA. (C) CV of the same detector as in curve A but in response to a complementary target DNA. Concentrations of DNA solutions used in these experiments were: (A) probe DNA, 1.97 µM; (B) noncomplementary target DNA, 2.01 µM; (C) complementary target DNA, 1.37 µM.
6
4
Current / A
Materials and Methods
D
2
E 0
−2
−4
0.6
0.4
0.2
0.0
−0.2
−0.4
−0.6
Potential / V Figure 4. Detection of the DNA hybridization event: Curve D is difference current that indicates that the hybridization event took place. Curve E represents the event when hybridization did not take place. The curve D results from subtraction of curve A − C, and curve E from the subtraction of curve A − B, shown in Figure 4.
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curve C from curve A shown as curve D. The areas under the curves represent the net quantity of charge that has been kinetically hindered at the electrode–electrolyte interface owing to exposure to the DNA target. One can easily see that the charge that has been injected by reduction in the forward sweep (from 0.5 V to ᎑0.5 V) and withdrawn by oxidation in the reversal sweep differs significantly for the two curves. Since the hybridization event hinders the Cl− ions from the electrode–electrolyte solution interface to move into the PPy layer, the curve E can be identified as a “false positive”. Curve D represents an event when the hybridization did take place. With only qualitative understanding of the experiment we saw that we could predict the general shape of the responses. However, we are ultimately interested in obtaining quantitative information about the electrode processes taking place during the hybridization. The quantitative determination of the response can be done by integration the area under the Curves A, B, and C shown in Figure 3. This type of evaluation is presented in the Supplemental Material.W The relative changes in the label-free DNA hybridization probe of 6.08 ± 2.47% can be qualified as negative response when hybridization does not take place. The response of 16.55 ± 3.45% can be qualified as a positive response when hybridization takes place. For the last three years, we have been offering a “capstone undergraduate” class that lasts for six half-day sessions. In the class, the students perform a research project using this method aimed at improving the efficiency of the procedure and at lowering the limits of detection of hybridization. The results of each class helped us to solve various problems with the technique and to suggest new ways to improve the procedure. The students enjoy exploring new techniques and doing work that has a real impact on the progress of this method of detection. The results have been consistent and encouraging, and there is plenty of room for future undergraduates to try different method variations and experience science at work. Summary The goal of the described laboratory experiment is to make an electrochemical label-free DNA hybridization detector and test its reliability. This set of experiments provides an opportunity to expose students to the principles of electrochemistry, electropolymerization, conducting polymers, electrically controlled ion exchange, and the properties and hybridization of DNA. The students get hands-on experience with electrochemical techniques, UV–vis spectrophotometry, basic principles of acquiring and processing electrochemical
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data, and data analysis using simple graphing programs. The experiments described here can be used as a flexible platform for developing more advanced techniques, small individual student projects, and to introduce undergraduate students to the principles of biochemical sensors. Acknowledgments This work has been supported by the National Science Foundation, Grant No. CHE 9816017 and, partially, by the U.S. Department of Energy. W
Supplemental Material
Detailed experimental procedures, student handout, instructor notes, and spectra are available in this issue of JCE Online. Note 1. To receive a small quantity of TPTC3PO3H2, contact Janusz Kowalik by email (
[email protected]).
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