Review

On-Demand Electrochemical Release of Nucleic Acids

Joseph Wang*

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA; e-mail: joewang@nmsu.edu

Received: June 2, 2000

Final version: September 20, 2000

Abstract

Electrochemical protocols for releasing DNA from electrode surfaces are discussed. These protocols make use of the negative potential ofnucleic acids for modulating electrically their interfacial properties. The miniaturization, surface-tailoring, safety, simplicity, convenience,automation, and low-cost advantages of electrochemical systems, coupled with the lack of immune reactions, make the electrical routea very attractive gene-delivery alternative. In particular, the use of DNA-modi®ed microelectrodes holds promise for delivering the geneticmaterial to speci®c locations at selected times. Such on-demand electrochemical removal of DNA should ®nd important applications,besides gene therapy, including the localization of DNA onto tiny surfaces, design of `active'scanning probe tips (e.g., SECM, STM) whichrelease the DNA locally while scanning over the surface, controlled introduction of DNA to various analytical systems, or fundamentalstudies related to DNA interactions. Several protocols for releasing DNA and DNA-lipid complexes from gold and carbon microelectrodesunder potential control are described, along with surface characterization techniques for monitoring the removal process. Fundamental andtechnological issues facing the development of DNA electrochemical carriers are discussed.

Keywords: DNA, Electrochemical release, Gene therapy, Desorption, Delivery

1. Background±DNA Delivery

Gene therapy, which involves the delivery of therapeutic genesinto cells, offers tremendous opportunities for the treatment andcure of a wide variety of genetic diseases [1±3]. Such technologywill almost certainly revolutionize the practice of medicine in the®rst part of the 21st century. Despite these opportunities, progressin developing effective gene-delivery clinical protocols has beenslow [3, 4]. This is due to major obstacles, such as safety=toxicity,low ef®ciency, high cost, and reproducibility of existing genedelivery vectors [3].

An ideal gene delivery vehicle would allow the introduction ofDNA into the desired cells=tissue in an ef®cient, timely, repro-ducible, convenient and safe manner, and with minimal sideeffects. There are two major classes of vehicles for gene transfer:viral and nonviral vectors [1±4]. Current methods for transferringgenes rely on the use of viruses to deliver the DNA. Such use ofretroviral and adenoviral vectors suffers from safety concerns,dif®culty of manufacturing, and has not yielded optimal results.Accordingly, recent attention has been shifted to the introductionof nonviral physical methods for delivering genes. Thesemethods include use of cationic lipids, liposome complexes,direct injection, gene gun, ultrasound, cell bombardment, orelectroporation. As most of these approaches suffer from variousde®ciencies discussed above [3], there are urgent needs fordesigning new and improved alternate gene delivery vehicles.

The electrochemical routes for delivering DNA, described inthis article, should ®nd important fundamental and technologicalapplications beyond gene therapy. These may include thelocalization and immobilization of DNA onto tiny, predeterminedsites (as needed, for example, for fabricating high-density genechip), the design of `active' (DNA-modi®ed) scanning probe tips(e.g., SECM, STM) which release their DNA locally whilescanning over the surface, removal of nonspeci®cally adsorbedDNA in hybridization biosensors, controlled introduction ofDNA to various analytical systems (e.g., ICP-MS, MS), orfundamental studies related to DNA interactions or conductivity.

2. Why Electrochemistry?

This article reviews recent efforts in our laboratory aimed atdesigning new electrochemical protocols for delivering DNA inhighly controllable and safe manner. The new electrochemicalroute offers several advantages for delivering therapeutic genesand for other applications requiring localized delivery of DNA.The physical nature of electrochemistry makes this type ofdelivery applicable to all types of tissues. The ability to fabricateultramicroelectrodes (down to the nanometer domain) has beenwidely used for the exploration of physiological processes inmicroenvironments [5], including the in vivo monitoring ofneurochemical events in living brains, time-resolved probing ofdynamic processes (e.g., secretion) in single cells, as well asother intracellular measurements. Microelectrodes have also beenwidely implanted in various tissues or the blood stream. Suchminiaturization is potentially attractive for localizing the delivery(to very small spaces) and minimizing tissue damage.

Electrochemistry has been used previously by Miller's group,for the controlled release of glutamate and dopamine inconnection to the undoping or cleavage of doped=functionalizedpolymers [6, 7]. Although the focus of Miller's activity has beenneurotransmitter delivery, the basic concept of releasing chemi-cals from surfaces has much broader implications. Electricallyerodable polymeric gels have also been proposed for thecontrolled release of small drugs [8].

The ability to deliberately modify electrode surfaces representsanother attractive advantage of the electrochemical approach. Inaddition to the above-mentioned polymer-tailored electrodes,several laboratories (including our own) have been interested inthe rational design of DNA-modi®ed electrodes. Various schemesfor deliberately and reproducibly attaching nucleic acids toelectrode surfaces have thus been developed for detecting DNAsequences, DNA interactions, or DNA damage [9, 10]. Thecoupling of the above miniaturization and modi®cation technol-ogies has resulted in the organized con®nement of hundredthousands DNA probes onto micrometer-sized surfaces [11].

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The high charge of DNA molecules, coupled with theirinterfacial properties, open up unique opportunities for usingelectrochemistry for controlling and regulating their af®nity toconducting electrode surfaces. Electrochemical protocols are alsocost-effective and can be readily automated under computercontrol. The miniaturization, surface-tailoring, safety, simplicity,convenience, automation, and low-cost advantages of electro-chemical systems, coupled with the lack of immune reactionsand the electrically modulated interfacial properties, make theelectrochemical route an extremely attractive gene-deliveryalternative.

In the following sections we will describe recently developedDNA electrochemical carriers and will address fundamental andtechnological issues facing the development of the electro-chemical gene delivery system.

3. Electrochemical Protocols for Delivering DNA

Over the past three years we have assessed several electro-chemical avenues for triggering the release of nucleic acids forelectrode surfaces. Different surface characterization techniqueshave been employed to characterize the desorption process.These include X-ray photoelectron spectroscopy (XPS), re¯ec-tion infrared spectroscopy (IR), energy dispersive X-ray analysis(EDX), electrochemical quartz crystal microbalance (EQCM),and voltammetry. EQCM has been particularly useful forstudying the electrochemical release, as it offers in situ moni-toring of mass changes associated with the desorption process.The XPS, EDX, and IR characterizations rely on monitoring the`disappearance' of DNA-related signals after the removal process(e.g., the DNA XPS nitrogen signal). Cyclic voltammetry andsquare-wave voltammetry are very useful for assessing the barrierproperties of the DNA layer, in connection to a proper redoxmarker (e.g., ferrocyanide).

Past activity on DNA electrochemistry [9] has paved the wayto our new electrically driven protocol for delivering nucleicacids. In particular, adsorptive-stripping voltammetric andpotentiometric protocols, developed ®rst for mercury electrodes[12] and then with solid electrodes [13], have demonstrated theability to modulate (accumulate=strip) the interfacial propertiesof DNA and RNA.

The basic concept of the new electrochemical routes is tocon®ne the nucleic acid molecules onto the surface, and to expelthem on demand under potential control (Fig. 1). These protocolsmake use of the negative charge of DNA for modulating andregulating its interfacial properties. Potentials more positive than

the PZC are used for con®ning the nucleic acid molecules to thesurface (Fig. 1A), while negative potentials are employed fortheir controlled release (Fig. 1B). Such electrostatic repulsion ofthe negatively charged nucleic acid molecules from the surface ofa DNA-modi®ed gold electrode is illustrated from the EQCM(frequency vs. potential) data of Figure 2. Such pro®le indicatesthe ability of tuning the desorption potential for controlling theamount of released DNA [14]. Low negative potentials can thusbe used for a prolonged and sustained DNA delivery.

We also demonstrated the electrochemically triggered releaseof nucleic acids from gold surfaces, based on the cathodicdesorption of thiolated DNA layers (i.e., a reductive cleavage ofthe sulfur-gold bond) [15]. Unlike an analogous desorption ofself-assembled alkanethiols layers (SAM) that employed alkaline(pH> 11) medium, the removal of the thiolated nucleic acid wascarried out in a physiological buffer (pH 7.4). A 350 bp longsegment of dsDNA was employed in connection to a 25 mmdiameter gold microelectrode. Figure 3 displays XPS spectra of adsDNA-coated crystal before (A) and after (B) applying apotential of 71.30 V for 10 min. The removal of the nucleic acidfrom the surface is indicated from the disappearance of the DNAnitrogen N1s peak (around 400 eV) and the dramatic increase ofthe Au 4f XPS signal (re¯ecting the greater exposure of thegold surface). EQCM measurements indicated the removal of466 ng dsDNA that correspond to surface densities of4.8610712 mol=cm2. Such measurements indicated also that theDNA-modi®ed crystals respond rapidly to the potential step (withcomplete removal of the dsDNA requiring 170 s).

Such potential control was also used to release dsDNA fromcarbon paste microelectrodes [16]. This protocol relied on theelectrostatic adsorption of the nucleic acid onto an electro-chemically pretreated carbon microelectrode (280 mm diameter),followed by a reductive desorption at 71.20 V (vs. Ag=AgCl).Square-wave voltammetric `blocking' experiments indicated acomplete removal of the adsorbed layer following 3 min atÿ1.20 V. These electrochemical observations were supported bymonitoring the DNA nitrogen and phosphorous peaks of EDXsurface analysis. Large surface-area (highly porous) carbonelectrodes are currently being assessed in our laboratory forincreasing the surface loading and hence the release ef®ciency.Particular attention is given to the integration of a reticulatedvitreous carbon (RVC) `plug' into a needle-type electrodecon®guration. Figure 4 demonstrates the use of adsorptivestripping potentiometry for measuring the DNA released from theRVC needle electrode. For this purpose, the DNA-modi®edelectrode was transferred to a blank solution in which the removalproceeded; a second carbon paste electrode (immersed in thissolution) was used for monitoring the released DNA. A well-de®ned guanine oxidation peak is thus observed after steppingthe potential of the DNA-modi®ed RVC electrode to 71.2 V for10 min (A). No peak is observed for a control experiment

Fig. 1. Schematic drawing showing the electrochemical (desorptive)removal of DNA layers from electrode surfaces.

Fig. 2. Effect of the desorption potential upon the EQCM frequencyresponse of a dsDNA-coated gold crystal. (See [14], for details.)

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employing a potential of � 0.5 V(B). The stripping potentio-metric peak of Figure 4A corresponds to 31 mg of released DNA.

Initially, we intended to exploit doping=undoping processes ofconducting polymers for the electrical release of DNA, in amanner analogous to Miller's electrochemical removal of smallanions [6]. We demonstrated that various oligonucleotides andchromosomal DNAs can be incorporated as dopants into poly-pyrrole (PPy) networks [17]. While the incorporation of nucleic

acids is similar to that of small inorganic anions, such largedopants could not be readily expelled from the polymer network(i.e., exchanged with the electrolyte anion). The EQCM data ofFigure 5, recorded during repetitive cyclic voltammograms in ablank electrolyte solution, clearly indicate that electrochemistryof the PPy=nucleic-acid ®lms is dominated by the movement ofthe electrolyte cation, i.e., insertion and ejection of the sodiumion (B), and not by doping=undoping process observed for thePPy=Cl7 coating (A). (Note the opposite directions of thefrequency response during the reduction process.) Such irrever-sible entrapment precludes the use of conducting-polymermodi®ed electrodes for gene delivery applications.

As desired for facilitating the transfer of DNA into cells, weexamined the on-demand electrical release of lipid-DNAcomplexes. For this purpose, we use complexes prepared bymixing the anionic nucleic acid with positively chargedDOTMA-containing liposomes. Such complexes possess anet negative charge and accordingly can be con®ned or removedfrom the surface under potential control. For example, Figure 6displays typical EQCM time pro®les for different modi®ed andunmodi®ed crystals upon stepping the potential to 71.0 V(vs. Ag=AgCl). The coated crystals display an increase oftheir resonance frequency upon the potential step. Differentdesorption kinetics and frequency changes are observed for theDNA-(B) and lipid-DNA (C) coated surfaces. Note also

Fig. 3. XPS spectra in the N 1s region (a) and Au 4f region (b) of adsDNA-coated gold surface before (A) and after (B) applying the de-sorption potential of 71.30 V for 10 min. (From [15].)

Fig. 4. Stripping potentiometric measurements of ssDNA released fromthe RVC-needle electrode using a second carbon electrode. (A) Afterholding the needle electrode for 10 min at 71.2 V; (B) same as A butusing a potential of � 0.5 V. Stripping detection with 5 min accumulationat � 0.5 V, and stripping current of � 5mA.

Fig. 5. EQCM frequency response of PPy=Cl7(A) and PPy=oligo(dG)20

(B) modi®ed electrodes (in a 1 M NaCl solution) during cyclic voltam-metric scans over the 70.5 V to � 0.5 V range. (From [17].)

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the absence of response for the control experiment using theuncoated crystal.

We are currently exploring new potential±time waveforms fora prolonged and controlled release of DNA. These includerepetitive normal-pulse or square-wave pulsations (of differentamplitudes, durations, and intervals), with no release at the initialpotential, as desired for an `on-off' mechanism. We are alsoassessing a simultaneous electroporative permeabilization of themembrane for facilitating transport through the cell membrane.The electrochemical removal will thus be integrated with anelectroporation of the cell membrane, on a single miniaturizedprobe, for an all-electrical release=access.

4. Conclusions

We have described several electrochemical protocols forreleasing DNA from gold and carbon electrodes under potentialcontrol. The miniaturization, surface-tailoring, safety, simplicity,convenience, automation, and low-cost advantages of electro-chemical systems, coupled with the lack of immune reactionsand the electrically modulated interfacial properties, make theproposed route a very attractive gene-delivery alternative. Suchuse of ultramicroelectrodes holds great promise for delivering thegenetic material to speci®c locations at selected times. The newelectrochemical route thus represents a useful addition to thearsenal of nonviral delivery vectors. In addition to gene therapy,the new electrochemical protocols should ®nd other importanttechnological applications, including localization of DNA ontotiny surfaces, the design of `active' scanning probe tips,

controlled introduction of DNA to various analytical systems, orfundamental studies related to DNA interactions or conductivity.

We are currently examining additional tools for obtaining newinsights into the release process, including dual-electrode (e.g.,ring-disk) and SECM protocols that offer direct detection of thereleased nucleic acid. Future efforts would require proper atten-tion to the challenges of higher ef®ciency and transport across thecell membrane and into the nucleus. Assessment of the chemicaland biological integrity of the released DNA, including possiblestructural or redox changes, would also be required. The reali-zation of new biological and technological applications based onthe electrochemical delivery route will also be pursued in the nearfuture.

5. Acknowledgements

The work was supported by a grant from the National Instituteof Health (NIH Grant No. RR14549-01). All members of theauthor's DNA team are gratefully acknowledged for their majorcontributions to the DNA delivery studies.

6. References

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Fig. 6. Time course of the EQCM frequency change of DNA-(B) andlipid-DNA (C) coated crystal, as well as of the uncoated (A) crystal uponstepping the potential to 71.0 V. Arrows indicate points of switching thepotential from � 0.4 to 71.0 V (vs. Ag=AgCl).

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