DNA electrochemical biosensors for environmental monitoring. A review

ANALYTICA CHIMKA ACTA ELSEVIER

Analytica

DNA electrochemical

Chimica

Acra 347 (1997) 1-8

biosensors for environmental A review’

monitoring.

J. Wang*, G. Rivas2, X. Cai, E. Palecek3, P, Nielsen4, H. Shiraish?,

C. Parrado7, M. Chicharro’, Department Received

P.A.M. Farias’, ES. Valera”, M.N. Flair

of Chemistry and Biochemistry, 12 September

N. Dontha, D. Luo6, D.H. Grant”, M. OZSOZ’~,

New Mexico State Universi@, Las Cruces, NM 88003, USA

1996: received in revised form 26 November

1996; accepted 2 December

1996

Abstract DNA sensing protocols, based on different modes of nucleic acid interaction, possess an enormous potential for environmental monitoring. This review describes recent efforts aimed at coupling nucleic acid recognition layers with electrochemical transducers, It considers DNA hybridization sensors for sequences related to microbial or viral pathogens, and DNA-modified carbon electrodes for monitoring low molecular weight priority pollutants interacting with the surfaceconfined DNA. Carbon strip or paste electrode transducers, supporting the DNA recognition layer, are used with a highly sensitive chronopotentiometric transduction of the DNA analyte recognition event. Factors influencing the performance of these new environmental biosensors are discussed, and their environmental utility is illustrated. While the use of DNA biosensors is at a very early stage, these and similar developments are expected to have a profound effect on environmental analysis. Keywords: DNA; Biosensors: Carbon transducers

Environmental

monitoring;

Nucleic

acid recognition;

*Corresponding author. Tel.: +1 505 646 2502; fax: +1 505 646 6033. ‘Presented as a Plenary Lecture at the 2nd Workshop on Biosensors and Biological Techniques in Environmental Analysis, September, 1996, Lund, Sweden. ‘Permanent address: Department0 Fisico Quimica, Universidad National de Cbdoba, Grdoba, Argentina. “Permanent address: Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Bmo, Czech Republic. ‘Permanent address: Center for Biomolecular Recognition, IMBG, Department of Biochemistry B, The Panum Institute, Blegdamsvej 3c, DK 2200 Copenhagen, Denmark. ‘Permanent address: Department of Chemistry, Ritsumeikan University. Kusatsu, Japan. 0003.2670/97/$17.00 ,I:; 1997 Elsevier Science B.V. All rights reserved P/I SOOO3-2670(96)00598-3

Hybridization:

Pollutants:

Pathogens;

Water quality;

‘Permanent address: Department of Chemistry, South-Central Institute for Nationalities, Wuhan 430074, China. ‘Permanent address: Department0 Quimica Analitica, Facultad de Ciencias Quimicas, Universidad Complutense. E-28040, Madrid, Spain. ‘Permanent address: Department0 Quimica Analitica y Analisis Instrumental, Universidad Aut6noma de Madrid, Madrid. Spain. ‘Permanent address: Department0 Chemistry, Pontificia Universidade Catolica do RJ, Rio de Janeiro, Brazil. “Permanent address: Institute of Chemistry. University of the Philippines, Philippines. “Permanent address: Department of Chemistry. Mount Allison University, Sackville, Canada, EOA 3C0. “Permanent address: Faculty of Pharmacy, Ege University, Izmir, Turkey.

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1. Introduction Biosensors hold great promise for the task of environmental monitoring and control [l-3]. The specific interaction of an immobilized biological layer with target pollutants provides the basis for analytical devices for laboratory or field use. While environmental applications of biocatalytic (enzyme) and immunosensors have greatly increased during the 1990s [4], little attention has been given to the development of nucleic acid probes for environmental surveillance. Advances in molecular biology and biotechnology have set the stage for exciting possibilities for DNA-based environmental biosensors. Unlike enzymes or antibodies, nucleic acid recognition layers are very stable, and can be readily synthesized or regenerated for repeated use. Such recognition layers add new and unique dimensions of specificity to our arsenal of environmental biosensors, and should play a major role in future environmental analysis. The aim of the present paper is to review recent research efforts in the area of DNA electrochemical sensors at the author’s laboratory. Various new strategies for environmental DNA biosensors will be examined, including the hybridization detection of nucleic acid sequences from infectious microorganisms and monitoring of small pollutants interacting with the immobilized DNA layer. Several carbon electrode transducers have been used to support the nucleic acid recognition layer, and to transduce the DNA-analyte interactions by means of a highly sensitive constant-current chronopotentiometric operation. As should prove useful for on-site environmental applications, the new DNA detection schemes have been combined with microfabricated thick-film carbon strips and easy-to-use miniaturized chronopotentiometric analyzers. Besides their monitoring capability, the new devices hold great promise for elucidating the interactions of pollutants with DNA, and hence may shed useful insights into the toxicological action of priority pollutants.

2. Chronopotentiometric

hybridization

biosensors

The detection of specific DNA sequences provides the basis for detecting a wide variety of microbial and viral pathogens. Traditional methods for DNA sequen-

Chimica Acta 347 (1997) 1-8

cing, based on the coupling of electrophoretic separations and radioisotopic (32P) detection, are labor intensive and time consuming, and are thus not well suited for routine and rapid environmental analysis, particularly for field screening tasks. Newly developed hybridization biosensors for the detection of DNA sequences may greatly reduce the assay time and simplify its protocol. Such fast on-site monitoring schemes are required for quick preventive action by water utilities, as would be needed to protect consumers during sudden contamination events. The basis for these nucleic acid hybridization devices is DNA base pairing. Namely, they rely on the immobilization of a short (20-40-mer) synthetic oligomer (the single-stranded DNA (ssDNA) “probe”), whose sequence is complementary to the sought-for target. Exposure of the sensor to a sample containing the target results in the formation of the hybrid on the surface. Electrochemical or optical monitoring of this duplex formation can result in a useful transducer signal. DNA hybridization biosensors, based on electrochemical transduction of hybridization events [5], hold great promise for the task of environmental monitoring. Such devices couple the high specificity of DNA hybridization reactions with the excellent sensitivity and portability of electrochemical transducers. For this purpose, the formation of the hybrid is commonly detected by exposing it to a solution of an electroactive indicator (e.g., a redox-active cationic metal complex), that strongly and yet reversibly binds to the hybrid (Fig. 1). The increased electrochemical response due to the indicator associated with the newly formed surface duplex thus serves as the hybridization signal. This section describes the characterization, optimization and environmental utility of new biosensing hybridization protocols for screening pathogens, based on carbon electrode transducers operated in the chronopotentiometric mode. We have been developing DNA hybridization biosensors for the detection of microorganisms in environmental samples. Particular attention was given for a rapid and simple assay for detecting the waterborne pathogen Cryptosporidium [6]. This protozoan microorganism, which exists in water supplies as a highly resilient, dormant and capsular form called oocyst, is recognized as a major cause of diarrhoeal disease in humans [7]. Over 11000 children die every day in

.I. Wang et al. /Analytica

?

Chimica Acta 347 (1997) 1-H

Probe Immobilization

0.5 sN

b

a

C

A(J_ Target hybridization

I 0.4

I -0.2 J

POTENTIAL (V)

indicator bindingl Transduction

Fig. 1. A scheme of sequence-specific hybridization detection using an electrochemical DNA biosensor. Ox represents a redoxactive indicator that associates with the surface duplex following the hybridization event.

developing countries from such diseases. Numerous outbreaks of Cryptosporidium infection have been reported in the United States, including a highly publicized case in Milwaukee involving the death of 104 people and the illness of 400 000 [8]. At this stage, no analytical method satisfactorily detects the Cryptosporidium pathogen, no drug therapy is effective in treating its infection and no water disinfecting procedure destroys Cryptosporidium oocysts. Our DNA Cryptosporidium biosensor relies on the immobilization of a 38-mer oligonucleotide probe, unique to the Cr)?ptosporidium parvum DNA, onto the carbon-paste transducer [6]. Fig. 2 displays the chronopotentiometric response of the sensor for an untreated drinking water sample spiked with increasing levels of the target Cryptosporidium sequence (between 0.5 and 1.5 pg ml-‘, (a)-(c)). The Co(phen)iindicator response for the unspiked sample, shown as the dotted line, reflects some association of the marker with the probe alone. The increased area of the indicator peak upon its stronger binding with the

Fig. 2. Detection of Cryptosporidium DNA sequences in drinking water. Chronopotentiograms for Co(phen):’ at the 3%mer probe modified carbon-paste electrode after 3 min hybridization with the 38.mer Cryptosporidium target with increasing concentration: 0.5 (a), 1.0 (b) and 1.5 (c) pg ml-‘. Dotted lines represent the response of the unspiked water sample. Details of the probe immobilization, hybridization step and indicator detection can be found in [9].

surface hybrid serves as the hybridization signal. Very short (3 min) hybridization periods give rise to welldefined hybridization signals at pg ml-’ concentrations of the Cryptosporidium target, while longer (2030 min) ones permit ng detection limits. A similar performance was obtained using microfabricated carbon strips in connection with a hand-held computerized chronopotentiometric analyzer. Such computerized chronopotentiometric transduction of hybridization events - involving passing of a constant current and monitoring the resultant change in potential as a function of time - couples high sensitivity with effective background compensation and data smoothing. The high signal-to-background characteristics of the chronopotentiometric operation thus result in improved performance compared to analogous voltammetric schemes [9]. Despite the very low detection limits of this DNA hybridization/chronopotentiometric operation, its coupling with an amplification step would be essential for environmental applications requiring reliable detection at the single-cell level. Compact microfabricated polymerase chain reaction (PCR) amplification units, developed recently for point-of-care clinical testing [lo], could be readily integrated with the new electrochemical sensing devices. Such compact integrated amplification/sensing systems would

4

J. Wang et al./Analytica

,E

0 1 2 3 CONCENTRATION(pg/mL)

I

0.2 SN

C

b

a

0.2

-d.2

POTENTIAL (V) Fig. 3. Detection of E. coli DNA sequence. Chronopotentiograms at the probe modified screen-printed carbon for Co(bpy);+ electrode after 2 mm hybridization with the E. coli target sequence with increasing concentration in 1.O pg ml-’ steps (a)-(c), along with the resulting calibration plot. Hybridization solution, phosphate buffer (20 mM, pH 7.0) containing 0.5 M NaCl. Other conditions as in [9].

offer on-site testing capabilities, in connection with a simple freeze-and-thaw procedure aimed to release the DNA from the oocysts. The portable system would thus provide rapid screening capability essential for quick corrective action by water utilities. Similar hybridization/chronopotentiometric schemes are currently being developed for other pathogens, such as Escherichia coli or Giardia [l 11. For example, Fig. 3 displays chronopotentiograms for the Co(bpy):+ indicator, obtained at the probe-coated microfabricated strip in the presence of increasing levels of the E. coli DNA target. The ultimate goal of this research is to design an array of microelectrodes on a chip (each electrode being coated with a different probe) for the simultaneous field monitoring of multiple pathogens in water supplies. Nucleic acid recognition was also employed in our laboratory in developing biosensors for screening sequences speci-

Chimica Acta 347 (1997) I-8

fit to HIV-l [ 121 or Mycobacterium tuberculosis [ 131 DNAs. The development of such hybridization biosensors requires careful control of the assay protocol. Systematic optimization of the probe-immobilization step, the hybridization event, and the indicator binding and detection is essential for meeting the requirements of high sensitivity, specificity, and speed. Particular attention is given to factors influencing the kinetics and efficiency of the hybridization event, including hybridization time, ionic strength, presence of accelerators, probe length or sequence, hybridization temperature, and operating potential [l I-141. We have also evaluated various probe immobilization schemes, including self-assembly of thiol-functionalized probes, binding of biotin-derivatized oligonucleotides with an avidin-coated surface, and adsorptive immobilization and have found the adsorptive immobilization to be the most useful. Adsorption conditions, leading to an optimal arrangement/conformation of the probe layer for most efficient hybridization, have otenalso been explored. We also screened numerous p+ tial redox indicators, and identified Co(phen), and to be most effective (as was similarly Co(bpy):+ reported by Mikkelsen [5]). The success of these markers is the net result of their low potential electrochemical response and effective binding with the probe-target duplex. While chemical (urea) or thermal treatments can be used to regenerate the singlesingle-use sensors are stranded probe, “one-shot” preferred for on-site environmental applications. The relative standard deviations for equivalently prepared sensors are typically below 10%. Additional improvements in the specificity have been achieved by using new probes based on peptide nucleic acids (PNA) [ 151. These DNA mimics represent a drastic departure from DNA structure, with the replacement of the sugar-phosphate backbone with a neutral pseudopeptide chain (Fig. 4). By avoiding the charged phosphate groups while maintaining a proper interbase spacing, these synthetic oligomers result in much more stable PNA-DNA duplexes compared to DNA-DNA ones, as indicated by the higher melting temperatures. In view of their very strong affinity for complementary DNA sequences, PNA-derived sensors offer greatly improved distinction between cloeffective related sequences (including sely discrimination against single-base mismatches), and

J. Wang et al./Analytica

r--------

B--fo iNg-JNY 0

;

j B-f0 “-YO

_HN-N-E o

iNFN-y 0 ,..-

j

--.-..-I

Repeat

unit

(B = Base) Fig. 4. Structure of peptide nucleic acid (PNA).

afford greater latitude in the selection of experimental conditions, including efficient hybridization in low ionic-strength solutions or at elevated temperatures, as well as the use of short probes.

3. DNA biosensing

of pollutants

Environmental monitoring can benefit from different modes of DNA recognition besides base-pairing hybridization events. In particular, various interactions of an immobilized dsDNA layer with low molecular weight pollutants can be utilized for detecting these substances. Since the toxic action of numerous pollutants (e.g. carcinogens and mutagens) is related to their interaction with DNA, it is logical to exploit these events for designing new environmental biosensors. The deliberate alteration of a transducer surface, through the judicious immobilization of a nucleic acid recognition layer, can thus form the basis for new sensing devices and provide solutions to various environmental problems. So far little work has been performed on the direct detection of pollutants in a biosensor format, i.e., where a real-time signal is obtained. Pandey and Weetall [ 161 reported on an evanescent wave biosensor for aromatic compounds based on their intercalative association with an immobilized dsDNA layer and displacement of a highly fluorescent marker. The decreased flow-injection response of the ethidium bromide indicator in the presence of the target aromatic pollutant served as a measure of the pollutant (based on the “intercalative binding strength”). Palecek’s group [ 17,l S] has shown that nucleic acid modified mercury drop electrodes can be prepared by immersing the electrode in a droplet of the modifier solution, and allowing the biomacromolecule to

Chimica Acra 347 (1997) 1-N

5

adsorb strongly and irreversibly at the surface. Transfer of the modified electrode into another solution was used for voltammetric studies of DNA interactions with drugs and proteins. Research in our laboratory has centered on the chronopotentiometric transduction of various pollutant-DNA interactions at nucleic acid modified carbon electrodes for the biosensing of toxic substances. The environmental utility of these new signal-generation schemes is described in this section. We have been exploring three schemes using analyte-DNA interactions for detecting toxic pollutants. These include the preferential accumulation of electroactive pollutants by the immobilized dsDNA layer, changes in the intrinsic oxidation signal of the nucleic acid coated electrode induced by the DNA-pollutant binding event, and the detection of nonelectroactive analytes via the competitive binding and displacement of a redox marker from the surface-bound DNA. Aromatic amines constitute a very important class of environmental pollutants. We exploited their binding with the immobilized dsDNA layer, and their inherent electroactivity in designing a new affinity electrochemical biosensor for these pollutants [ 191. The enhanced sensitivity accrued from the DNA collection process has thus been coupled to a new dimension of selectivity provided by the structural requirements for such accumulation. Nanomolar detection limits were obtained after 10 min accumulation. Fig. 5 demonstrates the applicability of the DNA sensor to the analysis of an untreated groundwater sample. With a short (3 min) preconcentration time, the sensor yields well-defined chronopotentiometric oxidation peaks following the two additions of 4x lop7 M 2-anthramine ((B) and (C); II). The natural constituents of the groundwater sample did not yield any detectable signal (A). Peak I, at ca. + 1.O V, corresponds to the oxidation of the DNAguanine residue. Note the gradual decrease of this anodic guanine peak in the presence of increasing levels of the aromatic amine. Such lowering of the DNA intrinsic response is attributed to changes in the accessibility of the guanine moiety to the surface upon binding of the aromatic amine to the dsDNA. Unlike voltammetric transduction modes, the chronopotentiometric operation results in a well-defined guanine oxidation signal, despite its extreme peak potential 1201.

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J. Wang et al. /Analytica

Chimica Acta 347 (1997) l-8

mNH2

I

I

1.2

0.6

POTENTIAL lV1

2-Anthramine t

(%I

0.5 SN

CONCENTRATION

i

(ng/mL)

Fig. 6. Chronopotentiometric response of the DNA carbon paste biosensor for increasing levels of dimethylhydrazine in I .2 pg 1-i steps (b)-(l), along with the resulting calibration plot. Also shown (a) is the response of the sensor prior to the hydrazine addition. Interaction time, 10 min. (See [21] for details.)

ik

I

I

1.15

0.50

POTENTIAL (V) Fig. 5. Chronopotentiometric response of the DNA carbon paste biosensor for a groundwater sample spiked with 0 (A), 4x lo-’ M (B) and 8x 10m7 M (C) 2-anthramine. Accumulation time, 3 min.

Such ability of chronopotentiometric carbon transducers to conveniently measure the DNA-guanine anodic response opens the door to a new class of solid-state environmental sensors. Changes in the intrinsic guanine signal of DNA modified electrodes, associated with chemical, structural, or conformational variations of the immobilized probe (e.g. Fig. 5, I (A)-(C)), may thus be exploited for environmental monitoring, provided that they display a useful concentration dependence. For example, we developed a highly sensitive DNA biosensor for the detection of hydrazine compounds [21]. The exposure of the dsDNA modified electrodes to such compounds results in diminution of the guanine peak due to the formation of N7-methylguanine. (Note that the oxidation of the guanine moiety proceeds through the N7 position [17].) Such suppression of the guanine response results in a well-defined concentration dependence and offers convenient quantitation of trace levels of different hydrazines. For example, the gradual decrease of the DNA-guanine signal in

the presence of increasing levels of dimethylhydrazine (in 1.2 ng ml-’ steps) leads to a distinct calibration plot (Fig. 6). Applicability to analysis of untreated groundwater and river-water samples was illustrated. Change in the intrinsic DNA response may be used also for detecting physical damage to DNA. There is an increasing interest in the development of assays for measuring DNA radiation damage [22]. Early studies by Palecek [23] and Nurnberg [24] illustrated the utility of polarography at mercury drop electrodes for assessing gamma and ultraviolet radiation damage to native DNA. This approach relies on the sensitivity of the electrochemical response of DNA to minor changes in the conformation and structure of the DNA double helix induced by the radiation dose. Current efforts in our laboratory are focusing on exploiting changes in the anodic DNA-guanine signal for developing microfabricated sensor chips for the detection of radiation damage [25]. For example, Fig. 7(A) displays the chronopotentiometric response of a screen-printed carbon strip electrode before (a) and after (b) irradiating the dsDNA solution with ultraviolet light. Similarly, Fig. 7(B) illustrates chronopotentiograms for a dsDNA modified strip electrode prior to (a) and after (b) 5 min irradiation. A distinct diminution of the DNA-guanine peak is observed following the irradiation of the solution-phase and surface-bound DNA. Such a decrease may be ascribed to the photoconversion of the guanine residue to 2,6-

J. Wung et al. /Analytica

a 1;

I

Chimica Acta 347 (1997) 1-R

1

date the specificity of such contaminant-DNA binding events, or explore DNA structural changes. Early polarographic studies [ 17,271 have illustrated the potential of electrochemistry for exploring DNA interactions with various biomolecules.

2.0 SN

b

4. Conclusions

.__.__-___ ....-_._____

-.

B

a

r 1.1

1

0.6

POTENTIAL (V) Fig. 7. Chronopotentiograms for solution-phase (A) and surfaceconfined (B) dsDNA prior to (a) and after (b) exposure to ultraviolet radiation. (A) Adsorptive stripping potentiometric measurements at the screen-printed carbon electrode, with I min pretreatment at +I.8 V and 2 min adsorptive accumulation at +0.2 V. Sample-lamp distance, 2.5 cm; wavelength, 254 nm: solution, 5 ug ml..’ dsDNA in 0.2 M sodium acetate buffer (pH S.0). (B) dsDNA-coated strip electrode, covered with a 50~1 droplet of 20 mM phosphate buffer, and placed 0.4 cm from the lamp. The dsDNA was immobilized on the pretreated strip via adsorptive accumulation (as in A). Radiation time, 15 min (A) and 5 min (B). Constant current, +4 PA.

diamino-4-hydroxy-5formamidopyrimidine [26]. A similar strategy is being explored for the screening of chemical agents, capable of inducing DNA damage. Besides their sensing utility, the DNA-coated carbon transducers can offer useful information regarding the dynamics and strength of pollutant-DNA interactions, and may thus shed useful insights into the toxic action of various pollutants. For example, such devices can probe in real time the kinetics of DNA reactions with chemical damaging agents, provide useful information on the relative strength of the association of aromatic compounds with DNA, eluci-

While the use of nucleic acid recognition layers is still in the embryonic stage, DNA biosensors offer many great prospects for environmental monitoring and control. Various possibilities of using such devices have been discussed in the previous sections, with special attention given to sequence-specific hybridization detection of viral or bacterial pathogens and to the sensing of low molecular weight pollutants interacting with the surface-bound DNA. Such DNA-based devices thus add new dimensions of selectivity to the arsenal of biosensors proposed for environmental analysis. Such realization of solid-state DNA sensing devices is attributed to the judicious coupling of solidstate transducers, proper selection and immobilization of the nucleic acid layer, and efficient transduction of the DNA recognition event. Yet, the pioneering studies of nucleic acid electrochemistry at mercury electrodes [ 171 did pave the way for several of the above developments. Further improvements in the biosensor protocols are needed for addressing the challenges that hinder the routine environmental utility of these devices. Besides the introduction of new sensing/hybridization schemes (e.g., based on enzyme labels for amplifying the hybridization signal, via direct electrochemical monitoring of the hybridization reaction without redox indicators, and use of strand displacement or sandwich hybridization), it is essential to develop improved schemes for immobilizing the DNA layer, and for obtaining a better understanding of the conformation and arrangement of such a surface layer for maximum accessibility of target analytes. Additional sensing possibilities may result from different modes of pollutant-DNA interactions. Fast detection of DNA damage may be accomplished using immobilized plasmid DNAs, based on changes in their oxidation signal at carbon electrodes [28]. Advances in microfabrication and microinstrumentation. involving micromachined chips for sample handling and multi-

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ple hybridization sensing (in connection with high density arrays of individually addressed micromolecules), and on-chip multichannel-potentiostats, should further facilitate the realization of on-site DNA environmental testing. There is no doubt that these and similar developments would result in low-cost, and yet powerful, devices capable of operation in a field environment, and would lead to a better protection of the public from potential contamination events.

Acknowledgements J.W. acknowledges the financial support from the Department of Energy (DOE) and the DOE-WERC program. E.P. acknowledges financial support from the US-Czechoslovak Science and Technology Program (Grant No. 930 11) and from the Grant Agency of the Czech Republic (Grant No. 20419611680). G.R., H.S., D.L., C.P., M.Ch., P.A.M.F., ES.V, D.H.G. and M.O. acknowledge fellowships from CONICET (Argentina), Ritsumeikan (Japan), The State Education Commission (P.R. China), DGICYS: PR95310 (Spain), UAM and CAM (Spain), CNPq (Brazil), UP and DOST-ESEP (Philippines), Mount Allison University (Canada) and Scientific and Technical Research Council (Turkey), respectively.

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