Biosensors and Bioelectronics 18 (2003) 529 /540 www.elsevier.com/locate/bios

New highly sensitive and selective catalytic DNA biosensors for metal ions Yi Lu *, Juewen Liu, Jing Li, Peter J. Bruesehoff, Caroline M.-B. Pavot, Andrea K. Brown Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Received 11 May 2002; received in revised form 7 November 2002; accepted 12 November 2002

Abstract While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a ‘negative selection’ strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring. # 2003 Published by Elsevier Science B.V. Keywords: Catalytic DNA; DNAzyme; DNA sensors; Metal biosensors; Fluorescent sensors; Colorimetric sensors

1. Introduction Detection and quantification of metal ions are important in many applications including household and environmental monitoring, waste management, developmental biology and clinical toxicology. Laboratory techniques routinely used for metal ion analysis, such as atomic absorption spectrometry (Bannon et al., 1994; Parsons and Slavin, 1993; Tahan et al., 1994),



This paper has received the Runner-up award for the Biosensor and Bioelectronics Award at the Seventh World Congress on Biosensors (Biosensor 2002), held May 15 /17, 2002 in Kyoto, Japan. * Corresponding author. Tel.: /1-217-333-2619; fax: /1-217-3332685. E-mail address: [email protected] (Y. Lu).

inductively coupled plasma mass spectrometry (Aggarwal et al., 1994; Bowins and McNutt, 1994; Liu et al., 1999), anodic stripping voltammetry (Feldman et al., 1994; Jagner et al., 1994), X-ray fluorescence spectrometry (Blank and Eksperiandova, 1998; Ellis et al., 1998; Toeroek et al., 1998) and microprobes (Carpenter and Taylor, 1991; Gordon et al., 1990; Rindby, 1993; Rivers et al., 1992; Sutton et al., 1995, 1994; Thompson et al., 1988; Wu et al., 1990) require sophisticated equipment, sample pretreatment, or skilled operators. Most techniques can detect the total amount of metal ions. However, several studies have established that only certain oxidation states of water-soluble or bioavailable metal ions pose the most risk to human health and the environment. For example, Cr(III) is an essential nutrient required in insulin action and sugar and fat

0956-5663/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0956-5663(03)00013-7

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metabolism, while Cr(VI) is believed to be highly toxic and carcinogenic (McCullough et al., 1999). Therefore, simple, rapid, inexpensive, selective, and sensitive methods that permit real-lime detection of bioavailable metal ions in their different oxidation states are very important in the assessment of concentration, speciation, and stability of these metal ions (Razek et al., 1999), In addition, due to the dangers that certain toxic metal ions may pose to operators, remote sensing devices are desirable (Arnold, 1989, 1990, 1992). Therefore, portable, sensitive and selective sensors for both beneficial and toxic metal ions are needed. To design effective metal sensors, several innovative methods have been developed (Arnold and Solsky, 1986; Blake et al., 1998; Da Silva and Oliveira, 1999; Engstrom and Karlberg, 1996; Herdan et al., 1998; Hirayama et al., 2000; Lin et al., 1994; Lin and Burgess, 1994; Schweyer et al., 1996; Shamsipur et al., 2000; Szurdoki et al., 1995; Vlasov et al., 1997; Walker et al., 1994; Wang and Wasielewski, 1997; Westhoff et al., 1999; Wrobel et al., 1997). Sensors based on organic chelators (Bandyopadhyay et al., 2000; Burdette et al., 2001; Czarnik, 1994, 1995; de Silva et al., 1997; Fabbrizzi et al., 1998; Fahmi and O’Halloran, 1999; Grandini et al., 1999; Hennrich et al., 1999; Hirano et al., 2000a,b; Koike et al., 1996; Mahadevan et al., 1996; Ramachandram and Samanta, 1998; Rurack et al., 2000; Tsien, 1993; Walkup et al., 2000; Watanabe et al., 2001; Winkler et al., 1998; Yoon et al., 1997), organic polymers (Wu et al., 2000), proteins (Blake et al., 2001; Bonagura et al., 1999; Bontidean et al., 1998; Hitomi et al., 2001; Miyawaki et al., 1997; Thompson and Jones, 1993; Thompson et al., 1998a,b, 1999; Wittmann et al., 1997), peptides (Deo and Godwin, 2000; Godwin and Berg, 1996; Imperiali et al., 1999; Walkup and Imperiali, 1996, 1997, 1998), or cells (Kress-Rogers, 1997; Ramanathan et al., 1998) have emerged as powerful tools toward achieving the above goals (McGown and Nithipatikom, 2000; Oehme and Wolfbeis, 1997; Oldham et al., 2000). While remarkable progress has been made in developing fluorosensors for metal ions such as Ca(II) (Adams et al., 1997; Lew et al., 1982; Miyawaki et al., 1997; Tsien, 1989, 1993, 1999), Zn(II) (Budde et al., 1997; Burdette et al., 2001; Fabbrizzi et al., 1996; Fahmi and O’Halloran, 1999; Frederickson et al., 1987; Godwin and Berg, 1996; Hirano et al., 2000a,b; Koike et al., 1996; Mahadevan et al., 1996; Thompson et al., 1998a,b; Walkup et al., 2000; Walkup and Imperiali, 1996; Zalewski et al., 1993), and Pb(II) (Deo and Godwin, 2000), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. For example, the widely used Ca(II) sensor, Fura-2, binds Zn(II) and Cd(II) 102-, and 105-fold more tightly, respectively, than it binds Ca(II) (Haugland, 1999). This and other similar Ca(II) sensors have been used successfully to monitor Ca(II) in

the presence of relatively low concentrations of free Zn(II) and Cd(II) in cells and have revolutionized the study of cell physiology (Lew et al., 1982; Tsien, 1989, 1993, 1999). However, these sensors may not be useful for detecting Ca(II) in samples containing similar concentrations of other interfering metal ions, such as in environmental monitoring (Thompson et al., 1998a). These results illustrate perhaps the biggest challenge in fluorosensor research, i.e. the design and synthesis of sensors capable of specific and strong binding of a metal ion of choice and not other competing metal ions. Since the knowledge of the design of metal ion specific sensors is limited, searching for sensors in a combinatorial way is particularly attractive. The concept and advantages of combinatorial searches for metal ion sensors have been demonstrated recently by several groups (Bergbreiter et al., 1999; Li and Lu, 2000; Szurdoki et al., 2000; Wu et al., 2000). Among these methods, in vitro selection of DNA/RNA from a library of 1014 /1015 random DNA/RNA sequences offers considerable possibilities (Breaker, 1997a,b; Breaker and Joyce, 1994a; Ellington and Szostak, 1990; Hesselberth et al., 2000; Joyce, 1999; Robertson and Joyce, 1990; Tuerk and Gold, 1990). Unique advantages of in vitro selection of DNA/RNA (when compared with combinatorial selection of organic ligands or peptides) are the ability to sample a larger pool of sequences, amplify the desired sequences by polymerase chain reaction (PCR), and introduce mutations by mutagenic PCR to improve performance. For example, in vitro selection methods have been used to obtain DNA/RNA aptamers (Cox and Ellington, 2001; Hamaguchi et al., 2001; Jhaveri et al., 2000a; Potyrailo et al., 1998) and aptazymes (or allosteric ribozymes) (Jose et al., 2001; Koizumi and Breaker, 2000; Koizumi et al., 1999; Robertson and Ellington, 1999, 2000; Seetharaman et al., 2001; Soukup and Breaker, 1999a,b,c; Soukup et al., 2001, 2000) that are responsive to small organic molecules or proteins. The use of DNA/RNA aptamers to separate different organic compounds (Kotia et al., 2000) or to transduce the molecular recognition of proteins (Hamaguchi et al., 2001; Lee and Walt, 2000; Potyrailo et al., 1998) or small organic molecules (such as adenosine) (Jhaveri et al., 2000a,b) to changes in fluorescence intensity has been demonstrated recently. While in vitro selection of DNA/RNA aptamers has shown great promise in diagnostic applications of these biosensors for organic molecules and biomolecules (Hesselberth et al., 2000; Jayasena, 1999; Marshall and Ellington, 1999; Soukup and Breaker, 1999c, 2000), the same strategy has not been applied to search for metal ion sensors until recently. It has been demonstrated that in vitro selection of catalytic DNA/RNA can change the metal specificity or binding affinity of existing catalytic RNA (ribozymes), or can lead to catalytic DNA/RNA specific for a target metal ion. For example, in vitro

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selected variants of the group I intron (Lehman and Joyce, 1993) and the RNase P ribozyme (Frank and Pace, 1997) have shown greatly improved activity with Ca(II), which is not an active metal ion cofactor for the native ribozyme. The Mg(II) concentration required for optimal hammerhead ribozyme activity has been lowered using in vitro selection to improve the enzyme performance under physiological conditions (Conaty et al., 1999; Zillmann et al., 1997). Similarly, catalytic DNA/RNA molecules that are highly specific for Pb(II) (Breaker and Joyce, 1994a; Pan and Uhlenbeck, 1992), Cu(II) (Carmi et al., 1996; Cuenoud and Szostak, 1995), and Zn(II) (Li et al., 2000; Santoro et al., 2000) have been obtained. Here we present concepts and procedures for in vitro selection and engineering of catalytic DNA that binds a metal ion of choice with strong affinity and high selectivity. The adaptation of the resulting catalytic DNA into a highly sensitive and selective fluorescent sensor, and the use of the sensors for detection and quantification of lead (Pb(II)) in lake water are also demonstrated.

2. Experimental 2.1. In vitro selection In vitro selection of catalytic DNA was performed using a protocol reported previously (Breaker and Joyce, 1994a; Li et al., 2000). Fig. 1 shows a schematic representation of the selection scheme used in this study. The initial DNA pool contained a 40-nueleotide randomized sequence (represented by rectangles in Fig. 1B) flanked by two conserved primer-binding domains (lines on either side of the N40 sequence in Fig. 1B). The 5?-

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biotin moiety and the single RNA base were introduced by a modified PCR primer. The pool containing the RNA/DNA chimer (a strand of nucleic acid that incorporates both RNA and DNA bases) was immobilized on a 100 ml NeutrAvidin column via the 5?-biotin moiety on the strand containing the single RNA base. Unbound strands were removed by 5 /100 ml washes with buffer A (50 mM N -[2-hydroxyethyl]piperazineN ?-[2-ethanesulfonic acid] (HEPES), 500 mM NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0). The non-biotinylated DNA was then washed off the column using 5/100 ml washes of 0.2 N NaOH containing 20 mM EDTA. The column was subsequently neutralized by 5/100 ml washes with buffer B (50 mM HEPES, 500 mM NaCl, pH 7.0). Cleavage of the singlestranded DNA containing the single ribonucleotide was accomplished with 3 /20 ml washes of reaction buffer (buffer B plus 1 mM metal ion). The eluted DNA was collected and co-precipitated with 50 pmol of PCR primers. Half of this DNA was amplified using PCR over eight thermal cycles. One tenth of the product was further amplified over eight thermal cycles. This PCR product was precipitated with 10% of 3 M sodium acetate and 2.5 volumes of ethanol and was used to initiate the next round of selection. To increase the cleavage efficiency of the selected sequences, the reaction time for each round of selection was steadily decreased from 60 min for round 1 to 30 s for round 16. To improve the metal binding affinity of the selected DNA molecules, the concentration of metal ion present in the reaction buffer was continuously decreased from 1 mM for round 1 to 50 mM for round 16, at which round the activity of the catalytic DNA stopped increasing. To reduce the observed activity with metal ions other than the metal ion of interest, a negative selection strategy was performed starting with round 6. After the DNA pool was allowed to bind the NeutrAvidin column and the non-biotinylated DNA strand was removed, the active strand was exposed to a ‘metal soup’ containing metals but not the metal of choice. The cleavage steps with the ‘metal soup’ were 3 /20 ml of reaction buffer over 1 h (20 min per wash). Following the cleavage, the column was washed with 3/100 ml of buffer A to remove any remaining metal ions and 5/ 100 ml buffer B to remove the EDTA. The cleavage reaction with the metal ion of interest was then performed as previously described (Li et al., 2000). 2.2. Biochemical study of in vitro selected catalytic DNA

Fig. 1. The starting pool and general selection scheme. Adapted from Breaker and Joyce (1994a).

Activity assays were performed in buffer B by adding equal volumes of the purified DNA strand and buffer B containing the metal ion of interest at twice the desired final concentration. Aliquots were taken at pre-determined time points, and the reactions were stopped by

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mixing the aliquots with a stopping buffer (containing 8 M urea, 50 mM EDTA, and 0.1% bromophenol blue and xylene cyanol as dyes). The substrate and product were separated by PAGE and quantified using a PhosphorImager. 2.3. Fluorescent sensors The catalytic DNA enzyme /substrate complex was prepared with 50 nM each of 17E-Dy (Dabcyl-labeled enzyme) and Rh-17DS (TAMRA-labeled substrate) in 50 mM HEPES (pH 7.5) with a volume of 600 ml. A final concentration of 50 mM NaCl was added to the sample to facilitate DNA hybridization unless otherwise noted. The sample was heated at 90 8C for 2 min and cooled to 5 8C over 15 min to anneal the deoxyribozyme and the substrate together. The steady-state and the slow kinetic fluorescence spectra were collected with a SLM 8000S photon-counting fluorometer. Polarization artifacts were avoided by using ‘magic angle’ conditions (Lakowicz, 1999). The steady-state fluorescence emission was measured from 570 to 700 nm (lex /560 nm). To initiate the catalytic DNA reaction, a concentrated divalent metal ion solution was injected into the cuvette using a 10 ml air-tight syringe while the DNA sample in the cuvette was being constantly stirred with a glass mixer connected to an electric motor. Meanwhile, the fluorescence intensity of the sample at 580 nm (lex /560 nm) was recorded at 2 s intervals. 2.4. Colorimetric sensors The 13 nm gold nanoparticles were prepared using literature methods. The size of the nanoparticle was verified by transmission electron microscopy (JEOL 2010), and thiol-modified DNA probes were attached to the gold nanoparticles for Pb(II) detecting functionality. The DNA modified gold nanoparticles were then mixed with the catalytic DNA and linking DNA that can be cleaved in the presence of target Pb(II) to make the DNA Pb(II) sensor. The quantitative monitoring of color changes was performed on a Hewlett /Packard 8453 spectrophotometer.

3. Results and discussion 3.1. In vitro selection of catalytic DNA with high affinity and selectivity for metal ions Catalytic DNA with high affinity and selectivity for metal ions can be obtained through in vitro selection with a protocol reported previously (Fig. 1) (Breaker and Joyce, 1994a; Li et al., 2000). The initial selection pool contains /1014 out of the possible 440 (/1024) DNA sequences. These molecules contain a random

sequence domain of 40 nucleotides flanked by two conserved primer-binding regions. A ribonucleic adenosine (rA) is embedded in the 5?-conserved sequence region (Fig. 1) and is intended to be the cleavage site due to the relative lability of the RNA bond toward hydrolytic cleavage. The DNA pool is then immobilized on an avidin column through the biotin moiety on the 5? of the DNA. The sequences that undergo cleavage at the internal RNA bond in the presence of the metal ion of our choice are eluted from the column, amplified via PCR, and used to seed the following round of selection. The activity of the selected enzymes can be improved by gradually using more stringent conditions (such as shorter incubation times) in each subsequent round of selection. The metal-binding affinity of the enzymes may also be improved by gradually decreasing the concentration of the metal ion. The selection continues until the generation at which improvement of activity stops. The catalytic DNA molecules can then be cloned and sequenced. We have successfully used this protocol to select several transition metal ion dependent catalytic DNA (Bruesehoff et al., 2002; Li et al., 2000). This protocol is adaptable to selection of any catalytic DNA that is dependent on any given metal ion. More importantly, this protocol can be used to select for different oxidation states of the same metal ion (such as Fe(II) vs. Fe(III)) since DNA is known to be specific to certain oxidation states of a given metal ion (e.g. catalytic DNA molecules are active only in the presence of Mn(II), and not with the manganese ions of higher oxidation states, Li et al., 2000; Santoro and Joyce, 1997, 1998). Finally, because the selection is carried out using a gradually decreasing concentration of metal ions at different rounds of selection, isolation of the DNA in both the middle rounds and in the last rounds may result in DNA with medium and high metal affinity, respectively. DNA biosensors with different affinity ranges are necessary to prevent early signal saturation of sensors with too high an affinity and to allow quantitative detection of metal ions in a wide range of concentrations. As observed in our lab (Li and Lu, 2000) as well as in others (Faulhammer and Famulok, 1997; Landweber and Pokrovskaya, 1999; Lehman and Joyce, 1993; Peracchi, 2000), in vitro selection of catalytic DNA/ RNA in the presence of one metal ion may result in a certain fraction of the enzymes being more active with a different metal ion. Several factors may contribute to the change of metal ion dependence of the catalytic DNA activity. First, due to the slight impurity of the chemical reagent, the primary metal ion solution may be contaminated by other metal ions, such as Pb(II). Therefore, even though the contaminating metal ions were not intentionally added in the selection, DNA molecules that are active with those metal ions could be mistakenly obtained. Although we cannot rule this out

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completely, we can minimize this possibility by using ultra pure reagents with desired metal content of 99.999%. In addition, the buffers used to make the metal ion solution were rendered metal-free by treatment with Chelex beads. Second, similarities between the primary metal and the contaminating metal ion could make substitution possible. For example, Zn(II) ˚ ) and similar and Co(II) have the same ionic radii (0.74 A pK a values in water in their hydrated forms (9.0 for hydrated Zn(II), and 9.6 for hydrated Co(II)) (Cotton et al., 1999), Zn(II) ions in many zinc proteins have been substituted by high spin Co(II) and the resulting proteins retain full activity (Banei et al., 1982; Holmquist et al., 1975; Makinen et al., 1985; Maret and Vallee, 1993). Third, the catalytic metal-binding site(s) in some of the selected catalytic DNA molecules are sufficiently flexible to accommodate other metal ions, such as lanthanide ions, in the same site or at similar locations. These ‘false-positive’ results are not uncommon in the selection process. For example, the so-called ‘selfish’ molecules have been observed in the selection process (Breaker and Joyce, 1994b). Strategies have been designed to overcome the problem (Breaker, 1997b; Breaker and Joyce, 1994b). To prevent the problem of ‘false-positive’ results in selecting metal ion-dependent catalytic DNA molecules, we have employed a strategy of ‘negative selection’ in combination with the normal selection process to remove those catalytic DNA molecules that are more active in other metal ions (Brueseh-

Fig. 2. Strategies for improving metal ion selectivity during in vitro selection. Adapted from Bruesehoff et al. (2002).

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off et al., 2002). In this strategy (Fig. 2), after a few rounds of normal ‘positive’ selection of Co(II)-dependent catalytic DNA molecules, we introduce one to two rounds of ‘negative selection’ in which the resulting catalytic DNA molecules are selected against a ‘metal soup’ containing various metal ions (e.g. Pb(II), Mg(II), Ca(II), Zn(II), Hg(II), etc.). Sequences that undergo selfcleavage in the presence of metal ions other than Co(II) are then eluted from the column and discarded. The remaining sequences are further selected with Co(II) as the cofactor. In this way, the fraction of catalytic DNA molecules that are active in the presence of other metal ions will be discarded and only those that are most likely active in Co(II) will be selected. We note that ‘false-positives’ are also common in the other types of metal ion sensors. For example, as mentioned in the Section 1, the widely used Ca(II) sensor, Fura-2, binds Zn(II) and Cd(II) 102- and 105fold more tightly, respectively, than it binds Ca(II) (Haugland, 1999). While positive design and selection has been emphasized in these and other rational and combinatorial design methods, our work indicated that ‘negative’ design (i.e. selecting against those molecules that bind other metal ions competitively) is an important aspect of metal sensor design and selection, and in vitro selection is well adaptable to the ‘negative selection’ purpose. 3.2. Biochemical studies of in vitro selected catalytic DNA Before the in vitro selected DNA can be used for sensor application, a detailed biochemical study is necessary to provide a framework for sensor design. Typical procedures include: (a) Enzyme length truncation. In the current system design (see Fig. 1), the catalytic DNA after in vitro selection is 107 bp long. As demonstrated previously (Breaker and Joyce, 1994a; Li et al., 2000; Santoro and Joyce, 1998), not all 107 bases are necessary for activity. It is necessary to determine the minimum number of bases required for activity by truncating the sequence at either the 3? or the 5?-end (Fig. 3A). The shortened catalytic DNA molecules are then chemically synthesized and their activity is tested in the presence of the metal ion in which the selection was carried out. Length truncation is necessary for costeffective studies and applications. (b) Cis to trans form transition. The catalytic DNA molecules obtained from in vitro selection are in the cis form, i.e. self-cleaving form. To convert the system to a true enzyme with catalytic turnover, the enzyme can be transformed to the trans form, with one strand defined as the enzyme and another as the substrate. The transformation usually requires some knowledge of the secondary structure of the selected DNA sequences, which can be predicted using programs such as DNA mfold (available at http://

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Fig. 3. (A) The primary structure of the selected DNA. The primer binding domains are in italics, the N40 region is in normal type, and the underlined regions form the three base-pair stem around nucleotide 60, shown in B. (B) The most stable secondary structure of in vitro selected DNA predicted by the DNA mfold program. (C) The final secondary structure of the in vitro selected DNA after truncation and cis -to trans transformation. The DNA bases in blue were determined to be the conserved sequence of the catalytic DNA and are observed as bases 62 /66 in part B.

www.bioinfo.math.rpi.edu/ /mfold/dna/form1.cgi) (Fig. 3B). (c) Conserved sequence identification. The starting point for identifying the conserved sequences is the invariant bases in different sequences within the same class of catalytic DNA molecules obtained from a selection. If mutations of these bases abolish enzyme activity, then they are part of the enzyme that must be conserved for the enzyme to be active. Fig. 3 shows how an in vitro selected DNA was truncated and transformed into a trans form, ready for sensor applications. 3.3. Design and demonstration of a catalytic DNA fluorescent sensor After selection and characterization of metal iondependent catalytic DNA, the next step is to transform a metal ion dependent conformational change or cleavage

into an observable signal. Fluorescence provides significant signal amplification since a single fluorophore can absorb and emit many photons, leading to strong signals even at very low concentrations of fluorescent probe or analyte. In addition, the fluorescence timescale is fast enough to allow real-time monitoring of concentration fluctuations. Fluorescent properties only respond to changes related to the fluorophore, and, therefore, can be highly selective. Furthermore, fluorimeters for field use are available (Jones et al., 1989; Milanovich et al., 1986; Savage, 1998). Fluorescence detection is also compatible with fiber-optic technology and well-suited for remote sensing applications (Arnold, 1989, 1990, 1992; Ferrel et al., 1992; Jung et al., 1998; Spalding et al., 1997; Thompson, 1991; Wolfbeis, 1988). The trans -cleavage catalytic DNA system consists of two parts, the enzyme and the substrate. As shown in

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Fig. 4B, the 5?-end of the substrate strand is covalently attached to a fluorophore such as 6-carboxytetramethylrhodamin (TAMRA). The 3?-end of the substrate strand can be labeled with a fluorescence quencher, such as 4-(4?-dimethylaminophenylazo)benzoic acid (Dabcyl). Dabcyl can quench the fluorescence of a nearby fluorophore very efficiently. When the substrate strand is alone, the TAMRA on its 5?-end can fluoresce (Fig. 4A). Upon formation of the substrate /enzyme complex by Watson /Crick base pairing, TAMRA and Dabcyl will be in close proximity; thus, the TAMRA fluorescence is quenched significantly by Dabcyl. However, as soon as the sensor is exposed to a solution containing sequence-sensitive metal ions, for example, Pb(II), the substrate will be cleaved and the products will dissociate. As a result, TAMRA is no longer in close proximity to Dabcyl. This will be reflected by the increase of the TAMRA fluorescence intensity (Fig. 4A). The above concept has been demonstrated with a lead-sensitive catalytic DNA (Fig. 4CD). When a fluorophore (TAMRA) is attached to the 5?-end of the substrate, the fluorescence signal at 580 nm is quenched by a fluorescence quencher (Dabcyl) at the nearby 3?-

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end of the catalytic DNA. In the presence of Pb(II), the fluorescence emission of TAMRA increased by about 400%, due to the cleavage of the substrate followed by product release (Fig. 4C). This catalytic DNA sensor is highly sensitive for Pb(II), with a quantifiable detection range from 10 nM to 4 mM (Fig. 4D). Even in the presence of equal concentrations of other metal ions (such as Mg(II), Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), and Cu(II)) and under simulated physiological conditions, this biosensor displays a remarkable sensitivity and selectivity (Fig. 4D). The principles demonstrated in this work can be used to obtain catalytic DNA sensors for other metal ions. 3.4. Application of catalytic DNA fluorescent sensors in sensing lead in lake water Compared with RNA and proteins, DNA molecules are much more robust. The stability of DNA makes the DNA-based sensor useful not only in the lab, but also for real world samples. To demonstrate the DNA Pb(II) sensors ability to function on real samples, water from Lake Michigan was tested. The fluorescent-based detection methods have shown ability to function properly in

Fig. 4. Catalytic DNA molecules as a new class of metal ion biosensors. (A) concept and design of metal ion biosensors, using lead sensors as an example. Adapted from C&EN News, 2000 (October 30), 78 (44), 9 /10; (B) the sequences and proposed secondary structure of the lead sensor (top), and examples of fluorescence tag (TAMRA) and quencher (Dabcyl); (c) steady-state fluorescence spectra of the substrate (Rh-17DS); alone (1), after annealing to the deoxyribozyme (17E-Dy) (II), and 15 min after adding 500 nM Pb(OAc)2 (III); (d) the fluorescence response rate (vfluo) of sensor in the presence of 500 nM of different divalent metal ions in 50 mM HEPES (pH 7.5). The inset shows the variation of initial rate vfluo with the concentration of Pb(II) (y ) or Co(II) (n ). Figures in C and D are adapted from Li and Lu (2000).

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Fig. 5. Detection of Pb(II) in Lake Michigan water. Lake Michigan water was used as real water for Pb(II) detection using fluorescence based DNA sensor design. The reporting fluorophore used in this experiment is FAM. The image was taken from a fluorescence image reader (Fuji). The excitation wavelength was set at 473 nm and the emission filter was set to cut wavelength shorter than 520 nm. Since FAM is a green fluorescence fluorophore, the image presented here is shown in green color. The more intense of the green color, indicates the larger fraction cleavage of the fluorophore labeled substrate (see Fig. 4).

real samples. The fluorescence image of a row of a 96well plate containing Lake Michigan water with different concentrations of added Pb(II) is shown in Fig. 5. The overall trend is the same as in pure water. The sensor detects Pb(II) in spite of the presence of other potentially interfering substances. This demonstrates that although there are many other ions and chemicals in water sample, the DNA Pb(II) sensor can still detect Pb(II).

4. Conclusions Long considered as simply a genetic material, DNA was shown in 1994 (Breaker and Joyce, 1994a) to carry out catalytic functions, and thus became the newest member of the enzyme family after proteins and RNA. The results presented here showed that one can take advantage of catalytic DNA to make highly sensitive and selective fluorescent or colorimetric sensors. Several features make catalytic DNA molecules an excellent choice for developing sensors for metal ions. First and perhaps the biggest advantage of choosing catalytic DNA molecules is that they can be subject to in vitro selection, which, when compared with other combinatorial methods based on organic chelators or peptides, can sample a larger pool of sequences (up to 100 billion), amplify the desired sequences by PCR, and introduce mutations to improve performance by mutagenic PCR. The metal selectivity of this method can be further improved by successfully introducing a negative selection strategy (Bruesehoff et al., 2002). This approach can overcome our limited knowledge about the metalbinding affinity and selectivity of different organic- or biomolecules, and a detailed study of the resulting metal-specific catalytic DNA molecules may provide insight into rational design of other metal sensors. In vitro selection can be carried out in short time and with limited cost (1 /2 days and a few dollars per round of selection). Second, under physiological conditions, DNA is nearly 1000-fold more stable to hydrolysis than proteins and nearly 100 000-fold more stable than RNA (Breaker, 1999). As seen from the recent crystal structure (Nowakowski et al., 1999), catalytic DNA molecules usually form a compact globular shape like proteins and are, therefore, not easily recognized by

endo- or exonucleases, and thus are more resistant to nuclease attack than single or even double-stranded DNA/RNA (Chow and Bogdan, 1997). When folded, the compact globular catalytic DNA molecules are also less likely to bind other biomolecules in the cells than the single- or double-stranded DNA/RNA. Third, unlike proteins, most catalytic DMA molecules can be denatured and renatured many times without losing binding ability or activity. They can be used and stored under rather harsh conditions. Fourth, DNA is adaptable to fiber optic and microarray technology (Ferguson et al., 2000; Taylor and Walt, 2000; Walt, 2000), which is important for on-site or remote sensing of multiple metal ions simultaneously. Finally, as demonstrated recently (Li and Lu, 2000), there are three additional advantages of catalytic DNA fluorescent and colorimetric sensor systems; (a) the metal sensing is achieved by both metal-binding and catalytic activity, allowing signal amplification through catalytic turnover; (b) the fluorophores or gold nanoparticles can be placed remotely from the binding and cleavage sites so that binding and sensing do not interfere with each other and can be optimized independently, and (c) the effective placement of the fluorophores or gold nanoparticles can be accomplished with little knowledge of the three dimensional structure of the system. These features make catalytic DNA one of the most promising systems for sensor design and application.

Acknowledgements We thank Professor Robert Clegg for advice and helpful discussions. This material is based upon work supported by the Natural and Accelerated Bioremediation Research (NARIR) program, Biological and Environmental Research (BHR), US Department of Energy (DHFG02-01-ER63179) and by Nanoscale Science and Engineering Initiative of the National Science Foundation (DMR-0117792). The experiments reported in this paper were performed at the Laboratory for Fluorescence Dynamics (LFD) at the University of Illinois at Urbana-Champaign (UIUC), The LFD is supported jointly by the Division of Research Resources of the National Institutes of Health (PHS 5 P41RRO3155) and UIUC.

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