Anal. Chem. 1998, 70, 3699-3702
Polishable and Renewable DNA Hybridization Biosensors Joseph Wang,* Joa˜o Roberto Fernandes,† and Lauro Tatsuo Kubota‡
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the singlestranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications. DNA hybridization biosensors offer considerable promise for obtaining sequence-specific information in a faster, simpler, and less expensive manner compared to traditional hybridization assay.1,2 The basis for these nucleic acid hybridization devices is the DNA base pairing, namely, the strong interaction between two complementary nucleic acid strands (the immobilized probe and sample target). As with other types of affinity biosensors, major attention should be given to the reusability of DNA biosensors, i.e., to the regeneration of the surface-bound singlestranded probe after each assay. Thermal and chemical regeneration schemes, involving prolonged incubation in hot water, concentrated urea, or sodium hydroxide solutions, are commonly used for “removing” the bound target in connection with different DNA biosensor formats.3,4 However, such regeneration schemes are time-consuming and inconvenient and are useful only for few measurement cycles (with a gradual loss of the hybridization activity thereafter). Simpler and faster regeneration procedures are thus desired to enhance the day-to-day practicality of nucleic acid biosensors. † Permanent address: Departamento de Quı´mica, FC/UNESP, Bauru-SP, Cxp 473, CEP 17033-360, Brazil ‡ Permanent address: Depto. de Quı´mica Analı´tica/IQ/UNICAMP/, Cxp. 6154, Campinas-SP, CEP 13083-970, Brazil (1) Mikkelsen, S. Electroanalysis 1996, 8, 15. (2) Wang, J.; Rivas, G.; Cai, X.; Palecek, E.; Nielsen, P.; Shiraishi, H.; Dontha, D.; Luo, D.; Parrado, C.; Chicharro, M., Farias, P.; Valera, F.; Grant, D.; Ozsoz, M.; Flair, M. Anal. Chim. Acta 1997, 347, 1. (3) Millan, K.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943. (4) Piunno, P.; Krull, U.; Hudson, R.; Damha, M.; Cohen, H. Anal. Chem. 1995, 67, 2635.
S0003-2700(98)00092-4 CCC: $15.00 Published on Web 06/26/1998
© 1998 American Chemical Society
This note describes a novel approach for the construction of reusable hybridization biosensors based on the mechanical exposure of a “fresh” probe-containing surface layer. Several studies have illustrated that the incorporation of biocomponents in carbon composite matrixes results in renewable amperometric enzyme or immunoelectrodes.5-8 The bulk of these composite bioelectrodes serves as a continuous source of the biorecognition activity; “fresh” bioactive surfaces can be easily obtained by polishing. Similarly, in the present study, the oligonucleotide probe was uniformly dispersed within a renewable carbon paste electrode matrix, which served as a continuous “reservoir” for the hybridization activity, so that each nucleic-acid measurement was performed on a new surface. The new regeneration scheme is simple, and fast and offers numerous hybridization/detection cycles with no apparent carry-over or loss in hybridization activity. The challenges of designing renewable carbon composite hybridization biosensors, along with their attractive performance characteristics, are illustrated below for the detection of a short DNA fragment specific to the deadly waterborne pathogen Cryptosporidium parvum. EXPERIMENTAL SECTION Apparatus. Chronopotentiometric measurements were performed with the TraceLab unit (PSU 20, Radiometer, Denmark) interfaced with an IBM PS/2 55SX. The derivative signal (dt/ dE) was recorded as a function the potential. The peak area following baseline fitting was used as the analytical signal. A diode array spectrophotometer (model 8452A, Hewlett Packard) was employed to measure the concentration of nucleic acids by monitoring the absorbance at 260 nm. The three-electrode arrangement consisted of a DNA-modified carbon paste working electrode, a Ag/AgCl reference electrode (model RE-1, BAS Inc., West Lafayette, IN), and a platinum wire auxiliary electrode. The electrodes entered the cell through holes in its Teflon cover. The electrochemical cell was a 4-mL vial (16 × 20 mm, Kimble Glass Inc.); it was cleaned with dilute nitric acid (1:4), rinsed thoroughly with water, and then dried before use. The conventional carbon paste was prepared by hand-mixing graphite powder (grade 38, Fisher Scientific) and mineral oil (Sigma Chemical Co., Catalog No. M5904, free of DNase, RNase, (5) (6) (7) (8)
Wang, J.; Varughese, K. Anal. Chem. 1990, 62, 318. Wang, J.; Gonzalez-Romero, E.; Ozsoz, M. Electroanalysis 1992, 4, 539. Algeret, S. Analyst 1996, 121, 1751. Santandreu, M.; Ce´spedes, F.; Alegeret, S.; Martinez-Fabregas, E. Anal. Chem. 1997, 69, 2080.
Analytical Chemistry, Vol. 70, No. 17, September 1, 1998 3699
and protease). The mass ratio of graphite powder to mineral oil was 70:30. The modified carbon paste electrodes contained different loadings (4-25% w/w) of the probe-coated silica-titania (ST) beads. The pistonlike working electrode configuration9 was prepared by tightly packing the resulting paste into a Teflon sleeve (1-mm, i.d.), filling the tip to a height of about 15 mm. Electrical contact and extrusion of the paste were obtained with a threaded 1-mm-diameter copper wire. The surface was renewed by turning the copper wire to extrude the 0.5-mm outer layer and was smoothed on a weighing paper prior to each use. Reagents and Materials. Silica gel particles (Merck), with specific surface area (SBET) of 463 m2 g-1, an average pore diameter of 6 nm, and a particle size of 0.2 mm, were activated and coated with titanium oxide using a previously described protocol.10,11 Sigma Inc. provided the sodium acetate buffer (3 M, pH 5.2 at 25 °C; Catalog No. S-7899), Tris-HCl buffer (1 M, pH 7.00 ( 0.05 at 25 °C; Catalog No. T-2413), sodium phosphate dibasic (Catalog No. S-3264), and sodium chloride (Catalog No. S-3014). All chemicals were free of RNase and DNase and were used as received. All water and pipet tips were sterilized by autoclaving for 30 min (120 °C). Single-stranded 38-mer probe and target DNA sequences, along with the 36-mer noncomplementary oligonucleotide, were acquired from Life Technologies. These had the following sequences.
immobilized probe: 5′-GGG GAT CGA AGA CGA TCA GAT ACC GTC GTA GTC TTA AC-3’ (1) target: 5′-GTT AAG ACT ACG ACG GTA TCT GAT CGT CTT CGA TCC CC-3’ (2) noncomplementary oligomer: 5′-GTC GTC AGA CCC AAA ACC CCG AGA GGG GAC GGA AAC-3′ (3) All probe and target stock solutions of the 38-mer oligomers (1000 mg/L) were prepared with a TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and kept frozen. More dilute solutions were prepared with autoclaved water prior to use. Immobilization of the Probe. ST beads (8 mg) were added to a 100-mL solution of the 38-mer probe (500 mg/L in TE buffer, pH 8, containing 5 mM tris and 0.5 mM EDTA); vigorous mixing (vortex) proceeded for 5 min. The resulting mixture was kept at 4 °C overnight. The excess of nucleic acid was removed, and the coated particles were rinsed thoroughly with autoclaved water and then were dried in low-pressure desiccator prior to their mixing with the ordinary carbon paste. Most experiments employed pastes containing 4%(w/w) DNA/ST particles. Procedure. (a) Pretreatment Step. The “fresh” carbon paste surface (containing the probe) was smoothed and pretreated by applying a potential of +1.7 V for 60 s in 2 mL of stirred acetate buffer solution (0.2 M, pH 5). (9) Wang, J.; Freiha, B. Anal. Chem. 1984, 56, 849. (10) Kubota, L. T.; Gouvea, F.; Anrade, A.; Milageres, B.; Neto, G. Electrochim. Acta 1996, 41, 1465. (11) Kubota, L. T.; Gushiken, Y.; Castro, S. C.; Moreira, J. C., Colloids Surf. 1991, 57, 11.
3700 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998
(b) Hybridization. The pretreated electrode was immersed in the stirred hybridization solution (20 mM phosphate buffer pH 7, 0.5 M sodium chloride), containing different levels of the target sequence, while the potential was held at +0.5 V for a given period (usually 2-5 min). (c) Indicator Association and Detection. The electrode was transferred from the hybridization solution to the stirred TrisHCl buffer detection solution (20 mM, pH 7) containing 0.1 mM Co(phen)33+ indicator for 60 s while the potential was held at +0.5 V. Subsequent measurement of the associated indicator was performed in unstirred Tris-HCl buffer (20 mM, pH 7) solution, following a 15-s rest time at +0.5 V, by applying a constant current of -4.0 µA. (d) Renewal. The surface was regenerated by turning the copper wire for extruding the 0.5-mm-thick outer paste layer and was smoothed on a weighing paper. Between each (electrodetransfer) step, the electrode was rinsed with the corresponding next solution. All experiments were conducted at room temperature (22.0 ( 0.5 °C). RESULTS AND DISCUSSION We examined different routes for designing renewable composite hybridization biosensors, based on dispersing the singlestranded oligonucleotide probe within the bulk of carbon composite electrodes. Our first effort focused on the use of modified graphite epoxy, in a manner analogous to the regenerable immunosensors of Santandreu et al.8 However, such use of polishable and rigid graphite/epoxy(Epotek)/nucleic acid composites resulted in a large background contribution that completely obscured the peak of the Co(phen)33+ redox indicator. Accordingly, we shifted our effort to the softer carbon paste composites, known for their favorable indicator response,1-3 and employed a pistonlike electrode configuration9 which allowed removal of the desired amount of the paste. Initial attempts, obtained with mixed probe/carbon paste sensors, resulted in poorly reproducible hybridization signals (associated with the nonuniform distribution of the probe). In contrast, reproducible and reusable hybridization response was obtained by immobilizing the single-stranded oligonucleotide onto ST beads, similar to those employed for the preparation of biocatalytic carbon paste electrodes.10 Apparently, such immobilization proceeds through the reaction of the titanium oxide monolayer (on the silica surface) with the phosphate backbone of the oligonuceotide probe, leaving the nucleobases readily accessible for the hybridization event. The affinity of ST particles to phosphate groups was documented earlier.10 Such use of highly porous microscopic ST beads results in homogeneous dispersion of the nucleic acid probe within the interior of the carbon paste composite and does not adversely affect the basepairing recognition process. The new renewable DNA biosensor format has been tested for the detection of short sequences specific to the DNA of the C. parvum pathogen,12 and in connection to a chronopotentiometric detection of the hybridization/redox indicator. Figure 1A displays chronopotentiograms for the Co(phen)33+ indicator, following 16 sequential exposures of individual ST-DNA/ carbon paste surfaces to the blank and Cryptosporidium target (2 mg/mL) solutions (in connection to a 2-min hybridization). (12) Wang, J.; Rivas, G.; Parrado, C.; Cai, X.; Flair, M. Talanta 1997, 44, 2003.
Figure 2. Effect of DNA/ST loading upon the hybridization signal (A) and on the target/probe peak ratio (B). Conditions, as in Figure 1.
Figure 1. (A) Chronopotentiograms for the Co(phen)33+ indicator following sequential exposures of individual sensor surfaces to the blank and Cryptosporidium target solutions. (B) Carry-over experiment involving three replicate target measurements, followed by three blank measurements, and return to the target solution (using a total of 27 individual surfaces). ST/DNA loading (in the paste), 4% (w/w); target concentration, 2 mg/L; hybridization time, 2 min; chronopotentiometric current, -4 mA. Solutions and other conditions are described in the Experimental Section.
Significantly larger indicator peaks are observed following immersions in the target solution. The renewable sensor responds rapidly to such “switching” between the target and blank solutions, with the use of fresh surfaces erasing memory effects and restoring the biorecognition activity. No deterioration in the hybridization activity is observed over the entire series of 16 hybridization/renewal cycles. The average blank and target responses correspond to 3.5 ( 0.6 and 23.5 ( 2.7 ms, respectively. Similarly, minimum carry-over effects and no loss of the hybridization signal are indicated from Figure 1B, involving three replicate target measurements, followed by three blank measurements, and a return to the target solution (for a total of 9 such target/blank cycles using 27 individual surfaces). Such use of fresh DNAmodified surfaces is not affected by residuals from the previous target signal, hence obviating the need for thermal or chemical regeneration steps (which are time-consuming and limited to a few measurement cycles). While the size of the carbon paste cavity/reservoir (used in the present study) results in a total of 30 individual surfaces, the electrode design permits easy and fast repacking of the modified paste to allow hundreds measurement cycles. The reproducible response to the target reflects the uniform dispersion of the DNA-coated ST beads in the carbon paste matrix. The DNA/silica loading in the paste has a profound effect upon the response of the renewable DNA carbon paste biosensor (Figure 2). Both the absolute hybridization signal (i.e., difference between the indicator peak in the presence of the target and blank solutions) (A) the ratio of the indicator peaks (in the presence of
the target and blank solutions) (B) are strongly dependent upon the weight percentage of the DNA-confined ST particles. The hybridization signal increases rapidly upon changing the modifier loading from 4 to 12% (w/w), and decreases sharply at higher loading. In contrast, the “target/blank” peak ratio decreases gradually upon raising the percentage of the DNA/ST particles from 4 to 25% (w/w). Such different profiles reflect not only changes in the hybridization capacity of the electrode but also the effect of the DNA/ST loading upon the Co(phen)33+ background response (that increases greatly at high modifier contents). As a large target/blank peak ratio provides a more attractive route for detecting the hybridization event, compared to the absolute hybridization signal, most subsequent work employed a DNA/ ST loading of 4% (w/w). The ability to mechanically regenerate the single-stranded probe modified surface was tested also in calibration experiments involving different concentrations of the 38-mer Cryptosporidium DNA target and the individual (freshly restored) biosensor surfaces (Figure 3). As expected for the saturation of the probe binding sites, the linear range and sensitivity are strongly dependent upon the hybridization time. For example, using a 2-min hybridization, the indicator peak increased linearly with the target concentration up to 2 mg/L and leveled off above 4 mg/L (B). Longer hybridization times offered higher sensitivity and convenient quantitation of lower concentrations. For example, with 5-min hybridization, the response increased linearly up to 0.6 mg/L, with slight curvature thereafter (B). The initial linear portions had sensitivities of 9.22 and 37.0 ms‚L/mg following 2and 5-min hybridization, respectively (correlation coefficients, 0.994 and 0.998). Overall, the data of Figure 3 demonstrate that the use of renewable DNA carbon paste surfaces has no detrimental effect upon the quantitative use of these devices and that the exposure of fresh probe layers results in calibration profiles analogous to those common at surface-modified hybridization biosensors.12 A detection limit of 20 mg/L was estimated from the signal-to-noise characteristics (S/N ) 3) of the response for a 50 mg/L target sequence in connection to a 20-min hybridization period (not shown). Control experiments were performed to assess whether the renewable sensor responds selectively, via hybridization, to the target DNA. For example, Figure 4A shows the response to sequential exposures of the device to the Cryptosporidium target Analytical Chemistry, Vol. 70, No. 17, September 1, 1998
3701
Figure 4. Chronopotentiograms for the Co(phen)33+ indicator following sequential exposures of individual sensor surfaces to (A) solutions of the noncomplementary (a) and target (b) oligonucleotides and to (B) blank (a) and target (b) solutions. Conditions and concentrations are similar to those of Figure 1, except that no immobilized probe was used in (B).
Figure 3. Calibration plots over different ranges of the Cryptosporidium DNA target following (A) 2- and (B) 5- min hybridization times. Other conditions, as Figure 1.
oligomer and to a 36-mer noncomplementary oligonucleotide. A large and reproducible indicator signal is observed following immersion in the target solution. In contrast, small peaks (approaching the blank response; e.g., Figure 1) are observed following exposure to the noncomplementary oligonucleotide. These observations demonstrate that the binding is primarily via hybridization. Yet, nonspecific adsorption effects (common to most electrochemical hybridization biosensors) may still occur following exposure to a large excess of noncomplementary DNA oligomers. Compared to common DNA sensors, the renewable sensor strategy circumvents memory effects associated with the continuous buildup of such adsorbed layers during repetitive assays. Further evidence that hybridization is responsible for the observed signals is obtained from another control experiment involving no immobilized probe. Figure 4B displays chronopotentiograms for the Co(phen)33+ indicator following six sequential exposures of the individual ST/carbon paste surfaces to the blank and target solutions. No indicator peaks are observed following the immersion in these solutions, indicating that the cobalt (13) Wang, J.; Palecek, E.; Nielsen, P. Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. J. Am. Chem. Soc. 1996, 118, 7667.
3702 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998
complex is not interacting with the ST beads or the carbon paste surface. Note the well-defined hybridization signals obtained under the same conditions in the presence of the immobilized probe (Figure 1A). The experiments described above illustrate an attractive avenue for the fabrication of renewable DNA hybridization biosensors. The fast mechanical regeneration appears to be a viable alternative to time-consuming, partly effective thermal or chemical renewal schemes. Since the bulk-confined probe retains its hybridization activity, numerous hybridization/regeneration cycles can be employed. The confinement of highly specific peptide nucleic probes13 may be required for distinction between closely related sequences. Proper attention to nonspecific adsorption effects (e.g., via potential reversal) would still be required in the presence of a large excess of noncomplementary strands. Because of their reusable nature, such bulk-modified nucleic acid electrodes hold great promise for routine biosensing applications (involving both sequence detection and DNA interactions with small molecules). Indicator-free assayssbased on the intrinsic redox activity of the target guanine residue and the use of guanine-free (inosinesubstituted) probessare currently being coupled to the new surface regeneration strategy. ACKNOWLEDGMENT This work was supported by the NM Water Resources Research Institute. J.R.F. acknowledges a fellowship from FAPESP (Brazil). Received for review January 29, 1998. Accepted May 25, 1998. AC980092Z
