Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization

Biosensors and Bioelectronics 22 (2007) 1798–1806

Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization Yiran Wang a,b , Manfred Stanzel b , Walter Gumbrecht b , Martin Humenik a , Mathias Sprinzl a,∗ a

Laboratorium f¨ur Biochemie, Universit¨at Bayreuth, Universit¨atsstr. 30, 95440 Bayreuth, Germany CT PS6, Siemens Corporate Technology, G¨unther-Scharowski-Str. 1, 91058 Erlangen, Germany

b

Received 18 May 2006; received in revised form 22 August 2006; accepted 25 August 2006 Available online 13 October 2006

Abstract A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol–gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 × 10−18 mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 ␮L. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter. © 2006 Elsevier B.V. All rights reserved. Keywords: Electrochemical; Hybridization; Esterase 2; Conjugate; Mismatch; Single nucleotide polymorphism

1. Introduction The detection of DNA and RNA sequences by microchip technologies has become important for diagnosis of diseases (Nebling et al., 2004), detection of pathogenic organisms in environmental- (Baeumner et al., 2003), food- (Ko and Grant, 2006) or clinical- (Mitterer et al., 2004) samples and for screening the high throughput systems (Heller et al., 1997; Schena et al., 1998). Any self-replicating organism can be discriminated from another on the basis of nucleic acid sequences unique to that particular organism (Baeumner et al., 2004). Although, various strategies to identify unique DNA sequences have been exploited, the nucleic acid-based biosensors are becoming more important as the demand for faster, simpler and cheaper methods for obtaining sequence-specific information increases (Wang, 2000; Nebling et al., 2004). Progress in biosensors has mainly been made by the improvement of the biological components and their implementation ∗

Corresponding author. Tel.: +49 921 55 2420; fax: +49 921 55 2432. E-mail address: [email protected] (M. Sprinzl).

0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.08.046

in microsystem technologies. Among the biological components, enzymes are the most appropriate recognition elements because they combine high chemical specificity and inherent catalytic signal amplification (Scheller et al., 2001). Recently, enzyme–oligodeoxynucleotide (ODN) conjugates (Caruana and Heller, 1999; Kukolka and Niemeyer, 2004; Fruk and Niemeyer, 2005) or streptavidin–enzyme conjugates (Carpini et al., 2004; Gabig-Ciminska et al., 2004) were developed and successfully applied for solid-phase hybridization and specific DNA detection. For purpose of electrochemical transduction of nucleic acid recognition event, soybean peroxidase–ODN and streptavidin–alkaline phosphatase have been employed in most cases as bioelectrocatalysts (Caruana and Heller, 1999; Carpini et al., 2004; Gabig-Ciminska et al., 2004). It has been concluded that covalent enzyme–ODN conjugates can improve the performance of DNA detection system provided enzyme remains stable under stringent hybridization conditions. However, the mostly used streptavidin–alkaline phosphatase is not suitable for these applications due to conditions and elevated temperatures needed to test the stringency of DNA hybridization (Kynclova et al., 1995). Moreover, in the case of alkaline phosphatase, at

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alkaline pH, optimal for the enzyme activity, the stability of the resultant product p-aminophenol is decreased (Nebling et al., 2004). Bearing these limitations of the existing reporter enzyme systems in mind, the objective of the present work was to develop a reporter system consisting of a single chain thermostable enzyme covalently coupled with ODN for application on electrochemical microchip. The esterase 2 (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable monomeric protein with a molecular mass of 34 kDa. The enzyme, characterized as a “B-type” carboxylesterase (EC 3.1.1.1), displays the maximal activity at 65 ◦ C and pH 7.1 and has a denaturation temperature above 90 ◦ C (Manco et al., 1998; Del Vecchio et al., 2002). Interestingly, it is also quite active at 20 ◦ C, an unusual feature for an enzyme isolated from a thermophilic microorganism (D’Auria et al., 2000). EST2 has also been used as a reporter enzyme to monitor EST2 fusion proteins in cell-free protein biosynthesis (Agafonov et al., 2005). Thermostability, monomeric structure and the maximal enzymatic activity at neutral pH make EST2 a promising electrochemical reporter enzyme. 2. Experimental 2.1. Materials All ODNs (sequence in Table 1 of Supplementary information) were synthesized by Biomers (Ulm, Germany). Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC) and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were from Pierce (Rockford, USA). MonoQ HR 5/5 was from Amersham Pharmacia Biotech (Freiburg, Germany). MPVA SAV1 magnetic beads were purchased from Chemagen (Baesweiler, Germany). Electrical DNA chip and multipotentiostat device were obtained from Siemens Corporate Technology (Erlangen, Germany). Silica gel Kieselgel 60, was from Merck (Darmstadt, Germany). Tween 20, Bovine serum albumin (BSA), p-nitrophenylbutyrate, biotin–maleimide and 2-iminobiotin-agarose were purchased from Sigma–Aldrich (Taufkirchen, Germany). Streptavidin was obtained from Roche (Mannheim, Germany). Plasmid DNA pIVEX-efts (catalog number T-1201-02) which contains the bacterial translational gene of elongation factor Ts (EF-Ts) from Escherichia coli, was obtained from RiNA GmbH (Berlin, Germany). Interface SCB-68 and software Labview 6.0 were purchased from National Instruments (Munich, Germany). Other reagents were analytically pure grade. 2.2. Buffers The buffers mostly used in this work were as follows: DNA immobilization buffer, 1 mM TCEP, 300 mM NaCl in 10 mM sodium phosphate, pH 7.0; hybridization buffer, 0.05% Tween 20, 1 mg/mL BSA, 300 mM NaCl, 2 mM EDTA, 20 mM NaH2 PO4 , pH 7.4; washing buffer, 0.05% Tween 20, 75 mM NaCl, 0.5 mM EDTA, 5 mM NaH2 PO4 , pH 7.4; chip buffer,

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100 mM NaCl, 10 mM sodium phosphate, pH 7.1; conjugation buffer, 100 mM NaH2 PO4 , 100 mM NaCl, pH 7.3; binding and washing buffer, 20 mM Tris–HCl, 1 mM EDTA, 2 M NaCl, pH 7.5; phosphate buffered saline (PBS), 10 mM NaH2 PO4, 137 mM NaCl, 2 mM KH2 PO4 and 2.7 mM KCl, pH 7.4; storage buffer, 50 mM Tris–HCl, 100 mM NaCl, 5 mM EDTA, pH 7.5. 2.3. Preparation, purification and characterization of EST2–A34 conjugates Plasmid DNA of EST2 mutant, EST2E118C, was constructed and expressed in E. coli BL21(DE3) and purified by a single step trifluoromethyl ketone Sepharose CL-6B (TFK-Sepharose, prepared as described (Hanzlik and Hammock, 1987)) affinity chromatography (Agafonov et al., 2005). Preparation of EST2–A34 conjugates were performed as follows: 300 ␮L of 200 ␮M ODN A34 were dissolved in conjugation buffer and incubated 1 h with 60 ␮L 120 mM sulfo-SMCC dissolved in dimethylformamide. Ethanol precipitation was used to remove the excess of sulfo-SMCC and the precipitate was dissolved in 100 ␮L conjugation buffer. One milliliter of 100 ␮M EST2E118C was incubated with 5 mM TCEP for 15 min at 37 ◦ C to reduce any disulfide bonds formed upon storage. The 100 ␮L activated A34 together with EST2E118C were combined and shook at 20 ◦ C for 1 h. EST2–A34 conjugates were purified by anion-exchange chromatography on a MonoQ HR5/5 column by linear gradient of NaCl from 0 to 0.7 M using 25 mM Tris–HCl, pH 8.0 as washing buffer. Peak fractions were pooled, dialyzed against storage buffer and concentrated. EST2–A34 conjugates were characterized by 10% SDSPAGE (Laemmli, 1970). The molecular mass of the conjugates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2.4. Preparation of EST2–biotin·streptavidin (EST2–biotin·SA) conjugate One milliliter of 60 ␮M EST2E118C in conjugate buffer was incubated with 5 mM TCEP for 15 min at 37 ◦ C. Then 150 ␮L of 20 mM biotin–maleimide in dimethyl sulfoxide was added and the mixture was incubated at 37 ◦ C for 2 h. Dialysis against PBS was used to remove excess biotin–maleimide. Two milliliters of 50% suspension iminobiotin-agarose were washed by 10 mL of 50 mM Na2 CO3 , 500 mM NaCl, pH 11.0, then incubated with 1 mL 28 ␮M streptavidin for 30 min with periodic mixing. The resulting agarose·streptavidin conjugates were washed with 5 mL PBS and incubated with EST2–biotin at 20 ◦ C for another 30 min. Agarose·streptavidin·biotin–EST2 was washed by 0.1 M NaOAc, pH 4.0 and eluted fractions were collected and dialyzed against PBS. The resultant EST2–biotin·SA conjugate was characterized by 10% SDS-PAGE to be one streptavidin labeled with one or two EST2. In this report, “–” in EST2–biotin·SA conjugate represents covalent coupling and “·” means streptavidin/biotin high affinity binding.

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2.5. Synthesis of p-aminophenylbutyrate (pAPB) pAPB was synthesized in analogy to described paminophenylphosphate preparation (Ram and Ehrenkaufer, 1984). p-Nitrophenylbutyrate (1.19 g, 0.336 mL, 5.688 mmol) was dissolved in dry methanol (12 mL) under N2 atmosphere and the solution was cooled to 0 ◦ C in ice bath. A 10% Pd/C (0.339 g) was added to the solution of p-nitrophenylbutyrate followed by ammonium formate (1.654 g, 26.23 mmol). The resulting reaction mixture was stirred at 0 ◦ C under N2 for 5 min and then 15 min at 20 ◦ C before diluted with ethyl acetate (20 mL) and filtered through short pad of silica. The filtrate was evaporated under reduced pressure, residue was extracted with toluene, and the insoluble part was removed by filtration. Toluene was evaporated under reduced pressure. Purification of crude product by chromatography on silica gel column (nhexane/ethyl acetate = 2:1) and recrystallization from ethanol gave 0.50 g pAPB (64%). The purity of pAPB was analyzed by C18 column to be 99.7%. The identity of produced pAPB was confirmed by mass spectrometric analysis (EI MS, m/z (%): 179 [M]+ (10), 109 (100)). 2.6. PCR amplification and magnetic beads preparation of single-stranded DNA (ssDNA) The PCR amplification was performed in a final volume of 50 ␮L in the DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA). The reaction mixture contained approximately 5 ng of pIVEX-efts plasmid DNA, 0.2 ␮M EFTS-F (5 -biotinGGCATCATTCTGGAAGTTAACTGCC-3 ) and EFTS-R (5 -CAATACCTGTATTCCTCGCCTGTCTTTTGCTTGGTTCCATAACGA-3 ) primers, 200 ␮M each dNTP and 1 U Taq DNA polymerase in 10 mM Tris–HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2 , 0.1% (v/v) Triton X-100. For amplification, after 2 min denaturation at 94 ◦ C, 16 cycles were used with 30 s denaturation at 94 ◦ C, 30 s annealing at 55 ◦ C, and 40 s extension at 72 ◦ C. A final extension step was done for 5 min at 72 ◦ C. Biotinylation of DNA was done during the PCR by the biotinylated primer EFTS-F and the whole length of dsDNA was 534 bp including 24 nucleotides complement to A34. The biotinylated dsDNA was directly incubated with pretreated 20 ␮L magnetic beads in binding and washing buffer at 20 ◦ C for 30 min, then washed thrice with binding and washing buffer and the beads were collected with external magnets. A 50 ␮L 0.3 M NaOH was applied to beads and incubated for 5 min before the supernatant was removed. The final beads were washed twice by binding and washing buffer and 25 ␮L of water was incubated with beads at 95 ◦ C for 5 min to dissociate biotinylated ssDNA from the beads. The final ssDNA was analyzed by agarose gel electrophoresis, and the concentration was assumed to be 0.2 ␮M (derived from the original primer EFTS-F concentration 0.2 ␮M). 2.7. Chip construction and instrumentation Chip and instrumentation used are as described (Nebling et al., 2004) with minor modification. Each chip (11 mm × 13 mm)

consisted of 8 individual 0.85 mm diameter (0.6 mm2 ) electrodes with spaces of 2.0 mm to the next positions. For measurement, the printed circuit board of the chip was connected to multipotentiostat device. The potentiostat was connected to a PC through a serial interface SCB-68. Software Labview 6.0 was applied to control the potentiostat, collect the data and plot figures. 2.8. Pretreatment of electrodes and capture ODN immobilization Gold electrodes were immersed in ethanol for 5 min, rinsed by deionized water and dried by 0.2 ␮m filtered air stream. For immobilization of thiolated capture ODNs, a thiol/gold interaction at gold surfaces was used. One microliter of 0.2 ␮M capture ODN in immobilization buffer were spotted onto electrodes and then incubated at 20 ◦ C for 30 min before rinsed by water. DNA immobilization and following hybridization procedures were performed in humidity chamber. 2.9. Hybridization For binding of EST2–A34 conjugates to immobilized ODNs, 1 ␮L of 20 nM EST2–A34 in hybridization buffer was applied onto each electrode and incubated for 30 min at 50 ◦ C. The chip was then washed three times with washing buffer. For estimation of detection limit, after stepwise dilution of EST2–A34 with hybridization buffer, 0.5 ␮L of diluents were applied onto each electrode that had been immobilized with the capture ODN-P, and incubated for 30 min at 20 ◦ C, then chip was washed thrice with 100 ␮L washing buffer. For determination of mismatch selectivity, 1 ␮L 100 nM EST2–A34 in hybridization buffer was incubated with electrodes which carrying immobilized capture PM, MM-13, MM-7 and MM-4, respectively, for 30 min and washed three times with 100 ␮L of washing buffer before measurement. To detect mismatches by ODN biotin-34, 1 ␮L of 100 nM biotin-34 in hybridization buffer was dropped onto electrodes and incubated for 30 min, followed by three times 100 ␮L washing buffer. Afterward, the electrodes were exposed to a 400 nM EST2–biotin·SA in hybridization buffer for 30 min and washed thrice with 100 ␮L washing buffer. To detect PCR product, 1 ␮L of 13 nM ssDNA together with 100 nM EST2–A34 in hybridization buffer were applied onto electrode-immobilized capture and incubated at 20 ◦ C for 30 min. Subsequently three washing steps were applied to remove abundant ssDNA and reporter. 2.10. EST2 activity assay After hybridization and washing steps, the chip was fixed onto the multipotentiostat device, and 1 mM pAPB in chip buffer was pumped into chamber at 0.2 mL/min for about 1 min. Slope of current after flow stopped was measured to assay the EST2 enzymatic activity. It was assumed that this value is proportional to the amount of analyte DNA on the measured positions.

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3. Results 3.1. Preparation and purification of EST2–A34 conjugates The EST2–A34 conjugates were prepared by covalent coupling following a described procedure (Kukolka and Niemeyer, 2004) of a 5 -amino modified 34mer ODN (A34) with EST2 mutant (EST2E118C) in which the 118th residue, a glutamate, was replaced by cysteine. Wild type EST2 contains one cysteine residue in position 97. This cysteine is, however, buried in the tertiary structure (De Simone et al., 2000) and does not react with sulfhydryl reagents. The introduction of an additional cysteine, on the surface of the molecule, allowed a specific reaction of the EST2 with a bifunctional cross-linking reagent, sulfo-SMCC, maleimide and activated ester (Fig. 1A). The EST2–A34 conjugates were purified by anion-exchange chromatography on a MonoQ HR5/5 column. The peak indicated by an arrow contained the expected conjugates and was eluted at about 0.5 M NaCl (Fig. 1B). SDS-PAGE was used to identify the EST2–A34 conjugate, which can be localized by both ethidium bromide and EST2 activity staining (Higerd and Spizizen, 1973) (Fig. 2A).

Fig. 2. Characterization of EST2–A34 conjugates on 10% SDS-PAGE and MALDI-TOF-MS. (A) SDS-PAGE Gel was stained by ethidium bromide (left) and by EST2 activity (right). Lane 1: crude product; lane 2: EST2E118C; lane 3: purified EST2–A34 from the arrowed peak in Fig. 1B, arrow indicates the position of EST2–A34 conjugates. (B) The molecular masses of the conjugate determined by MALDI-TOF MS. Artifact intensity (a.i.) shows intensity of signal.

SDS-PAGE of the purified conjugate revealed a main band with an apparent mass of about 58 kDa (Fig. 2A, lane 3), which is larger than theoretically expected value of 44 kDa. MALDI-TOF MS, however, shows that the conjugate has a molecular mass of about 44.2 kDa (Fig. 2B), which is consistent with the anticipated value. As expected, one A34 coupled to one EST2, i.e. the buried sulfhydryl group of the Cys97 remained unmodified under applied conditions. 3.2. Sensitivity of the detection

Fig. 1. Preparation and purification of EST2–A34 conjugate (A) covalent coupling of 5 -NH2 modified A34 to EST2E118C and (B) purification of conjugate by ion-exchange chromatography. Peak of EST2–A34 conjugate is indicated by an arrow.

To demonstrate the application of EST2–A34 as a reporter for binding to complementary ODNs, we performed a two components DNA hybridization experiment depicted in Fig. 3A. A capture ODN with a sequence complementary to EST2–A34 was covalently immobilized via 5 -SH group to the gold surface of the electrode and the EST2 was brought to its vicinity by means of hybridization with EST2–A34 conjugate. Then the p-aminophenylbutyrate started to be hydrolyzed. A redox recycling mode (Nebling et al., 2004) was applied to measure the amount of produced p-aminophenol (Fig. 3B). The pAPB was selected among several p-aminophenyl esters with different

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Fig. 3. (A) Schematic diagram of DNA hybridization assay with EST2–A34 reporter. (B) The esterase immobilized on the electrode by DNA hybridization catalyzes the hydrolysis of p-aminophenylbutyrate to electrochemical active p-aminophenol and results in current of redox recycling between p-aminophenol and quinoneimine. (C) Currents measured from the differently treated electrode positions. During first 75 s, 1 mM pAPB was continuously delivered to the electrode with a flow rate of 0.2 mL/min. Then, the substrate flow was stopped. The slope of the burst from first 5 s was used to measure the amount of the immobilized enzyme. Solid, dashed and doted lines represent electrodes with perfectly matched ODN (ODN-P), noncomplementary ODN (ODN-N) and blank electrode (Blank), respectively. (D) Relative amounts of immobilized esterase as determined by the value of slope under stop-flow mode in (C).

length of acyl chain (C2 to C8) as an optimal substrate possessing stability and maximal EST2 specific substrate activity (data not shown). A typical time versus amperometric signal plot generated with pAPB at 20 ◦ C is shown in Fig. 3C. During first 75 s, 1 mM pAPB was continuously delivered over the chip with immobilized EST2 placed in a chamber of about 20 ␮L volume. After about 30 s a steady state situation, under which supply of p-aminophenylbutyrate and release of p-aminophenol at the electrode position is in equilibrium, is reached. The slow decrease of the current at steady state is probably due to EST2 deactivation on the electrode surface and/or redox recycling’s damage to p-aminophenol. Increase of the signal was registered under stop-flow conditions (76–120 s). Theoretically, a linear increase of the signal is expected when the redox recycling is fully efficient and a p-aminophenol is permanently regenerated. Probably, as a result of EST2 deactivation, quinoneimine hydrolysis and substrate consumption, the current becomes constant after about 45 s of the stop-flow mode. Therefore, we used the slope derived from the signal at the beginning of the stopped-flow modus as a measure for EST2 activity. Compared to capture ODN-P, which was complementary to EST2–A34, a noncomplementary capture ODN, ODN-N, gave rise to only a marginal signal. By omitting the capture ODN

completely, only a very low, barely measurable signal could be registered (Fig. 3C), which probably resulting from an unspecific interaction of EST2–A34 with the gold surface. Reports about noncovalent interactions between the DNA backbone and the gold surface support this interpretation (Kimura-Suda et al., 2003; Lao et al., 2005). Fig. 3D shows the numerical presentation of the data collected in experiments presented in Fig. 3C. The value of current slope for the complementary capture, noncomplementary capture and blank were 3.20, 0.25 and 0.06 nA/s, respectively. This means that EST2 can be immobilized exclusively on electrodes by Watson-Crick base pairs formed between the EST2–A34 and its corresponding capture. The detection limit of the reporter was determined by serial dilution of the EST2–A34 in hybridization buffer, followed by hybridization. The lowest amount of EST2–A34 that could be detected under the used experimental arrangements with an analytical standard of signal/noise ratio >3, was estimated to be 1.5 × 10−18 mol (Fig. 4), corresponding to 0.5 ␮L of 3 pM EST2–ODN conjugate or ODN. 3.3. Selectivity of the detection DNA hybridization on a solid support is usually used to detect a mismatch in biosensor based systems. However, the compre-

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Fig. 4. Detection limit of EST2–A34 conjugates on a gold electrode of 0.6 mm2 area. Shown are mean values from three independent experiments.

hensive studies of hybridization conditions, such as temperature and different washing steps in order to achieve an optimal fidelity and stringency of DNA hybridization are limited by properties and stability of ODN–enzyme conjugates. In order to improve this situation we compared the hybridization results obtained by two different types of EST2 reporters, a covalent EST2–A34

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conjugate and an EST2–biotin·SA·biotin-34 complex. The latter was outcome of EST2–biotin·SA binding to ODN biotin-34, which had hybridized to its surface-immobilized capture. Experiments were carried out simultaneously on one chip containing four working electrodes array with immobilized capture ODNs. PM is perfect matched capture to EST2–A34, while MM-13, MM-7 and MM-4 are mismatched captures that each containing one mismatched nucleotide at number indicated position of 24mer nucleotides. Two different hybridization temperatures were compared. At 50 ◦ C EST2–A34 was able to distinguish the perfect match and single mismatch (Fig. 5A), while EST2–biotin·SA·biotin-34 complex failed to do so (Fig. 5B). However, at 20 ◦ C, shown in Fig. 5C and D, both EST2–A34 and EST2–biotin·SA·biotin-34 were able to detect the mismatch, though the mismatch discrimination (Fig. 5D) is not as good as that for experiments shown in Fig. 5C. Clearly, the selectivity provided by covalent EST2–A34 conjugate is superior to the selectivity achieved via streptavidin/biotin conjugation. Washing the electrodes with a buffer containing 30 mM NaCl and 10 mM Tris–HCl, pH 7.5 did not improve the stringency of mismatch recognition (Fig. 5C and D). It is known that base mismatches have influence on the stability of helices and exert a strong effect on solid-phase hybridization (Hughes et al., 2001; Peterson et al., 2002). These effects

Fig. 5. Discrimination between immobilized, perfectly matched ODN and single base mismatched ODNs. (A) and (C) hybridization of EST2–A34 at 50 ◦ C and 20 ◦ C, respectively; (B) and (D) hybridization of biotin-34 at 50 ◦ C and 20 ◦ C, respectively, followed by binding of EST2–biotin·streptavidin conjugate. Empty histogram in (C) and (D) shows the effect of an additional thrice 20 min washing step with 30 mM NaCl, 10 mM Tris–HCl, pH 7.5. Reporter EST2–biotin·SA·biotin-34 is the outcome of hybridization of biotin-34 with capture and subsequent binding of EST2–biotin·SA.

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were also observed in Fig. 5A and C when the hybridization was performed at 50 and 20 ◦ C. In the case of short-length capture ODNs, mismatch discrimination can be achieved by rinsing the initially formed complex with low salt buffer, which leads to preferential dissociation of the less stable complex. In experiments presented in Fig. 5C and D with 24mer captures, we did not observe such an effect. Nevertheless, mismatch discrimination achieved by EST2–A34 without additional stringent washing was sufficient enough to detect the interaction (Fig. 5A and C). The solid-phase hybridization appears to have a mechanism preventing the mismatched capture from binding to the EST2–A34. It seems that this energy barrier was eliminated at 50 ◦ C, leading to increased density of immobilized EST2–A34 and high current response. However, ability to differentiate mismatches is better at 20 ◦ C. There is a 10-fold increase in the current intensity in Fig. 5A as compared with Fig. 3D. This is probably due to a low capture density on electrode surface, and to a long thymidine spacer used in experiment depicted in Fig. 5. Thus, a low steric crowding on the electrode increases the hybridization efficiency (Peterson et al., 2001, 2002) and gives rise to a strong signal. Under identical conditions the covalent EST2–A34 conjugate gave much stronger signal (Fig. 5A and C) than that of EST2–biotin·SA bounding formation of an EST2–biotin·SA·biotin-34 complex. The molecular mass and dimension of streptavidin being 60 kDa and 8.4 nm × 8.4 nm × 4.65 nm, respectively (Scheuring et al., 1999), probably hinders the access of the reporter to the electrodes. The low signal intensity and mismatch selectivity observed with streptavidin/biotin mediated conjugation of ODN with EST2 (Fig. 5B and D) demonstrate the advantage of direct covalent conjugation of ODN with the reporter enzyme (Fig. 5A and C). 3.4. Electrochemical detection of a mismatch in a single gene by EST2–A34 reporter Mismatch detection during DNA hybridization is an important research topic of molecular biology and medicine. Ultimate goal is to develop an easily applicable platform for the detection of single nucleotide polymorphism or points mutations (Marrazza et al., 2000; Nakamura et al., 2005; Tombelli et al., 2000). High sensitivity, simple instrumentation, low price and a possibility of on-line applications are the main advantages of the electric detection systems. A 510 nucleotides sequence of EF-Ts was chosen as model to investigate discrimination in hybridization between perfectly matched and single-base mismatched DNA duplexes by EST2–A34 conjugate as a reporter. The hybridization was performed in an array of four electrodes, each endowed with a different capture, and signal was collected in parallel under nearly simultaneous response of the current from all four electrodes. As demonstrated in Fig. 6A and B, the hybridization occurred with all capture ODNs and its extent could be determined by EST2–A34 reporter. Investigations using microarray technology show that the secondary structures of long DNA can prevent hybridization to ODNs on microarray (Chien et al., 2004; Lane et al., 2004). In this work we also observed a significant position effect upon hybridiza-

Fig. 6. Simultaneous measurements of EF-Ts ssDNA on a four electrodes arrayed chip. (A) Scheme of ssDNA preparation and hybridization. The sequences of particular capture ODNs are listed in Table 1 of Supplementary information. (B) Diagrams of current intensities obtained by perfectly matched capture ODNs to different position of ssDNA sample. (C) Diagrams of current intensities obtained for four types of mismatched base-pair in the 90–110 region of 510 nucleotides EF-Ts segment. Shown are the mean values of three independent experiments.

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tion. As shown in Fig. 6B, different signal intensities depicted by different captures are due to effects of secondary structure. To determine the ability for single mismatch identification, as shown in Fig. 6C, captures (C-TS2A, C-TS2C and C-TS2G) with single nucleotide variation gave less than 50% signal values as compared with that given by perfectly matching capture, CTS2. This indicates that identification of point mutation in long PCR product can be realized using EST2 as a reporter. 4. Discussion Thermostable esterase 2 from A. acidocaldarius was used as a reporter enzyme to detect DNA hybridization on gold electrodes equipped with 20–25 nucleotide-long ODNs. The main purpose of the work was to test the limits of electrochemical detection using this new reporter enzyme. Another objective of the present investigation was to compare a chemically defined covalent conjugation of ODN to a single chain polypeptide of the EST2 with the widely used streptavidin/biotin mediated conjugation of ODNs to the alkaline phosphatase. Alkaline phosphatase is a dimer reporter enzyme commonly used in enzyme arrays for spectrophotometric or amperometric detections (Gabig-Ciminska et al., 2004; Lucarelli et al., 2005). Electrochemical detection of DNA hybridization by paminophenol coupled reaction on gold electrodes requires only very small reaction volumes, limited only by the properties of the available microfluidic systems. The detection limit for electrochemical identification of p-aminophenol is 20-fold better than that of spectrophotometric detection of p-nitrophenol (Thompson et al., 1991). These merits render possibility to use the method for sensitive, routine analysis. The optimal sensitivity range as estimated in this work is about 105 to 108 molecules EST2–ODN conjugate/0.6 mm2 gold electrode overlaid with 0.5 ␮L solution (Fig. 4). Bellow this range (<105 molecules/electrode) the signal intensity is low and the signal/noise ratio decreases. In order to go below this detection limit, the size of the electrode and the volume of the analyzed solution have to be decreased, which can be reached by high density electrode arrays and improved microfluidics. The most common enzyme reporter, used in numerous assays previously, is streptavidin–alkaline phosphatase conjugated via glutaraldehyde cross-linking between ␧-amino of lysine residues. DNA hybridization using a bulky streptavidin–alkaline phosphatase conjugates, might be disguised by unspecific interaction of the protein with the electrode surface or by steric hindrance for the access of the biotin modified ODNs. Due to statistical character of all accessible ␧-amino groups of lysine residues in streptavidin and alkaline phosphatase, large number of different complexes can be formed giving rise to broad distribution of steric hindrance. Application of a single chain enzyme as a reporter allows a controlled chemical modification and conjugation with an ODN, a predictable molecular structure of the reporter enzyme and definitely enzymatic activity. Correspondingly, the reproducibility and the selectivity of hybridization had been expected to be improved in the case of ODN covalent linked to EST2 as compared with unspecific streptavidin/biotin type of

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conjugation. This was indeed observed in the present investigation. Surprising and not entirely understood is the temperature effect observed during hybridization with EST2–A34 conjugates. The selectivity at 50 ◦ C in respect of single base replacement was relatively low, compared with selectivity detected at 20 ◦ C. Differences in the thermodynamic stabilities of the perfectly matched and mismatched complexes should be actually larger near the melting temperature than that at 20 ◦ C. The low selectivity at 50 ◦ C can be possibly explained by unspecific interaction of the protein or ODN with the gold electrode (Kimura-Suda et al., 2003; Lao et al., 2005). Under optimal condition, at 20 ◦ C, even a detection of a single nucleotide polymorphism in 510 nucleotide ssDNA is feasible by electrode arrays equipped with ODN captures complementary to successive DNA sequence segments. This application had been demonstrated in the present work. It became, however, again clear that the selectivity of hybridization and the signal to noise ratio may critically depend on the secondary structure of the long DNA strand (Chien et al., 2004; Lane et al., 2004). Application of high-density microelectrode arrays allowing a parallel measurements and internal controls may be, therefore, essential for this type of routine applications. 5. Conclusion In this study, we introduced a sensitive enzyme–ODN reporter which is promising in electrochemical chip detection of DNA hybridization. The esterase 2 from A. acidocaldarius combined with its optimized electrochemical substrate paminophenylbutyrate has a detection limit at attomolar level, using the available microelectrode. Furthermore, as shown in this study, EST2–ODN conjugate exhibits superior mismatch discrimination to mostly adopted streptavidin–phosphatase conjugate. Finally, detection of single nucleotide polymorphism can be achieved and simplified by using EST2 conjugated with ODN implemented in highly integrated microsystem. Acknowledgements This work was supported by HighTec Offensive Bayern and Fonds der Chemischen Industrie. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2006.08.046. References Agafonov, D.E., Rabe, K.S., Grote, M., Huang, Y., Sprinzl, M., 2005. FEBS Lett. 579 (10), 2082–2086. Baeumner, A.J., Cohen, R.N., Miksic, V., Min, J., 2003. Biosens. Bioelectron. 18 (4), 405–413. Baeumner, A.J., Pretz, J., Fang, S., 2004. Anal. Chem. 76 (4), 888–894. Carpini, G., Lucarelli, F., Marrazza, G., Mascini, M., 2004. Biosens. Bioelectron. 20 (2), 167–175. Caruana, D.J., Heller, A., 1999. J. Am. Chem. Soc. 121 (4), 697–774.

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