2456
Bioconjugate Chem. 2008, 19, 2456–2461
Enhancement of Electrochemical Signal on Gold Electrodes by Polyvalent Esterase-Dendrimer Clusters Martin Humenik, Christopher Po¨hlmann, Yiran Wang, and Mathias Sprinzl* Laboratorium fu¨r Biochemie, Universita¨t Bayreuth, Universita¨tstrasse 30, 95440 Bayreuth, Germany. Received August 18, 2008; Revised Manuscript Received October 14, 2008
5′-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterasecatalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.
INTRODUCTION Electrochemical sensors for the detection of DNA hybridization combine nucleic acid layers with electrochemical reporters to generate electrical signals. This type of sensors simplifies the data collection and makes it suitable for construction of portable DNA biosensors (1, 2). The improvement of biosensor sensitivity remains, however, a challenging task. Enhancement of the signal has been achieved by optimization of sensor design and performance of enzyme reporters. The physical optimization via minimization of the sensor surface (3, 4) and redox-recycling of electro-active substrate (5, 6) has improved detection limits dramatically. Nowadays, most frequently used reporter enzymes are alkaline phosphatase and horseradish peroxidase (7). The important task is to increase stability and specific activity of enzymes (8, 9), and to improve the quality of enzyme conjugation to the DNA hybridization event. Conjugation can be achieved either via interaction of streptavidin-enzyme and biotinylated ODN1 (6, 10) or directly via covalently conjugated enzyme-ODN (8, 11). We recently developed a sensitive enzyme-ODN reporter for electrochemical detection of DNA hybridization (8), which uses thermostable esterase 2 from Alicyclobacillus acidocaldarius (12) as a reporter enzyme. Single-chain esterase 2 provides the possibility for site-specific conjugation of the protein and ODN. EST-ODN conjugate with a 1:1 ratio of protein to oligonucleotide allowed the detection of attomoles of DNA (8). It is expected that the sensitivity of detection will be substantially improved if several reporter enzyme molecules are attached to one ODN. In the present study, we describe a site-specific chemistry for the preparation of ODN reporters equipped with multiply coupled esterase via dendrimer branch* Author to whom correspondence should be addressed. Fax: +49921-55-2066; e-mail:
[email protected]. 1 Abbreviations: EST, esterase 2 (C97S, E118C); G3/G5, generation 3/generation 5; FPLC, fast protein liquid chromatography; ODN, oligodeoxynucleotide; PAGE, polyacrylamide gel electrophoresis; PAMAM, poly(amidoamine) dendrimer; sulfo-SMCC, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; SMEG, succinimidyl-[(N-maleimidopropionamido) diethylene glycol]; TCEP, tris(2carboxyethyl)phosphine.
ing. We employed commercially available PAMAM dendrimers of generation 3 and generation 5 (Figure 1A), which differ in diameter, multivalency, and surface group density (13, 14). These polyvalent EST-dendrimer-ODN clusters were used in a DNA-directed immobilization assay on a gold electrode (Figure 1B). The capacity of signal enhancement and the ability of mismatch discrimination were determined and compared to monovalent EST-ODN conjugate (8).
MATERIALS AND METHODS General. The organic bifunctional reagents sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidyl-[(N-maleimidopropionamido)-diethylene glycol] ester were purchased from Pierce (Rockford, IL, USA); G3 and G5 cystamine core PAMAM dendrimers and tris(2-carboxyethyl)phosphine were from Aldrich (Taufkirchen, Germany). 5′Amino and 5′-sulfhydryl modified ODNs, equipped with C6 linker, were obtained from Biomers (Ulm, Germany). BiogelP6 was from Biorad (Mu¨nchen, Germany). SepPak C18 columns (Waters Corp., Eschborn, Germany) were used for desalting of oligonucleotides. Microcon YM10 centrifuge tubes (Millipore, Schwalbach, Germany) were used for desalting and concentration of dendrimer-ODN probes. Gel permeation chromatography was performed by using Pharmacia FPLC system on Sephacryl S-200 HR column (Pharmacia, Freiburg, Germany). Preparation of Esterase 2 (C97S, E118C). Plasmid pET30aEstC97S_E118C was derived from pT7-SCII-Est2 plasmid DNA (12) to replace Cys97 to Ser and Glu118 to Cys in esterase 2. The gene was expressed in E. coli BL21 (DE3). Esterase was purified according to published procedure (12) and stored at -20 °C in 100 mM Tris/HCl, pH 7.5, 20 mM MgCl2, 200 mM KCl, 1 mM β-mercaptoethanol, and 50% glycerol. The esterase was dialyzed against 0.1 M Na-Pi, pH 7.2, before conjugation with oligonucleotides. 5′-Maleimide-ODN. 5′-Amino-modified ODN (50 nmol) in 0.1 M Na-Pi, pH 8.0, (100 µL) was treated with sulfo-SMCC (2.5 µmol, 50 µL in DMF). The reaction mixture was left at RT for 1 h and the 5′-maleimide-ODN was precipitated with ethanol and isolated by centrifugation. The pellet was dissolved in 0.1 M Na-Pi buffer, pH 7.4, (500 µL) and desalted on a Biogel P6 column. The procedure yielded typically 90% of 5′-
10.1021/bc800353z CCC: $40.75 2008 American Chemical Society Published on Web 11/19/2008
Electrochemical Signal on Gold Electrodes
Bioconjugate Chem., Vol. 19, No. 12, 2008 2457
Figure 1. (A) Structure of the PAMAM containing a cystamine core (for simplicity, only the branching of G1 dendrimer is depicted). (B) Principle of the signal enhancement on a gold electrode; multivalent attachment of esterase 2 to ODN via PAMAM increases the amount of reporter enzyme per hybridization event in the vicinity of particular electrode.
maleimide-ODN as determined by A260 absorption. The purity and identity of the product were analyzed by HPLC and MALDI-TOF (Supporting Information). Dendrimers-ODN Conjugates. Dendrimer G3 (100 nmol, 7.7 µL, in methanol) was added into triethylamine solution in DMF (25 µL, 123 mM) followed by solution of acetic anhydride in DMF (25 µL, 93 mM). The reaction mixture was left at RT for 2 h and used without purification for next steps. The acetylation of G5 PAMAM was performed under similar conditions, where 100 nmol of dendrimers (33.6 µL in methanol) was diluted with DMF to the end volume of 100 µL and treated with 0.9 µL of acetic anhydride (9.5 µmol) and 1.5 µL of triethylamine (11.5 µmol). The reaction mixture of acetylated dendrimer was then diluted with 3 volumes of buffer (50 mM Na-Pi, 150 mM NaCl, 5 mM EDTA, pH 6.3) treated by 1 µmol of TCEP (100 mM solution in the same buffer), followed by incubation at 37 °C for 1 h. The freshly prepared reaction mixture of sulfhydryl-activated dendrimer was combined with 100 nmol of 5′-maleimide-ODN (40 µM in 50 mM Na-Pi, 150 mM NaCl, 5 mM EDTA, pH 6.3). The reaction mixture was left at RT for 1 h and subsequently filtered through YM10 centrifuge tubes. The retained product was washed twice with 0.1 M Na-Pi buffer, pH 8.0, and collected in the same. Typically, 70% yield was obtained for dendrimer-ODN conjugates of generations 3 and 5. The reaction course was followed by ureaPAGE and HPLC. Product was characterized by MALDI-TOF (Supporting Information). Maleimide Activation of Dendrimer-ODN Conjugates. Dendrimer-ODN conjugates in 0.1 M Na-Pi, pH 8.0, (15 nmol, 150 µL) were treated with bifunctional reagent SMEG (4.5 µmol, 90 µL in DMF) at RT for 1 h. Excess of the reagent was removed by a Biogel-P6 column affording 50% of maleimidedendrimer-ODN of generation 3. G5-derived conjugate was isolated by 4× repeated filtration in YM10 centrifuge tubes against 50 mM Na-Pi, 150 mM NaCl, 5 mM EDTA, pH 6.3, yielding typically 70% maleimide-dendrimer-ODN conjugate. Preparation of EST-Dendrimer-ODN Cluster. Esterase 2 (C97S, E118C) in 0.1 M Na-Pi buffer, pH 7.2, (70 µΜ) was
treated with 1.4 mM TCEP at 37 °C for 30 min and isolated by filtration in YM10 centrifuge tubes removing TCEP. The esterase was collected in 50 mM Na-Pi, 150 mM NaCl, 5 mM EDTA, pH 6.3, and 360 µL of the protein solution (0.2 mM) was treated with G3 maleimide-dendrimer-ODN conjugate (0.1 mM, 90 µL in the same buffer) at 37 °C for 16 h. Product was purified by gel permeation chromatography on Sephacryl S-200 HR column (2 × 70 cm), eluted with 10 mM Tris/HCl, 10 mM KCl, pH 7.5, at a flow rate of 1 mL/min. The main peak of the product was collected (10-12 mL), concentrated under vacuum to one-fifth of the volume, and reloaded on the same column for the second round of chromatography. Electrochemical Detection on Gold Electrodes. Chip modules and potentiostat were obtained from Siemens Corporate Technology (Erlangen, Germany). Each chip (11 mm × 13 mm) consisted of 8 individual 0.85-mm-diameter (0.6 mm2) gold electrodes with spaces of 2.0 mm between the positions. Four electrodes were treated with a silver/silver chloride solution and served as reference electrodes. For measurements, a U-shaped 10 µL flow chamber was placed over the electrodes, and the printed circuit board of the chip was connected to the multipotentiostat device. The potentiostat was connected to a PC through a serial interface SCB-68. LabView 6.0 software was used to control the potentiostat, collect the data, and plot figures. Immobilization of DNA on Gold Electrodes. The gold electrodes were rinsed with water and ethanol, and dried under air stream. The thiol group of the capture ODN (0.2 µM) was first reduced by treatment with 2 mM TCEP in 10 mM Na-Pi, 300 mM NaCl, pH 7.0, at 37 °C for 5 min. Activated capture ODN (1 µL) was spotted onto electrodes and incubated at ambient temperature in a humidity chamber for 16 h. Individual electrodes were washed three times with the same buffer. The sequences of the used capture ODNs are listed in Table 1S (Supporting Information). EST Activity Measurements on the Gold Electrodes. Detection limits were estimated by serial dilution of EST-ODN conjugate or EST-dendrimer-ODN clusters in 20 mM Na-Pi, 300 mM NaCl, 2 mM EDTA, 0.05% Tween 20, 1 mg/mL BSA,
2458 Bioconjugate Chem., Vol. 19, No. 12, 2008
Humenik et al.
Table 1. Apparent Michaelis-Menten Constants Obtained from the Kinetic Measurement of Esterase Reporter Immobilized via Hybridization of Corresponding ODN Conjugate or Dendrimer-ODN Cluster on the Gold Electrode EST-ODN KMapp [mM] Imax [nA s-1] kcat [nA s-1 nM-1]
0.7 ( 0.1 6.6 ( 1.8 1.3 ( 0.3
EST-dendrimer-ODN G3
G5
0.9 ( 0.1 31.7 ( 3.6 6.3 ( 0.7
1.1 ( 0.4 57.8 ( 12.6 11.5 ( 2.5
pH 7.4. Different concentration of EST conjugates (1 µL) was applied onto each electrode equipped with immobilized perfect match capture ODN and incubated at 25 °C for 20 min. The electrodes were washed three times with 100 µL of 5 mM NaPi, 75 mM NaCl, 0.5 mM EDTA, 0.05% Tween 20, pH 7.4. The same experimental procedure was used for mismatch determination. Different capture ODNs comprising 1 to 3 mismatches were used. After hybridization at 50 °C and washing steps, the chip was placed into a potentiostat and 1 mM substrate, p-aminophenylbutyrate, in 0.1 M Na-Pi, pH 7.0, was pumped through a flow chamber at flow rate of 50 µL/min for 1 min. The flow was stopped and the current slope was measured to assay the EST activity (8). Measurement of enzyme kinetics was conducted by using EST-ODN conjugate and EST-dendrimer-ODN clusters (5 nM) to hybridize with perfect match capture ODN on gold electrodes. Different concentrations of p-aminophenylbutyrate in 0.1 M NaPi, pH 7.0, were delivered over gold electrodes, and signal was recorded as described above.
RESULTS AND DISCUSSION Preparation and Characterization of EST-DendrimerODN Clusters. Primary amino groups on the surface of G3 and G5 PAMAM cystamine core dendrimers (14) had to be partially acetylated (15) to prevent the unspecific binding of the positively charged dendrimers to oligonucleotide polyanion. Approximately 60% acetylation ratio, estimated by MALDITOF (Supporting Information, Figures 3S and 4S), was found as an optimal shield of positive charge on the dendrimer surface and at the same time provides sufficient amount of free amino groups for further multiple EST coupling. The partially acetylated dendrimers were reduced and conjugated to 5′-maleimideODN (16). Bifunctional linker sulfo-SMCC was used to activate 5′-amino-ODN for monovalent conjugation as previously described (8). The free amino groups on the surface of dendrimer-ODN conjugate allow further modification with a maleimide linker. Multiple binding of nonpolar linkers such like SMCC decreases the solubility in aqueous environment. Therefore, ethylene glycol linker SMEG, which provides both better solubility and spacing between dendrimer and protein surface, was used. The formation of dendrimer products with different extent of maleimide activation was evident from smear on ureaPAGE and from peak broadening on HPLC (Supporting Information, Figures 1S and 2S). MALDI-TOF spectra also revealed broad molecular peaks, typical for dendrimers (17, 18) and dendrimer-ODN conjugates (16) and did not allow precise molecular mass determination of the product (Supporting Information, Figures 3S and 4S). The wild-type esterase 2 contains only one cysteine at position 97, which is buried inside the structure and is not accessible for modification (19). Thus, replacements of cysteine 97 by serine and glutamine 118 by cysteine were performed to expose a reactive sulfhydryl group on the protein surface. ESTdendrimer-ODN clusters (Figure 2) were prepared by reaction of 8- and 16-fold excess of EST with maleimide-activated dendrimer-ODN of generations 3 and 5, respectively. Statistical mixtures of clusters (Figure 3A,B, lane 1), as reported also for
other protein-dendrimer conjugates (20, 21), were obtained. The higher molecular mass clusters were separated from unreacted esterase and dendrimer-ODN conjugate by gel permeation chromatography on Sephacryl S 200 HR (Figure 3A,B). Monovalent EST-ODN conjugate was also prepared and purified as described (8). The relative protein content of the purified clusters was determined from absorbance ratio at 260 and 280 nm, defined as 260 A260 ε260 p cp + εo co ) 280 A280 ε c + ε280c p
p
o
(1)
o
where A is absorbance at 260 or 280 nm and ε represents extinction coefficient, while cp and co represent the concentration of protein and ODN, respectively. Experimental A260 and A280 values from different mixtures of esterase 2 and 5′-aminomodified ODN (from 0:1 to 18:1) were fitted to a hyperbolic curve (Figure 4). The resulting calibration curve allowed us to deduce the composition of the G3 cluster (7:1) and the G5 cluster (10:1). G3 and G5 PAMAMs significantly differ in the number of surface amino groups (4-fold) (14), whereas the observed loading difference between G3 and G5 dendrimer-ODN with the esterase was less than 2-fold. The different reactivities of smaller G3 and larger G5 maleimide-dendrimer-ODN conjugate toward bulky protein are probably reasons for observed deviations. The amount of the esterase anchored on the gold electrode via hybridization between capture ODN and either monovalent EST-ODN conjugate or polyvalent G3/G5 clusters (Figure 1B) was tested. The intensities of the electrical signal in the presence of varied concentrations of p-aminophenylbutyrate were measured. Apparent kinetic parameters (Table 1) were calculated from the electrochemical version of the Lineweaver-Burk equation (eq 2) (22). app KM 1 1 + ) × Islope Imax [S] Imax
1
(2)
The apparent Michaelis-Menten constant (KMapp) is a reflection of both the enzymatic affinity and the ratio of microscopic constants including diffusion of the product to the electrode surface. Islope represents the increase of the current after the addition of substrate (this value was determined the first 5 s in stopped flow mode (8)), [S] is the bulk concentration of substrate, and Imax is the maximum current measured under substrate saturation. Different values of current slope against substrate concentration, with obvious similarity to Michaelis-Menten kinetics (Supporting Information, Figure 5S), were observed using 5 nM EST-ODN, G3 and G5 EST-dendrimer-ODN clusters. With the G3 and G5 clusters, the increase in maximal current Imax (Table 1) demonstrates that more esterase is brought to the electrode surface by a hybridization event if compared with monovalent EST-ODN. Additionally, catalytic efficiency kcat corresponds with EST/ODN ratio obtained from the A260/A280 dependence depicted in Figure 4. Signal Enhancement on Gold Electrodes Using Multivalent EST-Dendrimer-ODN Clusters. The influence of polyvalent G3 and G5 EST-dendrimer-ODN clusters on signal enhancement was compared with monovalent EST-ODN conjugate (8). Different concentrations of EST bound ODN probes were assayed on the gold electrodes uniformly modified with perfectly matched capture ODN and resulted in linear dependences in all experiments (Figure 5). The lowest detectable ODN concentration using the EST-ODN conjugate was 3 pM, which is in agreement with results reported previously (8). The G3 and G5 EST-dendrimer-ODN clusters revealed upon the same experimental arrangement the lowest detectable target
Electrochemical Signal on Gold Electrodes
Bioconjugate Chem., Vol. 19, No. 12, 2008 2459
Figure 2. Schematic representation of esterase-dendrimer-ODN cluster. SMCC linker connects ODN and sulfhydryl group of PAMAM. Esterase is attached to the dendrimer surface via ethylene glycol linker in a site-specific manner (Cys118).
Figure 4. Estimation of the molecular EST/ODN ratio for ESTdendrimer-ODN cluster derived from G3 and G5 PAMAM; UV absorption was recorded at 260 and 280 nm for different mixtures of esterase 2 and 5′-amino-ODN ([). The data were fitted to the hyperbolic plot (s). UV absorption of G3 (full line with arrows) and G5 cluster (dashed line with arrows) correspond to EST/ODN ratio 7:1 and 10:1, respectively.
Figure 3. Purification of the EST-dendrimer-ODN cluster by gel permeation chromatography on Sephacryl 200 HR; clusters derived from G3 (A) and G5 PAMAM (B) were eluted in the void volume of the column. First (- -) and second (s) round of purification are presented. Inserts show 10% SDS-PAGE of crude reaction products (lane 1) and purified clusters (lane 2) after second round of gel permeation chromatography.
concentrations of 0.3 pM (Figure 5A) and 0.02 pM (Figure 5B), respectively. Strong enhancement of the signal were achieved at low concentrations of clusters (10 nM, no signal enhancement was observed. It is conceivable that at higher concentrations the
advantage of the multiple esterase attachment was compromised by a large hydrodynamic volume of polyvalent EST-dendrimer moiety. On the other hand, at lower concentration levels, where most of the capture ODNs became unoccupied, the multiple enzyme binding significantly enhanced the signal. G3 and G5 clusters have comparable protein content (Figure 4), whereas diameters and corresponding areas of dendrimers differ significantly (4× and 8×, respectively) (14). Apparently, the dense protein loading of the G3 dendrimer surface may alter proper protein folding and substrate accessibility/diffusion summed in observed enzyme activity. Thus, G3 and G5 EST-dendrimerODN clusters differ by 10× in their signal enhancement abilities. The controlled spotting of G3 and G5 clusters either onto a bare gold electrode or onto an electrode layered with nonmatched capture ODN excluded any possible unspecific binding of higher PAMAMs onto the gold surface (23) or to capture oligonucleotides (24).
2460 Bioconjugate Chem., Vol. 19, No. 12, 2008
Humenik et al.
Figure 6. Mismatch discrimination ability of the EST-ODN conjugate and G3 and G5 clusters compared in the amperometric detection of immobilized capture probes. Perfectly matched capture and one, two, and three mismatches are presented as black, dashed, open, and crossed bars, respectively; the signal values for perfectly matched capture was set as 100%.
respectively. These detection limits are comparable with alternative methods of electrochemical DNA detection, which use horseradish peroxidase bound liposomes (30), ferrocene multiple labeling of gold nanoparticles (31), and cyclic DNA probes (32). The simple and efficient preparation and fairly improved detection limits emphasize the potential of the presented esterase 2-dendrimer-ODN construct in electrochemical detection of DNA.
ACKNOWLEDGMENT Figure 5. Detection of DNA hybridization on the gold electrodes equipped with uniform capture ODN. (A) Correlation of the signal with ODN concentration of G3 EST-dendrimer-ODN clusterd (•) and monovalent EST-ODN conjugate (O). (B) Analogous comparison for the G5 cluster.
Specificity of DNA hybridization and mismatch detection are important parameters of DNA analysis on biosensors. In order to test these parameters, gold electrodes were prepared with immobilized capture ODNs resulting in a perfect match and one, two, and three mismatched base pairs. The EST-ODN conjugate and EST-dendrimer-ODN clusters were then hybridized to these immobilized capture ODNs. Monovalent EST-ODN and polyvalent G3 and G5 clusters distinguished the perfect from the mismatched probes (Figure 6). The discrimination efficiency increased with the number of the nonmatched base pairs when EST-ODN and G3 clusters were assayed. However, the G5 cluster was not able to discriminate among the numbers of mismatches as efficiently as the G3 cluster.
CONCLUSION In summary, we prepared EST-dendrimer-ODN clusters derived from G3 and G5 cystamine core PAMAM dendrimers. Efficient sulfhydryl-maleimide chemistry to connect ODN and dendrimer, which was polyvalently functionalized with esterase 2, was exploited. Although mutivalency of dendrimers was already applied in the construction of protein (20, 21, 25-27) and ODN (16, 28, 29) nanoscale devices, according to our knowledge the synthesis of polyvalent enzyme-dendrimer-ODN clusters had not yet been reported. DNA-directed hybridization of the presented polyvalent clusters on gold electrodes revealed 10- and 100-fold shifts of the detection limit to lower concentrations if compared among G3 and G5 clusters and monovalent EST-ODN conjugate (8). Hence, the multivalent binding of esterase to capture ODN resulted in detection limits of 3 × 10-13 M and 2 × 10-14 M for G3 and G5 dendrimer clusters,
This work was supported by the Forschungskreis der Erna¨hrungsindustrie, AiF 230 ZN. Supporting Information Available: Figures 1S-5S and Table 1S as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED (1) Drummond, T. G., Hill, M. G., and Barton, J. K. (2003) Electrochemical DNA sensors. Nat. Biotechnol. 21, 1192–1199. (2) Sassolas, A., Leca-Bouvier, B. D., and Blum, L. J. (2008) DNA biosensors and microarrays. Chem. ReV. 108, 109–139. (3) Paeschke, M., Wollenberger, U., Kohler, C., Lisec, T., Schnakenberg, U., and Hintsche, R. (1995) Properties of interdigital electrode arrays with different geometries. Anal. Chim. Acta 305, 126–136. (4) Zhang, Y., Kim, H. H., and Heller, A. (2003) Enzyme-amplified amperometric detection of 3000 copies of DNA in a 10-microL droplet at 0.5 fM concentration. Anal. Chem. 75, 3267–3269. (5) Niwa, O., Morita, M., and Tabei, H. (1990) Electrochemicalbehavior of reversible redox species at interdigitated array electrodes with different geometries - consideration of redox cycling and collection efficiency. Anal. Chem. 62, 447–452. (6) Nebling, E., Grunwald, T., Albers, J., Schafer, P., and Hintsche, R. (2004) Electrical detection of viral DNA using ultramicroelectrode arrays. Anal. Chem. 76, 689–696. (7) Bakker, E. (2004) Electrochemical sensors. Anal. Chem. 76, 3285–3298. (8) Wang, Y., Stanzel, M., Gumbrecht, W., Humenik, M., and Sprinzl, M. (2007) Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization. Biosens. Bioelectron. 22, 1798–1806. (9) Zhang, Y., Pothukuchy, A., Shin, W., Kim, Y., and Heller, A. (2004) Detection of approximately 10(3) copies of DNA by an electrochemical enzyme-amplified sandwich assay with ambient O(2) as the substrate. Anal. Chem. 76, 4093–4097.
Electrochemical Signal on Gold Electrodes (10) Hwang, S., Kim, E., and Kwak, J. (2005) Electrochemical detection of DNA hybridization using biometallization. Anal. Chem. 77, 579–584. (11) Caruana, D. J., and Heller, A. (1999) Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7-µm-diameter microelectrode. J. Am. Chem. Soc. 121, 769–774. (12) Manco, G., Adinolfi, E., Pisani, F. M., Ottolina, G., Carrea, G., and Rossi, M. (1998) Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem. J. 332, 203–212. (13) Svenson, S., and Tomalia, D. A. (2005) Dendrimers in biomedical applications-reflections on the field. AdV. Drug DeliVery ReV. 57, 2106–2129. (14) Tomalia, D. A., Huang, B., Swanson, D. R., Brothers, H. M., and Klimash, J. W. (2003) Structure control within poly(amidoamine) dendrimers: size, shape and regio-chemical mimicry of globular proteins. Tetrahedron 59, 3799–3813. (15) Majoros, I. J., Keszler, B., Woehler, S., Bull, T., and Baker, J. R. (2003) Acetylation of poly(amidoamine) dendrimers. Macromolecules 36, 5526–5529. (16) DeMattei, C. R., Huang, B. H., and Tomalia, D. A. (2004) Designed dendrimer syntheses by self-assembly of single-site, ssDNA functionalized dendrons. Nano Lett. 4, 771–777. (17) Shi, X. Y., Lesniak, W., Islam, M. T., Muniz, M. C., Balogh, L. P., and Baker, J. R. (2006) Comprehensive characterization of surface-functionalized poly (amidoamine) dendrimers with acetamide, hydroxyl, and carboxyl groups. Colloids Surf., A 272, 139–150. (18) Lesniak, W. G., Kariapper, M. S. T., Nair, B. M., Tan, W., Hutson, A., Balogh, L. P., and Khan, M. K. (2007) Synthesis and characterization of PAMAM dendrimer-based multifunctional nanodevices for targeting alpha(v)beta(3) integrins. Bioconjugate Chem. 18, 1148–1154. (19) De Simone, G., Galdiero, S., Manco, G., Lang, D., Rossi, M., and Pedone, C. (2000) A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. J. Mol. Biol. 303, 761– 771. (20) Kluger, R., and Zhang, J. (2003) Hemoglobin dendrimers: Functional protein clusters. J. Am. Chem. Soc. 125, 6070–6071. (21) van, B. I., Malda, H., Synowsky, S. A., van Dongen, J. L., Hackeng, T. M., Merkx, M., and Meijer, E. W. (2005) Multivalent peptide and protein dendrimers using native chemical ligation. Angew. Chem., Int. Ed. 44, 5052–5057. (22) Kamin, R. A., and Wilson, G. S. (1980) Rotating-ring-disk enzyme electrode for biocatalysis kinetic-studies and character-
Bioconjugate Chem., Vol. 19, No. 12, 2008 2461 ization of the immobilized enzyme layer. Anal. Chem. 52, 1198– 1205. (23) Rahman, K. M. A., Durning, C. J., Turro, N. J., and Tomalia, D. A. (2000) Adsorption of poly(amidoamine) dendrimers on gold. Langmuir 16, 10154–10160. (24) Bielinska, A. U., Kukowska-Latallo, J. F., and Baker, J. R. (1997) The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA. Biochim. Biophys. Acta, Gene Struct. Exp. 1353, 180–190. (25) Fischer-Durand, N., Salmain, M., Rudolf, B., Juge, L., Guerineau, V., Laprevote, O., Vessieres, A., and Jaouen, G. (2007) Design of a new multifunctionalized PAMAM dendrimer with hydrazide-terminated spacer arm suitable for metal-carbonyl multilabeling of aldehyde-containing molecules. Macromolecules 40, 8568–8575. (26) Patri, A. K., Myc, A., Beals, J., Thomas, T. P., Bander, N. H., and Baker, J. R. (2004) Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem. 15, 1174–1181. (27) Wu, G., Barth, R. F., Yang, W. L., Chatterjee, M., Tjarks, W., Ciesielski, M. J., and Fenstermaker, R. A. (2004) Site-specific conjugation of boron-containing dendrimers to anti-EGF receptor monoclonal antibody cetuximab (IMC-C225) and its evaluation as a potential delivery agent for neutron capture therapy. Bioconjugate Chem. 15, 185–194. (28) Choi, Y. S., Mecke, A., Orr, B. G., Holl, M. M. B., and Baker, J. R. (2004) DNA-directed synthesis of generation 7 and 5 PAMAM dendrimer nanoclusters. Nano Lett. 4, 391–397. (29) Choi, Y., Thomas, T., Kotlyar, A., Islam, M. T., and Baker, J. R. (2005) Synthesis and functional evaluation of DNAassembled polyamidoamine dendrimer clusters for cancer cellspecific targeting. Chem. Biol. 12, 35–43. (30) Alfonta, L., Singh, A. K., and Willner, I. (2001) Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen-antibody or oligonucleotide-DNA sensing processes by the precipitation of an insoluble product on electrodes. Anal. Chem. 73, 91–102. (31) Wang, J., Li, J., Baca, A. J., Hu, J., Zhou, F., Yan, W., and Pang, D. W. (2003) Amplified voltammetric detection of DNA hybridization via oxidation of ferrocene caps on gold nanoparticle/streptavidin conjugates. Anal. Chem. 75, 3941–3945. (32) Patolsky, F., Weizmann, Y., and Willner, I. (2002) Redoxactive nucleic-acid replica for the amplified bioelectrocatalytic detection of viral DNA. J. Am. Chem. Soc. 124, 770–772. BC800353Z
