Alkaline Phosphatase Nanoarchitectures for

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Dendritic-like Streptavidin/Alkaline Phosphatase Nanoarchitectures for Amplified Electrochemical Sensing of DNA Sequences Fausto Lucarelli, Giovanna Marrazza, and Marco Mascini* Department of Chemistry, UniVersity of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy ReceiVed NoVember 24, 2005. In Final Form: January 19, 2006 This study describes the development and characterization of a novel dendritic-like signal amplification pathway. Such an analytical strategy relies on the use of streptavidin and biotinylated alkaline phosphatase, which can be simply and conveniently self-assembled to build nanoarchitectures rich in enzyme labels.The performance of this enzymebased amplification route was demonstrated in connection with the electrochemical sensing of DNA sequences. Compared to the commercially available streptavidin-conjugated alkaline phosphatase labels, a single generation of the streptavidin/biotinylated alkaline phosphatase assembly allowed a 15-20-fold enhancement of the electroanalytical signals. The higher sensitivity allowed by the dendritic-like route was attributed to the lower steric hindrance of the proteins employed for this amplification path. As low as 50 pmol/L of a 388-bp-long amplicon identifying Salmonella spp. was easily detected. The experimental results additionally demonstrated that the sensitivity of the method could be further increased in a linear fashion with the number of protein-enzyme generations.

1. Introduction Over the past few years, researchers have been challenged to push the sensitivity of analytical bioassays down to subnanomolar values while keeping these procedures as simple, reliable, and cost-effective as possible. In particular, the formation of supramolecular assemblies as well as the use of enzymes and inorganic tracers as a means to enhance the sensitivity of genoassays have attracted substantial research effort. Functionalized liposomes,1,2 multiple enzyme labels,3,4 arrays of gold5,6 and CdS7 nanoparticles, metal tracers,8 redox marker-,9 gold-,10 and enzyme-loaded11 microspheres, and enzyme-carbon nanotubes architectures12,13 have been extensively exploited, in combination with optical,7 microgravimetric,2,3,5,6 and electrochemical transducers.1,4,8-13 Outstandingly low detection limits were achieved. However, such extreme sensitivities often refer to the detection of synthetic oligonucleotide targets. This means that all the complications that arise from the manipulation of longer and double-stranded sequences (e.g., slower diffusion of the sample, reannealing of the two complementary strands, etc.) were completely ignored. Additionally, most of these procedures rely on the use of special reagents that are not commercially available and must be prepared * Corresponding author. E-mail: [email protected]. Tel. +39 055 4573283. Fax: +39 055 4573384. (1) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940. (2) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 5194. (3) Patolsky, F.; Lichtenstein, A.; Kotler, M.; Willner, I. Angew. Chem., Int. Ed. 2001, 40, 2261. (4) Dominguez, E.; Rincon, O.; Narvaez, A. Anal. Chem. 2004, 76, 3132. (5) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025. (6) Nicu, L.; Guirardel, M.; Chambosse, F.; Rougerie, P.; Hinh, S.; Trevisiol, E.; Francois, J.-M.; Majoral, J.-P.; Caminade, A.-M.; Cattan, E.; Bergaud, C. Sens. Actuators, B 2005, 110, 125. (7) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem., Int. Ed. 2001, 40, 1861. (8) Wang, J.; Liu, G.; Zhu, Q. Anal. Chem. 2003, 75, 6218. (9) Wang, J.; Polsky, R.; Merkoci, A.; Turner, K. L. Langmuir 2003, 19, 989. (10) Kawde, A.-N.; Wang, J. Electroanalysis 2004, 16, 101. (11) Wang, J.; Kawde, A.-N.; Jan, M. R. Biosens. Bioelectron. 2004, 20, 995. (12) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010. (13) Munge, B.; Liu, G.; Collins, G.; Wang, J. Anal. Chem. 2005, 77, 4662.

or synthesized by the user. Clearly, this aspect severely limits the widespread application of such signal-amplification pathways. Addressing these issues, this article describes the development of an electrochemical genoassay based on a novel signal amplification route. As an alternative to the commercially available and widely used streptavidin/alkaline phosphatase conjugates, the use of streptavidin and biotinylated alkaline phosphatase as building blocks to generate dendritic-like (and enzyme-rich) nanoarchitectures is suggested (Figure 1). Such an analytical strategy was illustrated in combination with the electrochemical sensing of 388-bp-long amplicons that identify the bacterial pathogen Salmonella spp. The new analytical method was developed by modifying disposable gold electrodes with a self-assembled monolayer of a thiol-tethered oligonucleotide probe and a spacer thiol.14-16 Both synthetic oligonucleotides and PCR-amplified sequences were analyzed according to a sandwich hybridization format in the presence of a biotinylated signaling probe. The resulting biotinylated hybrid was then alternately exposed to streptavidin and biotinylated alkaline phosphatase solutions, thus generating the desired number of streptavidin/alkaline phosphatase “layers”. After incubation with the enzymatic substrate, R-naphthyl phosphate, DPV was used to detect the R-naphthol signal. The advantageous analytical performance of this novel amplification pathway is illustrated within the next sections and compared with that of an enzyme-linked assay based on the use of streptavidin/alkaline phosphatase conjugates. 2. Experimental Section 2.1. Reagents. Streptavidin (S4762, Strept) and streptavidin/ alkaline phosphatase conjugate (S2890, Strept2-AP, 2:1 conjugation stoichiometry) were obtained from Sigma-Aldrich. Biotinamidocaproyl-labeled alkaline phosphatase (B000-05, biot-AP, 5-10 biotins/ enzyme molecule) was purchased from Rockland. All other reagents (14) Carpini, G.; Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2004, 20, 167. (15) Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2005, 20, 2001. (16) Del Giallo, M. L.; Lucarelli, F.; Cosulich, E.; Pistarino, E.; Santamaria, B.; Marrazza, G.; Mascini, M. Anal. Chem. 2005, 77, 6324.

10.1021/la053187m CCC: $33.50 © 2006 American Chemical Society Published on Web 04/01/2006

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Figure 1. Schematic representation of the conventional (A) and dendritic-like (B) assays. (a) Surface-confined biotinylated hybrid. (b) Binding of 1 streptavidin/alkaline phosphatase conjugate. (c) Binding of 1 streptavidin molecule. (d) Binding of several biotinylated alkaline phosphatase molecules. (e) Further binding of streptavidin. (f) Further binding of biotinylated enzyme labels.

The concentration of the amplicons was determined by fluorescence measurements using Picogreen dye and a TD-700 fluorometer (Analytical Control). No purification of these samples was required prior to analysis. 2.2. Biomodification of the Sensor Surface and Hybridization with Synthetic and PCR-Amplified Samples. Materials and procedures to screen print the transducers, biomodify the gold surfaces, and hybridize synthetic and PCR-amplified targets are described in our previously published papers.14-16 Briefly, the gold surface of the working electrodes was modified by interaction with 10 µL of the thiolated probe solution (0.25 µmol/L in 0.5 mol/L sodium phosphate buffer at pH 7.0, overnight). According to our experience,16 a surface-immobilized probe that overlapped the top primer was used. In such a way, interfacial hybridization involved one of the termini of the amplicon, the region less sterically impeded in approaching the probe-modified surface. Minimal inhibition of the probe/amplicon hybridization process was observed despite the presence of excess unincorporated primers within the amplified sample. The immobilization step was followed by treatment with the mercaptohexanol (MCH) spacer thiol (10 µL, 1 mmol/L aqueous solution; 30 min). Prior to the hybridization reaction, the modified electrodes were washed twice with 15 µL of sodium phosphate buffer. Both synthetic oligonucleotides and PCR products were analyzed by sandwich hybridization. The samples were diluted to the desired concentration using a 0.5 mol/L sodium phosphate buffer that contained 100 nmol/L of the biotinylated signaling probe. Doublestranded amplicons were thermally denatured using a boiling water

bath (5 min at 100 °C); amplicon strand reannealing was retarded by cooling the sample in an ice-water bath (1 min). A 10 µL aliquot of these solutions was finally allowed to interact with the probemodified electrodes for 30 min. Noncomplementary oligomers and PCR products were used as negative controls. After hybridization, the electrode surface was washed twice with 15 µL of DEA buffer (0.1 mol/L diethanolamine, 1 mmol/L MgCl2, 0.1 mol/L KCl; pH 9.6) to remove nonspecifically adsorbed sequences. 2.3. Labeling with Alkaline Phosphatase and DPV Detection. Strept2-AP-Based Assay. The biotinylated hybrid was reacted with 10 µL of a solution containing 3.1 × 10-15 mol/µL (7.7 × 10-4 U/µL) of Strept2-AP conjugate and 10 mg/mL of BSA (as the blocking agent) in DEA buffer. After 10 min, the sensors were washed twice with 15 µL of DEA buffer. Dendritic-like Route. The biotinylated hybrid was alternately exposed to Strept (10 µL, 3.3 × 10-14 mol/µL + 10 mg/mL of BSA in DEA buffer, 10 min) and biot-AP (10 µL, 3.9 × 10-15 mol/µL + 10 mg/mL of BSA in DEA buffer, 10 min) solutions until the desired number of protein-enzyme generations was obtained. After each step, nonspecifically adsorbed proteins were washed away using 2 × 15 µL of DEA buffer. Note that when used at the above-mentioned dilutions the Strept2AP conjugate and the biotin-labeled AP possessed identical hydrolytic activity. The planar electrochemical cell was then incubated with 110 µL of an R-naphthyl phosphate solution (1 mg/mL in DEA buffer). After 20 min, the oxidation signal of the enzymatically produced R-naphthol was measured by DPV (modulation time ) 0.05 s, interval time ) 0.15 s, step potential ) 5 mV, modulation amplitude ) 70 mV, potential scan: from 0 to +0.6 V). All electrochemical measurements were performed with an Autolab PGSTAT10 (Eco Chemie). The experiments were carried out at room temperature (23 ( 1 °C). Each result is the mean and standard deviation of at least three measurements. 2.4. ac Impedance Measurements. ac impedance measurements were performed using the FRA2 module of the Autolab PGSTAT10. An alternating potential with an amplitude of 10 mV and a frequency of 113 Hz18 was superimposed on the bias potential of +0.11 V (a value that corresponds to the open circuit potential). Both the real (Z′) and imaginary (Z′′) components of the impedance were monitored. For these experiments, the probe-modified electrodes were first hybridized with an excess of the complementary or noncomplementary sequence (50 nmol/L + 100 nmol/L of the biotinylated signaling probe). The working electrode surfaces were then blocked by treatment with 10 µL of a solution containing 10 mg/mL BSA. After 20 min, the screen-printed sensors were washed with 0.5 mol/L sodium phosphate buffer and introduced into a homemade flow-

(17) Manzano, M.; Cocolin, L.; Astori, G.; Pipan, C.; Botta, G. A.; Cantoni, C.; Comi, G. Mol. Cell. Probes 1998, 12, 227.

(18) Dijksma, M.; Kamp, B.; Hoogvliet, J. C.; van Bennekom, W. P. Anal. Chem. 2001, 73, 901.

were of analytical grade. Milli-Q water was used throughout this work. Synthetic oligonucleotides were obtained from MWG Biotech AG: surface-immobilized probe: target: noncomplementary: signaling probe:

5′- HS-(CH2)6-GCCGCGCGCGA ACGGCGAAGCGTAC-3′ 5′- CCGCCAATAAAGTTCACA AAACGGTACGCTTCGCCGT TCGCGCGCGGC-3′ 5′-GGCAGAGGCATCTTCAACG ATGGCC-3′ 5′-TTTGTGAACTTTATTGGCG G-TEG-biotin-3′

The genomic DNA of Salmonella spp. was obtained from Promochem. A 388-bp region of the gene inVA (GenBank NC•003197) was then amplified according to the procedure described by Manzano et al.17 using the following primers: Fw: 5′-GCCGCGCGCGAACGGCGAAG-3′ Rev: 5′-ATCCCGGCAGAGTTCCCATT-3′

StreptaVidin/Alkaline Phosphatase Nanoarchitectures

Figure 2. Detection of 1 nmol/L of a 48-mer synthetic oligonucleotide target (T) using either the Strept2-AP conjugate or a single generation of the Strept/biot-AP assembly. A noncomplementary oligo (NC) was used as the negative control. Further details are described in the Experimental Section. through cell where the Strept/biot-AP interactions were monitored in real time. Both Strept and biot-AP solutions (3.3 × 10-14 mol/ µL and 3.9 × 10-15 mol/µL, respectively, with no BSA added) were prepared in 0.5 mol/L phosphate buffer (pH 7). A flux of 160 µL/ min of phosphate buffer was established, and the impedimetric measurement was started. The interaction of each protein with the previously assembled biolayer was followed for ∼6 min; after each step, the cell was rinsed with phosphate buffer for ∼10 min to remove nonspecifically adsorbed proteins. Electrodes that interacted with the noncomplementary sequence (and therefore lack the biotinylated hybrid) were used for reference measurements.

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Figure 3. Detection of 2 nmol/L of a 48-mer synthetic oligonucleotide target (T) using the Strept2-AP conjugate, a single generation of the Strept2-AP/(biot-AP)n assembly, or a single generation of the Strept/(biot-AP)k complex. A noncomplementary oligo (NC) was used as the negative control. The Strept2-AP solution used for the conventional assay and for assembling the Strept2-AP/(biotAP)n architectures was 10 µL of 3.1 × 10-15 mol/µL Strept2-AP + 10 mg/mL BSA in DEA buffer; 10 min of interaction. Further details are described in the Experimental Section.

3. Results and Discussion This article describes the development of a dendritic-like route that allows the sensitivity of enzyme-linked assays to be improved by ∼ 2 orders of magnitude. Such an analytical strategy relies on the use of the commercially available streptavidin and biotinylated alkaline phosphatase that can be conveniently selfassembled to build an enzyme-rich network. The performance of the new method was illustrated in connection with the electrochemical sensing of the sequences that identify the bacterial pathogen Salmonella spp. As shown by Figure 2, assembling of the first Strept/biot-AP generation resulted in a dramatic 15-20-fold enhancement of the electroanalytical signals. The higher sensitivity allowed by the dendritic-like route was likely to be attributed to the different steric hindrance of the proteins involved in conventional and new schemes, respectively. In an attempt to determine the protein size, dynamic light scattering measurements were performed. However, the sensitivity of these measurements was relatively low, and significant scattering was observable only at high proteins concentrations, when the molecules do form aggregates in solution. Theoretical calculations19 were then used to predict the hydrodynamic radius of each protein. Such calculations predicted for streptavidin (MW ≈ 60 kDa), biotinylated alkaline phosphatase (MW ≈ 160 kDa), and streptavidin/alkaline phosphatase conjugate (MW ≈ 280 kDa) hydrodynamic radii of 3.48, 5.21, and 6.55 nm, respectively (footprint: 38.0, 85.2, and 134.7 nm2). The footprint value calculated for Strept is in good agreement with that reported by Kim and Wyckoff in 1991 (i.e., 33.6 nm2).20 Concerning the Strept2-AP conjugate, its calculated footprint fits quite well with the value recently determined by Cremer’s group through fluorescence measurements (134 nm2).21 (19) http://www.doe-mbi.ucla.edu/∼sangho/SECURE/calc.html (20) Kim, E. E.; Wyckoff, H. W. J. Mol. Biol. 1991, 218, 449.

Figure 4. ac impedance-time traces for the stepwise construction of the first and second protein-enzyme generations. Further details are described in the Experimental Section.

Owing to a capture probe surface coverage of (1.7 ( 0.1) × 1012 molecules/cm2 (estimated by chonocoulometric measurements in the presence of the [Ru(NH3)6]3+ complex22), an average area of ∼59 nm2 is available for each tethered and biotin-labeled hybrid. This means that, when a Strept molecule binds, most of the surrounding space is still unoccupied and there are no steric constrains inhibiting the affinity coupling of other proteins to neighboring duplexes. The formation of such a nondensely packed Strept layer thus leaves the remaining biotin binding sites of each molecule available for interaction with up to three biotinlabeled enzymes. In contrast, the binding of a single Strept2-AP conjugate requires ∼135 nm2, the surface normally occupied by an average of 2.3 duplexes. Therefore, more than 1 biotinylated hybrid is sterically compelled to interact with the same enzymatic conjugate, determining a significant loss of sensitivity. The assumption that steric factors govern the surface assembling of the different proteins and consequently the sensitivity of the two labeling strategies was confirmed by another (21) Mao, H.; Yang, T.; Cremer, P. S. Anal. Chem. 2002, 74, 379. (22) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670.

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Figure 5. Electroanalytical signals as a function of the assembled Strept/biot-AP generations: (A) target-related differential pulse voltammograms; (B) measured peak heights for both complementary and noncomplementary amplicons. The concentration of both sequences was 5 nmol/L. Further details are described in the Experimental Section.

experiment. When bound to the biotinylated hybrid, the Strept2AP conjugate possesses a total of seven remaining biotin binding sites. Some of them are certainly inaccessible because of the conjugation procedure, but others (“n”, for example) are likely to occupy accessible positions suitable for binding biot-AP molecules. Hence, a hypothetical Strept2-AP/(biot-AP)n assembly, which incorporates (1 + n) AP molecules, should theoretically provide a higher amplification factor compared to the enzyme-poor Strept/(biot-AP)k complex (the case n g k is considered). Nevertheless, use of the Strept2-AP conjugate as a building block to assemble Strept2-AP/(biot-AP)n architectures resulted in less efficient signal amplification (Figure 3). Clearly, the smaller Strept is prone to form more densely packed layers at the modified interface, determining the actual number of biotin binding sites per surface unit to be much higher. One of the most attractive advantages of the proposed dendriticlike route is that its sensitivity, if required, can be further increased in an extremely simple manner. After binding the first Strept “layer”, each biot-AP still possesses several free biotins that can efficiently cross link a second layer of Strept molecules. By sequentially repeating this self-assembling process, one can in principle generate dendritic-like nanoarchitectures with the desired content of AP labels. The layer-by-layer assembly of the Strept/biot-AP nanoarchitectures was characterized in situ by ac impedance measurements. The formation of the first and second protein/enzyme generations is illustrated in Figure 4, where an increase in the real component of the impedance (Z′) upon assembling the protein and enzyme layers is shown. The impedimetric signal monitored at 113 Hz exclusively described nonfaradic phenomena occurring at the electrodeelectrolyte interface because there were no species in solution that undergo an electrochemical conversion at +0.11 V. The increase in Z′ was attributed to the hydrophobic insulation of the electrode by the large biomolecules that perturb the flux of ions toward the conductive surface2. Interestingly, the ∆Z′ values associated with the assembly of the biot-AP (2.3 ( 0.9 Ω) are smaller than those associated with the binding of Strept (5.6 ( 0.1 Ω). This behavior is likely to reflect the different size of the two proteins. Being much larger, the biot-AP associates form loosely packed layers that can be easily permeated by solutionphase ions. Figure 5 shows how the assembly of up to five proteinenzyme generations influenced the electroanalytical signals. The response increased linearly, demonstrating that even the R-naphthol produced by AP molecules bound on the top of the network could easily diffuse through the protein layers becoming electrochemically accessible. Leveling off of the signal, which

Figure 6. Calibration plot for 388-bp-long Salmonella amplicons. Further details are described within the text and the Experimental Section.

probably occurs when assembling even more Strept/biot-AP layers, was not observed under all investigated experimental conditions. Despite the decrease in the target/noncomplementary signal ratio (from 62 for the first to 10 for the fifth generation), the relatively moderate level of nonspecific signals confirmed the supramolecular specificity of binding (Figure 5B). The linear increase in the signal also suggests that a typical dendritic growth of the Strept/biot-AP nanostructures probably occurs only for lower target concentrations, when each “nucleation point” (i.e., biotinylated hybrid) is sufficiently distant from the others2. For relatively higher target concentrations, all nucleation points immediately merge, and the growth of the dendritic nanostructure is possible only along a single axis (approximately normal to the electrodic plane). Figure 6 shows the detection of the sequences amplified from the Salmonella genomic DNA upon assembling a single Strept/ biot-AP generation. The genosensor response increased linearly (R2 ) 0.977) with the target concentration between 0 and 1 nmol/L; a detection limit of 50 pmol/L was easily achieved for this 388-bp-long target. Further lowering of the detection limit is expected in connection with the assembly of two, three, or more Strept/biot-AP generations. However, the prevention of the nonspecific adsorption of both Strept and biot-AP is of critical importance for the successful completion of the assay, and the use of BSA (as the blocking agent) certainly plays a crucial role. For example, when using Strept or biot-AP at concentrations much higher than those suggested, their physisorption becomes severe and cannot be completely suppressed however high the concentration of BSA is (up to 100 mg/mL). Nonspecific adsorption phenomena

StreptaVidin/Alkaline Phosphatase Nanoarchitectures

practically limited the sensitivity of the method to 1-5 pmol/L at the 5th Strept/biot-AP generation.

4. Conclusions This article described the development of a novel dendriticlike signal-amplification pathway. Such an analytical strategy relied on the use of streptavidin and biotinylated alkaline phosphatase, which can be simply and conveniently self-assembled to build nanoarchitectures rich in enzyme labels. The performance of this enzyme-based amplification route was demonstrated in connection with the electrochemical sensing of DNA sequences. Compared to the traditional streptavidin-conjugated alkaline phosphatase labels, a single generation of the streptavidin/ biotinylated alkaline phosphatase assembly allowed a 15-20fold enhancement of the electroanalytical signals. The higher sensitivity allowed by the dendritic-like route was attributed to the lower steric hindrance of the proteins employed for this amplification path. As low as 50 pmol/L of a 388-bp-long amplicon identifying Salmonella spp. was easily detected. The experimental results additionally demonstrated that the sensitivity of the method could be further increased in a linear fashion with

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the number of protein-enzyme generations. The amplification potential of five generations of the Strept/biot-AP assembly could be evaluated in an ∼100-fold increase of the analytical signals. Although still insufficient to detect nonamplified samples of genomic DNA, this post-amplification protocol might be particularly useful when the yield of the PCR process is limited (e.g., because of the presence of traces of inhibitors of Taq polymerase in samples from complex matrixes). Although the use of streptavidin and biotinylated alkaline phosphatase as novel signal amplification units has been demonstrated for the electrochemical detection of DNA sequences, any other enzyme-linked assay (including immunoassays) could benefit from this analytical strategy. Moreover, because the assembly of these nanobioarchitectures certainly influences the mass associated with the sensor and the optical properties of the transducer/solution interface, significant advantages in terms of sensitivity could be additionally found using microgravimetric (QCM) and SPR-based systems. LA053187M