DNA-Encoded Signal Conversion for Sensitive Microgravimetric

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DNA-Encoded Signal Conversion for Sensitive Microgravimetric Detection of Small Molecule−Protein Interaction Yue-Hua Fei,† Dengyou Liu,‡ Zai-Sheng Wu,*,† Guo-Li Shen,*,† and Ru-Qin Yu† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, P R China ‡ Science College of Hunan Agricultural University, Changsha 410128, PR China S Supporting Information *

ABSTRACT: Identification and quantification of small organic molecules capable of binding to a protein of interest with reasonable affinity and specificity is a central problem. Via developing DNA-encoded recognizing probe, we validate a proofof-principle for constructing of small target-to-DNA conversion that screens the small molecule−protein interaction. Successful identification of β-indole acetic acid, abscisic acid, or 2,4dichlorophenoxyacetic acid/corresponding antibody binding implies its fascinating potential for interrogating small molecule/ protein interaction.

T

modified oligonucleotide probes upon binding to specific target molecules, utilizing microgravimetric quartz-crystal-microbalance, we proposed a novel signaling scheme for investigating the interaction of small molecule with specific protein. This screening scheme is based on the design of a target moleculelinked DNA hybrid that is responsible for the transduction of common immunoreaction into the specific oligonucleotide detection. The present “signal-on” microgravimetric biosensor can not only screen the small target molecule, but also provide quantitative analysis with desirable analytical features (e.g., low detection limit, high sensitivity, and selectivity, as well as a wide dynamic range and cost-effectiveness). Phytohormone screening is considerably significant to agriculture, horticulture, and other related fields. Considering the pressing needs for the precise and sensitive measurements, β-indole acetic acid (IAA), a small plant hormone molecule regulating the plant growth was used as the model target molecule for testing proposed analytical technique. It is wellknown that the characteristics of sensing platform are highly dependent on the signaling format. Namely, the “signal-on” reporting mechanism is superior to the “signal-off” one in terms of the detection sensitivity and working range. Although the sandwich immunoassay format is promising for sensitively detecting the target antigen, small molecules cannot simultaneously interact with two antibodies, precluding their inter-

he identification and quantification of small organic molecules capable of binding to a protein of interest with reasonable affinity and specificity is a central problem in chemistry, biology, and medicine. Although spectroscopic and chromatographic measurements for the exploration of smallmolecule/protein interaction and detection of small molecules have achieved impressive results,1−5 these traditional techniques generally require expensive instrument, specific signal reporters, or cumbersome assay procedures. Thus, the development of a universal protocol for the convenient, costeffective, sensitive, and selective detection of small molecules remains a compelling need. Considering the enormous capabilities6−11 of oligonucleotides in site-specific labeling, sequence-specific biobarcoding, versatile amplification, and isolation, the DNA-encoded oligonucleotide probe has emerged as an appealing tool for the construction and screening of large chemical libraries.12 However, for the existing aptameric screening systems, the ability of aptamer probes to convert the probes/target molecule binding into a measurable signal is highly dependent on their conformational change originating from the formation of the target binding pockets.5,13−17 Moreover, aptamers are the greatly appreciated binding molecules against the corresponding targets. To meet the requirements of many technological applications, it would be desirable to introduce a universal signaling scheme for detecting some organic ligands with low molecular weight (e.g., oligonucleotides and non-nucleic acid biomolecules).18 Inspired by the biological behavior change (for example, susceptibility to enzymatic cleavage6,19 or polymerization20) of original or © 2011 American Chemical Society

Received: February 16, 2011 Revised: September 9, 2011 Published: October 13, 2011 2369

dx.doi.org/10.1021/bc200086c | Bioconjugate Chem. 2011, 22, 2369−2376

Bioconjugate Chemistry

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DNA duplex is easier to immobilize onto the gold substrate in a spatially ordered manner than a flexible single-stranded DNA molecule. Naturally, during this step, not only is the intrinsic self-assembly behavior of the thiolated probe maintained, but hybridization of this probe with the recognition probe can also improve the capability to self-assembly on the QCM surface. Subsequently, the biotin-labeled probe can be immobilized on the resulting surface of QCM (also used as an electrode when performing the electrochemical measurements to characterize the sensing interfaces at different stages of target detection) via specific hybridization to the fragment close to the 3′ end. As a result, the avidin-HRP is capable of being adsorbed onto the resultant QCM via the biotin−avidin bridge. In the presence of H2O2, the HRP-biocatalyzed oxidation of 4-chloro-1-naphthol occurs and the precipitation of the insoluble product is produced on the support surfaces, generating an enhanced mass or electrochemical signal (this process is called the signal amplification step, only involved during evaluation of the features of the proposed microgravimetric sensing platform). In contrast, in the absence of IAA in the sample, the assembling probe/recognition probe conjugate can interact with antibody protein, forming the triplex. Because the thiol group is designed to be in close proximity to the terminal IAA of recognition probe, binding of the recognition probe to the antibody protein, a large biomolecule, can cause steric hindrance serving as the barrier to the self-assembly of a thoil-labeled assembling probe/recognition probe hybrid. As a result, encoding DNA is not adsorbed onto the QCM surface. This is capable of inhibiting the subsequent transducing reaction and amplification process. In this case, no signal is observed. Apparently, via encoding the information on the IAA/antibody interaction and decoding the appearance of DNA recognition probe, a versatile amplification route for exploring small molecule/protein interaction in a “signal-on” manner is developed. The developed small target-to-DNA conversion scheme is described in detail in the text; the characterization of the resulting microgravimetric transducer is represented; the screening ability is validated and primary application is evaluated to afford convincing evidence for the reliability and practicality of the proposed fluorescence assay. The rationality of the proposed design for screening the small molecule/protein interaction is validated by the impedance data. As shown in Figure 1, even if the biomolecules confined on the electrode surfaces are different from each other, each attachment of the molecules involved can consistently induce a considerable increase in the electrochemical impedance, indicating the expected attachment and bioactivity of surface-confined biomolecules. Considering the intrinsic feature of biomolecules and binding affinity at different stages, it is reasonable that a different change in impedance is observed. For example, compared with the difference between lines a and b, line c does not exhibit a significant increase. This should be attributed to the fact that the signaling probe has no large moiety (e.g., HRP) and is immobilized via hybridizing to the surface-confined recognition probe rather than selfassembling directly onto the electrode surface as the thiolated probe/recognition probe hybrid does. Moreover, due to the shorter base sequence, the amount of its negative charge is smaller than the overall value of assembling probe/recognition probe conjugate, generating the comparatively weak electrostatic repulsion between dissolved ferricyanide and resultant electrode. In contrast, substantial increase in impedance is observed for line e compared with all other lines. This is

rogation by “signal-on” two-site immunoassay including the powerful “immuno-PCR” amplification technique.21 In the present communication, the change in the self-assembly behavior of thiolated small molecule-tethered DNA hybrid, which is closely related with the presence of target analyte in sample, was employed to translate the interaction of antibody with small molecules into microgravimetric sigal. The DNA sequences involved in the encoding oligonucleotide-based sensing are detailed in Table 1. The screening principle of Table 1. Oligonucleotides Designed in This Studya

a

The assembling probe is modified with thiol group at the 3′ end to self-assemble onto the surface of quartz-crystal-microbalance (QCM). The steric hindrance-related probe is functionalized with amino groups at the 5′ end in order to prepare recognition probes by being covalently attached to IAA molecule containing a COOH moiety; the signaling probe is biotinylated at the 5′ end to capture the avidin-HRP conjugate. Because two underlined segments are complementary to each other as the shaded parts behave, the three sequences are designed to form the sandwich hybrid complex via hybridization reaction.

the microgravimetric assay platform that converts the presence of target small molecules into the appearance of DNA probes (defined as small target-to-DNA conversion), as well as the signaling procedure, is illustrated in Scheme 1. The proposed Scheme 1. Schematic Diagram of the Amplified Detection of IAA

scheme is essentially a competitive assay between the labeled and free small molecule, which compete for binding the antibody protein. When adding target molecule sample, the free IAA can bind to the anti-IAA antibody protein by preincubating in a solution, inhibiting the formation of antibody/IAA-labeled probe (also called recognition probe)/assembling probe complex in the subsequence step. Namely, the IAA attached to recognition probe/assembling probe hybrid cannot interact with antibody because the binding site of antibody is occupied by free IAA. In this case, the thiol of assembling probe can randomly run into the quartz-crystal-microbalance (QCM) surface, resulting in the s-Au bond. Thus, the recognition probe is easily captured on the surface. It is well-known that the rigid 2370



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Figure 1. Nyquist diagram of Faradic impedance measurements collected for the same QCM with different modified surfaces corresponding to the following: (a) the bare QCM; (b) the self-assembly of the mixture of tube 1 and tube 2 onto the QCM (see the experimental section for the details); (c) hybridization of the surface b with signaling probe; (d) exposure of interface c to HRP-avidin; (e) the same as interface d but with the biocatalyzed deposition in the presence of 1 × 10−3 M 4-chloro-1-naphthol and 1.5 × 10−4 M H2O2. The IAA concentration of 20 μM is involved in this section.

Figure 2. Capability of microgravimetric quartz-crystal-microbalance transducer. The lines indicate typical characteristics of frequency responses in the absence (a,b) and presence of 0.2 μM IAA (c), 0.4 μM IAA (d), 2 μM IAA (e), and 20 μM IAA (f) under given conditions. Note that line a corresponding to the blank sample has no HRP-avidin compared with line b.

measured data are shown in Figure 2. The frequency change directly reflects the amount of biocatalyzed insoluble precipitation by avidin-HRP, which is associated with the content of surface-confined assembling probe/recognition probe hybrid that is highly dependent on the number of target in solution. Thus, the frequency shift is employed to evaluate the small molecule/protein interaction. The experimental data clearly shows that the presence of IAA can rapidly induce the frequency shift especially in the first period of time (e.g., line c), and the higher concentration of IAA results in the larger

because the biocatalyzed precipitation of the insoluble product can directly electrically insulate the electrode surface. As an effective technique to probe the interface properties of functionalized electrodes, the measured data by impedance spectroscopy provide the strong evidence that the proposed screening strategy can efficiently work. To confirm the feasibility of the DNA encoding-based sensing scheme, the interactions between the IAA at the different concentrations and the constant concentration of antibody protein are monitored in a real-time manner. The 2371



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frequency change (e.g., line e), indicating a success in screening the interaction of small molecules with protein. Assuming there is a dynamic equilibration between IAA/antibody complex and free IAA and antibody, increasing the concentration of IAA in the sample can promote the formation of antibody/IAA complex. Naturally, this decreases the amount of antibody that can bind to the IAA-labeled assembling probe/recognition probe hybrid. Consequently, more recognition probes can be immobilized onto the substrate surfaces, finally generating a larger frequency shift. Although the blank sample produces a slight frequency shift (line b), it can be easily distinguished from target samples even at the low concentration (line c). The unwanted frequency change should be attributed to the nonspecific adsorption avidin-HRP onto the QCM surface and/or other factors. Frequency shift upon the blank is expected to be alleviated (see the following section for the further discussion). Taking into account the observation that frequency shift corresponding to target samples especially at the high concentration (e.g., line f) tends to stabilize after 800 s, this incubation time is selected in the subsequent experiments (this data-processing method is called the steady-state measurement) though the blank sample (line b) can make the crystal frequency further rise after this response point. To exactly evaluate the utility of the small target-to-DNA conversion-based sensing protocol for the quantification of small molecule, the IAA at various concentrations were detected under identical conditions and a dose−response curve was constructed. The results are described in Figure 3.

observed to be from 0.1 to 20 μM, suggesting a substantially improved screening capability. The more detailed comparison with two or more other literature works in terms of other assay features is shown in Table S1 of Supprting Information. It is noteworthy that those measurements were carried out under randomly given conditions. The further improved analytical characteristics are expected to be achieved after optimizing the parameters influencing the response signal. The assay selectivity against several other molecules is depicted in Supporting Information Figure S1. The frequency shift corresponding to nontarget molecules is less than 8.0% assuming that the IAA-triggered signal (the difference between the frequencies of blank and IAA) is 100%. The recovery results provide the immediate evidence that the newly developed piezoelectric transducer shows good quality in accuracy and stability (shown in Supporting Information Table S2). To validate the universality of the proposed screening strategy, utilizing the same DNA probes, the interactions of the other two small molecules, abscisic acid (ABA) and 2,4dichlorophenoxyacetic acid (2,4-D), with their respective antibodies were investigated according to the procedure similar to that for IAA assay. The results are shown in Figure 4. Regardless of target species, the frequency change of QCM increases with increasing target concentration (Figure 4A and B). The rise of the frequency shift originates from the fact that more and more assembling probe/recognition probe hybrids are freed. Taking into account the competition between free targets and labeled targets for antibodies, this reflects that the increasing number of target molecules interact with the antibody proteins. The measured results also indicate that the different concentrations of target species in samples can be detected via the developed strategy. Namely, quantification of ABA and 2,4-D can be implemented via the DNA-encoded signal conversion strategy. Strikingly, although no substantial difference (not more than 5%) between the frequency changes at high concentration (e.g., 20 μM) of IAA (Figure 3), ABA (Figure 4A), and 2,4-D (Figure 4B) is observed, the low concentration of ABA or 2,4-D more easily induces the frequency change of QCM. ABA (Figure 4A Inset) and 2,4-D (Figure 4B Inset) at concentration of 0.4 μM can cause the signal intensities of 38% and 35%, respectively, while IAA at the same concentration only leads to the signal intensity of 24% (Figure 4C). From this, one can infer that more desirable analytical characteristics could be achieved when the DNAencoded signal conversion strategy is employed to detect ABA and 2,4-D. These experimental results, as well as comparative data, validate to a great extent the universality and intriguing assay capability of the developed screening strategy. In the present work, a successful “signal-on” sensing strategy possessing the desirable signal amplification capability is proposed for screening the interaction of anti-IAA antibody with IAA and quantitative detection of IAA. Along this line, a proof of concept is for the first time presented for other small molecules of interest including chemical and biological species via utilizing steric hindrance-mediated behavior change (e.g., self-assembly and intermolecular binding) of DNA oligonucleotides. Oligonucleotides serve as the information-encoded molecules during the small target-to-DNA conversion. Decoding the oligonucleotides involved can unambiguously screen the small target/protein interaction. This not only achieves a “signal-on” response mechanism, but also circumvents the drawbacks usually encountered by the conventional small molecule assays, facilitating the design of a sensing

Figure 3. Calibration curve describing the relationship between the frequency responses and the concentration of IAA under given conditions. The curves are expressed as the frequency decreases against the logarithm of IAA concentration. The regression equation is Y = 217.6 + 83.57X − 13.71X 2 with a correlation coefficient of 0.9947, where Y represents the difference between recorded frequency values after and before injecting biocatalytic deposition solution, while X is the logarithm of IAA concentration, respectively. The largest relative standard deviation of three measurements performed for samples is not more than 7.5%. The detection limit is 0.1 μM, which is defined as three times the standard deviation corresponding to the blank sample measurement.

The detection limit is 0.1 μM, indicating a value improved by 9 times to 2 orders of magnitude compared with the previously reported biosensors on the basis of both optical 22 and electrochemical23 measurements for the detection of the same target molecule. The dynamic response range for IAA is 2372



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Figure 4. DNA-encoded signal conversion for screening the interaction between ABA (A), 2,4-D (B), and their respective antibody protein. The signaling procedure similar to that for IAA assay was employed. The error bars represent standard deviations of three measurements performed for each sample. To evaluate the capability of the sensing strategy to investigate the different small molecules, the largest frequency shift induced by the highest concentration of targets was assigned as 100%. On the basis of this assumption, the frequency changes corresponding to the low concentration of targets were accurately calculated. Utilizing this method, the data obtained from the ABA, 2,4-D, and IAA assays are depicted in A Inset, B Inset, and C, respectively. The data in C are estimated from Figure 3.

spectroscopy, simultaneous detection of different small molecules is expected to be readily achieved after designing a proper signaling scheme (e.g., labeling DNA probes with different fluorescent dyes as described in the following section). Moreover, after converting the interaction of small molecules with proteins into the emergence of DNA sequences, extracting the information on small molecules can be accomplished via screening the DNA sequences. This signaling strategy represents an appealing avenue for screening the small molecules through various preexisting toolkits available for DNA manipulation. Besides the methods to manipulate DNAs, this makes the seeming impossible measurement techniques for small molecule assays, as well as signal amplification methods, possible. For example, microgravimetric transducer for the detection of molecules with small molecular weight could not be achieved unless anti-immune complex antibodies were devised.29 However, the detection of IAA based on microgravimetric measurement can be achieved via the developed sensing scheme even without any anti-immune complex antibodies, demonstrating the promising signaling strategy for small molecules of interest. Note that other techniques (e.g., electrochemical and fluorescent measurements) are also capable of executing such a small target-to-DNA conversion and could possibly offer other advantages (for example, assay convenience and high sensitivity). For example, compared with the microgravimetric transducer that only records the frequency change during the signal amplification step, the electrochemical measurement can obtain the overall signal intensity value corresponding to several steps (difference between line a and line e of Figure 1 in which each of the impedance changes from line b to line e are closely associated with the content of IAA in the sample). In this case, the signal-to-noise ratio can increase from 3.6 to 7.0. This intrinsic, attractive feature endows the current sensing scheme with additional advantages, making it a potentially robust tool suitable for screening small molecules. Polymerase chain reaction (PCR) and rolling circle amplification (RCA) activated by DNA polymerase can dramatically replicate the linear and circularized oligonucleotide strands under specific conditions, respectively, affording

scheme and a screening procedure. For example, a considerable amount of immunosensing systems for the detection of small molecules rely usually on anti-immune complex antibodies. Not only are those antibodies difficult to produce, but the systems also exhibit high cross-reactivity with the unliganded primary antibody.24 Additionally, aptamer probes are attracting increasing attention from analysts and biochemists, and substantial research efforts have been focused on devising aptameric systems to obtain the information on a wide variety of target species including small molecules. However, even if the sandwich-type assay is not considered that requires each target analyte to be simultaneously bound by two antibodies, the oligonucleotides responsible for recognizing small molecules must have the unique features so that the structureswitching upon the addition of analytes is ensured to occur5,25−27 or the binding activity can be maintained even after being split into two half-sites.17,28 In contrast, because the oligonucleotides used in the proposed signaling protocol are the common strands, the encoding DNAs require nothing about the base sequence or second structure. This indicates a general and desired tool holding the appreciated potential for targeting applications. As an attractive sensing strategy for transducing the small molecule/protein interaction into a detectable signal, the small target-to-DNA conversion scheme exhibits other several distinct advantages as listed below. Because the small molecule/protein interaction is independent of the oligonucleotide sequences as indicated in Scheme 1, the assay systems for different targets could be cost-effectively designed by sharing the DNA probes except for substituting other species for IAA when preparing the recognition probes. The detection of ABA and 2,4-D has preliminarily proven this intrinsic advantage. Moreover, oligonucleotide is suitable for the construction of various recognition probes for specific targets due to its outstanding features, such as huge diversity of unique sequences, sufficient biostability, easy synthesis, and modification. Additionally, different DNA sequences can be used to prepare recognition probes for recognizing target molecules in a one-to-one manner. In this case, employing other measurement techniques, for example, fluorescence 2373



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of the anchoring nature, residual HRP−avidin conjugates can slowly catalyze the oxidation of 4-chloro-1-naphthol in the presence of H2O2, generating a continuous decrease of crystal frequency. Presumably, the former should be cured via optimizing the incubation time for the interaction between HRP−avidin and biotin of the signaling probe on the QCM surface or via substituting other stronger blockers for glycin molecules, and the latter should be capable of being inhibited via increasing the concentration of antibody protein involved. Another efficient method to suppress the effect of the blank frequency change on the sensing performance is to pursue the maximum reaction rate-based data. Namely, the optimal time response dot (OPD), which is defined as the datum point corresponding to the maximum slope in the real-time measurement curve, is employed for quantitative detection of target molecule. To confirm the rationality of this alternative, the frequency values corresponding to the 250 s response dot, at which the frequency value in line b approaches that in line a, are used to characterize the sensing platform. The calculated data show that the signal-to-noise ration is increased from 3.6 to 15.5, exhibiting superior properties over the steady-state measurement technique. Clearly, it is natural that the screening capability of the small target-to-DNA conversion-based microgravimetric transducer can be further improved if additional efforts are made along the aforementioned directions. Additionally, the essential requirement for the DNA-encoded signal conversion is that the binding of labeled small molecules to antibody proteins can successfully proceed. Presumably, attachment of oligonucleotides, especially double-stranded oligonucleotides, to the small molecules, could more or less inhibit the immunoreaction between small molecules and antibodies, compromising the capability to transfer the recognition event into the detectable signal. Thus, although three small molecules (IAA, ABA, and 2,4-D) were sensitively detected, the developed signaling strategy could not possibly be applied to analyze any small molecules. In summary, DNA oligonucleotides are used to mediate the transition of interaction between IAA and specific antibody into the detectable signal, elegantly developing a microgravimetric biosensor for the small molecule detection. Utilizing the transducer, not only can the efficient investigation of IAA/ antibody interaction and sensitive quantification of IAA be accomplished compared with the conventional assays, but also the analytical capability is expected to be further improved. More importantly, via encoding and decoding with the aid of the DNA probes, a novel small target-to-DNA conversion concept is represented, paving the way to develop the various sensing platforms for the expansion of IAA detection methods to other small targets and analytes. It seems plausible that the success in developing DNA-encoded microgravimetric transducer for IAA assay provides the proof-of-principle evidence that validates the DNA strand-encoded small molecule/protein recognition process and promotes the application of versatile oligonucleotide-based signaling schemes and relative signal amplification biotechnologies in basic and applied research besides in the genomics.

powerful tools for the sensitive immuoassay. ImmunoPCR30−33 and immuno-RCA34,35 based immunosensing systems exhibited impressive assay performances (allowing detection of protein analytes with zeptomole36 or subzeptomole35 sensitivity), indicating a dominant direction for developing ultrasensitive immunoassays. However, both amplification techniques require cumbersome antibody−DNA conjugation processes to convert the antigen−antibody immunoreaction events into DNA strand detection. To circumvent the aforementioned limitations, although aptamer sequences able to directly recognize their target proteins with high affinity and specificity have recently been used to implement the binding event conversion,37−39 the formation of sandwich structure is yet the prerequisite for designing the detection schemes. The small target-to-DNA conversion strategy described in the present work facilitates the applications of DNA-based signal amplification biotechnologies (e.g., PCR and RCA) in the small molecule recognition. Additionally, the small target-to-DNA conversion is based on the change of self-assembly behavior of thiolated assembling probe/recognition probe hybrid due to the increase in steric hindrance between terminal thiol group and the QCM surface upon the IAA/antibody interaction. However, the increase in steric hindrance could essentially inhibit the binding of recognition probe-labeled small species to specific receptors (for instance, interaction between recognition probe-labeled biotin and avidin or enzyme-conjugated avidin), offering the excellent opportunity to develop a variety of optical or electrochemical signaling schemes for sensitively investigating the small molecule/protein interaction. As a conceptual model system for screening the interaction between small molecules and proteins in a homogeneous manner, the small target-toDNA conversion-based fluorescent assay scheme is described in Supporting Information Scheme S1. Small molecules in sample can compete with the recognition probe-labeled ones for antibody protein, freeing the DNA hybrid. This promotes the binding of biotin conjugated to assembling probe to the QD surface-confined avidin, generating the FRET from QDs to fluorescent dyes. In the absence of target, antibody protein may easily bind to the recognition probe-conjugated small molecule, leading to the increase of steric hindrance that prohibits the interaction between surface-confined avidin and biotin of assembling probe. In this case, the distance between fluorescent dye and QD is great. Thus, FRET does not occur and no optical signal is observed. If encoding DNA sequences modified with different fluorescent dyes are involved in this system, the simultaneous detection of different small molecules could be implemented. Nevertheless, addition efforts should be made to further improve the analytical capability of the proposed system. As shown in Figure 2, the blank sample can make the crystal frequency continuously decrease even if slowly. The failure to suppress the blank-induced frequency change could be attributed to two possible causes. First, the HRP−avidin conjugates could be adsorbed onto the QCM surface presumably via replacing the preadsorbed glycin molecules rather than the specific avidin/biotin binding reaction. Second, the small amount of assembling probe/recognition probe hybrid could not bind to the antibody protein event in the absence of target under given conditions. This results in the unwanted immobilization of assembling probe/recognition probe hybrid that, in turn, causes the adsorption of HRP− avidin conjugates during the subsequent assay step. Regardless

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Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 2374



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displacement amplification detection of cocaine. Anal. Chem. 82, 1358−1364. (16) Wu, Z. S., Guo, M. M., Zhang, S. B., Chen, C. R., Jiang, J. H., Shen, G. L., and Yu, R. Q. (2007) Reusable electrochemical sensing platform for highly sensitive detection of small molecules based on structure-switching signaling aptamers. Anal. Chem. 79, 2933−2939. (17) Zuo, X., Xiao, Y., and Plaxco, K. W. (2009) High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices. J. Am. Chem. Soc. 131, 6944−6945. (18) Melkko, S., Scheuermann, J., Dumelin, C. E., and Neri, D. (2004) Encoded self-assembling chemical libraries. Nat. Biotechnol. 22, 568−574. (19) Zhang, S., Metelev, V., Tabatadze, D., Zamecnik, P. C., and Bogdanov, A. Jr. (2008) Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein−DNA interactions. Proc. Natl. Acad. Sci. U.S.A. 105, 4156− 4161. (20) Zhu, C. F., Wen, Y. Q., Li, D., Wang, L. H., Song, S. P., Fan, C. H., and Willner, I. (2009) Inhibition of the in vitro replication of DNA by an aptamer−protein complex in an autonomous DNA machine. Chem.Eur. J. 15, 11898−11903. (21) Kobayashi, N., Iwakami, K., Kotoshiba, S., Niwa, T., Kato, Y., Mano, N., and Goto, J. (2006) Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv−enzyme fusion protein and anti-idiotype antibodies. Anal. Chem. 78, 2244−2253. (22) Jiao, C. X., Niu, C. G., Chen, L. X., Shen, G. L., and Yu, R. Q. (2003) 4-Allyloxy-7-aminocoumarin as a fluorescent carrier for optical sensor preparation and indole-3-acetic acid assay. Sens. Actuators, B 94, 176−183. (23) Li, J., Xiao, L. T., Zeng, G. M., Huang, G. H., Shen, G. L., and Yu, R. Q. (2003) A renewable amperometric immunosensor for phytohormone β-indole acetic acid assay. Anal. Chim. Acta 494, 177− 185. (24) González-Techera, A., Vanrell, L., Last, J. A., Hammock, B. D., and González-Sapienza, G. (2007) Phage anti-immune complex assay: general strategy for noncompetitive immunodetection of small molecules. Anal. Chem. 79, 7799−7806. (25) Nutiu, R., and Li, Y. (2003) Structure-switching signaling aptamers. J. Am. Chem. Soc. 125, 4771−4778. (26) Zuo, X., Song, S., Zhang, J., Pan, D., Wang, L., and Fan, C. (2007) A target-responsive electrochemical aptamer switch (TREAS) for reagentless detection of nanomolar ATP. J. Am. Chem. Soc. 129, 1042−1043. (27) Zhang, S. B., Hu, R., Hu, P., Wu, Z. S., Shen, G. L., and Yu, R. Q. (2010) Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule. Nucleic Acids Res. 38, e185. (28) Fahlman, R. P., and Sen, D. (2002) DNA conformational switches as sensitive electronic sensors of analytes. J. Am. Chem. Soc. 124, 4610−4616. (29) Jin, X. F., Jin, X. Y., Liu, X. P., Chen, L. G., Jiang, J. H., Shen, G. L., and Yu, R. Q. (2009) Biocatalyzed deposition amplification for detection of aflatoxin B1 based on quartz crystal microbalance. Anal. Chim. Acta 645, 92−97. (30) Sano, T., Smith, C. L., and Cantor, C. R. (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120−122. (31) Ruzicka, V., Marz, W., Russ, A., and Gross, W. (1993) ImmunoPCR with a commercially available avidin system. Science 260, 698− 699. (32) Javaherian, S., Musheev, M. U., Kanoatov, M., Berezovski, M. V., and Krylov, S. N. (2009) Selection of aptamers for a protein target in cell lysate and their application to protein purification. Nucleic Acids Res. 37, e62. (33) Gullberg, M., Gú stafsdó ttir, S. M., Schallmeiner, E., Jarvius, J., Bjarnegård, M., Betsholtz, C., Landegren, U., and Fredriksson, S.

AUTHOR INFORMATION Corresponding Author *Phone: 86-731-88821916; Fax: (+86) 731-88821916; E-mail address: [email protected] (Z.S. Wu); [email protected] (G.L. Shen).

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ACKNOWLEDGMENTS Financial assistance is gratefully acknowledged from the National Natural Science Foundation of China (Grants No. 90817101, 20905022, 20775023, and 20865006) and “973” National Basic Research Program of China (No. 2007CB310500).

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REFERENCES

(1) Tagliaro, F., Antonioli, C., De Battisti, Z., Ghielmi, S., and Marigo, M. (1994) Reversed-phase high-performance liquid chromatographic determination of cocaine in plasma and human hair with direct fluorimetric detection. J. Chromatogr, A. 674 (1−2), 207−215. (2) Trachta, G., Schwarze, B., Saegmuller, B., Brehm, G., and Schneider, S. (2004) Combination of high-performance liquid chromatography and SERS detection applied to the analysis of drugs in human blood and urine. J. Mol. Struct. 693 (1−3), 175−185. (3) Buryakov, I. A. (2004) Express analysis of explosives, chemical warfare agents and drugs with multicapillary column gas chromatography and ion mobility increment spectrometry. J. Chromatogr., B 800 (1−2), 75−82. (4) Strano-Rossi, S., Molaioni, F., Rossi, F., and Botre, F. (2005) Rapid screening of drugs of abuse and their metabolites by gas chromatography/mass spectrometry: application to urinalysis. Rapid Commun. Mass Spectrom. 19 (11), 1529−1535. (5) Baker, B. R., Lai, R. Y., Wood, M. S., Doctor, E. H., Heeger, A. J., and Plaxco, K. W. (2006) An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids. J. Am. Chem. Soc. 128, 3138−3139. (6) Wu, Z., Zhen, Z., Jiang, J.-H., Shen, G.-L., and Yu, R.-Q. (2009) Terminal protection of small-molecule-linked DNA for sensitive electrochemical detection of protein binding via selective carbon nanotube assembly. J. Am. Chem. Soc. 131, 12325−12332. (7) Huh, Y. S., Lowe, A. J., Strickland, A. D., Batt, C. A., and Erickson, D. (2009) Surface-enhanced raman scattering based ligase detection reaction. J. Am. Chem. Soc. 131, 2208−2213. (8) Hill, H. D., and Mirkin, C. A. (2006) The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange. Nat. Protoc. 1, 324−336. (9) Huang, Y., Zhang, Y. L., Xu, X. M., Jiang, J. H., Shen, G. L., and Yu, R. Q. (2009) Highly specific and sensitive electrochemical genotyping via gap ligation reaction and surface hybridization detection. J. Am. Chem. Soc. 131, 2478−2480. (10) Schweitzer, B., and Kingsmore, S. (2001) Combining nucleic acid amplification and detection. Curr. Opin. Biotechnol. 12, 21−27. (11) Xu, X. Y., Georganopoulou, D. G., Hill, H. D., and Mirkin, C. A. (2007) Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes. Anal. Chem. 79, 6650−6654. (12) Mannocci, L., Zhang, Y., Scheuermann, J., Leimbacher, M., De Bellis, G., Rizzi, E., Dumelin, C., Melkko, S., and Neri, D. (2008) Highthroughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries. Proc. Natl. Acad. Sci. U.S.A. 105 (46), 17670−17675. (13) Stojanovic, M. N., Prada, P., and Landry, D. W. (2001) Aptamer-based folding fluorescent sensor for cocaine. J. Am. Chem. Soc. 123, 4928−4931. (14) Stojanovic, M. N., Prada, P., and Landry, D. W. (2000) Fluorescent sensors based on aptamer self-assembly. J. Am. Chem. Soc. 122, 11547−11548. (15) He, J. L., Wu, Z. S., Zhou, H., Wang, H. Q., Jiang, J. H., Shen, G. L., and Yu, R. Q. (2010) Fluorescence aptameric sensor for strand 2375



Communication

(2004) Cytokine detection by antibody-based proximity ligation. Proc. Natl. Acad. Sci. U.S.A. 101, 8420−8424. (34) Schweitzer, B., Wiltshire, S., Lambert, J., O’Malley, S., Kukanskis, K., Zhu, Z., Kingsmore, S. F., Lizardi, P. M., and Ward, D. C. (2000) Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. U.S.A. 97, 10113−10119. (35) Schweitzer, B., Roberts, S., Grimwade, B., Shao, W., Wang, M., Fu, Q., Shu, Q., Laroche, I., Zhou, Z., Tchernev, V. T., Christiansen, J., Velleca, M., and Kingsmore, S. F. (2002) Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359− 365. (36) Nam, J. M., Thaxton, C. S., and Mirkin, C. A. (2003) Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 1884−1886. (37) Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C., Pietras, K., Gú stafsdo ́ ttir, S. M., östman, A., and Landegren, U. (2002) Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473−477. (38) Di Giusto, D. A., Wlassoff, W. A., Gooding, J. J., Messerle, B. A., and King, G. C. (2005) Proximity extension of circular DNA aptamers with real-time protein detection. Nucleic Acids Res. 33, e64. (39) Zhou, L., Ou, L. J., Chu, X., Shen, G. L., and Yu, R. Q. (2007) Aptamer-based rolling circle amplification: a platform for electrochemical detection of protein. Anal. Chem. 79, 7492−7500.

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DNA-Encoded Signal Conversion for Sensitive Microgravimetric

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