Enzyme-Linked Small-Molecule Detection Using Split Aptamer

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Enzyme-Linked Small-Molecule Detection Using Split Aptamer Ligation Ashwani K. Sharma, Alexandra D. Kent, and Jennifer M. Heemstra* Department of Chemistry and the Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah 84112, United States ABSTRACT: Here we report an aptamer-based analogue of the widely used sandwich enzyme-linked immunosorbent assay (ELISA). This assay utilizes the cocaine split aptamer, which is comprised of two DNA strands that only assemble in the presence of the target small molecule. One split aptamer fragment is immobilized on a microplate, then a test sample is added containing the second split aptamer fragment. If cocaine is present in the test sample, it directs assembly of the split aptamer and promotes a chemical ligation between azide and cyclooctyne functional groups appended to the termini of the split aptamer fragments. Ligation results in covalent attachment of biotin to the microplate and provides a colorimetric output upon conjugation to streptavidin−horseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM−100 μM in buffer and 1−100 μM human blood serum. The detection limit of 1 μM in serum represents an improvement of two orders of magnitude over previously reported split aptamer-based sensors and highlights the utility of covalently trapping split aptamer assembly events.

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avert signal loss and thus enable development of a colorimetric split aptamer sandwich assay. In sandwich ELISA, a capture antibody is covalently attached to a polystyrene microplate. If the target analyte is present, it binds to the capture antibody and recruits a detection antibody that is functionalized with a reporter enzyme such as horseradish peroxidase (HRP). A chromogenic substrate is then added and is converted by HRP into an optically observable signal that can be quantified using an absorbance plate reader.17 To construct an aptamer-based sandwich assay for cocaine, we utilized chemically modified fragments of the cocaine split aptamer18,19 in place of the “capture” and “detection” antibodies used in ELISA. The capture strands have an azide at one terminus and an amine at the opposite terminus, and the detection strands have a biotin at one terminus and a cyclooctyne at the opposite terminus. First, a capture strand is attached to an N-hydroxysuccinimide (NHS)-functionalized microplate via amide bond formation, then unreacted NHS groups on the microplate surface are chemically blocked using bovine serum albumin (BSA). In the next step, a test sample is added containing a detection strand and varying concentrations of the cocaine target. If present, cocaine directs assembly of the split aptamer fragments, bringing the azide and cyclooctyne groups into close proximity and thus promoting a cycloaddition to ligate the two fragments.20 The resulting ligation yield is dependent upon cocaine concentration. Thus, the concentration of cocaine present in the test sample is translated into dose-dependent covalent pull-down of biotin to the microplate

ucleic acid aptamers have emerged as a promising alternative to antibodies, as they are capable of binding to a specific protein or small-molecule target with high selectivity and affinity, and have the benefit of being chemically synthesized rather than produced in vivo.1−6 Aptamer-based analogues of “sandwich” enzyme-linked immunosorbent assay (ELISA) have been reported for protein detection.7−9 However, these assays are not directly applicable for smallmolecule detection, as they rely on the ability of two separate aptamers to bind simultaneously to the protein target, and small molecules lack the surface area required to accommodate such interactions. While aptamers are not capable of forming the necessary “sandwich” interaction with small molecules, split aptamers are capable of this task, as they are comprised of two nucleic acid strands that only bind to one another in the presence of a specific small-molecule or protein target.10 DNA split aptamers have recently been used to construct sandwich-format electrochemical11−14 and surface plasmon resonance12 sensors for small molecules. However, there are no examples of split aptamer-based sandwich assays that utilize the reporter enzyme−chromogenic substrate format, which is the current standard for use in clinical diagnostics laboratories.15 This standard ELISA format provides a convenient colorimetric output and is highly amenable to multiplexing for high-throughput analysis. However, ELISA does require washing steps, during which the signal can be degraded via interruption of target binding. This is especially problematic for split aptamers, which have binding constants in the high μM range. We recently demonstrated novel Split Aptamer Proximity Ligation (StAPL) technology in which attachment of reactive groups to the termini of split aptamer fragments enables translation of a small-molecule signal into the output of DNA ligation.16 We envisioned that StAPL could be used to © 2012 American Chemical Society

Received: April 15, 2012 Accepted: June 18, 2012 Published: June 18, 2012 6104

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surface. In the final readout step, a streptavidin-HRP conjugate is added, which binds to biotin and converts a colorless tetramethylbenzidine (TMB) substrate into an optically observable blue product (Figure 1).

functionalized DNA-BIND microplates were purchased from Fisher. Streptavidin−HRP conjugate (catalog no. 21130) was purchased from Thermo scientific. TMB substrate (product code: TMBW) was purchased from SurModics (MN, USA). DNA was purchased from the University of Utah DNA/Peptide Synthesis Core Facility. Human blood serum was purchased from Aldrich. Mass spectra were obtained through the Mass Spectrometry Core Facility, University of Utah. PAGE gels were analyzed for Cy3 fluorescence using a Typhoon 9400 scanner (Amersham Biosciences) with a 532 nm excitation laser and 580 BP 30 emission filter. Fluorescence volumes were corrected for background by subtracting the fluorescence volume of an identically sized area of the gel in which no bands were present. Absorbance values were recorded on a Biotek Synergy MX microplate reader. Modifiers Used for DNA Synthesis. All modified phosphoramidites and CPG cartridges were purchased from Glen Research. Azide functionality was installed using bromohexyl phosphoramidite (10-1946), which was converted to azide on resin according to supplier protocols using NaI and NaN3. Amine functionality was installed using C6-amino CPG cartridges (20-2956). Biotin functionality was installed using BiotinTEG phosphoramidite (10-1955). Cy3 functionality was installed using Cy3 phosphoramidite (10-5913). Cyclooctyne Modification of DNA. DNA 2a. ALO carboxylic acid was coupled to amine-terminated DNA following previously reported procedures.16 MALDI-TOF (linear positive mode) calcd [M+H]+ 4949.1, found 4950.2. DNA 2b. A solution of DIBAC-NHS ester (100 μL, 200 mM in DMF) was added to a solution of DNA (350 μL, 85 μM in 100 mM sodium phosphate buffer, pH 7.2) in a 1.7 mL microcentrifuge tube. The reaction proceeded for 2 h at room temperature, then the reaction mixture was desalted using a NAP-5 column (GE Healthcare). DNA was purified using reverse phase HPLC (Agilent ZORBAX Eclipse XDB-C18, 5 μm particle size, 4.6 mm × 150 mm) with a binary mixture of 0.1 M TEAA:acetonitrile. MALDI-TOF (linear positive mode) calcd [M+H]+ 5158.5, found 5159.2. Preparation of Microplate for Enzyme-Linked Assay. To each well of a 96-well NHS-functionalized DNA-BIND plate was added 100 μL of capture strand 1 (1 μM) in binding buffer (0.5 M NaPi, pH 8.5). The plate was agitated on a shaker for 24 h, then each well washed with 3 × 100 μL wash buffer (10 mM NaPi, pH 7.4, 150 mM NaCl, 0.05% Tween 20) followed by 3 × 100 μL water. Unreacted NHS groups were blocked by addition of 100 μL blocking solution (3% BSA in binding buffer) to each well followed by incubation at 37 °C for 18 h. Each well was washed with 3 × 100 μL water, 3 × 100 μL wash buffer, and then 3 × 100 μL water. The plate was wrapped in foil and stored dry at 4 °C until use. Cocaine Detection Using Enzyme-Linked Assay. A 100 μL solution containing 100 pmol of detection strand 2 along with the specified concentration of cocaine and buffer or serum was added to each microplate well. The plate was agitated on a shaker for the specified incubation time, then each well was washed with 3 × 100 μL water, 3 × 100 μL wash buffer, then 3 × 100 μL water. A total of 100 μL of SA-HRP solution (1:1000 streptavidin−HRP, 10% BSA in binding buffer) was added to each well, and the plate was agitated on a shaker for 1 h. Each well was washed with 3 × 100 μL water, 3 × 100 μL wash buffer, then 3 × 100 μL water. A total of 100 μL of TMB solution was added to each well, and the absorbance value was read after 10 min (ALO) or 20 min (DIBAC) using an

Figure 1. Enzyme-linked cocaine detection using split aptamer ligation. The presence of cocaine results in covalent pull-down of biotin to the microplate surface. Cocaine binding can be interrupted during washing steps without impacting the outcome of the assay. Biotin is detected by conjugation to streptavidin−horseradish peroxidase (SA-HRP) followed by addition of chromogenic TMB substrate.

In principle, this type of sandwich assay could be executed without the covalent DNA ligation step. However, the binding affinity of the cocaine split aptamer is modest at about 100 μM,18 and aptamer assembly is highly susceptible to variables such as salt concentration.16 Thus, the conversion of cocaine binding into the outcome of covalent ligation via StAPL improves both detection sensitivity and reproducibility, as the binding interaction between cocaine and the split aptamer can be altered or disrupted in washing and analysis steps without impacting the outcome of the assay. Here we report the first aptamer-based analogue of traditional sandwich ELISA capable of small-molecule detection. Our StAPL technology is a critical aspect of this assay format, as a detectible signal is only observed when split aptamer assembly events are trapped by covalent ligation. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM−100 μM in buffer and 1−100 μM human blood serum. The detection limit of 1 μM in serum represents and improvement of two orders of magnitude over previously reported split aptamer-based sensors. Thus, we propose that StAPL is not only a critical aspect of the ELISA format reported here, but could be utilized to improve detection sensitivity in a wide variety assay formats. Moreover, DNA split aptamers can be generated for a variety of small-molecule targets, making this research broadly applicable for detection of small-molecule analytes in biological samples.

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EXPERIMENTAL SECTION General Techniques. Unless otherwise noted, all starting materials were obtained from commercial suppliers and were used without further purification. Cocaine was purchased from Sigma-Aldrich as a 1 mg/mL solution in acetonitrile, which was diluted with water, lyophilized, and redissolved in water at a concentration of 1 mg/mL. DIBAC(DBCO)-NHS ester was purchased from Click Chemistry Tools, Inc. Clear NHS6105

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absorbance plate reader. Net absorbance values were calculated by subtracting the absorbance for a control having no cocaine present from the absorbance for each test solution. Solution-Phase Ligation Reactions. Solutions of DNA 4, DNA 3, and cocaine were diluted into 50 mM Tris buffer to give the final concentrations specified for each reaction. The reaction mixture was allowed to stand at room temperature for the specified reaction time, then was diluted into 2× PAGE loading buffer containing 7 M urea. The reaction mixture was separated by denaturing PAGE on a 12% TBE/urea polyacrylamide gel. Denaturing polyacrylamide gels were imaged as described above, and ligation yields were calculated using eq 1 %Yield = 100 × [Vp/(Vp + VR )]

Figure 2. Chemical structures and relative rate constants for ALO and DIBAC cyclooctyne reagents.

100 μM provided no detectible ligation. The slower relative reaction kinetics in the microplate format may result from reduced accessibility of the azide strand when immobilized on the microplate surface. Extending the incubation time to 20 h provided dose-dependent ligation (Figure 3), but this length of incubation is not competitive with the standard 1 h incubation time used in antibody-based ELISA.

(1)

where VR is the fluorescence volume of the band for reactant 4 and VP is the fluorescence volume of the band for 3 + 4 product. Calculation of Metabolite Cross-Reactivity. Solutionphase or enzyme-linked assays were carried out using cocaine, metabolite, or no small molecule. Ligation yield or absorbance at 360 nm was measured, and the percent cross-reactivity was calculated using eq 2 %Cross‐reactivity = 100 × [(Ymet − Ynsm)/(Ycoc − Ynsm)] (2)

where Ymet is the yield or absorbance for reactions using metabolite, Ycoc is the yield or absorbance for reactions using cocaine, and Ynsm is the yield or absorbance for reactions with no small molecule present.

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Figure 3. Enzyme-linked assay using capture strand 1a and ALOfunctionalized detection strand 2a. Assay was tested using incubation time of 4 h (red squares) or 20 h (blue circles). Conditions: 100 pmol 2a (1 μM), 25 mM Tris, pH 8.2, 5 mM NaCl. Data reflects one trial.

RESULTS AND DISCUSSION In our initial attempt at assay development, we utilized capture strand 1a and detection strand 2a (Table 1). Strand 2a is functionalized with the same ALO cyclooctyne that had been employed previously for solution-phase split aptamer ligation reactions (Figure 2).20 In our previous studies, ALO provided strong dose-dependent ligation after a reaction time of 4 h. However, in the microplate format, a 4 h incubation using strand 2a in the presence of cocaine concentrations as high as

Fortunately, a number of cyclooctyne reagents having enhanced reactivity for strain-promoted azide−alkyne cycloaddition have been reported recently in the literature.21,22 From these, we chose to pursue DIBAC for use in our assay, as it reacts approximately 240-fold faster than ALO.23 We initially studied solution-phase reactions between DNA strands 3a and 4b to evaluate the suitability of DIBAC for use in cocainedependent split aptamer ligation. After a reaction time of 30 min, significant background ligation was observed in the absence of cocaine (Figure 4a, lane 2). Similar background ligation has been observed in the presence of human blood serum, where high salt concentrations can increase the affinity of the DNA strands for one another such that they anneal and subsequently react even in the absence of the small-molecule target. In the case of DIBAC, we postulate that the increased lipophilicity relative to ALO22 may be acting analogously to promote DNA annealing and subsequent ligation via hydrophobic packing interactions between the appended reactive groups. To overcome the background ligation with DIBAC, we subtly mutated the azide strand to 3b to decrease the inherent binding affinity of the split aptamer fragments for one another. Specifically, a GC base pair at the terminus of a duplex region was mutated to a CC mismatch and a GT wobble pair was mutated to a GC base pair (Figure 4b). This net change of less than one base pair was sufficient to reduce the unwanted background ligation but still

Table 1. Split Aptamer Sequences Used for Enzyme-Linked and Solution-Phase Cocaine Detectiona

a

number

sequence (5′-3′)

1a 1b 1c 1d 2a 2b 3a 3b 3c 3d 3e 3f 3g 3h 4a 4b

N3-GTT CTT CAA TGA AGT GGG ACG ACA-NH2 N3-CTT CTT CAA CGA AGT GGG ACG ACA-NH2 N3-CTT CTT CAA CGA AGT GGG ACG ACA-A10-NH2 N3-GTC CTT CAA CGA AGT GGG ACG ACA-A10-NH2 Biotin-GGG AGT CAA GAA C-NH-ALO Biotin-GGG AGT CAA GAA C-NH-DIBAC N3-GTT CTT CAA TGA AGT GGG ACG ACA N3-CTT CTT CAA CGA AGT GGG ACG ACA N3-GCT CTT CAA TGA AGT GGG ACG ACA N3-GCT CTT CAA CGA AGT GGG ACG ACA N3-GTC CTT CAA TGA AGT GGG ACG ACA N3-GTC CTT CAA CGA AGT GGG ACG ACA N3-GCC CTT CAA TGA AGT GGG ACG ACA N3-GCC CTT CAA CGA AGT GGG ACG ACA Cy3-GGG AGT CAA GAA C-NH-ALO Cy3-GGG AGT CAA GAA C-NH-DIBAC

Mutated bases are underlined. 6106

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phase studies and is comparable to that of the most sensitive split aptamer-based assay reported to date.14 We next investigated the selectivity of the enzyme-linked assay for cocaine versus its structurally similar metabolites. Previous studies using the cocaine split aptamer have demonstrated that aptamer assembly is not significantly induced by ecgonine (EC), benzoylecgonine (BE), or ecgonine methyl ester (EME).18,24 However, norcocaine (NC) does bind to the split aptamer and induce ligation in the solution phase with yields nearly as high as those observed for cocaine.16 Our previous studies of metabolite-dependent ligation had been carried out in the solution phase using ALO cyclooctyne with sequences 3a and 4a. Thus, in the research described here, we sought to elucidate whether the selectivity of split aptamer ligation would be impacted by the sequence changes made to accommodate DIBAC or by immobilization of the split aptamer in the microplate format. Solution-phase reactions and enzymelinked assays were performed using sequences 3b:4b and 1c:2b, respectively, with 100 μM cocaine or metabolite. Figure 6 compares metabolite cross-reactivity for solution-phase

Figure 4. (a) Azide 3b reduces background ligation with DIBACfunctionalized 4b but is capable of cocaine dose-dependent ligation. Conditions: 0.5 μM 4b, 2.0 μM 3a or 3b, 25 mM Tris, pH 8.2, and 30 min. (b) Base pairing in split aptamer sequences 3a:4b, 3b:4b, and 3f:4b. Mutated bases are highlighted in yellow.

enable dose-dependent ligation for cocaine concentrations of 1 μM−1 mM in the solution phase (Figure 4a, lanes 3−7). Having established the feasibility of DIBAC for use in solution-phase reactions, we sought to replace ALO with DIBAC in the enzyme-linked assay. Initial experiments using capture strand 1b (having the same sequence as 3b) and DIBAC-functionalized detection strand 2b showed only modest dose dependence after a 30 min incubation time. However, the use of capture strand 1c having an A10 linker inserted between the aptamer sequence and the microplate resulted in vastly improved signal, presumably by providing increased access of the detection strand to the immobilized capture strand. As shown in Figure 5, the enzyme-linked assay using capture strand 1c and DIBAC-functionalized detection strand 2b provided dose-dependent signal for cocaine concentrations of 100 nM−100 μM with a total assay time of less than 2 h. Importantly, this detection limit in buffer is an order of magnitude better than that achieved in our previous solution

Figure 6. Metabolite cross-reactivity for solution-phase reactions using ALO-functionalized 3a:4a (blue), solution-phase reactions using DIBAC-functionalized 3b:4b (green), and enzyme-linked assays using DIBAC-functionalized 1c:2b (red). Conditions: 3a:4a − 0.5 μM 4a, 2 μM 3a, 25 mM Tris, pH 8.2, 5 mM NaCl, 1 mM metabolite, 4 h; 3b:4b - 0.5 μM 4b, 2.0 μM 3b, 25 mM Tris, pH 8.2, 100 μM metabolite, 30 min; and 1c:2b - 100 pmol 2b (1 μM), 25 mM Tris, pH 8.2, 5 mM NaCl, 100 μM metabolite, 30 min incubation time. Error bars represent standard deviation of three (ALO) or four (DIBAC) independent trials.

Figure 5. Enzyme-linked assay using capture strand 1b (red squares) or 1c (blue circles) and DIBAC-functionalized detection strand 2b. Conditions: 100 pmol 2b (1 μM), 25 mM Tris, pH 8.2, 5 mM NaCl, 30 min incubation time. Error bars represent standard deviation of three independent trials. Inset shows expansion of net absorbance at 360 nm for cocaine concentrations of 0.1−10 μM.

reactions using ALO- or DIBAC-functionalized split aptamer and enzyme-linked assays using DIBAC-functionalized split aptamer. As anticipated, EC, BE, and EME show low crossreactivity, but NC demonstrates nearly identical reactivity compared with cocaine. Importantly, the relative crossreactivities for the DIBAC-functionalized split aptamer in solution or microplate format are not significantly different from those observed using the ALO-functionalized split aptamer in the solution phase. This demonstrates that the selectivity of small-molecule detection is not affected by minor sequence changes25 or transition to the enzyme-linked format. For our aptamer-based assay to find optimum utility in clinical diagnostics, it should be capable of functioning not only in buffer but also in complex biological fluids such as human blood serum. As noted above, the high salt concentration in serum can increase background ligation between the aptamer fragments. We were therefore not surprised to find that the DIBAC-functionalized 3b:4b aptamer sequence that had worked well in buffer gave significant background ligation 6107

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we and others have found this mutation to enhance the cocaine binding affinity of the split aptamer.26 Interestingly, DNA sequences 3g and 3h have two consecutive AC mismatches in the duplex region, resulting in a more pronounced bulge relative to 3f. However, as shown in Figure 7, significantly more background ligation is observed for these two sequences relative to their less-mutated counterparts, 3e and 3f. Studies are underway to elucidate the source of this increased DNA assembly despite the increase in mutation severity. To test the ability of re-engineered azide sequence 3f to support enzyme-linked cocaine detection in serum, we appended an A10 linker to 3f to give capture strand 1d. Strand 1d was then utilized with DIBAC-functionalized detection strand 2b to perform the enzyme-linked assay in 50% human blood serum. The data in Figure 7b show dose-dependent signal for cocaine concentrations of 100 nM−100 μM. The signal for 100 nM cocaine is above the baseline but not outside of error. Thus, the detection limit of the enzyme-linked assay is 1 μM for cocaine in human blood serum. However, the detection of 1 μM cocaine in serum represents a 10-fold improvement relative to our previous solution-phase studies and a 100-fold improvement relative to the best reported split aptamer sandwich assay.14 Table 2 compares the detection limits of our enzyme-linked assay to those of other split aptamer-based assays as well as antibody-based assays. While the assay reported here has the lowest serum detection limit of any split aptamer-based assay, it is not as sensitive as the antibody-based assays. This is due to the inherently lower binding affinity of the cocaine split aptamer relative to the corresponding antibody. Our laboratory is currently developing new SELEX methodology to enable generation of split aptamers having improved target binding affinity, as this would improve detection sensitivity. Additionally, research is underway to select aptamers for new drug targets such as opioids. The antibodies currently available for opioids suffer from low target selectivity, limiting the utility of immunoassay for detection of these commonly abused drug molecules.27 Aptamers offer increased target selectivity relative to antibodies, as they are generated in vitro where negative selection rounds can be performed to increase binding of the desired target over structurally similar molecules.28 For opioids, the positive screening cutoff concentration is 7 μM,27 which is well within the sensitivity of our split aptamer-based assay. Thus, in the case of opioids, the increased selectivity offered by aptamers would outweigh any decrease in sensitivity relative to antibodies. The research reported here provides a critical

when tested in 50% human blood serum (Figure 7a, lane 1). However, as described above, this background ligation can be

Figure 7. (a) Screening azide sequences 3b−h in solution phase to reduce background ligation with DIBAC-functionalized 4b in serum. Conditions: 0.5 μM 4b, 2.0 μM 3b-h, 50% human blood serum in water, 30 min. (b) Enzyme-linked assay in human blood serum using capture strand 1d and DIBAC-functionalized detection strand 2b. Conditions: 100 pmol 2b (1 μM), 75 mM NaCl, 50% serum, 30 min incubation time. Error bars represent standard deviation of three independent trials.

circumvented via rational engineering of the split aptamer sequence, and thus, we evaluated a series of mutated azide strands (3c−h) to identify a new sequence that would be compatible with both the DIBAC cyclooctyne and human blood serum (Figure 7a, lanes 2−7). DNA 3f showed only minimal background ligation in solution phase reactions containing 50% serum and, thus, was selected for use in the microplate format. In the sequence of 3f, an AT base pair is mutated to an AC mismatch. However, this mismatch is located near the middle of a duplex region, as opposed to at the end of a duplex region, as is the case for 3b. As a result, the mutation in 3f causes a bulge, rather than a fray, leading to a more pronounced impact on binding affinity between the split aptamer fragments (Figure 4b). Sequence 3f also has the GT wobble pair to GC base pair mutation that was used in 3b, as

Table 2. Comparison of Dynamic Range for Cocaine Detection Using Antibody- And Split Aptamer-Based Sensors and Assays

a

recognition element

output

dynamic range (buffer)

dynamic range (human blood serum)

reference

antibody (STC ELISA) antibody (Immunalysis ELISA) antibody split aptamer split aptamer split aptamer split aptamer split aptamer split aptamer split aptamer

absorbance absorbance piezoelectric/amperometric electrochemical photoelectrochemical surface plasmon resonance absorbance electrochemical gel electrophoresis absorbance

n.d. n.d. 1 nM−100 nMa 1 μM−10 mM 1 μM−1 mM 1 μM−1 mM 500 nM−1 mM 33 nM−1 mM 1 μM−1 mM 100 nM−100 μM

20 nM−1 μMa,b 20 nM−1 μMa,b n.d. 1 mM n.d. n.d. n.d. 100 μM 10 μM−1 mM 1 μM−100 μM

15 15 29 11 12 12 13 14 16 this work

detection of benzoylecgonine. bdetection in whole blood. 6108

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(3) Strehlitz, B.; Nikolaus, N.; Stoltenburg, R. Sensors 2008, 8, 4296− 4307. (4) Cho, E. J.; Lee, J.-W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241−264. (5) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948−1998. (6) Mascini, M.; Palchetti, I.; Tombelli, S. Angew. Chem., Int. Ed. 2012, 51, 1316−1332. (7) Drolet, D. W.; Moon-McDermott, L.; Romig, T. S. Nat. Biotechnol. 1996, 14, 1021−1025. (8) Higuchi, A.; Siao, Y.-D.; Yang, S.-T.; Hsieh, P.-V.; Fukushima, H.; Chang, Y.; Ruaan, R.-C.; Chen, W.-Y. Anal. Chem. 2008, 80, 6580− 6586. (9) Chen, J.; Zhang, J.; Li, J.; Yang, H.-H.; Fu, F.; Chen, G. Biosens. Bioelectron. 2010, 25, 996−1000. (10) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656−665. (11) Zuo, X.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131, 6944−6945. (12) Golub, E.; Pelossof, G.; Freeman, R.; Zhang, H.; Willner, I. Anal. Chem. 2009, 81, 9291−9298. (13) Freeman, R.; Sharon, E.; Tel-Vered, R.; Willner, I. J. Am. Chem. Soc. 2009, 131, 5028−5029. (14) Wen, Y.; Pei, H.; Wan, Y.; Su, Y.; Huang, Q.; Song, S.; Fan, C. Anal. Chem. 2011, 83, 7418−7423. (15) Kerrigan, S.; Phillips, W. H., Jr. Clin. Chem. 2001, 47, 540−547. (16) Sharma, A. K.; Heemstra, J. M. J. Am. Chem. Soc. 2011, 133, 12426−12429. (17) Engvall, E.; Perlmann, P. Immunochemistry 1971, 8, 871−874. (18) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2000, 122, 11547−11548. (19) Zhang, J.; Wang, L.; Pan, D.; Song, S.; Boey, F. Y. C.; Zhang, H.; Fan, C. Small 2008, 4, 1196−1200. (20) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. ACS Chem. Biol. 2006, 1, 644−648. (21) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974−6998. (22) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805−815. (23) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Chem. Commun. 2010, 97−99. (24) Li, T.; Li, B.; Dong, S. Chem.Eur. J. 2007, 13, 6718−6723. (25) Sequence changes were made to regions of the split aptamer known to not directly participate in cocaine binding. Small-molecule binding preferences can be significantly altered by sequence changes made to the core region of the cocaine aptamer: Reinstein, O.; Neves, M. A.; Saad, M.; Boodram, S. N.; Lombardo, S.; Beckham, S. A.; Brouwer, J.; Audette, G. F.; Groves, P.; Wilce, M. C.; Johnson, P. E. Biochemistry 2011, 50, 9368−9376. (26) Neves, M. A. D.; Reinstein, O.; Johnson, P. E. Biochemistry 2010, 49, 8478−8487. (27) Reisfield, G. M.; Salazar, E.; Bertholf, R. L. Ann. Clin. Lab. Sci. 2007, 37, 301−313. (28) Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science 1994, 263, 1425−1429. (29) Teller, C.; Halámek, J.; Ž eravík, J.; Stöcklein, W. F. M.; Scheller, F. W. Biosens. Bioelectron. 2008, 24, 111−117.

foundation for the development of improved assays for detection of these commonly abused drug molecules.

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CONCLUSIONS Using the cocaine split aptamer, we report here the first DNAbased analog of sandwich ELISA capable of small molecule detection via standard colorimetric readout. By exchanging the ALO cyclooctyne for the more reactive DIBAC, enzyme-linked cocaine detection is achieved with only a 30 min incubation time and a total assay time of less than 2 h. Cross-reactivity for cocaine metabolites is not significantly impacted by subtle changes to the split aptamer sequence or transition to the enzyme-linked format, demonstrating the robust selectivity of DNA split aptamers. The assay is capable of detecting cocaine concentrations as low as 100 nM in buffer or 1 μM in human blood serum, making it the most sensitive split aptamer-based assay reported to date. The significant improvements in detection sensitivity reported here demonstrate the utility of StAPL to increase assay robustness by covalently trapping split aptamer assembly events. We chose to demonstrate the use of StAPL in the context of ELISA, as this is the standard for use in clinical diagnostics laboratories and had not previously been adapted for use with split aptamers, likely because of signal loss in the requisite washing steps. However, StAPL technology can potentially be applied to improve detection sensitivity in multiple split aptamer-based sensing formats. Additionally, after covalent trapping of an aptamer assembly event, it may be possible to intentionally denature and reassemble the split aptamer fragments, as this would enable further signal amplification via substrate turnover. Our laboratory is currently investigating alternative ligation chemistries that would be compatible with the thermocycling required to achieve such turnover. The replacement of antibodies with nucleic acid aptamers in diagnostic assays is anticipated to lower cost, as aptamers are chemically synthesized rather than produced in vivo. Moreover, aptamers offer increased target selectivity relative to antibodies, enabling individual detection of structurally similar molecules. Our laboratory is currently exploring the selection of new split aptamers for small-molecule targets where lack of selectivity on the part of antibodies currently limits the utility of ELISA. Selection of these new split aptamers and their subsequent incorporation into the enzyme-linked assay format described here is anticipated to provide improved tools for convenient and selective detection of small molecules in biological samples.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the University of Utah.

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REFERENCES

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