Antibody-Bridged Beacon for Homogeneous Detection of Small

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Antibody-bridged Beacon for Homogeneous Detection of Small Molecules Xiaowen Yan, X. Chris Le, and Hongquan Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02510 • Publication Date (Web): 02 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Analytical Chemistry

Antibody-bridged Beacon for Homogeneous Detection of Small Molecules Xiaowen Yan, X. Chris Le,* and Hongquan Zhang* Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2G3, Canada ABSTRACT: In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecule beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences was required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmark, e.g., binding energy of -11 kcal/mole, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated for the analyses of two small molecule targets, biotin and digoxin. Limits of detection of low nM were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.

Sandwich immunoassays are widely used bioanalytical techniques, especially for the analysis of proteins.1,2 However, for the detection of small molecules (SM), it is difficult to accommodate simultaneous binding of two large antibody molecules to a small target molecule.3-6 Therefore, immunoassays for SM usually use a competitive format, in which an antibody is immobilized to a solid phase, and tedious blocking and washing steps are needed.7 Although several homogeneous methods have been developed for SM, complicated conjugation and purification steps are required to label antibodies on micro-/nano-particles,8,9 or with fluorescent dyes.10,11 There is a need for simple homogeneous assays that avoid immobilization or labeling of antibodies. Heyduk et al. used the bivalent nature of the antibody to develop homogeneous immunosensors for the detection of antibodies and protein antigens.12,13 These sensors utilized the binding of two DNA-tethered antigen molecules to the same antibody molecule to induce hybridization of short complementary DNA domains, in a similar process to that of proximity ligation assays.4-6,14 Ricci et al. developed several sensors for detection of antibodies by further using the binding to two DNA-tethered antigen molecules to a single antibody molecule to cause structure changes of molecular beacons,15 or to induce assembly of split aptamers and DNA switches.16,17. Although the concept of DNA switches was further applied to the detection of SM, the assay relied on detection of fluorescence turn-off and required expensive internal conjugation of the quencher and fluorophore.18 Homogeneous assays for SM that take advantage of the bivalent nature of antibodies, remain very limited.

The primary objective of the present study was to develop a fluorescence turn-on assay enabling homogeneous detection of SM without the need for immobilizing or labeling antibodies. We report here the construction and application of an antibody-bridged beacon (AbB) which can translate the event of antibody binding with small molecules into turn-on fluorescence signals (Figure 1). Similar to conventional molecular beacons (MB),19-21 AbB has a stem-loop structure where a pair of fluorophore (F) and quencher (Q) molecules are respectively conjugated to the ends of two stem sequences. A main difference of AbB from MB is that the loop of AbB is composed of a symmetrical antibody-SM ternary complex, which is formed by simultaneous binding of two SMs to the same antibody molecule. Because each SM is conjugated to oligonucleotides (oligos) containing a short complementary sequence of the stem, the formation of the ternary complex induces the stem-loop structure of AbB. In the presence of the SM target, the competitive binding releases the oligo probes from the antibody, destructs the stem-loop structure of AbB and restores the fluorescence. To construct the AbB (Figure 1), we designed a pair of oligos each containing a poly-thymine (25 nt) spacer and a short complementary sequence serving as the stem. The spacers were used to provide sufficient spatial distance allowing two short stem sequences to interact with each other after binding to the same antibody molecule.22-24 One of the oligos was labeled with a fluorophore (F) at the 3’ end, and the other oligo was labeled with a quencher (Q) at the 5’ end. Simultaneous binding of the two oligo probes to the same antibody molecule brings the two probes into the same

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complex molecule, dramatically increasing the local effective concentrations of the two short complementary sequences in the probes. These two short complementary sequences therefore hybridize together, resulting in the formation of a stem-loop structure of the AbB. Consequently, the close proximity of the quencher to the fluorophore resulted in fluorescence quenching. When the AbB encounters target SM, the competitive binding of SM to the antibody induces the dissociation of the two oligo probes from the antibody. Consequently, the stemloop structure of the AbB is destructed because hybridization of the two short stem sequences becomes unstable. The quenched fluorescence is then restored, giving rise to the fluorescence detection for the analysis of the SM target.

resulting in efficient intramolecular fluorescence quenching and minimum background. The complementary hybridization of the stem provides additional binding affinity that drives the preferential binding of a pair of F and Q probes to the antibody. In contrast, if the two probes were not hybridized together, random binding of the two free probes to the Ab could lead to the unwanted formation of two quencher probes or two fluorophore probes on the same Ab. These complexes are undesirable because they do not achieve intramolecular fluorescence quenching. The AbB beacon design with optimum length of the complementary sequences overcomes this problem, resulting in efficient fluorescence quenching and minimum background. To test the feasibility of the AbB, we chose the anti-biotin monoclonal IgG as a model antibody to construct an AbB for the detection of biotin (Figure 2). To study the effect of the length of the complementary stem on the formation of the desired stem-loop structure of AbB, we designed three pairs of oligo probes containing complementary stem sequences of 6, 8 or 10 nucleotides (nt), respectively (Table S1). We compared three pairs of oligo probes for the formation of the desired AbB. We incubated 2.0 nM each pair of oligo probes with varying concentrations (0, 1.0, 2.0 5.0, 10.0, and 20.0 nM) of the anti-biotin antibody in 1×PBS buffer containing 0.05% Tween-20. Tween-20 was used to eliminate nonspecific adsorption of oligo probes and antibodies to the surface of vial. The fluorescence quenching efficiency was then determined, which is proximately equivalent to the percentage of the total oligo probes in the form of AbB.

Figure 1. Schematic illustrating the construction of antibodybridged beacon (AbB) and its application to small molecule (SM) detection. A pair of oligo probes, conjugated to the small molecule target and modified with either a fluorophore (F) or a quencher (Q), bind simultaneously to the same antibody molecule, forming an AbB. F is brought in close proximity to Q by the stable intramolecular hybridization between the stem portions of the two oligos. As a result, the fluorescence of F is efficiently quenched by Q, leaving the AbB probe in a fluorescence off state. Competitive binding of SM target to the antibody causes the dissociation of F and Q from the AbB, resulting in detectable fluorescence. Throughout the assay, native antibody is used without any immobilization or labeling step. Hybridization of the complementary stem sequences in the fluorophore probe and the quencher probe is a unique feature of the AbB beacon. In our design, when a pair of the fluorophore probe and the quencher probe are hybridized to each other, their binding to the Ab forms the AbB beacon that has a precise 1:1 ratio of the fluorophore and the quencher,

Longer stem length led to higher quenching efficiency (Figure 2b), suggesting that increasing stem length enhances the formation of the desired AbB. With the probe of a 6-nt complementary stem and 1-20 nM Ab, the quenching efficiency was only 1-13.7%. As expected, increase of the antibody concentration increases the formation of AbB. When the stem length was increased to 8-nt, the maximum quenching efficiency was 54.3%, suggesting that about half of the oligo probes remained unbound. The use of the 10-nt stem resulted in an 89.4% quenching efficiency, acceptable for the subsequent applications. Increasing the antibody concentration from 5 nM to 20 nM did not further improve the quenching efficiency. The incomplete formation of the desired AbB using probes of 6-nt and 8-nt complementary sequences could be because the binding energy of stem sequences is not sufficient to preferentially drive a pair of fluorophore and quencher probes to bind to the same antibody molecule. We further estimated the binding energy (∆G) of the three pairs of stem sequences. The ∆G values corresponding to the 6-nt, 8-nt, and 10-nt stems are -5.3 kcal/mole, -9.0 kcal/mole, and -11.1 kcal/mole, respectively. Our results in Figure 2b shows that hybridization of the 10-nt complementary stem sequences (∆G = -11.1 kcal/mole) lead to efficient formation of the desired stem-loop structure and good quenching efficiency. If the stem sequences are too long, they can spontaneously hybridize together in the absence of the antibody binding to


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Analytical Chemistry the target. Such intermolecular hybridization would reduce the detection sensitivity. To avoid this potential problem by optimizing the design, we tested the quenching efficiency of the three pairs of probes in the absence of the antibody. As expected, longer stems resulted in stronger intermolecular hybridization and fluorescence quenching (Figure 2c). The use of the 10-nt stem sequences resulted in a 26.0% fluorescence quenching efficiency, as compared to negligible and 1.8% of fluorescence quenching efficiency when the stem sequences were 6-nt and 8-nt, respectively.

Figure 2. (a) Schematic illustrating the process of forming the stem-loop structure of AbB. A pair of F and Q probes first interact with each other through hybridization of stem sequences. The hybridization of stem sequences drives the preferential binding of the F and Q probes to the same antibody molecule, forming the desired stem-loop structure. (b) The measured quenching efficiency due to the formation of AbB after 2.0 nM Bio-F/Q complex was incubated with 1.0 – 20.0 nM anti-biotin IgG. The quenching efficiency was obtained by determining the fluorescence of the reaction solution as compared to the fluorescence of 2.0 nM Bio-F. The reaction buffer was composed of 1x PBS and 0.05% Tween20. Error bars show mean ± s.e.m. (n = 3). (c) Intermolecular hybridization in the absence of antibody. Quenching efficiency was evaluated by determining the fluorescence of 2.0 nM BioF/Q mixture compared to 2.0 nM Bio-F alone. To confirm that a sufficient binding energy from stem hybridization is required for efficient formation of the desired AbB, we further tested the formation of the AbB by hybridization of the 6-nt and 8-nt stem sequences, with the addition of Mg2+ to the PBS buffer (Figure 3a). Mg2+ is known to stabilize DNA hybridization through neutralizing the negative charges of phosphate groups of DNA backbones.25 The quenching efficiency (equivalent to the formation percentage of AbB) was improved from 4.2% to 34.4% along with increasing Mg2+ from 0 mM to 50 mM for the 6-nt stem

sequences. With the use of the AbB containing the 8-nt stem sequence, the quenching efficiency was increased from 54.3% in the absence of Mg2+ to 81.3% in the presence of 20 mM Mg2+ (Figure 3a). We estimated ∆G of stem sequences using 1×PBS containing 20 mM Mg2+ as the buffer. In the presence of 20 mM Mg2+, the ∆G values of 6-nt and 8-nt stem sequences were increased from -5.3 kcal/mole to -6.2 kcal/mole and from -9.0 kcal/mole to -10.2 kcal/mole, respectively. Therefore, the use of Mg2+ enhanced DNA hybridization, further improving the formation of the desired AbB. The quenching efficiency (81.3%) of the AbB with the 8-nt stem tested in the presence of Mg2+ is slightly lower than that (89.4%) of the AbB with the 10-nt stem tested in the absence of Mg2+. These results are consistent with the estimated ∆G values: -10.2 kcal/mole for the AbB containing the 8-nt stem in the presence of Mg2+, and -11.1 kcal/mole for the AbB containing the 10-nt stem in the absence of Mg2+.

Figure 3. (a) Impact of Mg2+ on the formation of AbB, evaluated by determining the quenching efficiency of 2.0 nM Bio-F/Q incubated with 5.0 nM anti-biotin IgG, compared to 2.0 nM Bio-F. (b) Impact of Mg2+ on intermolecular hybridization between Bio-F and Bio-Q, evaluated by determining the quenching efficiency of 2.0 nM Bio-F/Q mixture, compared to 2.0 nM Bio-F. Measurements were performed in buffers composed of 1x PBS, 0.05% Tween-20, and varying concentrations of Mg2+ as indicated. Error bars show mean ± s.e.m. (n = 3). Increasing binding energy can also enhance intermolecular hybridization of the stem sequences in the absence of antibody, which would decrease the detection sensitivity. We tested such hybridization of three pairs of stem sequences in the presence of Mg2+ (Figure 3b). The presence of Mg2+ increased the binding energy of stem sequences, and consequently their intermolecular hybridization. In the presence of 20 mM Mg2+, the 10-nt stem sequences resulted in



a quenching efficiency of 84.0%. The corresponding ∆G of 10-nt stem sequences is -12.7 kcal/mole. This high intermolecular hybridization of stem sequences is undesirable because of it reduces the detection sensitivity. Taken together, a ∆G value of about -11 kcal/mole from stem hybridization is adequate for efficient formation of the desired AbB. Estimation of ∆G can be used as a guideline for designing the stem sequences of AbB, to minimize background and maximize sensitivity. In addition to hybridization of stem sequences, interaction between the fluorophore and the quencher also contributes to the formation of the desired AbB. ∆G has not been reported for any fluorophore-quencher pairs, although the binding energy has been demonstrated to differ for different fluorophore-quencher pairs.26 Therefore, the difference in binding energy between fluorophore and quencher should be taken into account when a different fluorophore-quencher pair is used. Having constructed AbB and demonstrated the proof of principle, we applied it to the detection of biotin. The signal generation of AbB in response to the biotin target involves two steps (Figure S1a): competitive binding of biotin to antibody first dissociates the binding of oligo probes from antibody; and the hybrid of stem sequences becomes unstable and takes apart, restoring the fluorescence that is used for quantification of biotin. We compared two types of AbB, containing 8-nt and 10-nt stems, for the detection of varying concentrations of biotin. The fluorescence signal was monitored in real-time. When the AbB with an 8-nt stem structure was used, 20 mM Mg2+ was included in the detection buffer. This AbB showed a fast signal generation (Figure 4a). The fluorescence signal reached to a plateau within 20 min (Figure 4a). With the use of the AbB containing a 10-nt stem structure and in the absence of Mg2+ in the detection buffer, 60 min were needed to achieve maximum signal (Figure 4b). Because both of these AbB designs involve the same competitive binding, we reason that the difference in signal generation is due to dehybridization of the stems. To test this possibility, we conducted a dilution experiment to monitor the dehybridization of two stems (Figure S1). The two pairs of oligo probes were first incubated at 1µM to enhance intermolecular hybridization of stem sequences in the absence of antibody. The solutions were then diluted by 500-times to induce the dehybridization of the stem sequences. The dehybridizaiton curves of stem sequences are similar to the signal curves of AbB except for a time shift between the two curves. This time shift, similar for both AbB constructs, was due to the competitive binding of biotin to its antibody. The dehybridization of 10-nt stem is slower than that of 8-nt stem, proving that dehybridization of stem sequences corresponds to the differences in the signal generation of the two AbB systems. Between 10 nM and 10 μM biotin, the fluorescence increased as a function of biotin concentrations for both AbB systems (Figure 4c, d). The detection limit of biotin was calculated to be 13 nM, based on 3σ of the signal-to-noise ratio.

Figure 4. (a) and (b) Time-dependent signal generation in response to varying concentrations of biotin. (a) 8-nt stem and (b) 10-nt stem were used for the AbB, and the concentration of the two probes was 2.0 nM each. (c) and (d) Fluorescence intensity as a function of biotin concentration. (c) 8-nt stem and (d) 10-nt stem were used for the AbB. Fluorescence signal of AbB in response to 0-200 nM biotin was fitted using a


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Analytical Chemistry standard calibration curve model.The AbB constructed with the 8-bp stem was used in buffers containing 1x PBS, 0.05% Tween-20, and 20 mM Mg2+. The AbB constructed with the 10-bp stem AbB was used in buffers containing 1x PBS and 0.05% Tween-20. Data and error bars represent mean ± s.e.m. (n = 3). Having established the AbB technique, using biotin as a model target, we further applied the AbB technique to the detection of digoxin, a therapeutic drug commonly used to treat various heart conditions. We designed a pair of F and Q oligos labeled with digoxigenin (Table S1), whose structure is identical to the steroid moiety of digoxin (Figure S2a, S2b). We constructed the AbB using the same pair of oligos containing the 8-nt stem sequences (as for the detection of biotin) and a monoclonal antibody against digoxigenin, so that digoxin can compete with the F and Q probes in binding with the antibody. By applying this AbB to digoxin detection, we observed concentration-dependent responses between 30 nM and 20 μM digoxin and a detection limit of 28 nM (Figure 5). A linear dynamic range from 30 to 1000 nM was obtained. To test any possible interference from complex sample matrix, we spiked 1 µM digoxin into human serum samples of varying dilutions and applied the AbB assay to the determination of digoxin in these serum samples (Figure S3). Although higher background was observed from the serum samples of lower fold of dilution, probably due to intrinsic fluorescence of the serum,12 the net fluorescence signal (fluorescence difference between sample and blank) remains similar for different serum sample, and for digoxin in the buffer solution, indicating that the diluted serum matrix had no interference on the determination of digoxin by the AbB method .

the need for immobilizing or labeling of antibodies. This beacon takes advantage of the bivalent nature of the antibody, which contains two identical SM-binding sites, allowing for simultaneous binding of two oligo probes. The binding energy of stem sequences is vital for efficiently forming the desired stem-loop structure of AbB to achieve maximum sensitivity and minimum background. We systematically studied the impact of the stem sequences on the construction of AbB. A binding energy of approximately -11 kcal/mole serves as a benchmark for designing the stem-loop sequences of an AbB. The AbB strategy is not limited to the detection of biotin and digoxin. Alternation of antibody and antigen motif in oligo probes can expand AbB to the detection of other small molecules.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed. E-mail: [email protected], Homepage: http://www.ualberta.ca/~xcle; [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was financially supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Research Chairs Program, Alberta Innovates, and Alberta Health.

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Figure 5. Fluorescence intensity as a function of digoxin concentration (0-10 µM). The inset shows a linear calibration range from 0 to 1000 nM digoxin. The AbB was constructed with the oligo probes containing the 8-bp stems. The buffer solution contained 1x PBS, 0.05% Tween-20, and 20 mM Mg2+. Data and error bars represent mean ± s.e.m. (n = 3).

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In summary, we have successfully developed a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of small molecules without

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Analytical Chemistry

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Antibody-Bridged Beacon for Homogeneous Detection of Small

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