Localized high probe density greatly improves the signaling stability of supramolecular electrochemical aptamer-based (Supra-EAB) sensors

Abstract

DNA aptamers have emerged as a promising class of probes for the development of biosensors. However, the only viable strategy thus far for adjustment of probe densities is tuning DNA concentrations. Herein, we constructed a class of Supra-EAB sensors to introduce localized high probe densities and achieved significantly improved stability against enzymes.

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Aptamers, also called artificial antibodies, are single-stranded RNA, DNA, or peptide molecules that can be artificially selected to bind a specific target analyte.^1–3 Due to rapid progress and the convenience and effectiveness of selection technologies, aptamers have been incorporated as recognition probes into a variety of affinity sensors.^4–7 To construct stable, efficient sensor platforms, probe densities have been extensively investigated for a variety of sensors, including solution- and surface-confined assays.^8–11

For example, Chen et al. reported a fluorescent assay by immobilizing interferon gamma-recognizing, fluorophore-labelled aptamers on the surface of gold nanoparticles and demonstrated the significant impact of their probe densities on the binding kinetics and thermodynamics.⁸ Various spherical nucleic acids (SNAs) with different packing densities have been extensively investigated for diagnostic and therapeutic uses. To develop a strategy for producing molecularly well-defined SNAs, Mirkin et al. demonstrated that highly controllable oligonucleotide density and orientation is of great importance to allow for the quantitative study of structure–function relationships.¹⁰

In contrast to the above-described fluorescent assays in solution, electrochemical aptamer-based (EAB) biosensors are a surface-confined platform, enabling the analysis of a broad range of target analytes, including small molecules, nucleic acids, proteins, and cells.^11–17

As it is true for the solution assays, probe densities likewise significantly impact their performance for this sensor class. Previously, Plaxco and co-worker reported the probe density dependency on kinetics and the signal changes of such sensors.¹⁸ Other studies on electrochemical DNA-based sensor platforms also reported the impact of probe densities on sensor performance, including stability against nucleases.^19–22 Most, if not all, of these studies relied on the use of a single DNA aptamer by varying its concentration. Although this strategy is simple, it may not be effective to rationally control the aptamer's localized densities, due to its random distribution and the heterogeneous morphology on the electrode surface.^23,24

Herein, in response, we designed a supramolecular electrochemical aptamer-based (Supra-EAB) sensor probe with multiple-strand modifications of high density to improve the localized densities and thus the stability against nuclease cleavage (Fig. 1). Specifically, we incorporated a supramolecular structure of beta-cyclodextrin (β-CD) modified with multiple DNA aptamer strands as the host and a thiol-modified adamantine (Ada-SH) as the guest, which are well-known supramolecular assemblies.^25,26 The former serves as the capture probe and the latter as the anchoring group.

Fig. 1 The class of supramolecular electrochemical aptamer-based sensors we constructed with high localized probe densities.

The use of our hypothetical DNA-based biosensors with localized high probe densities would achieve optimal sensor performance due to the synergetic effect of the adjacent capture strands, including high signals and nuclease-resistant stabilities. To this end, we employed a host molecule, β-CD, with a total of seven azide functional groups that allows for precise chemical modification of a single DNA (e.g., mono-) or multiple DNA strands (e.g., tri-, penta-, hepta-). These strands further hybridize with an aptamer for target recognition. We employed a redox reporter-modified, thrombin-recognizing aptamer as a proof-of-principle study.

Using the supramolecular interaction between thiol-modified adamantine (Ada-SH) and β-CD, we then anchored the DNA-substituted β-CD onto a gold electrode surface. The recognition of target protein (i.e., thrombin) altered the electron transfer efficiency between the redox reporter and the electrodes, thus generating a measurable change in electrochemical signals for target analysis. It is likely that the numbers of binding sites on such a supramolecular construct would play a significant role in manipulating surface properties and sensor performance.

To synthesize mono-, tri-, penta-, and hepta-DNA-substituted β-CD, we exploited copper-free click chemistry between azide-modified β-CD molecules and dibenzocyclooctyne (DBCO)-modified nucleic acids (Fig. 2 and Fig. S1, ESI†).²⁷ To achieve these DNA-modified conjugates with a high yield, we optimized the ratio of β-CD to DNA, ranging from 2 : 1 to 1 : 18. To obtain a monostrand-substituted conjugate, we employed an excess of β-CD in comparison to DNA, with the major fraction being the expected one and side products of di- and tri-DNA-substituted conjugates (Fig. 2, lane 3). When we increased the amount of DNA, we obtained major products with higher DNA substitutions (Fig. S1, ESI†). For example, using the β-CD/DNA ratio of 1 : 4 (Fig. 2, lane 4), we achieved the main products of tetra-, penta-, and hexa-DNA-substituted conjugates. After further adjusting the β-CD/DNA ratio to 1 : 7 and 1 : 18 (Fig. 2, lanes 5 and 6, respectively), we obtained the as-expected hepta-DNA-substituted conjugate as the major product.

Fig. 2 (a) We employed click chemistry to precisely synthesize DNA-substituted β-CD derivatives. (b) Gel electrophoresis and yield analysis of the reaction mixture with different feed ratios of β-CD and DNA (lane 1: marker, lane 2: DBCO-DNA); lane 3–6: the ratios of 2 : 1, 1 : 4, 1 : 7, and 1 : 18. (c) The gel analysis of products after purification.

Fig. 3 Supra-EAB sensors with higher DNA-substituted strands exhibit improved current densities. (a) Taking the hepta-DNA derivative as an example, the sensor fabrication is a two-step process consisting of assembly in solution and surface immobolization. (b) and (c) Supra-EAB sensors with mono-, tri-, penta- and hepta-DNA strands exhibited significant differences in their current densities (p values: ***p < 0.001. The data are expressed as the mean ± SD, n = 3).

Upon the synthesis of our product, we further employed polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC) to purify these mixtures and subsequently obtain a pure fraction of mono-, tri-, penta- and hepta-DNA-substituted conjugates. The purified products were characterized by gel electrophoresis and mass spectroscopy (Fig. 2 and Fig. S2 (ESI†), respectively).²⁸ An Ada-SH derivative was synthesized using a protocol that was similar to that described in a previous report,²⁹ and characterized by NMR and mass spectroscopy (Fig. S3, ESI†). The size of DNA-substituted conjugates is also dependent on the number of strands. In our very recent study, we employed dynamic light scattering (DLS) and atomic force microscopy (AFM) to probe the strand number dependency on molecular size, observing that the derivative with the greater DNA strand number exhibited an increase in size.²⁸

Prior to the surface immobilization of the Supra-EAB sensors, we first demonstrated the assembly of Ada and β-CD in free solution using a fluorescent assay. To do so, we employed a Förster resonance energy transfer (FRET) mechanism between hepta-DNA-substituted β-CD with cyanine 3 (Cy3) modification at the far terminus of the DNA and a cyanine 5 (Cy5)-modified Ada derivative (Fig. S4 and S5, respectively, ESI†), which serve as donor and acceptor fluorophores, respectively. Upon supramolecular interaction between these two components, the two fluorophores were displaced at closer distances that enabled the energy transfer.^30–32

The substantial overlap between the absorption spectrum of Cy5 and the emission spectrum of Cy3 enable the occurrence of FRET (Fig. S4, ESI†). The supramolecular constructs between Ada and β-CD rapidly formed with an emission peak at 660 nm that was attributed to Cy5, achieving equilibria within 1 min (Fig. S5b and c, ESI†). We observed a decrease in the fluorescence intensity of the Cy3 residues (peak at 560 nm), with concomitant enhancement in the Cy5 fluorescence intensity (peak at 660 nm) as the concentration of Ada-Cy5 increased (Fig. S5d and e, ESI†). A steady peak was achieved when we used the equivalent molar concentrations for both components (i.e., 1 μM). These results demonstrate the successful assembly of Ada and β-CD in free solution, which would facilitate its immobilization onto the surface of sensors by such interactions.

The success of the supramolecular assembly inspired us to fabricate the electrochemical biosensors of mono-, tri-, penta- and hepta-DNA-substituted β-CD using a two-step protocol of immobilizing a pre-organized supramolecular structure on an electrode surface. In the first step, we constructed a fully assembled supramolecular structure compromising thiol-functionalized Ada and a DNA hybrid in the buffer solution. In the second step, we then immobilized such supramolecules onto an electrode surface via gold–thiol bonds (Fig. 3a). Alternatively, we also attempted to construct our sensors via a step-by-step assembly protocol (Fig. S6, ESI†), which was inefficient because the sensor did not exhibit a measurable current (data not shown). Thus, for all of the following studies, we exploited the two-step protocol for sensor fabrication. The electrode surfaces are hydrophilic, with contact angles ranging from 66.0° to 83.1° due to the fact that DNA molecules are hydrophilic, and the hepta-DNA-substituted sensor exhibited the highest hydrophilicity (Fig. S7, ESI†).

Fig. 4 Supra-EAB sensors with higher densities exhibited enhanced stability and robust responses against their target. (a) The stability test of these sensors collected in whole blood and (b) against nuclease enzyme. (c) The binding kinetics studies and (d) target titration results exhibited a robust response upon target recognition.

We fabricated our supramolecular sensors using as-prepared mono-, tri-, penta- and hepta-DNA-substituted supramolecular constructs and tested them by square wave voltammetry. As expected, the sensors fabricated from a higher substituted DNA construct exhibited stronger signals (Fig. 3b). To minimize the variation of effective electrochemical area between electrodes, we employed the parameter of current density to evaluate the signals from these sensors. For example, the hepta-DNA-based sensor exhibited approximately 7-fold enhanced current densities in comparison to the mono-DNA-based sensor. This is due to the higher grafted DNA (and thus the modified redox reporters), which corresponds to their improved localized packing densities (9.56 × 10¹⁰ molecules per cm²versus 1.39 × 10¹⁰ molecules per cm² for the hepta- and mono-DNA sensors, respectively, Fig. S8, ESI†).

The supramolecular sensors with higher localized DNA densities exhibited enhanced stability during electrochemical tests or against DNA enzymes. Specifically, the hepta-DNA-based sensor exhibited the highest baseline stability, with the current remaining above 50% over the course of 10 hours. In contrast, the mono-DNA-based sensor remained at merely 25% signal of the original current. The tri- and penta-DNA-based sensors were between these two. Likewise, these sensors exhibited a similar trend of stability when they were challenged against enzyme (e.g., DNase I), with higher localized DNA densities exhibiting greater stabilities (i.e., greater K_m values).

The Supra-EAB sensors with higher densities exhibited improved target responses as well as affinity despite their altered binding kinetics (Fig. 4). The mono-DNA-based sensor exhibited the fastest kinetic but lowest signal response, with a kinetic value 8-fold greater than that of the penta- and hepta-DNA-based sensors, and 6-fold greater than the value of the tri-DNA-based sensor. This is probably due to the steric effect of these increased probes, which hinders the recognition between protein and DNA probes.

Nevertheless, when we titrated our sensors using a series of increased target concentrations, the hepta-DNA-based sensor exhibited the highest signal change of approximately 80% and a K_D value of approximately 100 nM, in contrast to the values (approximately 50% and 130 nM, respectively) for mono-DNA-based sensor (Fig. S9, ESI†). The penta- and tri-DNA-based sensors exhibited almost indistinguishable affinities against target proteins, with their K_D values ranging from 80 nM with similar signal changes. The improved binding affinities of these derivatives in comparison to the mono-DNA-based sensor was probably due to the synergetic effect among high grafting probes.

In summary, we designed and constructed a series of Supra-EAB sensors employing the molecular interactions between DNA-modified β-CD as the host and thiol-modified adamantine (Ada) as the guest. By incorporating a variety of DNA strands into the host molecules, we achieved tuned localized densities of DNA probes. We demonstrated that localized high probe densities of DNA-based biosensors achieved optimal sensor performance due to the synergetic effect of the captured strands, including high signals and enhanced stabilities against nuclease enzymes, without perturbing the binding affinities.

We also demonstrated that the nuclease-resistant ability of the DNA monolayer can be greatly improved by decreasing the probe lateral distance so that it is smaller than the radius of the nuclease. By conjugating onto the host molecule β-CD with a variety of nucleic acid strands (e.g., mono-, tri-, penta-, and hepta-DNA), we were able to rationally manipulate the localized densities of these sensors, which in retrospect affect the surface wettability and sensing performance. It is likely that such a strategy would be beneficial for a wide variety of surface-confined sensor platforms.

SL and HL conceived the idea of this study. SL, SM, MC, and YZ performed the experiments. SL and HL supervised the implementation of this study. SL wrote the manuscript. All authors critically reviewed and revised the manuscript draft and approved the final version for submission.

This work was supported by the National Natural Science Foundation of China (22122410, 22474130, 22274144, 22090050), the National Key Research and Development Program of China (2021YFA1200403), the joint NSFC-ISF Research Grant Program (22161142020), and the Natural Science Foundation of Shenzhen (JCYJ20220530162406014).

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4cc05396f

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