Abstract

Sensitive and selective quantification of actinomycin D (ActD), a clinically important anticancer agent, remains analytically challenging due to its strong DNA-binding affinity and its presence at low concentrations in complex biological matrices. Here, we report a label-free nanopore biosensor employing an engineered α-hemolysin (M113F)₇ pore and a designed GpC-rich single-stranded DNA probe (GC₂) to profile ActD–DNA interactions at the single-molecule scale. Binding of ActD stabilizes the GC₂ hairpin, producing a characteristic population of long-lived ionic current blockades (>1 s) that is absent in unbound DNA. Quantification is achieved by measuring the frequency of these long events under optimized conditions. The sensor displays a linear analytical range from 2.5 to 250 nM, with a limit of detection of 1.20 nM, and demonstrates high selectivity against structurally relevant DNA-binding drugs. Application to spiked human serum—after mild pretreatment—yields recoveries of 95–103%, indicating suitability for therapeutic monitoring. This work establishes a robust, label-free analytical strategy for ActD and highlights nanopore biosensing as a practical tool for profiling DNA-interacting chemotherapeutics.

Keywords

Actinomycin D detection; nanopore biosensor; α-hemolysin; single-molecule sensing; DNA–drug interaction; label-free assay; therapeutic monitoring; serum analysis.

1. Introduction

Actinomycin D (ActD) is an essential chemotherapeutic drug widely administered for cancers such as gestational trophoblastic disease, sarcomas, and certain pediatric tumors. Its strong intercalation at GpC motifs and concurrent minor-groove binding allow ActD to inhibit transcription with exceptional potency. Owing to its narrow therapeutic window, precise measurement of ActD in biological samples is critical for pharmacokinetic evaluation, toxicity mitigation, and individualized treatment planning.

Contemporary analytical platforms—primarily liquid chromatography–mass spectrometry (LC–MS/MS), fluorescence assays, and electrochemical sensors—offer valuable performance but often require extensive sample preparation, expensive instrumentation, or molecular labeling. Nanopore single-molecule sensing provides a compelling alternative: it directly monitors the dynamic signature of molecular complexes as they modulate ionic currents through nanometer-sized pores, offering real-time insights with minimal chemical modification.

In this work, we advance a label-free nanopore biosensor built around an engineered α-hemolysin (α-HL) pore and a GpC-rich DNA probe specifically tailored to exploit the binding thermodynamics of ActD. This synergistic pairing provides distinctive kinetic fingerprints that enable fast, selective, and sensitive detection.

2. Literature Review

Early nanopore research demonstrated that α-HL pores can discriminate nucleotides, oligonucleotides, and protein–DNA complexes by virtue of current blockade signatures. Subsequent studies leveraged mutant α-HL pores to optimize constriction geometry and surface charge, thereby tuning sensitivity toward DNA-drug complexes.

ActD’s molecular mechanism has been extensively studied: crystallographic and thermodynamic analyses reveal its phenoxazone chromophore intercalates precisely at GpC steps, while the cyclic pentapeptides dock into the minor groove. This dual-mode interaction drastically slows DNA unzipping kinetics—an advantageous feature for nanopore sensing, as dwell-time changes serve as a robust analytical marker.

Recent fluorometric and electrochemical assays have improved ActD detection, yet many require fluorescent analogs, harsh reaction conditions, or suffer from poor matrix tolerance. Nanopore biosensors bypass these limitations, offering single-molecule resolution, minimal labeling chemistry, and compatibility with physiological environments. Nonetheless, selective detection of ActD in serum using nanopores had not been demonstrated prior to this approach.

3. Methodology

3.1 Nanopore Architecture

Experiments employed the (M113F)₇ α-HL mutant, a pore previously demonstrated to enhance resolution of DNA–drug complexes by strengthening hydrophobic interactions within the vestibule. A planar lipid bilayer was formed across a Teflon aperture using the Montal–Mueller technique, and ionic currents were recorded under +180 mV in 1 M NaCl + 10 mM MgCl₂ + 10 mM Tris (pH 7.5).

3.2 DNA Capture Probe

The GC₂ probe (5′-ATGCCGCATTGTATGCGGCAT-3′) was designed with concentrated GpC motifs to maximize ActD affinity. Its intrinsic hairpin structure translocates rapidly through the nanopore, producing short-lived blockades (tens of milliseconds). Binding of ActD stabilizes the hairpin, yielding long-duration events because the complex resists electrophoretic unzipping.

3.3 Event Analysis and Quantification

Events were filtered at 5 kHz and sampled at 10 kHz. Any event with a dwell time >1 s was classified as a GC₂–ActD complex. Frequency (f) of these events per unit time served as the quantification parameter.

Optimization studies varied:

• Voltage (120–200 mV)
• NaCl concentration (1–3 M)
• Mg²⁺ / Zn²⁺ concentrations (0–500 mM)

The conditions yielding maximum complex event frequency while preserving bilayer integrity were selected.

3.4 Serum Sample Preparation

Human serum was treated with Cleanascite to remove lipoproteins and prevent nonspecific pore fouling. Treated serum was spiked with ActD at defined concentrations for recovery studies.

4. Results

4.1 Distinctive Current Signatures

GC₂ alone produced characteristic short translocation events. Upon ActD addition, a new event class emerged: blockades exceeding 1 s, attributable to the strongly stabilized GC₂–ActD complex. ActD alone did not generate detectable signals.

4.2 Analytical Performance

Under optimized conditions, event frequency correlated linearly with ActD concentration:$$f = 0.0025c + 0.0029 \quad (R^2 = 0.9603)$$f=0.0025c+0.0029(R2=0.9603)

yielding:

• Detection limit: 1.20 nM
• Linear range: 2.5–250 nM

This performance surpasses classical fluorescence assays and approaches mid-tier LC–MS sensitivity but with significantly reduced complexity and measurement time.

4.3 Selectivity Studies

At 250 nM—fivefold higher than ActD’s lowest tested concentration—Netropsin, DAPI, Ethidium Bromide, and Daunomycin failed to generate comparable long-lived blockades:

• Minor-groove binders (Netro, DAPI): no significant event change
• EB: negligible interaction
• Daunomycin: weak events (~12 ms), roughly three orders of magnitude weaker binding

Only ActD produced the hallmark multi-second blockades, confirming the probe’s high molecular specificity.

4.4 Serum Compatibility

Spiked serum samples yielded:

Spike (nM)	Found (nM)	Recovery (%)
12.5	12.3 ± 2.3	99%
70	77 ± 12	103%
200	189 ± 40	95%

These results underscore the biosensor’s suitability for therapeutic drug monitoring in clinically relevant matrices.

5. Discussion

The proposed sensing mechanism derives its strength from ActD’s dual-mode DNA binding, yielding a kinetic barrier sufficiently large to be unambiguously resolved at the single-molecule level. Unlike label-dependent assays, this nanopore approach directly measures native binding behavior, enhancing biological relevance and simplifying sample preparation.

The method’s modest precision—typical of single-channel nanopore systems—reflects the stochastic nature of molecular capture events. Still, its accuracy and selectivity outperform many small-molecule biosensors, particularly those lacking a structural recognition element. The approach can be expanded by designing DNA probes targeted at other intercalators or minor-groove ligands, enabling a modular platform for analyzing DNA-binding chemotherapeutics.

Further enhancement through salt gradients, adoption of automated bilayer systems, or transition to solid-state nanopores may yield higher throughput and robustness.

6. Conclusion

This study establishes a highly selective, label-free nanopore biosensor for detecting Actinomycin D in both buffer and serum. Leveraging a GpC-rich ssDNA probe and engineered α-HL pore, the platform converts molecular binding kinetics into quantifiable electrical signatures. With a 1.20 nM detection limit, broad linear range, and strong matrix tolerance, the sensor offers a compelling avenue for pharmacokinetic studies, therapeutic drug monitoring, and drug-interaction research.

Its simplicity, analytical rigor, and biological specificity position this method as a foundational tool for future nanopore-based diagnostics targeting DNA-interactive therapeutics.

References

(Representative; may be expanded to full citations.)

1. Lo, Y.-S. et al. Nucleic Acids Research, DNA binding by Actinomycin D.
2. Veal, G. J. et al. J. Chromatogr. B, LC–MS analysis of ActD.
3. Ying, Y.-L. et al. Nat. Nanotechnol., advances in nanopore sensing.
4. Guan, X. et al., foundational work on α-HL nanopore drug–DNA studies.
5. Fu, Z. et al., nanopore characterization of small-molecule–DNA complexes.

Last Updated on 11/12/2025 by Jota Machinery