A label-free nanopore platform uses programmable DNA circuits to build versatile molecular logic gates, forming a universal basis for scalable DNA computing and advanced biosensing applications.
(Nanowerk Spotlight) Inside every cell, countless molecules move and interact, passing information that keeps life functioning. Understanding those molecular signals has become a central goal of modern biology because it underpins disease diagnosis, genetic analysis, and synthetic biology. The tools able to read these signals quickly and accurately are shaping the next generation of biomedical technology.
Nanopores are among the most powerful of those tools. A nanopore is a tiny opening in a membrane that lets single molecules pass through while an electric current records the disturbance they create. Each molecule leaves a characteristic trace, allowing scientists to identify it directly in solution.
This principle turned DNA sequencing from a laboratory procedure into a portable and continuous process. Solid-state nanopores built from materials such as silicon nitride or polymer films have made these sensors tougher and easier to integrate with electronics.
Even so, their signals remain limited. When target molecules are scarce, the changes in current are faint and difficult to distinguish from background noise. The sensors can detect a molecule but cannot interpret complex combinations of inputs. They measure, yet they do not decide. Researchers have looked for ways to give nanopores that missing logic by coupling them with programmable DNA reactions that can amplify or process molecular information.
DNA circuits offer that possibility. By designing specific sequences, scientists can create strands that recognize targets and then assemble or trigger measurable responses. Two such reactions, hybridization chain reaction and catalytic hairpin assembly, can multiply a weak signal into a strong one at a constant temperature without enzymes. These systems work, but they usually amplify in only one direction and lack the internal feedback needed for logical control.
The work points to a future in which nanopores function not only as sensors but also as decision-making molecular devices that analyze information directly within a membrane.
The new system joins solid-state nanopores with a DNA circuit that amplifies its own signal in two directions. It converts faint biochemical inputs into steady, measurable outputs and performs Boolean logic without the need for fluorescent markers or complex redesign for each operation. The approach moves nanopore sensing toward programmable computation and low-power diagnostic devices.
At the center of the design is an autocatalytic hybridization reaction, called AHR. Autocatalytic means that the products of the reaction promote their own formation. The researchers connected two DNA amplification methods, hybridization chain reaction and catalytic hairpin assembly, into a single feedback loop.
A) Diagram depicting the mechanism of the autocatalytic hybridization reaction employed for bidirectional signal amplification. B) Steps in nanopore surface modification: a) A conical nanopore initially bearing carboxyl and hydroxyl functionalities along its inner surface, b) Grafting of PAMAM onto the pore surface through EDC–NHSS-mediated covalent attachment, c) The modified nanopores, now functionalized with PAMAM, serve to recognize and quantify products from the AHR. (Image: Reprinted with permission by Wiley-VCH Verlag) (click on image to enlarge)
A short DNA strand called T1 starts hybridization chain reaction by opening a sequence of hairpins labeled H1 through H4. Those hairpins assemble into long double-stranded DNA wires. This process also reconstructs a second trigger strand, T2, from partial segments. T2 activates catalytic hairpin assembly using two more hairpins, H5 and H6, which repeatedly open each other to create many short duplexes.
The products of this second reaction release new T1 strands, which restart the first reaction. Together, the two pathways reinforce one another and generate large amounts of DNA product under constant temperature without enzymes.
Because either T1 or T2 can start the process, the circuit can amplify signals from both directions. Gel electrophoresis confirms that both triggers produce the same combined set of long and short DNA products. This symmetry makes the circuit robust and allows it to function as a bidirectional amplifier.
The electrical readout relies on ionic current rectification, a property of nanopores that conduct ions more easily in one voltage direction than the other. The researchers built conical pores in polymer membranes and coated their inner surfaces with a positively charged poly(amidoamine) dendrimer called G4-PAMAM. The positive charge attracts negatively charged DNA products. When these products adhere to the surface, they neutralize the positive charge and weaken the rectification.
The result is a clear drop in current that can be measured steadily at a fixed voltage. Because the signal comes from a stable current difference rather than brief translocation spikes, it is less affected by noise.
The AHR loop generates stronger and more reliable signals than either hybridization chain reaction or catalytic hairpin assembly alone. Hybridization chain reaction produces long DNA wires with high negative charge, which attach strongly to the pore surface. Catalytic hairpin assembly produces smaller duplexes that contribute less to charge change. When both processes operate together, they create dense networks of long and short DNA fragments that amplify the surface response and strengthen the signal.
The same mechanism can detect enzymes. The researchers used it to build an assay for polynucleotide kinase, an enzyme essential to DNA repair. Polynucleotide kinase modifies the ends of broken DNA strands by adding or removing phosphate groups so that other repair enzymes can join the DNA back together. Measuring its activity can indicate the state of cellular repair systems.
For this test, the team designed a DNA hairpin called HPNK that hides the T1 trigger inside its stem. Lambda exonuclease, an enzyme that digests DNA from the end with a phosphate group, acts only on strands that are phosphorylated. Polynucleotide kinase first adds that phosphate group to HPNK, allowing lambda exonuclease to digest the strand and release the T1 trigger. Once free, T1 starts the autocatalytic hybridization reaction. The resulting DNA products adhere to the nanopore surface and cause the measurable current drop. Control experiments confirm that neither enzyme alone can release T1. Only the combined action of both enzymes generates the signal.
The assay shows a linear relationship between signal strength and the logarithm of polynucleotide kinase concentration from 0.0001 to 0.2 units per milliliter, with a correlation coefficient of 0.9982. The detection limit is 0.0001 units per milliliter. Tests with other proteins show negligible interference, demonstrating high selectivity.
The method does not require fluorescent dyes or labels. Unreacted single strands are removed with graphene oxide to lower background noise. The full process, including enzyme incubation, amplification, and cleanup, takes about 190 minutes and operates at one temperature without specialized equipment.
Beyond detection, the nanopore platform performs digital logic using DNA strands as inputs. In this framework, each input represents the presence or absence of a specific DNA strand, and the output is a current change relative to a fixed threshold of 40 nA. Above that value represents logical one, below it logical zero.
Because both hybridization chain reaction and catalytic hairpin assembly can activate the feedback loop, the same molecular components can be rearranged to create multiple logic operations simply by selecting which input strands are provided.
The researchers demonstrated six standard Boolean gates within a single nanopore system: OR, YES, AND, INHIBIT, NOR, and NAND. In the OR gate, either T1 or T2 activates the loop, creating a current drop above threshold. The AND gate requires two inputs; only when both are present does the circuit release T1 and start amplification. The INHIBIT gate uses T1 and its complement T1*, which cancels the reaction when both appear. NOR and NAND gates use complementary blocking strands to suppress their triggers, producing outputs consistent with their logical definitions. Each gate produces clear, binary-like outputs without any change to the nanopore itself.
The design offers several practical strengths. It is enzyme-free during amplification, runs at a constant temperature, and uses standard salt buffers. It produces steady signals that can be averaged and compared easily across devices. The PAMAM surface coating enhances DNA adsorption and can be applied with well-known surface chemistry. Using a fixed current threshold simplifies interpretation and allows different gates to operate on the same device. Because new targets can be connected to the trigger strand through custom DNA hairpins or aptamers, the system is adaptable to many biomolecules.
When compared with fluorescence or electrochemical assays for polynucleotide kinase, the nanopore system achieves comparable or better sensitivity with fewer steps. It avoids labels, optical instruments, and complex hardware. Its compact setup and simple electrical readout suit portable testing devices.
While this work demonstrates single-pore experiments, the chemistry is compatible with nanopore arrays that could measure several targets at once. That scalability would allow more advanced logic circuits capable of combining multiple biochemical signals into unified decisions.
This study links DNA amplification chemistry and nanopore sensing into one coherent system. It addresses two central limitations: weak signal strength and the lack of digital logic in nanopore sensors. The autocatalytic feedback loop amplifies low concentrations with high precision, while the logic functions provide stable, reproducible outputs. The demonstrated sensitivity for polynucleotide kinase reaches 0.0001 units per milliliter, and the logic repertoire covers six Boolean operations with a single nanopore type and a unified readout method.
The work outlines a practical route toward molecular devices that detect and process information in the same physical structure. By merging programmable DNA reactions with steady-state electrical measurements, it moves nanopore technology toward a new class of biosensors that think as they measure.
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