1 Introduction

A logic gate is a physical device implementing a Boolean function. It performs a logical operation on one or more logical inputs, and produces a single logical output. Enzymes and nucleic acids are promising for constructing molecular logic gates for computational purposes [1-11], and DNA is advantageous in its high stability, suitable for bio-imaging, and precise programmability [12-17], hence an excellent candidate for building logic operating systems.

Since Adleman’s invention of bio-computations in 1994 using just DNA molecules [18], a diverse range of DNA logic devices have been designed [19-24]. Willner and co-workers developed fluorescent logic gates, such as “AND”, “OR”, and “SET-RESET”, constructed with ions as inputs and the fluorescence intensity of a G-quadruplex as outputs [25]. Wang and co-workers constructed an optical-output “INHIBIT” logic gate utilizing structural switched DNA probe with the addition of targets (K⁺ or Pb²⁺) [26, 27]. Kevin and co-workers reported a reagentless, molecular logic gates with electrochemical signal outputs by using electrochemical E-DNA sensor architectures on a millimeter-sized electrode [28].

So far, most of DNA-based logic gates were constructed in bulk solution or on a macroscopic electrode at millimeter level, which hinders their in vivo applications. An electrode in micrometer or even nanometer diameter can be readily matched to the dimensions of cells and suitable for implantation in vivo [29, 30]. At SINAP, we studied the application of gold nanoparticles in radiotherapy and synthesized gold nanostructures in different shapes [31-33]. Besides, we developed the protocol to prepare gold flower microelectrode. By immobilizing with biological probes, the gold flower microelectrode can be successfully used for small molecule (such as cocaine) detection, with fast response and high specificity [34].

Scheme 1.Full-screen View

The design principle of electrochemical logic gates. In our design, the microelectrode with flower-like nanostructure was used to immobilize bio-probes. Here, a stem-loop structured DNA probe and an aptamer of ATP, both modified with methylene blue (MB), were employed. When the target DNA (cDNA) was introduced, the electron transfer (eT) of MB was blocked due to the conformational change of DNA structure; while, when ATP was introduced, the eT was facilitated due to the conformational structure change of aptamer.

In this work, we used gold flower microelectrode and developed DNA XOR logic gates, which were based on two electrochemical biosensors for ATP and DNA detections, respectively. The targets (ATP and DNA) and the denaturant urea were used as inputs, and changes in faradic current observed via square wave voltammetry (SWV) caused by the presence or absence of targets were used as outputs (Scheme 1).

2 Experimental section

3 Results and Discussion

We investigated the target binding kinetics of the two electrochemical biosensors, respectively. The target binding processes were monitored by continuously recording the SWV current peak at −0.25 V (Fig. 1). The SWV current of ATP biosensor increased upon the ATP binding since the redox moiety (MB) approached the electrode surface to facilitate the electron transfer. As shown in Fig.1, the ATP sensor demonstrated fast kinetics with a saturated time of ~5 min. The SWV current of DNA biosensor decreased upon the DNA hybridization since the redox moiety (MB) was forced to separate from the electrode surface to block the electron transfer. Although the DNA sensor demonstrated relatively slower target-binding kinetics than that of ATP sensor, we could still obtain the saturated signal within 30 min.

Fig 1.Full-screen View

Kinetics for the ATP (a) and DNA (b) sensors in the presence of 500 nM of cDNA targets and 1 mM of ATP, respectively.

A two-analyte XOR logic device was hen designed by immobilizing the two bioprobes on a single gold flower microelectrode, which compressed two input states into one output state, and the concentration of ATP and cDNA was defined as inputs and the signal change of the faradic current resulted from methylene blue (MB) was defined as outputs. For input, we defined the presence of 1000 μM ATP and 200 nM cDNA as the ‘1’ states, and lower (to 0 M) concentrations as the ‘0’ states. As outputs, we defined signal changes of greater than 5% and less than 5% as the ‘1’ and ‘0’ states, respectively. Thus, we could control the inputs (the concentration of ATP and DNA) to obtain a logic signal output and produce the truth table of our designed logic gate. The logic gate is shown schematically in Fig. 2. From the truth table, if one, and only one, of the inputs to the gate is at ‘1’ state, the output is ‘1’; while if both inputs are ‘0’ states or both are ‘1’ states, the logic gate produced an output ‘0’. Thus, this label-free, dual-analyte device serves as a XOR logic gate. The logic device can be designed to monitor the ratio of two inputs. For example, when two inputs (ATP and cDNA) are controlled in optimal concentration ration of 1000 μM ATP: 200 nM cDNA, the logic gate output is “0”, indicating the system work well and the ratio of the two chemicals is under control. When the ratio of the two inputs shifts to a concentration ration of 0 μM ATP: 200 nM cDNA, the output changes to “1”, indicating a state out of control that should be adjusted.

Fig. 2.Full-screen View

(a) A schematic presentation of an XOR gate. To fabricate this two-input logic device, two electrochemical DNA probes described previously, i.e. the E-DNA sensor [38-40] composed of a stem-loop oligonucleotide and E-AB sensor [38] composed of a ATP-binding DNA aptamer, attached together to a single gold flower microelectrode via an alkane thiol and modified with a redox reporter. When interrogated via square wave voltammetry (SWV), the probes responded to their respective targets (a complementary DNA and ATP, respectively) via decreasing and increasing faradic current respectively. (b) The probes of the logic device for which four input combinations lead to different electrochemical output currents via SWV. (c) The truth table for this two-input logic gate. (d) The symbol of this logic gate.

Based on the two-analyte logic device, we have also designed a three-input logic gate by using urea to this system as the third input. Urea can denature the structure of DNA probes and change the ability to transfer electrons. For input, we also defined the presence of 1 M urea as the “1” state, and lower (to 0 M) concentrations as the “0” state. As before, we defined signal changes greater than 5% and less than 5% as “1” and “0” states, respectively. As a result, a three-input logic operation was constructed by controlling the concentrations of ATP, DNA, and urea. The truth table and schematic representation of this logic gate were presented in Fig. 3.

Fig 3.Full-screen View

Adding denaturant urea into this two-input gate as the third input to produce a three input logic gate. It could unfold DNA probe inducing decrease in faradaic current upon SWV interrogation. (a) A schematic representation of this three-input logic gate and the activation of the gate using varying concentrations of ATP, DNA, and urea as inputs (b) The eight input combinations induced different changes of electrochemical currents. (c) Truth table for the three-input logic gate. (d) The symbol of this logic gate.

Regeneration and stability of the logic gate were studied. The signal changed at the presence of the targets (ATP or DNA). Interestingly, through a simple rinsing by 1×PBS, the signal could be recovered for more than 90%. Furthermore, the logic gate performed well in relatively complex sample matrices (20% FBS) and (Fig. 4). The logic device showed similar signal change both in buffer solution and diluted FBS. Thus the designed logic gates showed potential for the continuous monitoring in complex system.

Fig 4.Full-screen View

(a) Rinsing with 1×PBS, signal regeneration of this dual-analyte sensor could recover more than 90%. (b) The dual-analyte device showed similar signal change both in buffer solution and in complex samples, such as 20% FBS (diluted with HEPES buffer).

4 Conclusion

In conclusion, we have constructed dual-analyte, DNA-based logic devices that functioned as an electrochemical XOR logic gate on the gold flower microelectrode. The combination of this logic gates and the gold flower microelectrode showed the advantages of electrochemical logic gate in signal readout. Most of the logic operations designed previously were realized through fluorescence output and in solution phase [2, 7, 12, 20-22, 43, 44], which may limit the connection between biological devices and electronics. Our design also reflected the application potential of ultramicroelectrode in applications of implantable device. Most of the electrochemical logic operations that designed previously were realized on microscopic electrode [28, 42]. For some biological applications such as applications in living cells and in-situ monitoring small molecules released by living cells, the scale of the electrode should be decrease to micrometers and even nanometers to fit the scale of living cells. The use of macroscopic electrode would not accomplish such tasks or result in damaging of living cells [41, 45-49].