Self-powered biomolecular keypad lock security system based on a biofuel cell

Abstract

The enzyme-based keypad lock was integrated with a biofuel cell yielding a self-powered biomolecular information security system. The correct “password” introduced into the keypad lock resulted in the activation of the biofuel cell, while all other “wrong” permutations of the enzyme inputs preserved the “OFF” state of the biofuel cell.

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Recent developments in unconventional chemical computing resulted in the formulation of molecular systems mimicking Boolean logic gates and networks.¹ One of the most important challenges in the chemical computing field is scalability of the systems. Combination of chemical logic gates in groups or networks resulted in simple computing devices performing basic arithmetic operations.² Chemical systems mimicking various components of digital electronic devices were designed.^1,2 The application of chemical logic networks for mimicking electronic security devices³ resulted in the design of molecular keypad lock systems⁴ activated only upon the correct combination of chemical or physical input signals.

The emerging research field of biocomputing, based on the application of biomolecular systems for processing chemical information, has achieved higher complexity of information processing due to the natural specificity and compatibility of biomolecules allowing their easy assembling in networks.⁵ Different biomolecular tools, including proteins/enzymes,⁶ DNA,⁷ RNA,⁸ and whole cells,⁹ were used to assemble computing systems processing biochemical information. Various Boolean logic operations were mimicked by enzyme systems¹⁰ allowing concerted operation of multi-enzyme assemblies performing simple arithmetic functions.¹¹ Although similar logic operations and arithmetic functions were also realized using non-biological chemical systems,² the advantage of the biomolecular systems has been the relative simplicity of the various assembled logic schemes. Scaling up such systems could result in artificial biocomputing networks with increased complexity, performing various logic functions and mimicking natural biochemical pathways.¹² The enzyme-based logic systems were applied to control states of switchable bioelectronic devices (modified electrodes¹³ and biofuel cells¹⁴). The switchable systems, controlled by biocomputing logic networks, laid the foundation for the advanced “smart” systems integrating a “decision” making part with an operating bioelectronic device. The application of the enzyme logic circuits in security systems allowed us to design a biomolecular keypad lock based on biocatalytic transformations which resulted in the lock “opening” only when the correct sequence of the enzyme-reactions was triggered by biochemical input signals.¹⁵ The present communication addresses a novel approach to the integrated bioelectronic devices where the enzyme-based keypad lock is connected to a biofuel cell, thus resulting in a self-powered biomolecular security system.

In order to demonstrate the concept, we designed a model biocatalytic system composed of three enzyme-catalyzed reaction steps resulting in pH changes only at the very last biocatalytic step. The enzymes were applied as the input signals triggering the biochemical transformations. When the enzyme-inputs were applied in the correct order the biocatalytic cascade—starting from starch and finishing with gluconic acid—was in succession, Scheme 1A. In the first reaction, β-amylase (βAm; from sweet potato, type 1-B, EC 3.2.1.2, 100 units mL⁻¹), being enzyme-input A, resulted in the biocatalytic hydrolysis of starch and yielded β-maltose. In the second step, maltose phosphorylase (MPh; from Enterococcus recombinant, expressed in E. coli, EC 2.4.1.8, 5.6 units mL⁻¹), coupled with acid phosphatase (AP; from wheat germ, type 1, EC 3.1.3.2, 0.8 units mL⁻¹) operating together as the enzyme-input B, produced glucose from maltose generated in the previous step. In this reaction step, MPh produced glucose and glucose-1-phosphate from maltose consuming phosphate, while AP converted glucose-1-phosphate to the second glucose molecule releasing phosphate. This resulted in the increased yield of glucose and re-circulation of phosphate ions in the solution, thus allowing to keep phosphate concentration low, which is important for having low buffer capacity in the solution. In the final step, glucose oxidase (GOx, type X-S from Aspergillus niger, EC 1.1.3.4, 70 units mL⁻¹) was used as the enzyme-input C and produced gluconic acid from glucose, thus resulting in the pH decrease. The initial solution, pH 6.7, before applying the enzyme-inputs included 8.3 mM starch as the primary substrate, 0.5 mM phosphate as a co-substrate for the second enzymatic reaction step, O₂ in equilibrium with air as a co-substrate for the last reaction step, and 10 mM K₃[Fe(CN)₆] as a redox probe for contacting a modified electrode after all enzyme-inputs are applied. The solution also included 0.1 M Na₂SO₄ to perform electrochemical measurements. To allow the stepwise application of the enzymes without their mixing, the reactions were performed in ultrafiltration tubes (cutoff 10 kDa) allowing separation of the low molecular products generated at each biocatalytic step from the enzyme-inputs, Scheme 1B. The product generated in the tube was applied to the next reaction step, while the enzyme-input was preserved in the reaction volume. This setup allowed easy exchange of the reaction volumes applying the enzyme-inputs in 6 different permutations: A–B–C, C–B–A, A–C–B, B–C–A, B–A–C and C–A–B where only the first one corresponded to the correct sequence of the biocatalytic reactions. All other combinations of the enzyme-inputs did not facilitate the whole biocatalytic cascade since the correct sequence of the products–reactants was not achieved. Thus the correct input-sequence A–B–C represented the “password” for the biocatalytic cascade to yield gluconic acid as the final product decreasing the solution pH value, while all other input-combinations did not result in significant pH changes, Fig. 1A. Note that the biocatalytic cascade was designed to have the acid as the final product, while all intermediate products do not have acidic properties.

Scheme 1 (A) The biocatalytic cascade triggered by the enzyme inputs when they are applied in the “correct” A–B–C sequence. (B) The reaction/separation setup allowing application of the enzyme inputs in different permutations (shown in the correct order A–B–C).

Fig. 1 (A) pH-changes generated at the final step of the reaction cascade for different permutations of the enzyme-inputs. (B) Cyclic voltammograms obtained on the P4VP–ITO-electrode in the presence of 10 mM [Fe(CN)₆]³⁻ at different pH values: (a) initial pH 6.7, (b) pH 4.2 generated upon A–B–C sequence of the enzyme-inputs. Potential scan rate, 100 mV s⁻¹.

The product-solution generated at the end of the three-step process was applied to switch the interfacial activity of a polymer-brush-modified electrode. As it was demonstrated earlier,¹³ an indium–tin oxide electrode (ITO; 20 ± 5 Ω/sq surface resistivity, geometrical area of 1.2 cm²) functionalized with poly(4-vinyl pyridine) (P4VP; MW 160 kDa) reveals electrochemical activity for soluble anionic redox species switchable by the solution pH values. The polymer-brush being in the non-protonated hydrophobic shrunk state at pH > 5.5 is impermeable for soluble redox species, thus inhibiting their electrochemical reactions. When pH < 4.5 is applied, the pyridine groups in the polymer-brush are protonated yielding a positively charged swollen hydrophilic thin-film permeable for anionic redox species, thus allowing their electrochemical reactions. The P4VP-brush was grafted to the surface of the ITO-electrode following the procedure detailed elsewhere.¹⁶ The thickness of the P4VP-brush in a dry state, 5.4 ± 0.3 nm, estimated by ellipsometry corresponds to the grafting amount of ca. 5.94 mg m⁻² (grafting density of ca. 2.2 × 10¹² cm⁻²) (note that ellipsometry measurements were performed on Si-wafers modified similarly to the ITO-electrode).

The electrochemical measurements on the P4VP-brush-modified electrode started at pH 6.7 when the electrode was in the “OFF” state for the soluble anionic redox probe, [Fe(CN)₆]³⁻, 10 mM, thus showing no waves in the cyclic voltammogram, Fig. 1B, curve a. When the enzyme-inputs were applied in the correct order A–B–C and gluconic acid was produced, the pH value in the last reacting solution was reaching ca. 4.2. This pH value corresponded to the “ON” state of the P4VP-brush-modified electrode. When this solution was applied to the electrode, a cyclic voltammogram showing the redox process of [Fe(CN)₆]³⁻ was obtained, Fig. 1B, curve b. The waves in the cyclic voltammogram were obtained only when the correct “password” A–B–C was used in the biocatalytic cascade, while all other permutations of the enzyme-inputs did not change the initial cyclic voltammogram of the electrode being in the “OFF” state.

After “reading” the “answer” of the enzyme-based keypad lock system by cyclic voltammetry, a biofuel cell was designed as a self-powered “reading” device. A simple model biofuel cell was composed of two ITO-electrodes. The cathode was modified with a pH-switchable P4VP polymer-brush operating in the presence of 10 mM K₃[Fe(CN)₆] used as a model oxidizer in a background solution composed of 0.1 M Na₂SO₄ and 0.5 mM phosphate. The anode was an unmodified ITO-electrode operating in the presence of soluble GOx (250 units mL⁻¹) which oxidized the glucose-fuel, 0.1 M, with the help of a diffusional redox mediator methylene blue, 0.1 mM in 100 mM phosphate buffer, pH 7.0, under Ar. The electrodes were separated with a Nafion® membrane (0.09 mm thick). The oversimplified design of the biofuel cell, Scheme 2, was specially selected to clearly demonstrate the power control by the keypad lock system without any complications from secondary effects related to the bioelectrocatalytic reactions. The experiment was started at pH 6.7 in the cathodic solution when the switchable cathode was in the “OFF” state and the entire biofuel cell was “mute” demonstrating low current-voltage, Fig. 2A, curve a, and low power, Fig. 2B, curve a. The final solution from the last step reaction in the keypad lock was applied to the cathodic compartment of the biofuel cell. When the correct “password” A–B–C was used, the produced acidic solution, pH ca. 4.2, resulted in the activation of the cathode, thus switching the biofuel cell “ON”. This resulted in the increasing current and power output generated by the biofuel cell, Fig. 2A and B, curves b. All other “wrong” permutations of the enzyme-inputs did not result in switching “ON” the biofuel cell, thus, keeping the power output low, Fig. 2B, inset. It should be noted that the difference between the current and power outputs generated by the biofuel cell can be increased upon appropriate optimization of the polymer-brush surface density. Since the reacting cocktail is consumed after application of three-step enzyme inputs, the additional inputs are not possible.

Scheme 2 The pH-switchable biofuel cell for “reading” the output signals generated by the keypad lock system. MB_ox and MB_red are the oxidized and reduced states of methylene blue mediator.

Fig. 2 The polarization functions (A) and power output (B) of the biofuel cell obtained at different pH values: (a) initial pH 6.7, (b) pH 4.2 generated upon A–B–C sequence of the enzyme-inputs.

The designed enzyme-based keypad lock system demonstrated the IMPLICATION logic function generating the final output “YES” only when the correct order of the input signals is applied (the A–B–C “password”). The “answer” “YES” was obtained in the form of the electrical power produced by the biofuel cell. All other permutations of the enzyme-inputs did not result in the activation of the biofuel cell preserving it in the initial “mute” state. The designed security system operated without the need of any external power source producing electrical power itself when the correct “password” is applied. It should be noted that the present example-device is aiming at concept demonstration only, while the real operating system should be based on a microfluidic system (lab-on-a-chip) allowing for its miniaturized design. The number of biocatalytic steps and the respective number of the enzyme-inputs might be easily increased up to 10 (this is the usual number of inputs in electronic keypad locks) without significant noise in the biocomputing network.¹⁷ Further advances of system complexity for the next generation of biomolecular security devices will include participation of immune-recognition components in the design of the biocomputing logic networks,¹⁸ while a biofuel cell controlled by immune-reactions was already designed¹⁹ and ready for the integration with the immune-based keypad lock system.

NSF grants DMR-0706209, CCF-0726698 and SRC award 2008-RJ-1839G are gratefully acknowledged. GS acknowledges the Wallace H. Coulter scholarship from Clarkson University.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details for the enzyme-inputs processing, modification of the switchable electrode and biofuel cell operation. See DOI: 10.1039/b925484f

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