Quantitative Evaluation of Biological Reaction Kinetics in Confined

Jul 18, 2014 - Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School ... purchased from Wuhan Boster Biological Technology Co., Ltd...
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Quantitative Evaluation of Biological Reaction Kinetics in Confined Nanospaces Jiachao Yu,† Peicheng Luo,† Chuanxian Xin, Xiaodong Cao, Yuanjian Zhang,* and Songqin Liu* Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China S Supporting Information *

ABSTRACT: Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

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kinetics in a quantitative way,4,22 most presumably due to the lack of theoretical models and the challenge of direct acquiring the kinetic parameters based on current available techniques. Herein, we developed a nanochannel-based electrochemical reactor and a model to describe the kinetics of immunological reaction in nanochannels quantitatively. As shown in Figure 1, the PAA nanochannels were modified with single-stranded DNA which each contained two covalently linked digoxin molecules. When antidigoxin flowed into the nanochannels and captured by digoxin, the flux of probe ions in nanochannels would be modulated, which was also influenced by many general experimental conditions, such as the ionic strength and pH of the solution, the pore size of the nanochannels, and the charge property of the probe molecules. On the basis of this, we proposed a facile model to quantitatively describe the immunological reaction in nanochannels, i.e., not only the reaction kinetic constant but also the move speed of antibody and the immunological reaction progress in nanochannels were successfully revealed. It provided us a deeper insight for the biological reaction that occurred in confined nanospaces.

ecently, construction of nanodevices for real-time evaluation of the molecular biological processes and their kinetic information in confined nanospaces has received growth interest. Nanospaces, such as the naturally occurring αhemolysin nanopore,1−3 aptamer-encoded glass nanopore,4,5 SiN nanopore,6,7 ligand-functionalized polymer nanochannel,8−10 and porous anodic alumina (PAA) nanochannel,11−14 have played important roles in the development of life analysis methodologies. For example, the recognition and detection of single molecule such as small molecules,15−17 DNA,18−20 proteins,21−23 organic polymers,24,25 and biological molecular complexes26−28 have been realized by using the nanopore/ nanochannel technology. Among these, PAA membrane has attracted much attention for its tunable nanopore diameter,29 well-defined nanochannel array,30 robust overall structure,11 easy surface modification,31 and physical and chemical stability.12 For instance, Merkoçi et al. reported a rapid PAAbased immunoassay to detect antigens, in which a redox probe was used to illustrate the steric effect induced by the proteins and nanoparticles in the nanochannels.13 Xia et al. demonstrated a facile method for DNA detection in the PAA nanochannels, in which a redox probe was utilized to show not only the steric effect but also the electrostatic effect before and after the hybridization of the target DNA and morpholino.18 The principle of these applications was based on the modulation of the probe molecule/ion transporting through nanochannels, and their applicability depended sensitively on the surface characteristics of the nanochannel inner walls.32−35 Although the applications of biological reactions in nanospaces have achieved considerable development, such as the detection of biomolecules and the measurement of apparent enzyme activity, there are still few studies on their reaction © 2014 American Chemical Society

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EXPERIMENTAL SECTION Materials. DNA (5′-CHO-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA T(Dig)T(Dig)-3′) and mouse monoclonal antidigoxin were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Goat antimouse IgG was obtained from Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China). Received: March 29, 2014 Accepted: July 18, 2014 Published: July 18, 2014 8129

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PBS. The functionalized PAA membrane with DNA immobilized on the inner wall of PAA nanochannels (PAA/DNA) was thus obtained and stored in PBS at 4 °C. Immunoassay and Regeneration of the PAA Membrane. The immunoassay was carried out using 100 μL of antidigoxin at various concentrations in PBS (10 mM, pH 7.0), which was dropped and incubated on DNA-functionalized PAA membrane (PAA/DNA) for 1 h.40−42 The residual antidigoxin was removed by washing with PBS thoroughly. The regeneration of the PAA membrane could be achieved by immersing it into 1 mL of glycine−NaOH solution (0.1 M, pH 10.5) and shaking gently for 10 min to interrupt the antigen− antibody immunocomplex,43−45 followed by rinsing with PBS thoroughly. Electrochemical Measurements. All the electrochemical measurements were performed with a homemade cell (Figure S-1 in the Supporting Information) containing 2 mL of electrolyte. Briefly, as working electrode, a gold disc combined with a copper pillar was inserted in one insulating block (i.e., poly(methyl methacrylate), PMMA). Then, a PAA membrane was put on the gold disc. After that, the other PMMA block was placed onto the PAA membrane and fixed with screws. Two silicone o-rings were used to avoid any liquid leakage. A CHI 660C electrochemical workstation (Chenhua Instrument, Shanghai, China) was used for all electrochemical measurements. An Au working electrode under PAA membrane, a Pt counter electrode, and a saturated calomel electrode (SCE) over the PAA membrane formed a three-electrode electrochemical system. To illustrate the electrostatic and steric effects in PAA nanochannels before and after immunological reaction, 1 mM of Fe(CN)63− was introduced into the electrolyte. The flux of Fe(CN)63− in the nanochannels was monitored by the cathodic current of Fe(CN)63− at 0 V. The experiments using PAA membranes with different pore diameters and PBS with different ion strength and pH values were carried out in the similar way. To monitor the immunological reaction and biosensing process in the nanochannels online, 100 ng mL−1 of antidigoxin was introduced into the electrolyte after the current of Fe(CN)63− reached a plateau.

Figure 1. Functionalization of porous anodic alumina (PAA) nanochannels with DNA and the time-course curves of electrochemical current in the absence (PAA/DNA) and presence (PAA/ DNA/AD) of antidigoxin (AD). The pore diameter of the nanochannels was 25 nm, the ionic strength and pH value of the solution were 2 mM and 8.0, respectively, and the concentration of the antidigoxin was 100 ng mL−1.

Rabbit antihuman IgG and rabbit polyclonal anti-TNF-α were purchased from Wuhan Boster Biological Technology Co., Ltd. (Hubei, China). Mouse monoclonal anti-α-HCG was received from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Mouse monoclonal anti-CEA was obtained from Meridian Life Science, Inc. (Maine, U.S.). Porous anodic alumina (PAA) was purchased from Hefei Puyuan Nanotechnology Co., Ltd. (Anhui, China). (3-Aminopropyl)triethoxysilane (APTES) and hexaammineruthenium(II) chloride were received from Sigma-Aldrich (Shanghai, China). All other chemicals were of analytical grade and used without further purification unless specified. Phosphate buffer solutions (PBS) with different pH and ionic strength were prepared based on NaH2PO4 and Na2HPO4 and adjusted by NaCl. Double distilled water was used throughout the study. Covalent Linking of DNA to PAA Nanochannels. The process for functionalization of PAA nanochannels with DNA was illustrated in Figure 1. The morphology and sizes of PAA membranes were characterized by a field emission scanning electron microscope (SEM, ULTRA PLUS, ZEISS, Germany) at an acceleration voltage of 5 kV. PAA membranes with thickness of 81 μm, diameter of 1.2 cm, geometry area of 1.13 cm2, and pore diameters of 25 ± 5, 55 ± 15, 90 ± 10, and 130 ± 20 nm were washed by ethanol and double distilled water to remove any impurities in the nanochannels. After drying in a nitrogen flow at room temperature, the PAA membranes were immersed into a 1 mL ethanol solution containing 5% APTES and shaken gently for 12 h.36−39 Then the PAA membranes were washed with ethanol to remove any residual silane in the nanochannels and last dried in a nitrogen flow at room temperature. After that, a 100 μL solution of DNA at a concentration of 10 μM prepared in PBS (10 mM, pH 7.0) was dropped onto the surface of the PAA membrane for 24 h. Thus, the DNA was assembled onto the inner wall of the nanochannels through a Schiff reaction between the −CHO group in DNA and the amino group on the nanochannels of PAA under mild reaction conditions.18,19 The remaining amino groups were further blocked by immersing the PAA membrane into 1 mL of PBS (10 mM, pH 7.0) containing 0.1% benzaldehyde and shaking gently for 12 h. The unbound DNA and residual benzaldehyde were removed by rinsing with

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RESULTS AND DISCUSSION Principle Verification. The modification of nanochannels with biomolecules may change the surface charge and the inner space of the nanochannels,8 which would result in a reduced flux of probe ions due to the steric effect and/or the electrostatic effect especially when the size of the biomolecules were comparable with the pore diameter of nanochannels. If the biorecognition reaction occurred, the steric effect and electrostatic effect could be more profound, limiting the flux of probe ions.13,18 Here, a 30-base single-stranded DNA containing two identical, covalently linked digoxin molecules was assembled to nanochannels through a Schiff reaction (see details in the Experimental Section). It was reported that when single-strand DNA lay on the inner wall of the nanochannels, it had negligible steric effect on the flux of Fe(CN)63−, since its lain size was much smaller than the pore diameter of nanochannels.18 However, if the DNA was negative charged (e.g., PI = 4.0−4.5 in a solution of pH 8.0), it would generate an extra electrostatic repulsion region toward anions in the nanochannels, limiting the flux of the negatively charged Fe(CN)63−. Consistently, the steady-state cathodic current of Fe(CN)63− at 0 V which corresponded to the amount of Fe(CN)63− passing through the DNA modified nanochannels 8130

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(25 nm PAA/DNA, ∼9.0 μA, Figure 1) was smaller than that of bare PAA nanochannels (25 nm PAA, ∼9.8 μA, Figure S-2 in the Supporting Information). When the target antidigoxin (AD, approximately 12 nm41) was introduced into the nanochannels, the immunological reaction between digoxin and antidigoxin throughout the nanochannels (as discussed in the Supporting Information) would make the antidigoxin conjugated DNA strand extend42 to a size that was comparable to the pore diameter (25 nm), thus increasing the steric effect on the flux of Fe(CN)63−. At the same time, the extended DNA strand in nanochannels would increase the electrostatic repulsion region toward Fe(CN)63−. Both the steric effect and electrostatic effect led the flux of Fe(CN)63− in the nanochannels to be blocked and the steadystate current to be decreased significantly (25 nm PAA/DNA/ AD, ∼5.9 μA, Figure 1). In contrast, only slight difference of current response was observed from the PAA nanochannels before (PAA) and after (25 nm PAA/AD, ∼9.6 μA, Figure S-2 in the Supporting Information) incubating with antidigoxin. This phenomenon was quite different to that of the DNA coated PAA nanochannels (PAA/DNA), which confirmed that (1) the nanochannels was successfully coated with DNA, (2) the digoxin in DNA strand could capture the antidigoxin in incubation solution to extend the DNA strand in nanochannels, and (3) more importantly, the immunological reaction in confined nanochannels influenced the diffusion flux of Fe(CN)63−, which could be monitored electrochemically. Experimental Condition Screening. The steric effect and/or the electrostatic effect were observed to be closely related to other common factors such as ionic strength (Figure S-3 in the Supporting Information), pH value (Figure S-4 in the Supporting Information), charge state of the electroactive probes (Figure S-5 in the Supporting Information), pore diameter of the PAA membrane (Figure S-6 in the Supporting Information), and concentration (Figure S-7 in the Supporting Information) and type (Figure S-8 in the Supporting Information) of antibody (see detailed discussion in the Supporting Information). Briefly, low ionic strength, high pH value, and a negatively charged electroactive probe were in favor of the electrostatic effect increasing, and small pore diameter of PAA membrane and high concentration of antibody were in favor of both steric and electrostatic effects increasing. Thus, the proposed system offered us a convenient example to study the kinetics of the biological reaction in the nanochannels. To simplify the investigation of such a immunological reaction in nanochannels, in general, a profound current drop in electrochemistry and a mild environment for the biomolecules were preferred. For this, an alkalescent solution (pH 8.0) with a low ionic strength of 2 mM, a negatively charged electroactive probe (Fe(CN)63−), and a PAA membrane with pore diameter of 25 nm were chosen in the following studies. Real-Time Monitoring of the Immunological Reaction. To explore the immunological reaction in the nanochannels (PAA/DNA) in real-time, the antidigoxin was introduced into the cell 300 s after Fe(CN)63− was added, and the current was measured in situ. It was found that the current decreased greatly with time and reached a plateau (percentage of total variation 36.1%) in ∼40 min (Figure 2A, PAA/DNA (exp.)). This phenomenon could be interpreted as that once the antidigoxin was introduced into the cell, it would diffuse into the PAA nanochannels and further couple with the digoxin on the DNA. As a result, the immunological reaction

Figure 2. (A) Experimental cathodic current with time indicated the flux of Fe(CN)63− in different nanochannels (PAA and PAA/ DNA).The fitted curves ranged from the time that antidigoxin was added until that a plateau was reached. Inset table: the fitted results of the kinetic parameters. The status of the antidigoxin in the nanochannels: PAA (B) and PAA/DNA (C). L is the length of the nanochannels, x is the distance that the movement of antidigoxin in nanochannels took, θ is the percentage of DNA bound with antidigoxin, and u is the moving speed of Fe(CN)63− ions in the nanochannels.

occurred and, subsequently, the steric and electrostatic effects on the flux of Fe(CN)63− in the nanochannels increased gradually, which made the current decrease. As a control, the PAA membrane without DNA was studied in the same way. As shown in Figure 2A (PAA (exp.)), the current decreased moderately after the introduction of antidigoxin and reached a plateau (percentage of total variation 5.3%) in ∼8 min. This was mainly attributed to the physical adsorption of antidigoxin on the inner wall of the nanochannels and the motion of antidigoxin in the nanochannels, both of which increased the steric effect on the flux of Fe(CN)63− in the nanochannels. These two factors should also exist in the case of PAA/DNA. Nevertheless, the effect they caused was much weaker than that caused by the specific immunological reaction. In this sense, the real-time current change could be regarded mostly as a result which was induced by the immunological reaction, and thus we could use the real-time current change to study the processes of the immunological reaction. Model Building. Inspired by the Darcy−Weisbach equation,46 the movement of Fe(CN)63− in nanochannels could be used to probe the inner state of the nanochannels. As discussed in the Supporting Information, the movement of Fe(CN)63− probe was primarily restricted by the steric hindrance and electrostatic repulsion from the DNA/AD biocomplexes on the inner wall of the nanochannels. To simplify this system, the diffusion of the probe, the electric field applied for probe detection, and the electrostatic repulsion from the probe product at the electrode surface47 were concluded as a general driving force on the movement of the probe. As the flow of solvent was negligible in this system, we assumed the transport of probe ions as a flow of ions in the 8131

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To associate u with I, the current of Fe(CN)63− reduction at the working electrode that could be obtained from the experiment, eq 2 was given as

solvent and the steric hindrance and electrostatic repulsion from the DNA/AD biocomplexes as the flow resistance throughout the nanochannels. The feasibility of the proposed theory was discussed and clarified in the Supporting Information. Generally, the steric effect and/or electrostatic effect on the flux of Fe(CN)63− in nanochannels, hereafter, were collectively named as the blockage ef fect, which came from the limited nanochannel space, the occupation of free antidigoxins in nanochannels, and the immunocomplexes on the inner wall of nanochannels. These three factors could be regarded as three resistance coefficients of the Fe(CN)63− ions in the nanochannels (λ0, λ1, and λ2). On the basis of this, we proposed a model to investigate the processes of immunological reaction in nanochannels. Interestingly, as a result, more detailed kinetic information could be successfully revealed. Without DNA, the diffusion of Fe(CN)63− probe ions in nanochannels (PAA) was schematically shown in Figure 2B. According to the Darcy−Weisbach equation, the flux of Fe(CN)63− ions could be described as Kt m =

λ 0L + (λ1 − λ 0)x 2 u λ0

(2)

I = neNAu

where n is the number of electrons for the reduction of one Fe(CN)63− ion on the working electrode, e is the elementary charge, N is the number of Fe(CN)63− ions in unit volume of the electrolytes, and A is the cross-sectional area of the nanochannels in the PAA membrane. Substituting eq 2 into eq 1 and differentiating eq 1 could yield (neNA)2 mKt m − 1 − (γ − 1)vI 2 dI = dt 2[L + (γ − 1)vt ]I

(3)

where γ is the relative resistance coefficient which is defined by γ = λ1/λ0, and v is the average moving speed of the antidigoxin which is supposed to be x/t. When DNA was modified in the nanochannels (PAA/DNA), as shown in Figure 2C, the diffusion of Fe(CN)63− probe ions could be divided into two coherent stages, i.e., x ≤ L and x > L. Accordingly, the Darcy−Weisbach equation could be given as

(1)

where K is an empirical correction factor for this system (depending on some factors in this system, such as the general driving force on the movement of the probe, the initial resistance coefficient on the flux of probe, the diameter of the nanochannel and the density of the solution), t and x are the time and distance that the movement of antidigoxin in the nanochannels took, m is the coefficient of t, L is the length of the nanochannels, λ0 and λ1 are the resistance coefficients of the Fe(CN)63− ions in the region where antidigoxin is absent and present in the nanochannels, and u is the moving speed of the Fe(CN)63− ions in the nanochannels.

where θ is the percentage of DNA bound with antidigoxin, λ0, λ1, and λ2 are the resistance coefficients of the Fe(CN)63− ions in the region where antidigoxin is absent, present but not bound with DNA, and bound with DNA in the nanochannels. Using eq 4 and differentiation, the kinetics of the antidigoxin and Fe(CN)63− in the nanochannels were given as eq 5:

where γ1 and γ2 are the relative resistance coefficients which are defined by γ1 = λ1/λ0 and γ2 = λ2/λ0, d is the diameter of the nanochannels, c0 is the concentration of antidigoxin, k is the reaction kinetic constant of antidigoxin with digoxin on DNA, NDNA is the mole number of DNA in a single nanochannel, which is determined by measuring ultraviolet absorption of the DNA solution before and after dropped onto the surface of the PAA membrane at 260 nm (as shown in Figure S-9 in the Supporting Information), θ0 is the percentage of DNA bound with antidigoxin at the end of the first stage, and t0 is the time of the antidigoxin passing through the nanochannels. It is noted that the values of K and m in eqs 5.1 and 5.2 are the same with those in eq 3. Application of the Model. By fitting the experimental data with the model, the parameters in the model could be calculated. As shown in Figure 2A, the fitted data matched the experimental data very well, which gave convincible evidence for the validity of the proposed models, and the values of the parameters were listed in the inset. The value of γ was ∼1.2,

which indicated that the introduced antidigoxin increased the blockage effect in PAA nanochannels by 1.2 times. It should be noted that the diffusion of Fe(CN)63− ions in nanochannels was restricted when γ > 1 and boosted when γ < 1. Similarly, γ1 (∼1.4) meant that the introduced antidigoxin which was free in PAA/DNA nanochannels made the blockage effect 1.4 times as large as the original PAA/DNA nanochannels. It was interesting that γ 1 was larger than γ, which implied PAA/DNA nanochannels more susceptible to free antidigoxin than PAA nanochannels. On the other hand, γ2 (∼3.1) suggested that the bound antidigoxin significantly increased the blockage effect in PAA/DNA nanochannels by 3.1 times. It was obvious that γ2 was much larger than γ1, which made γ2 the main factor on the variation of the blockage effect in PAA/DNA nanochannels. More importantly, γ1 and γ2 qualitatively indicated the amount of antidigoxin and immunocomplex in the nanochannels, and thus the relationship between γ1 and γ2 could reveal the progress of such biological reaction in nanochannels. Another parameter, v, which suggested the average move speed of 8132

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Figure 3. Relationships between the concentration of antidigoxin and the model parameters: the relative resistance coefficients γ1 and γ2 (A, B), the moving speed of antidigoxin v (C), and the reaction kinetic constant k (D). The error bars represent the confidence intervals of fitting parameters (see the detailed discussion in the Supporting Information).

was bound, which indicated a strong immunological reaction. Besides, as shown in Figure 3C, the average moving speed of antidigoxin through PAA/DNA nanochannels decreased from ∼450 nm s−1 to ∼90 nm s−1, while the concentration of antidigoxin increased from 1 ng mL−1 to 1 μg mL−1, which was mainly attributed to the increased blockage effect in PAA/DNA nanochannels. Moreover, as shown in Figure 3D, we found that k decreased from ∼1500 s−1 to ∼20 s−1 when the concentration of antidigoxin increased from 1 ng mL−1 to 1 μg mL−1, while k (2600 s−1) was kept the same in homogeneous solution.37 The reason was that the blockage effect in nanochannels restricted the diffusion of antidigoxin and made the antidigoxin difficult to react with digoxin on DNA, which would be enhanced when the antidigoxin concentration increased. It was interesting to find that all the aforementioned kinetic parameters had a tendency to become stable at high antidigoxin concentration except γ2. The reason might be that the amount of DNA was much larger than antidigoxin, and both of the digoxin molecules on one DNA would be captured by antidigoxin at its high concentration, which made the blockage effect much more significant. In summary, when the concentration of antidigoxin varied, (1) all of the parameters had reasonable values under each concentration, (2) each parameter had a variation tendency which was consistent with the actual situation, and (3) the values and variation tendencies of the parameters had good correlations between each other. These factors demonstrated that the proposed model was valid in this system and the fitted parameters had high reliabilities. Therefore, the proposed model successfully provided a quantitative and comprehensive way to reveal the kinetic process of the immunological reaction in nanochannels, simply

antidigoxin in such PAA/DNA nanochannels, had a value of ∼135 nm s−1 here. Furthermore, k (∼121 s−1) showed the rate of the immunological reaction in such nanochannels. The rate constant of this immunological reaction in homogeneous solution was 2600 s−1.48 It was obvious that the immunological reaction rate in such nanochannels was much lower than that in homogeneous solution. The main reason was that the diffusion of antidigoxin was restricted by the blockage effect in the nanochannels, which made the antidigoxin difficult to react with digoxin on DNA. Thus, these parameters could reveal the quantitative kinetic information on the biological reaction in confined nanospaces. To verify the general validity of the proposed model, more experiments were carried out using different concentrations of antidigoxin while the other conditions were kept the same (see time-course curve of the immunological reaction in Figure S-10 in the Supporting Information). By fitting the experimental data with the model, the parameters at different concentrations of antidigoxin were calculated and plotted in Figure 3. At low antidigoxin concentration (1 ng mL−1), the blockage effect in PAA/DNA nanochannels increased slightly after introducing antidigoxin (γ1 ≈ 1.0, γ2 ≈ 1.2). This indicated that antidigoxin was sparse in the PAA/DNA nanochannels and only a small number of antidigoxin was bound, which suggested a week immunological reaction. When increasing the concentration of antidigoxin, the blockage effect in PAA/DNA nanochannels were increased accordingly, as shown in Figure 3A,B. Once the concentration of antidigoxin reached 1 μg mL−1, the blockage effect in PAA/DNA nanochannels increased significantly (γ1 ≈ 1.9, γ2 ≈ 10.3). This suggested that antidigoxin was crowded in the PAA/DNA nanochannels and a large number of antidigoxin 8133

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based on electrochemical techniques. It should be noted although many pioneering works have been developed on the detection of biomolecules and measurement of apparent enzyme activity, the quantification of the reaction kinetics was scarcely investigated. And in the very few works, only the association and dissociation rate constant (k) of biomolecular binding events in nanopores were reported.4,22 In this regard, our work demonstrated a simple and effective strategy for the much more comprehensive quantitative evaluation of the biological reaction kinetics in confined nanospaces.

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CONCLUSIONS In this work, we developed a nanochannel-based electrochemical reactor and proposed a model to investigate the immunological reaction in confined nanospaces. As a result, except for the reaction kinetic constant (k), more insightful kinetic information such as the move speed of antibody (v) and the biological reaction progress (γ1, γ2) in nanochannels were successfully revealed by fitting the electrochemical data with the proposed model. It offered us a facile way to quantify the kinetic process of the immunological reaction in nanochannels simply based on electrochemical methods. This work assessed the molecular biological processes in vitro and revealed the fundamentals of biological reactions in confined nanospaces simply by electrochemistry and theoretical fitting. It would help us design novel nanodevices for life science and promote the research of biological reactions in confined nanospaces.

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ASSOCIATED CONTENT

S Supporting Information *

Influences of various conditions on the current response of the electrochemical reactor and the scheme of the assembled electrochemical cell. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

J.Y. and P.L. had an equal contribution to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project is supported by the Key Program (Grant 21035002) from the National Natural Science Foundation of China, the National Basic Research Program of China (Grant No. 2010CB732400), and the National Natural Science Foundation of China (Grant Nos. 21375014 and 21203023).

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