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A three-way junction aptasensor for lysozyme detection
Biosensors and Bioelectronics 39 (2013) 250–254
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A three-way junction aptasensor for lysozyme detection Yunfeng Xia, Siwen Gan, Qinghao Xu, Xiaowen Qiu, Peiyi Gao, Shasheng Huang n Life and Environmental Science College, Shanghai Normal University, Guilin Road 100, Shanghai 200234, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 May 2012 Received in revised form 23 July 2012 Accepted 25 July 2012 Available online 11 August 2012
A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called ‘‘conductive path’’, is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2 nM with a dynamic range extending to 100 nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure. & 2012 Elsevier B.V. All rights reserved.
Keywords: Three-way junction Aptasensor Lysozyme Conductive path
1. Introduction At present, most electrochemical aptasensors are formed mainly based on the conformational change of the aptamers induced by binding the specific target to the probes (Xiao et al., 2005; Baker et al., 2006; Shen et al., 2007). The specific binding of target to the surface-confined and redox-labeled aptamer essentially induces the conformational change of the aptamer probe, and leads to the change of distance between the labeled redox moiety and the surface of electrode (Radi et al., 2006), eventually resulting in the large variation of voltammetric signal used for electrochemical sensing of analytes. Although this kind of electrochemical aptasensors with aptamer as probe has so far been employed for selective and sensitive electrochemical sensing of targets such as thrombin (Xiao et al., 2005), cocaine (Baker et al., 2006), ATP (Zuo et al., 2007), and theophyline (Ferapontova et al., 2008), those aptasensors generally required a large-scaled conformational change of the aptamer probe induced by the specific target binding. Moreover, the electrochemical aptasensors with surface-confined aptamer as probe may suffer from the limitation of the complex procedure for sensor regeneration presumably due to the strong binding affinity of aptamers toward analytes. In order to avoid this limitation, a relatively general approach for the electrochemical aptasensor using the aptamer-complementary
n
Corresponding author. Tel.: þ86 021 64321828; fax: þ 86 021 64321828. E-mail address: [email protected] (S. Huang).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.053
DNA (cDNA) oligonucleotides as probe was studied (Lu et al., 2008). The labeled cDNA probe hybridized with aptamer to form a double-stranded oligonucleotide but allowed dehybridization when target bound to aptamer, leaving the single-stranded cDNA oligonucleotide alone on the surface of electrode. In other studies, electron transfer (eT) through DNA has experimentally shown that the existence of conductive path in duplex depends on its conformational state (Giese, 2000; Schuster, 2000). The consensus of opinion is that efficiency of charge transfer is perturbed by duplex containing mismatch (Boon et al., 2000) and bulge (Hall and Barton, 1997). However not all these discontinuous base stacks affect the electron transfer, as it has been observed in helix containing abasic sites (Gasper and Schuster, 1997) and short single-stranded overhangs (Kan and Schuster, 1999). However, DNA structures are not restricted to be double-helices only. Welch and his coworkers studied another DNA structure, three-way helical junction, which is an important element in nucleic acid folding. Such three-way helical junctions in DNA containing two unpaired bases, i.e., bulges, undergo a folding in the presence of metal ions to get a structure in which there is coaxial stacking of two helices, with the third helix at an acute angle (Welch et al., 1995). In other words, it is not possible to model a folded structure that includes coaxial stacking of two helices without significant disruption of base-pairing in the perfect Y-shaped three-way junction (TWJ) DNA with full base-pairings (Duckett and Lilley, 1990). In this paper, we developed a novel TWJ-DNA aptasensor based on the conception of ‘‘conductive path’’ using the
Y. Xia et al. / Biosensors and Bioelectronics 39 (2013) 250–254
aptamer-complementary DNA (cDNA) as probe to form quasidouble-stranded oligonucleotides for simplification of regeneration procedures, and penetratingly tap the potential advantages of the special structure as sensing device. Our strategy for sensor design utilized a trial analyte, lysozyme, which binds weakly to double-stranded DNA; but for 30-mer DNA aptamer sequence, lysozyme shows a high-affinity (Kd ¼31 nM) (Cox and Ellington, 2001; Kirby et al., 2004). Fig. 1 illustrates that binding of aptamer to lysozyme breaks the conductive path in the TWJ-DNA, and does not allow electron transfer between the redox-labeled moiety and the surface of electrode. As a consequence, the disappearance of such conductive path resulting from the target binding-induced dissociation of aptamer is conceived to be utilized for voltammetric sensing.
2. Experimental section 2.1. Materials The synthetic anti-lysozyme aptamer (Kirby et al., 2004), 50 –ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-30 , its partitioned complementary strand containing A2 bulges, 50 -H2N-(CH2)6-CTGC ACTCTTAAACGCTTTAATAGCCCTGAT-(CH2)3-SH-30 (cDNA1 31-mer), and control oligonucleotide, 50 -H2N-(CH2)6-CTGCACTCTTTAGCCCTGAT-(CH2)3-SH-30 (cDNA2 20-mer) were purchased from Sangon Biotech (Shanghai) Co., Ltd., formamide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide sodium (NHSS), 6-mercapto-1-hexanol (MCH), chloroauric acid (HAuCl4), hen egg white lysozyme (Lyso), bovine serum albumin (BSA), immunoglobulin G (IgG), hemoglobin (Hb) and equine heart cytochrome c (cyto c) were purchased from Sigma-Aldrich (Shanghai, China). Milli Q 18.2 MO water was used throughout the experiments. Phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) were used as buffer solution.
2.2. Assemblies of aptasensors. The electrochemical aptasensors were prepared as follows. The glassy carbon electrodes (GCE, 3.0 mm in diameter, CH Instrument.) were polished successively with 0.3 and 0.05 mm alumina powder to produce a smooth, shiny surface. Then it was ultrasonically cleaned in acetone and pure water (each for 3 min). The GCE working electrode was cycled between 0.1 and 0.3 V (vs. SCE) for 300 s
251
in 1 mM HAuCl4 to form gold film, carefully washed with water, and then dried with nitrogen (denoted as Au/GCE). The synthetic cDNA1 (and/or cDNA2) was hybridized with anti-lysozyme aptamer by mixing 12.5 mM aptamers (overdose) and 6.3 mM cDNA into 40 mL of distilled water, followed by addition of 10 mL annealing buffer containing 100 mM Tris-HCl (pH 7.4), 0.5 M NaCl. The resulting mixture was heated to 94 1C for 3 min, and the solution temperature was then gradually lowered from 94 1C to 25 1C to give 5 mM TWJ-DNA solutions. The TWJ-DNA oligonucleotides were immobilized through the formation of Au–S bonds on the surface of the Au/GCE. The electrode was then washed with water several times to remove the unbound DNA oligonucleotides. After TWJ-DNA was immobilized on the electrode, the resulting electrode was denoted as TWJ-DNA/Au/GCE. 2.3. Attachment of redox labels to cDNA oligonucleotides. A method for label of DNA oligonucleotides was reported (Ferapontova et al., 2008). Briefly, NHS-labeled Fc was conjugated to the 50 -end of amino-modified cDNA oligonucleotide through succinimide ester coupling. EDC (0.14 mmol), NHSS (0.13 mmol) and Fc (0.11 mmol) were dissolved in 5 mL of DMF, then stirred, and allowed to react for 2 h at room temperature. After 5 mL of the sample solution mixed with 5 mL of 0.1 M PBS was dropped onto the surface of TWJ-DNA modified electrode, the conjugation was kept for 3 h at room temperature. After that, the open area on the electrode is passivated with inert MCH to reduce nonspecific adsorption and current leakage. 2.4. Electrochemical measurements. The Fc-labeled TWJ-DNA/Au/GCE was incubated with 100 mL sample containing different concentrations of lysozyme in the microcentrifuge tube for 20 min. Following incubation, the electrode was rinsed with water and then immersed in 0.1 M PBS for electrochemical measurements. The electrochemical measurements were conducted in a 10 mL electrochemical cell with a three electrode system consisting of an Fc-labeled TWJ-DNA/Au/GCE working electrode, a saturated calomel reference electrode and a platinum wire counter electrode. Electrochemical impedance spectroscopy (EIS) was performed in 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) within a frequency range from 105 to 0.01 Hz. Square wave voltammetry (SWV) was performed with an initial potential of 0.45 V, amplitude of 0.025 V, and step potential of 0.003 V with a frequency of 20 Hz. 2.5. Regeneration procedures. A 20% formamide aqueous solution as a denaturant was used to clean the electrode incubated. As a crucial step of regeneration, the denatured electrode was immersed into annealing buffer containing 10 mM of the aptamers under 94 1C for 3 min, and the solution temperature was then gradually lowered from 94 to 25 1C at a rate of 1.5 1C/min, in order to rebuild the TWJ-DNA. The regenerated aptasensors were finally rinsed with pure water before reuse.
3. Results and discussion 3.1. Formation of TWJ-DNA structure. Fig. 1. Schematic representation of the aptasensor for detection of lysozyme and regeneration procedure.
To confirm the formation of TWJ structure of synthetic cDNA1 with its complementary aptamer under the present conditions,
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Y. Xia et al. / Biosensors and Bioelectronics 39 (2013) 250–254
1200 1000
Rs
a d
W Ret Zw
800 -Z'' / ohm
C
600
c
b
400 200 0 0
400
800
1200
1600
2000
Z' / ohm Fig. 2. EIS (Nyquist plots) of (a) bare GCE, (b) Au/GCE, (c) TWJ-DNA/Au/GCE and (d) the denatured cDNA1/Au/GCE. The data were obtained in 10 mM [Fe(CN)6]3 /4 and 0.1 M PBS (pH 7.4). The biased potential was 0.19 V. The frequency was from 100 kHz to 0.01 Hz and the amplitude was 5.0 mV. The inset shows the equivalent circuit applied to fit the EIS. Fitted data (solid line).
we compared the EIS of the sensor before and after the denaturation procedure. Fig. 2 shows the Nyquist plots obtained by (a) bare GCE, (b) Au/GCE, (c) TWJ-DNA/Au/GCE and (d) the denatured cDNA1/Au/GCE in 0.1 M PBS (pH 7.4) containing 10 mM [Fe(CN)6]3 /4 . A modified Randle’s equivalent circuit was utilized to model the EIS data for the present system, as shown in the inset of Fig. 2, where Rs represents solution resistance, C is the interface capacitance, Ret is the electronic transfer resistance and W is the Warburg element. Values for the parameters were obtained by fitting the experimental data to the equivalent circuit using ZSimpWin software. The plot for the bare GCE showed small resistance (plot a), with a fitting Ret of 135 O. When the bare GCE was coated with gold film, the Ret decreased to 27 O (plot b). This is attributed to the good conductivity of gold film. When the TWJ-DNA aptasensor was constructed, the Ret increased to 1262 O (plot c). After this sensor was denatured in 20% formamide aqueous solution, the Ret of cDNA1/Au/GCE declined to 970 O (plot d), indicating that the denaturation treatment of the electrode resulted in the decrease in electron transfer resistance. This confirmed the formation of TWJ-DNA structure on the electrode. 3.2. Effects of surface density of cDNA1 on sensing It is possible that different surface densities of DNA on the surface of the electrode may render different kinetics and thermodynamics of binding of the analyte (Herne and Tarlov, 1997; Cheng et al., 2007), probably due to conformational effects. For example, DNA hybridization efficiency on the electrode is significantly influenced by the strand density. Thus, here, we investigated two strategies for the attachment of TWJ-DNA onto a gold surface where different surface densities of cDNA1 oligonucleotides were located. The first is stepwise formation of a TWJ-DNA onto a gold surface: cDNA1 (2 mM and 5 mM were used, respectively) selfassembly, followed by adding 10 mM aptamer to form the folded strands (seen in Fig. 3, Method 1-1 and Method 1-2). The second is direct formation of a TWJ-DNA via adsorption from an annealed solution of cDNA1 oligonucleotides and aptamers (seen in Fig. 3 Method 2). To make sure the effects of cDNA1 surface density on the response of the sensor, chronocoulometry was applied to determine cDNA1 surface density loaded on the electrode. The relationship between the total charge (Q) and the surface density of the Fc-labeled cDNA1 (G) is described as Q¼ nFAG, where n is the number of electrons transferred per Fc moiety (n¼1), A is the electrode surface area of 0.069 cm2, and F is the Faraday constant. Fig. 3 shows the schematic representation of different fabrication
methods and their corresponding SWV performance. When Method 1-1 was applied, cDNA1 surface density was determined to be (1.06 70.15) 1012 molecules/cm2. In this case, the SWV signal of sensors in the ‘‘eT on’’ and ‘‘eT off’’ state shows no difference (Fig. 3A), which means a low cDNA1 surface density loaded, mainly due to both conformations of oligonucleotides facilitating electron transfer. When Method 1-2 was used, cDNA1 density was estimated to be (2.1270.23) 1012 molecules/cm2. SWV signal of sensor exhibits a distinguished redox peak in the ‘‘eT off’’ state, but a relatively diminished one in the ‘‘eT on’’ state (Fig. 3B), which runs counter to what we desired. It is probably because intermolecular mishybridization of oligonucleotides occurs in a space of high strand density. Moreover, the formation of this structure where there was no conductive path gave rise to increasing negative charge on the surface, and thus the charge current, as well as inclination of baseline, raised accordingly. Compared with the stepwise method mentioned above, the aptasensor fabricated via the single-step method (Method 2) displays the largest voltammetric attenuation (Fig. 3C) with a cDNA1 surface density of (1.42 70.20) 1012 molecules/cm2. From the cDNA1 surface density determined, density of (1.4270.20) 1012 molecules/cm2 is in the middle among the three data. We hold the view that sensor fabricated via the singlestep method (Method 2) could provide a better voltammetric response for sensing. 3.3. Characteristic of TWJ-DNA aptasensor. The strategy described here (Fig. 1) is essentially based on the target binding-induced conformational change of cDNA oligonucleotide, i.e., oligonucleotide underwent an unfolding from a double-strand to a single-strand. For this reason, we employed a 20-mer cDNA2 to assemble a standard double-stranded DNA (dsDNA) aptasensor in order to compare it with TWJ-DNA one. In both aptasensors, same number of complementary base pairs exists in the conductive path. To demonstrate the characteristic of these two in electrochemical response, here a signal attenuation factor, the ratio of the Ioff to Ion has been defined and investigated, where Ioff represents the peak current in the ‘‘eT off’’ state, Ion is the SWV peak current in the ‘‘eT on’’ state. Fig. 4 shows the signal attenuation factor of the dsDNA and TWJ-DNA aptasensors. From Fig. 4, the dsDNA aptasensors exhibited a ratio about 42%, and the TWJ-DNA ones indicated a lower ratio about 34%, which reveals the fact that TWJ-DNA aptasensors have a significant signal attenuation factor. In the inset of Fig. 4, SWV curve of the two types of aptasensors is shown. When the cDNA1 and 2 surface density is approximate the same, there is no big difference between Ion on the TWJ-DNA aptasensor and on the dsDNA one, however, the Ioff on the former one is smaller than that on the latter one, obviously. This is attributed to the existence of the third-way double-helical stem and bulge abutted on the conductive path, which provides enough space for cDNA1 itself to form a stable third-way helical structure and keeps Fc moiety a longer distance (11-mer difference between 20-mer cDNA2 and 31-mer cDNA1) away from the electrode in the ‘‘eT off’’ state. In comparison with the dsDNA aptasensor described here, our TWJ-DNA aptasensor with a significant signal attenuation factor should equip a higher sensitivity of current per unit concentration. 3.4. Aptasensor responses to lysozyme. Fig. 5 depicts square wave voltammograms (SWVs) for increasing concentrations of lysozyme from 0 to 100 nM at the Fc-labeled TWJ-DNA/Au/GCE. The electrode initially exhibits high voltammetric response in PBS, and shows an obvious redox peak at the apparent potential of approximate 0.13 V, corresponding to
Y. Xia et al. / Biosensors and Bioelectronics 39 (2013) 250–254
253
1 µA
Current
1 µA
1 µA
0.4 0.3 0.2 0.1 0.0 -0.1 Potential / V Fig. 3. Schematic representation of different fabrication methods and their corresponding SWV performance: (A) Method 1-1, (B) Method 1-2 and (C) Method 2. Blue curve represents SWV signal in the ‘‘eT on’’ state, and red curve represents that in the ‘‘eT off’’ state. SWV was performed with an initial potential of 0.45 V, an amplitude of 0.025 V, a step potential of 0.003 V and a frequency of 20 Hz in 0.1 M PBS (pH 7.4). (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
50
Current / µA
Attenuation factor, ratio/%
4 45
40
3 2 1 0
35
0.3 0.2 0.1 0.0 Potential / V 30 dsDNA TWJ-DNA
Fig. 4. Bar chart of the signal attenuation factor of dsDNA and TWJ-DNA aptasensor. Inset shows the SWV curve of the two types of aptasensor in the ‘‘eT on’’/‘‘eT off’’ state. Blue curve represents dsDNA, and red curve represents TWJ-DNA. Other experimental conditions are the same as those in Fig. 3. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
S¼
4.0 Normalized decrease
3.5 3.0 Current / µA
the redox process of the labeled Fc on a quasi-reversible (or irreversible) electrode (Hosseini et al., 2010; Long et al., 2003). After being incubated in the 0.5 nM lysozyme for 20 min, the voltammetric signal of the electrode decreases. The change in voltammetric response of electrode with the exposure of lysozyme could be understood in terms of the conformational change of cDNA described above and shown in Fig. 1: the stronger binding affinity of the aptamers with their targets than that with cDNA essentially destroyed the conductive path of TWJ-DNA assembled on the electrode, resulting in the dissociation of the aptamers from their cDNA into solution. Inset of Fig. 5 displays calibration plots of normalized signal decrease vs. lysozyme concentration for aptasensor. The effective dissociation constants (Kd,eff) were determined using nonlinear least-squares fitting to the Hill equation (Eq. (1)), with the Hill coefficients (n) set as variable.
2.5 2.0 1.5
ð1Þ
1.0 0.8
a
0.6 0.4 0.2 0.0 100p 1n
1.0
10n 100n 1µ
g
[Lysozyme] / M
0.5 0.0 0.6
½Ln ðK d,ef f Þn þ ½Ln
0.5
0.4
0.3 0.2 Potential / V
0.1
0.0
Fig. 5. The relationship between SWV current and concentration of lysozyme. The concentrations of lysozyme are: (a) 0.0 nM, (b) 0.5 nM, (c) 1.0 nM, (d) 5.0 nM, (e) 10 nM, (f) 50 nM and (g) 100 nM. The red curve was indicated after denaturation. Inset shows the plot of the normalized decrease of peak current as a function of the lysozyme concentration. Other experimental conditions are the same as those in Fig. 3. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
where S is the normalized SWVs signal decrease measured as a function of the concentration of lysozyme, [L]; Kd,eff represents the effective dissociation constant of the complex of interest (Hu and Easley, 2011). This simple model, extensively used in biochemistry, physiology, and pharmacology to analyze binding equilibria, provides flexibility in measuring systems of unknown stoichiometry (Weiss, 1997; Kim et al., 2010). Nonlinear least squares analyses revealed Kd,eff value of 5.770.2 nM and n value of 1.370.1. The limit of detection is 0.2 nM (S/B ¼3) corresponding to 20 fmol lysozyme in the 100 mL sample. 3.5. Specificity of aptasensor To demonstrate specificity, the aptasensor was challenged with nonspecific proteins such as immunoglobulin G (IgG), bovine serum albumin (BSA), hemoglobin (Hb), and cytochrome c (cyto c) as well as with the analyte of interest, lysozyme (Lyso). Fig. 6 compares relative signal decrease of SWV responses of our biosensor
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Y. Xia et al. / Biosensors and Bioelectronics 39 (2013) 250–254
4. Conclusions
Relative decrease
1.0 0.8 0.6 0.4 0.2 0.0 A
B
C
E
D
Fig. 6. Relative decrease of peak current on TWJ-DNA aptasensor after it reacted with lysozyme and other four control proteins. Column A is 10 nM lysozyme, column B is 100 nM BSA, column C is 100 nM Hb, column D is 100 nM IgG, and column E is 100 nM cyto c. Other experimental conditions are the same as those in Fig. 3.
Acknowledgments
3.5 3.0
This work was supported by the Project of the Foundation of Shanghai Municipal Government (08520510400), Shanghai Leading Academic Discipline Project (S30406) and Key Laboratory of Resource Chemistry of Ministry of Education.
"eT on" 2.5 Ion , Ioff / µA
We developed an electrochemical three-way junction aptasensor with a significant signal attenuation factor for detection of lysozyme. The as-prepared aptasensor was specific to lysozyme and showed a detection limit of 0.2 nM with a dynamic range extending to 100 nM. Compared with other lysozyme EIS-based aptasensors, our TWJ-DNA aptasensor has better stability and reproducibility though the sensitivity is similar, because EIS measurements suffer from many factors contributing to resistance, particularly, when the nonspecific targets foul the surface of electrode. On the contrary, Fc-labeled TWJ-DNA aptasensor relies on measurement of changes in Faradaic current that occur upon target binding. This novel method opens new promising routes for future applications in proteomics and diagnostics.
2.0 1.5
References
"eT off "
1.0 0.5 0.0 2
4
6 8 Time / days
10
12
Fig. 7. The time dependence of the Ion and Ioff. Error bar was obtained from three independent experiments on the same sensor.
to 10 nM lysozyme (A), 100 nM BSA (B), 100 nM Hb (C), 100 nM IgG (D) and 100 nM cyto c (E), respectively. The results showed that aptasensor did not respond to a high concentration of nonspecific proteins but did respond to a low concentration of lysozyme, indicating a good specificity of the TWJ-DNA aptasensor.
3.6. Stability and reproducibility of aptasensor The long term stability of the system should be investigated because the self-assembly monolayers are not always ideal. A leaching study has been carried out by our group (Fig. 7). The results indicate that, during 2 weeks re-usage, the Ion and Ioff declined by 7.9% and 18.5%, respectively, and the ratio approximately increased from 35% to 40%. It is probably because DNA and/or ferrocene were stripped from the electrode in the process of frequent regeneration.
Baker, B.R., Lai, R.Y., Wood, M.S., Doctor, E.H., Heeger, A.J., Plaxco, K.W., 2006. Journal of the American Chemical Society 128, 3138. Boon, E.M., Ceres, D.M., Drummond, T.G., Hill, M.G., Barton, J.K., 2000. Nature Biotechnology 18, 1096. Cheng, A., Ge, B., Yu, H., 2007. Analytical Chemistry 79, 5158. Cox, J.C., Ellington, A.D., 2001. Bioorganic and Medicinal Chemistry 9, 2525. Duckett, D.R., Lilley, D.M.J., 1990. EMBO Journal 9, 1659–1664. Ferapontova, E.E., Olsen, E.M., Gothelf, K.V., 2008. Journal of the American Chemical Society 130, 4256. Gasper, S.M., Schuster, G.B., 1997. Journal of the American Chemical Society 119, 12762. Giese, B., 2000. Accounts of Chemical Research 33, 631. Hall, D.B., Barton, J.K., 1997. Journal of the American Chemical Society 119, 5045. Herne, T.M., Tarlov, M.J., 1997. Journal of the American Chemical Society 119, 8916. Hosseini, A., Collamn, J.P., Devadoss, A., 2010. Langmuir 26, 17674–17678. Hu, J., Easley, C.J., 2011. Analyst 136, 3461. Kan, Y., Schuster, G.B., 1999. Journal of the American Chemical Society 121, 10857. Kim, J., Hu, J., Sollie, R.S., Easley, C.J., 2010. Analytical Chemistry 82, 6976. Kirby, R., Cho, E.J., Gehrke, B., Bayer, T., Park, Y.S., Neikirk, D.P., McDevitt, J.T., Ellington, A.D., 2004. Analytical Chemistry 76, 4066. Long, Y., Li, C., Sutherland, T.C., Chahma, M., Lee, J.S., Kraatz, H., 2003. Journal of the American Chemical Society 125, 8724–8725. Lu, Y., Li, X., Zhang, L., Yu, P., Su, L., Mao, L., 2008. Analytical Chemistry 80, 1883. Radi, A.E., Sanchez, J.L.A., Baldrich, E., O0 Sullivan, C.K., 2006. Journal of the American Chemical Society 128, 117. Schuster, G.B., 2000. Accounts of Chemical Research 33, 253. Shen, L., Chen, Z., Li, Y., Jing, P., Xie, S., He, S., He, P., Shao, Y., 2007. Chemical Communications 21, 2169. Weiss, J.N., 1997. FASEB Journal 11, 835. Welch, J.B., Walter, F., Lilley, D.M.J., 1995. Journal of Molecular Biology 251, 507–519. Xiao, Y., Lubin, A.A., Heeger, A.J., Plaxco, K.W., 2005. Angewandte Chemie International Edition 44, 5456. Zuo, X., Song, S., Zhang, J., Pan, D., Wang, L., Fan, C., 2007. Journal of the American Chemical Society 129, 1042.
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A three-way junction aptasensor for lysozyme detection Yunfeng Xia, Siwen Gan, Qinghao Xu, Xiaowen Qiu, Peiyi Gao, Shasheng Huang n Life and Environmental Science College, Shanghai Normal University, Guilin Road 100, Shanghai 200234, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 May 2012 Received in revised form 23 July 2012 Accepted 25 July 2012 Available online 11 August 2012
A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called ‘‘conductive path’’, is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2 nM with a dynamic range extending to 100 nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure. & 2012 Elsevier B.V. All rights reserved.
Keywords: Three-way junction Aptasensor Lysozyme Conductive path
1. Introduction At present, most electrochemical aptasensors are formed mainly based on the conformational change of the aptamers induced by binding the specific target to the probes (Xiao et al., 2005; Baker et al., 2006; Shen et al., 2007). The specific binding of target to the surface-confined and redox-labeled aptamer essentially induces the conformational change of the aptamer probe, and leads to the change of distance between the labeled redox moiety and the surface of electrode (Radi et al., 2006), eventually resulting in the large variation of voltammetric signal used for electrochemical sensing of analytes. Although this kind of electrochemical aptasensors with aptamer as probe has so far been employed for selective and sensitive electrochemical sensing of targets such as thrombin (Xiao et al., 2005), cocaine (Baker et al., 2006), ATP (Zuo et al., 2007), and theophyline (Ferapontova et al., 2008), those aptasensors generally required a large-scaled conformational change of the aptamer probe induced by the specific target binding. Moreover, the electrochemical aptasensors with surface-confined aptamer as probe may suffer from the limitation of the complex procedure for sensor regeneration presumably due to the strong binding affinity of aptamers toward analytes. In order to avoid this limitation, a relatively general approach for the electrochemical aptasensor using the aptamer-complementary
n
Corresponding author. Tel.: þ86 021 64321828; fax: þ 86 021 64321828. E-mail address: [email protected] (S. Huang).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.053
DNA (cDNA) oligonucleotides as probe was studied (Lu et al., 2008). The labeled cDNA probe hybridized with aptamer to form a double-stranded oligonucleotide but allowed dehybridization when target bound to aptamer, leaving the single-stranded cDNA oligonucleotide alone on the surface of electrode. In other studies, electron transfer (eT) through DNA has experimentally shown that the existence of conductive path in duplex depends on its conformational state (Giese, 2000; Schuster, 2000). The consensus of opinion is that efficiency of charge transfer is perturbed by duplex containing mismatch (Boon et al., 2000) and bulge (Hall and Barton, 1997). However not all these discontinuous base stacks affect the electron transfer, as it has been observed in helix containing abasic sites (Gasper and Schuster, 1997) and short single-stranded overhangs (Kan and Schuster, 1999). However, DNA structures are not restricted to be double-helices only. Welch and his coworkers studied another DNA structure, three-way helical junction, which is an important element in nucleic acid folding. Such three-way helical junctions in DNA containing two unpaired bases, i.e., bulges, undergo a folding in the presence of metal ions to get a structure in which there is coaxial stacking of two helices, with the third helix at an acute angle (Welch et al., 1995). In other words, it is not possible to model a folded structure that includes coaxial stacking of two helices without significant disruption of base-pairing in the perfect Y-shaped three-way junction (TWJ) DNA with full base-pairings (Duckett and Lilley, 1990). In this paper, we developed a novel TWJ-DNA aptasensor based on the conception of ‘‘conductive path’’ using the
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aptamer-complementary DNA (cDNA) as probe to form quasidouble-stranded oligonucleotides for simplification of regeneration procedures, and penetratingly tap the potential advantages of the special structure as sensing device. Our strategy for sensor design utilized a trial analyte, lysozyme, which binds weakly to double-stranded DNA; but for 30-mer DNA aptamer sequence, lysozyme shows a high-affinity (Kd ¼31 nM) (Cox and Ellington, 2001; Kirby et al., 2004). Fig. 1 illustrates that binding of aptamer to lysozyme breaks the conductive path in the TWJ-DNA, and does not allow electron transfer between the redox-labeled moiety and the surface of electrode. As a consequence, the disappearance of such conductive path resulting from the target binding-induced dissociation of aptamer is conceived to be utilized for voltammetric sensing.
2. Experimental section 2.1. Materials The synthetic anti-lysozyme aptamer (Kirby et al., 2004), 50 –ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-30 , its partitioned complementary strand containing A2 bulges, 50 -H2N-(CH2)6-CTGC ACTCTTAAACGCTTTAATAGCCCTGAT-(CH2)3-SH-30 (cDNA1 31-mer), and control oligonucleotide, 50 -H2N-(CH2)6-CTGCACTCTTTAGCCCTGAT-(CH2)3-SH-30 (cDNA2 20-mer) were purchased from Sangon Biotech (Shanghai) Co., Ltd., formamide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide sodium (NHSS), 6-mercapto-1-hexanol (MCH), chloroauric acid (HAuCl4), hen egg white lysozyme (Lyso), bovine serum albumin (BSA), immunoglobulin G (IgG), hemoglobin (Hb) and equine heart cytochrome c (cyto c) were purchased from Sigma-Aldrich (Shanghai, China). Milli Q 18.2 MO water was used throughout the experiments. Phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) were used as buffer solution.
2.2. Assemblies of aptasensors. The electrochemical aptasensors were prepared as follows. The glassy carbon electrodes (GCE, 3.0 mm in diameter, CH Instrument.) were polished successively with 0.3 and 0.05 mm alumina powder to produce a smooth, shiny surface. Then it was ultrasonically cleaned in acetone and pure water (each for 3 min). The GCE working electrode was cycled between 0.1 and 0.3 V (vs. SCE) for 300 s
251
in 1 mM HAuCl4 to form gold film, carefully washed with water, and then dried with nitrogen (denoted as Au/GCE). The synthetic cDNA1 (and/or cDNA2) was hybridized with anti-lysozyme aptamer by mixing 12.5 mM aptamers (overdose) and 6.3 mM cDNA into 40 mL of distilled water, followed by addition of 10 mL annealing buffer containing 100 mM Tris-HCl (pH 7.4), 0.5 M NaCl. The resulting mixture was heated to 94 1C for 3 min, and the solution temperature was then gradually lowered from 94 1C to 25 1C to give 5 mM TWJ-DNA solutions. The TWJ-DNA oligonucleotides were immobilized through the formation of Au–S bonds on the surface of the Au/GCE. The electrode was then washed with water several times to remove the unbound DNA oligonucleotides. After TWJ-DNA was immobilized on the electrode, the resulting electrode was denoted as TWJ-DNA/Au/GCE. 2.3. Attachment of redox labels to cDNA oligonucleotides. A method for label of DNA oligonucleotides was reported (Ferapontova et al., 2008). Briefly, NHS-labeled Fc was conjugated to the 50 -end of amino-modified cDNA oligonucleotide through succinimide ester coupling. EDC (0.14 mmol), NHSS (0.13 mmol) and Fc (0.11 mmol) were dissolved in 5 mL of DMF, then stirred, and allowed to react for 2 h at room temperature. After 5 mL of the sample solution mixed with 5 mL of 0.1 M PBS was dropped onto the surface of TWJ-DNA modified electrode, the conjugation was kept for 3 h at room temperature. After that, the open area on the electrode is passivated with inert MCH to reduce nonspecific adsorption and current leakage. 2.4. Electrochemical measurements. The Fc-labeled TWJ-DNA/Au/GCE was incubated with 100 mL sample containing different concentrations of lysozyme in the microcentrifuge tube for 20 min. Following incubation, the electrode was rinsed with water and then immersed in 0.1 M PBS for electrochemical measurements. The electrochemical measurements were conducted in a 10 mL electrochemical cell with a three electrode system consisting of an Fc-labeled TWJ-DNA/Au/GCE working electrode, a saturated calomel reference electrode and a platinum wire counter electrode. Electrochemical impedance spectroscopy (EIS) was performed in 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) within a frequency range from 105 to 0.01 Hz. Square wave voltammetry (SWV) was performed with an initial potential of 0.45 V, amplitude of 0.025 V, and step potential of 0.003 V with a frequency of 20 Hz. 2.5. Regeneration procedures. A 20% formamide aqueous solution as a denaturant was used to clean the electrode incubated. As a crucial step of regeneration, the denatured electrode was immersed into annealing buffer containing 10 mM of the aptamers under 94 1C for 3 min, and the solution temperature was then gradually lowered from 94 to 25 1C at a rate of 1.5 1C/min, in order to rebuild the TWJ-DNA. The regenerated aptasensors were finally rinsed with pure water before reuse.
3. Results and discussion 3.1. Formation of TWJ-DNA structure. Fig. 1. Schematic representation of the aptasensor for detection of lysozyme and regeneration procedure.
To confirm the formation of TWJ structure of synthetic cDNA1 with its complementary aptamer under the present conditions,
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1200 1000
Rs
a d
W Ret Zw
800 -Z'' / ohm
C
600
c
b
400 200 0 0
400
800
1200
1600
2000
Z' / ohm Fig. 2. EIS (Nyquist plots) of (a) bare GCE, (b) Au/GCE, (c) TWJ-DNA/Au/GCE and (d) the denatured cDNA1/Au/GCE. The data were obtained in 10 mM [Fe(CN)6]3 /4 and 0.1 M PBS (pH 7.4). The biased potential was 0.19 V. The frequency was from 100 kHz to 0.01 Hz and the amplitude was 5.0 mV. The inset shows the equivalent circuit applied to fit the EIS. Fitted data (solid line).
we compared the EIS of the sensor before and after the denaturation procedure. Fig. 2 shows the Nyquist plots obtained by (a) bare GCE, (b) Au/GCE, (c) TWJ-DNA/Au/GCE and (d) the denatured cDNA1/Au/GCE in 0.1 M PBS (pH 7.4) containing 10 mM [Fe(CN)6]3 /4 . A modified Randle’s equivalent circuit was utilized to model the EIS data for the present system, as shown in the inset of Fig. 2, where Rs represents solution resistance, C is the interface capacitance, Ret is the electronic transfer resistance and W is the Warburg element. Values for the parameters were obtained by fitting the experimental data to the equivalent circuit using ZSimpWin software. The plot for the bare GCE showed small resistance (plot a), with a fitting Ret of 135 O. When the bare GCE was coated with gold film, the Ret decreased to 27 O (plot b). This is attributed to the good conductivity of gold film. When the TWJ-DNA aptasensor was constructed, the Ret increased to 1262 O (plot c). After this sensor was denatured in 20% formamide aqueous solution, the Ret of cDNA1/Au/GCE declined to 970 O (plot d), indicating that the denaturation treatment of the electrode resulted in the decrease in electron transfer resistance. This confirmed the formation of TWJ-DNA structure on the electrode. 3.2. Effects of surface density of cDNA1 on sensing It is possible that different surface densities of DNA on the surface of the electrode may render different kinetics and thermodynamics of binding of the analyte (Herne and Tarlov, 1997; Cheng et al., 2007), probably due to conformational effects. For example, DNA hybridization efficiency on the electrode is significantly influenced by the strand density. Thus, here, we investigated two strategies for the attachment of TWJ-DNA onto a gold surface where different surface densities of cDNA1 oligonucleotides were located. The first is stepwise formation of a TWJ-DNA onto a gold surface: cDNA1 (2 mM and 5 mM were used, respectively) selfassembly, followed by adding 10 mM aptamer to form the folded strands (seen in Fig. 3, Method 1-1 and Method 1-2). The second is direct formation of a TWJ-DNA via adsorption from an annealed solution of cDNA1 oligonucleotides and aptamers (seen in Fig. 3 Method 2). To make sure the effects of cDNA1 surface density on the response of the sensor, chronocoulometry was applied to determine cDNA1 surface density loaded on the electrode. The relationship between the total charge (Q) and the surface density of the Fc-labeled cDNA1 (G) is described as Q¼ nFAG, where n is the number of electrons transferred per Fc moiety (n¼1), A is the electrode surface area of 0.069 cm2, and F is the Faraday constant. Fig. 3 shows the schematic representation of different fabrication
methods and their corresponding SWV performance. When Method 1-1 was applied, cDNA1 surface density was determined to be (1.06 70.15) 1012 molecules/cm2. In this case, the SWV signal of sensors in the ‘‘eT on’’ and ‘‘eT off’’ state shows no difference (Fig. 3A), which means a low cDNA1 surface density loaded, mainly due to both conformations of oligonucleotides facilitating electron transfer. When Method 1-2 was used, cDNA1 density was estimated to be (2.1270.23) 1012 molecules/cm2. SWV signal of sensor exhibits a distinguished redox peak in the ‘‘eT off’’ state, but a relatively diminished one in the ‘‘eT on’’ state (Fig. 3B), which runs counter to what we desired. It is probably because intermolecular mishybridization of oligonucleotides occurs in a space of high strand density. Moreover, the formation of this structure where there was no conductive path gave rise to increasing negative charge on the surface, and thus the charge current, as well as inclination of baseline, raised accordingly. Compared with the stepwise method mentioned above, the aptasensor fabricated via the single-step method (Method 2) displays the largest voltammetric attenuation (Fig. 3C) with a cDNA1 surface density of (1.42 70.20) 1012 molecules/cm2. From the cDNA1 surface density determined, density of (1.4270.20) 1012 molecules/cm2 is in the middle among the three data. We hold the view that sensor fabricated via the singlestep method (Method 2) could provide a better voltammetric response for sensing. 3.3. Characteristic of TWJ-DNA aptasensor. The strategy described here (Fig. 1) is essentially based on the target binding-induced conformational change of cDNA oligonucleotide, i.e., oligonucleotide underwent an unfolding from a double-strand to a single-strand. For this reason, we employed a 20-mer cDNA2 to assemble a standard double-stranded DNA (dsDNA) aptasensor in order to compare it with TWJ-DNA one. In both aptasensors, same number of complementary base pairs exists in the conductive path. To demonstrate the characteristic of these two in electrochemical response, here a signal attenuation factor, the ratio of the Ioff to Ion has been defined and investigated, where Ioff represents the peak current in the ‘‘eT off’’ state, Ion is the SWV peak current in the ‘‘eT on’’ state. Fig. 4 shows the signal attenuation factor of the dsDNA and TWJ-DNA aptasensors. From Fig. 4, the dsDNA aptasensors exhibited a ratio about 42%, and the TWJ-DNA ones indicated a lower ratio about 34%, which reveals the fact that TWJ-DNA aptasensors have a significant signal attenuation factor. In the inset of Fig. 4, SWV curve of the two types of aptasensors is shown. When the cDNA1 and 2 surface density is approximate the same, there is no big difference between Ion on the TWJ-DNA aptasensor and on the dsDNA one, however, the Ioff on the former one is smaller than that on the latter one, obviously. This is attributed to the existence of the third-way double-helical stem and bulge abutted on the conductive path, which provides enough space for cDNA1 itself to form a stable third-way helical structure and keeps Fc moiety a longer distance (11-mer difference between 20-mer cDNA2 and 31-mer cDNA1) away from the electrode in the ‘‘eT off’’ state. In comparison with the dsDNA aptasensor described here, our TWJ-DNA aptasensor with a significant signal attenuation factor should equip a higher sensitivity of current per unit concentration. 3.4. Aptasensor responses to lysozyme. Fig. 5 depicts square wave voltammograms (SWVs) for increasing concentrations of lysozyme from 0 to 100 nM at the Fc-labeled TWJ-DNA/Au/GCE. The electrode initially exhibits high voltammetric response in PBS, and shows an obvious redox peak at the apparent potential of approximate 0.13 V, corresponding to
Y. Xia et al. / Biosensors and Bioelectronics 39 (2013) 250–254
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1 µA
Current
1 µA
1 µA
0.4 0.3 0.2 0.1 0.0 -0.1 Potential / V Fig. 3. Schematic representation of different fabrication methods and their corresponding SWV performance: (A) Method 1-1, (B) Method 1-2 and (C) Method 2. Blue curve represents SWV signal in the ‘‘eT on’’ state, and red curve represents that in the ‘‘eT off’’ state. SWV was performed with an initial potential of 0.45 V, an amplitude of 0.025 V, a step potential of 0.003 V and a frequency of 20 Hz in 0.1 M PBS (pH 7.4). (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
50
Current / µA
Attenuation factor, ratio/%
4 45
40
3 2 1 0
35
0.3 0.2 0.1 0.0 Potential / V 30 dsDNA TWJ-DNA
Fig. 4. Bar chart of the signal attenuation factor of dsDNA and TWJ-DNA aptasensor. Inset shows the SWV curve of the two types of aptasensor in the ‘‘eT on’’/‘‘eT off’’ state. Blue curve represents dsDNA, and red curve represents TWJ-DNA. Other experimental conditions are the same as those in Fig. 3. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
S¼
4.0 Normalized decrease
3.5 3.0 Current / µA
the redox process of the labeled Fc on a quasi-reversible (or irreversible) electrode (Hosseini et al., 2010; Long et al., 2003). After being incubated in the 0.5 nM lysozyme for 20 min, the voltammetric signal of the electrode decreases. The change in voltammetric response of electrode with the exposure of lysozyme could be understood in terms of the conformational change of cDNA described above and shown in Fig. 1: the stronger binding affinity of the aptamers with their targets than that with cDNA essentially destroyed the conductive path of TWJ-DNA assembled on the electrode, resulting in the dissociation of the aptamers from their cDNA into solution. Inset of Fig. 5 displays calibration plots of normalized signal decrease vs. lysozyme concentration for aptasensor. The effective dissociation constants (Kd,eff) were determined using nonlinear least-squares fitting to the Hill equation (Eq. (1)), with the Hill coefficients (n) set as variable.
2.5 2.0 1.5
ð1Þ
1.0 0.8
a
0.6 0.4 0.2 0.0 100p 1n
1.0
10n 100n 1µ
g
[Lysozyme] / M
0.5 0.0 0.6
½Ln ðK d,ef f Þn þ ½Ln
0.5
0.4
0.3 0.2 Potential / V
0.1
0.0
Fig. 5. The relationship between SWV current and concentration of lysozyme. The concentrations of lysozyme are: (a) 0.0 nM, (b) 0.5 nM, (c) 1.0 nM, (d) 5.0 nM, (e) 10 nM, (f) 50 nM and (g) 100 nM. The red curve was indicated after denaturation. Inset shows the plot of the normalized decrease of peak current as a function of the lysozyme concentration. Other experimental conditions are the same as those in Fig. 3. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)
where S is the normalized SWVs signal decrease measured as a function of the concentration of lysozyme, [L]; Kd,eff represents the effective dissociation constant of the complex of interest (Hu and Easley, 2011). This simple model, extensively used in biochemistry, physiology, and pharmacology to analyze binding equilibria, provides flexibility in measuring systems of unknown stoichiometry (Weiss, 1997; Kim et al., 2010). Nonlinear least squares analyses revealed Kd,eff value of 5.770.2 nM and n value of 1.370.1. The limit of detection is 0.2 nM (S/B ¼3) corresponding to 20 fmol lysozyme in the 100 mL sample. 3.5. Specificity of aptasensor To demonstrate specificity, the aptasensor was challenged with nonspecific proteins such as immunoglobulin G (IgG), bovine serum albumin (BSA), hemoglobin (Hb), and cytochrome c (cyto c) as well as with the analyte of interest, lysozyme (Lyso). Fig. 6 compares relative signal decrease of SWV responses of our biosensor
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4. Conclusions
Relative decrease
1.0 0.8 0.6 0.4 0.2 0.0 A
B
C
E
D
Fig. 6. Relative decrease of peak current on TWJ-DNA aptasensor after it reacted with lysozyme and other four control proteins. Column A is 10 nM lysozyme, column B is 100 nM BSA, column C is 100 nM Hb, column D is 100 nM IgG, and column E is 100 nM cyto c. Other experimental conditions are the same as those in Fig. 3.
Acknowledgments
3.5 3.0
This work was supported by the Project of the Foundation of Shanghai Municipal Government (08520510400), Shanghai Leading Academic Discipline Project (S30406) and Key Laboratory of Resource Chemistry of Ministry of Education.
"eT on" 2.5 Ion , Ioff / µA
We developed an electrochemical three-way junction aptasensor with a significant signal attenuation factor for detection of lysozyme. The as-prepared aptasensor was specific to lysozyme and showed a detection limit of 0.2 nM with a dynamic range extending to 100 nM. Compared with other lysozyme EIS-based aptasensors, our TWJ-DNA aptasensor has better stability and reproducibility though the sensitivity is similar, because EIS measurements suffer from many factors contributing to resistance, particularly, when the nonspecific targets foul the surface of electrode. On the contrary, Fc-labeled TWJ-DNA aptasensor relies on measurement of changes in Faradaic current that occur upon target binding. This novel method opens new promising routes for future applications in proteomics and diagnostics.
2.0 1.5
References
"eT off "
1.0 0.5 0.0 2
4
6 8 Time / days
10
12
Fig. 7. The time dependence of the Ion and Ioff. Error bar was obtained from three independent experiments on the same sensor.
to 10 nM lysozyme (A), 100 nM BSA (B), 100 nM Hb (C), 100 nM IgG (D) and 100 nM cyto c (E), respectively. The results showed that aptasensor did not respond to a high concentration of nonspecific proteins but did respond to a low concentration of lysozyme, indicating a good specificity of the TWJ-DNA aptasensor.
3.6. Stability and reproducibility of aptasensor The long term stability of the system should be investigated because the self-assembly monolayers are not always ideal. A leaching study has been carried out by our group (Fig. 7). The results indicate that, during 2 weeks re-usage, the Ion and Ioff declined by 7.9% and 18.5%, respectively, and the ratio approximately increased from 35% to 40%. It is probably because DNA and/or ferrocene were stripped from the electrode in the process of frequent regeneration.
Baker, B.R., Lai, R.Y., Wood, M.S., Doctor, E.H., Heeger, A.J., Plaxco, K.W., 2006. Journal of the American Chemical Society 128, 3138. Boon, E.M., Ceres, D.M., Drummond, T.G., Hill, M.G., Barton, J.K., 2000. Nature Biotechnology 18, 1096. Cheng, A., Ge, B., Yu, H., 2007. Analytical Chemistry 79, 5158. Cox, J.C., Ellington, A.D., 2001. Bioorganic and Medicinal Chemistry 9, 2525. Duckett, D.R., Lilley, D.M.J., 1990. EMBO Journal 9, 1659–1664. Ferapontova, E.E., Olsen, E.M., Gothelf, K.V., 2008. Journal of the American Chemical Society 130, 4256. Gasper, S.M., Schuster, G.B., 1997. Journal of the American Chemical Society 119, 12762. Giese, B., 2000. Accounts of Chemical Research 33, 631. Hall, D.B., Barton, J.K., 1997. Journal of the American Chemical Society 119, 5045. Herne, T.M., Tarlov, M.J., 1997. Journal of the American Chemical Society 119, 8916. Hosseini, A., Collamn, J.P., Devadoss, A., 2010. Langmuir 26, 17674–17678. Hu, J., Easley, C.J., 2011. Analyst 136, 3461. Kan, Y., Schuster, G.B., 1999. Journal of the American Chemical Society 121, 10857. Kim, J., Hu, J., Sollie, R.S., Easley, C.J., 2010. Analytical Chemistry 82, 6976. Kirby, R., Cho, E.J., Gehrke, B., Bayer, T., Park, Y.S., Neikirk, D.P., McDevitt, J.T., Ellington, A.D., 2004. Analytical Chemistry 76, 4066. Long, Y., Li, C., Sutherland, T.C., Chahma, M., Lee, J.S., Kraatz, H., 2003. Journal of the American Chemical Society 125, 8724–8725. Lu, Y., Li, X., Zhang, L., Yu, P., Su, L., Mao, L., 2008. Analytical Chemistry 80, 1883. Radi, A.E., Sanchez, J.L.A., Baldrich, E., O0 Sullivan, C.K., 2006. Journal of the American Chemical Society 128, 117. Schuster, G.B., 2000. Accounts of Chemical Research 33, 253. Shen, L., Chen, Z., Li, Y., Jing, P., Xie, S., He, S., He, P., Shao, Y., 2007. Chemical Communications 21, 2169. Weiss, J.N., 1997. FASEB Journal 11, 835. Welch, J.B., Walter, F., Lilley, D.M.J., 1995. Journal of Molecular Biology 251, 507–519. Xiao, Y., Lubin, A.A., Heeger, A.J., Plaxco, K.W., 2005. Angewandte Chemie International Edition 44, 5456. Zuo, X., Song, S., Zhang, J., Pan, D., Wang, L., Fan, C., 2007. Journal of the American Chemical Society 129, 1042.