High-Performance Ratiometric Electrochemical Method Based on

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A high-performance ratiometric electrochemical method based on the combination of signal probe and inner reference probe in one hairpin-structured DNA Chunyan Deng, Xiaomei Pi, Pin Qian, Xiao-Qing Chen, Wuming Wu, and Juan Xiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04209 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Analytical Chemistry

A high-performance ratiometric electrochemical method based on the combination of signal probe and inner reference probe in one hairpin-structured DNA Chunyan Deng1, Xiaomei Pi1, Pin Qian1, Xiaoqing Chen1, Wuming Wu2, Juan Xiang*1 1

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, P. R. China

2

College of Optoelectriconic Science and Engineering, National University of Defense Technology, Changsha 410073, China

Corresponding Author * Tel.: +86-731-88876490; Fax: +86-731-88879616 E-mail: [email protected]

ABSTRACT: In this work, the dual signal-tagged hairpin structured DNA (dhDNA)-based ratiometric probe was developed by the combination of ferrocene-labeled signal probe (Fc-sP) and methylene blue-modified inner reference probe (MB-rP) in one hairpinstructured DNA. On basis of this, a high-performance ratiometric electrochemical method was proposed for biomarker detection. In contrast to the conventional ratiometric electrochemical probe, this dhDNA ratiometric probe integrated sP and rP into one structure, which ensured the completely same modification condition and the interdependence of sP and rP on one sensing interface. As a result, the dhDNA ratiometric probe possesses a stronger ability to eliminate the disturbance of environmental change, which was proven by the fact that the changes of the surface roughness and pH value had no significant effects on the reproducibility and stability of the sensor. Moreover, in the proposed strategy, the initial ratio responses of Fc-sP to MB-rP ((IFc-sP/IMB-rP)0) are controllable and can be kept constant at 1:1, which is favorable for the increase in signal-to-noise ratio and sensitivity. When the sequence of Fc-sP is designed as the aptamer of mucin 1 (MUC1), the dhDNA ratiometric sensor with signal amplification of Au nanoparticles becomes feasible for the sensitive detection of MUC1 by one-step incubation procedure. Compared with the conventional ratiometric sensor, the proposed dhDNA sensor has higher reproducibility, accuracy, stability, sensitivity, and simplicity, which are significant for the development of the sensor in various fields for practical applications.

considered impractical for potential point-of-care devices because the electrode operation and instruments are complicated and ensuring the close surface/interface conditions of the signal probe (sP) and inner reference probe (rP) is difficult, which leads to significant error in quantitative determination between two electrodes14,15. In view of these defects, another kind of electrochemical ratiometric sensor was developed by independent co-immobilization of sP and rP at one sensing interface16,17. Compared with the two-channel ratiometric detection, the conventional ratiometric sensor is relatively simple and accurate. However, the completely same modification conditions of sP and rP still cannot be ensured because of the different interface microenvironments. The disturbances in the initial current ratio responses ((IsP/IrP)0) from the different sensing microenvironments are inevitable, which seriously reduce the reproducibility and stability of the conventional ratiometric probe. Moreover, rP cannot provide the in situ information of sP because of the independency between sP and rP. As a result, the assumption that the observed signal changes are due to target binding or deterioration of the sensing surface is doubtful. All of these shortcomings would affect the performance of the ratiometric electrochemical probe and limit the practical applications of the ratiometric sensor.

INTRODUCTION A variety of sensors have been explored to achieve the accurate detection of disease-related markers and meet the growing demand for point-of-care medical diagnostics1–3. However, most of sensors encountered various problems in their practical applications, including poor reproducibility, robustness, reliability, and accuracy. Many attempts have been made to overcome these shortcomings. Recently, the ratiometric method has been proven to be a good choice to improve the practical applications of sensors because it reduces environmental influence and provides a more accurate signal through their self-referencing capability4–7. The ratiometric method has been extensively employed in various analytical techniques (e.g., fluorescence, electrochemistry, and chemiluminescence)8–10. Among these techniques, the ratiometric electrochemical sensor has been the focus of considerable interest because of its remarkable advantages, including high sensitivity, simplicity, relatively low cost, and amenability to miniaturization11–13. The reported ratiometric electrochemical sensors can be approximately divided into two types. Two-channel ratiometric detection was conducted by employing two working electrodes. Two-channel ratiometric detection is inconvenient and 1

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Therefore, exploring an efficient ratiometric electrochemical probe that can solve the existing problems and achieve higher reproducibility, robustness, and accuracy for ratiometric detection is urgently needed. Notably, a new class of ratiometric electrochemical probe based on the integrated dual signal-tagged hairpin-structured DNA (dhDNA)was developed. Herein, thiolated methylene blue-modified as inner reference probe (MB-rP) and ferrocene-labeled single-strand DNA as signal probe (Fc-sP) were involved. Mucin 1 (MUC1) as a tumor marker model, which is significant for the early diagnosis, differentiating diagnosis, curative effect monitoring, and follow-up examinations of patients with tumors or carcinomas, has been sensitively measured with signal amplification of Au nanoparticles (AuNPs). The most remarkable advantage of the proposed strategy is the combination of Fc-sP and MB-rP in one dhDNA as ratiometric probe (see Scheme 1A). In this case, the completely same modification conditions and the direct interdependent relationship between MB-rP and Fc-sP can be ensured. Therefore, the disturbances from environmental changes can be significantly reduced. Moreover, MB-rP can reflect the information of immobilized Fc-sP in situ, and the deterioration of the signal probe or sensing surface can be confirmed by the response of MB-rP, improving reproducibility and accuracy. Finally, the proposed electrochemical sensor Table 1. Names and Sequences of All the Oligonucleotides Used in This Worka Name

posed strategy is more feasible and more advantageous than the conventional ratiometric electrochemical probe, and the application of the dhDNA ratiometric probe would be significantly improved and expanded. EXPERIMENTAL SECTION Reagents. 6-Mercaptohexanol (MCH, 97%), NaCl, KCl, MgCl2, CaCl2, Tris, human serum albumin (HSA), and human immunoglobulin G (HIgG) were purchased from Sigma (Shanghai, China). Human serum samples (HSS) were obtained from Amyjet Scientific Inc. (Wuhan, China). Chloroauric acid (HAuCl4·4H2O, ≥99.9%) was obtained from Shanghai Chemical Reagent Company (Shanghai, China). MUC1 was synthesized by Shanghai Apeptide Co., Ltd. (Shanghai, China) and suspended in 10 mM phosphate-buffered saline (PBS; pH 7.4). The thiolated MB-modified dhDNAs and Fc-modified DNAs were used in this study. The DNAs used in this study were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) and were used as received. The names and sequences of all of the oligonucleotides used in this work are shown in Table 1. All of the prepared solutions were stored at 4°C as stock solution. Unless otherwise noted, all other chemicals were of analytical grade purity and used without further purification. Milli-Q water (18.2 MΩ·cm; Millipore MQ System Inc., Milford, MA, USA) was used in all experiments. Formation of the dhDNA Ratiometric Probe. MB-rP and Fc-sP were dissolved in 34 mM Tris–HCl buffer (pH 7.4; 233 mM NaCl, 8.5 mM KCl, 1.7 mM MgCl2, and 1.7 mM CaCl2). Afterward, MB-rP (5 µM) was mixed with equal volume of Fc-sP (10 µM). Then, the mixture was incubated in a water bath at 70°C for 5 min and gradually cooled down to room temperature to achieve hybridization between Fc-sP and MBrP, forming the dhDNA as ratiometric probe. The resulting solutions were stored at 4°C for further use. MB-rP was also mixed with Fc-sP1 and Fc-sP2 to obtain the corresponding ratiometric probes (dhDNA1 and dhDNA2) for the control experiments. Preparation of the Ratiometric Sensor. Prior to electrode fabrication, bare glassy carbon electrode (GCE; 2 mm in diameter) was polished carefully using aluminum powder (0.3 and 0.05 µm), followed by ultrasonic cleaning with ethanol and double distilled water for 5 min to remove the residual Al2O3 powder. Afterward, the GCE was electrochemically cleaned in 0.5 M H2SO4 by a series of oxidation and reduction cycles from 0 V to 1.6 V at a scan rate of 100 mVs−1 until a steady-state redox wave was obtained. Finally, the treated GCE was washed with ultrapure water and dried under nitrogen stream. Subsequently, the cleaned GCE was immersed in 0.5 M sulfuric acid solution containing 5 mM HAuCl4 with constant potential at −0.25 V (vs. Ag/AgCl) to produce the AuNPsplated GCE (AuNPs/GCE)17. The resulting AuNPs/GCE was rinsed with double distilled water, allowed to dry under nitrogen stream, and characterized by cyclic voltammograms. The real area of the AuNPs/GCE was determined from the charge associated with the gold oxide stripping peak obtained from the cyclic voltammograms18. Afterward, 15 µL of the dhDNA solution was dropped onto the cleaned AuNPs/GCE electrode surface. Then, the end of the electrode was covered with a plastic cap to prevent the

Sequence (5’-3’)

MB-rP

SH-(CH2)6-CGA CCA TAC CAG GGT ATC CAT TTT TTTTTT AAC TTA TTT ATG GTC G-MB

MB-crPb

SH-(CH2)6-CGA CCA TAT TTC ACA TGTTAT TTT TTTTTT AAC TTA TTT ATG GTC G-MB

Fc-sP

GCA GTT GAT CCTTTG GAT ACC CTG GTTTTTT-Fc

Fc-sP1

GCA GTT GAT CCT TTG GAT ACC CTG GTTT-Fc

Fc-sP2

GCA GTT GAT CCT TTG GAT ACC CTG G-Fc

Fc-csPb

SH-(CH2)6-GCA GTT GAT CCT TTG GAT ACC CTG G TTTTTT-Fc

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a In all sequences, Fc-sP containing the aptamer sequence (underline part) and the extended sequence is used as signal probe, which has twelve nucleotides sequence (italic portion) complementary to the bold portion of MB-rP. bMB-crP is an inner reference probe which was used to be co-immobilized with Fc-csP onto the AuNPs/GCE and investigate the general reproducibility of the conventional ratiometric probe.

fabrication and target detection are simplified by one-step assembly of the ratiometric probe and one-step incubation procedure, respectively, which significantly simplifies the experimental operation and reduces the random errors. The ratiometric detection of MUC1 with high reproducibility, sensitivity, accuracy, and stability was achieved because of the aforementioned distinguishing features. Therefore, the pro2


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solution from evaporating. The assembly was kept for 15 h in the dark and rinsed with PBS thoroughly to obtain the dhDNA/AuNPs/GCE. Afterward, dhDNA/AuNPs/GCE was immersed in 1 mM MCH solution for 1 h at room temperature to block the uncovered spots and displace nonspecifically bound oligonucleotides to obtain MCH/dhDNA/AuNPs/GCE for the sensing interface. Meanwhile, for the control experiments, dhDNA1 and dhDNA2 were formed from the hybridization between MB-rP and Fc-sP1, and between MB-rP and Fc-sP2, respectively. Subsequently, dhDNA1 and dhDNA2 were employed as ratiometric probe to fabricate MCH/dhDNA1/AuNPs/GCE and MCH/dhDNA2/AuNPs/GCE, respectively. MB-crP was also co-immobilized onto the same AuNPs/GCE together with FccsP via one-step self-assembly to obtain MCH/MB-crP+FccsP/AuNPs/GCE, which was used as conventional ratiometric probe for the sensing interface. Apparatus and Measurements. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV) were conducted with a Gamry Reference 600 electrochemical workstation (Gamry Instruments Co., Ltd., Warminster, PA, USA). A conventional threeelectrode system was used in this work. Different modified GCEs were used as the working electrodes, and a platinum wire electrode and an Ag/AgCl electrode were used as the counter and reference electrodes, respectively. All of the potentials in this study were in respect to the Ag/AgCl electrode. For each step of immobilization, EIS and CVs were conducted in the presence of equimolar [Fe(CN)6]3−/4− as the redox probe. The impedance spectra were plotted in the form of Nyquist plots. For the measurements of the target, SWV responses of the ratiometric probe before and after target detection were monitored in PBS. First, the initial SWV responses of MCH/dhDNA/AuNPs/GCE were investigated in PBS. Afterward, 15 µL PBS containing different concentrations of MUC1 were dropped onto the sensing surface and incubated for 120 min. After the resulting electrode was thoroughly rinsed with purified water and dried under nitrogen stream, its electrochemical performance was investigated by SWV in 10 mM PBS. The SWV parameters adopted were as follows: potential ranging from −0.4 V to 0.6 V with a step potential of 4 mV, a frequency of 10 Hz, and an amplitude of 25 mV. Each measurement was repeated at least three times, and the control experiments were also conducted under the same conditions. All of the measurements were conducted at room temperature (approximately 25 °C). RESULTS AND DICUSSION The Design Strategy. Scheme 1A shows the design strategy of the ratiometric electrochemical probe. In this protocol, the Fc-labeled DNA as signal probe (Fc-sP) and the thiolated MB-modified dhDNA as internal reference probe (MB-rP) are involved. Fc-sP contains a target-binding aptamer (in orange) and an extended sequence (in blue). MB-rP contains a link sequence (in red), which is complementary to a part of the aptamer sequence in Fc-sP. Therefore, Fc-sP can hybridize to MB-rP, forming the dhDNA, which will serve as the ratiometric probe for electrochemical sensing. As shown in Scheme 1B, the obtained dhDNA was selfassembled onto AuNPs/GCE to fabricate the sensing interface

Scheme 1. (A) Schematic representation of the formation of dhDNA that serve as electrochemical ratiometric probe. (B) The detection mechanism of the biomarker based on this dhDNA ratiometric probe, which is illustrated by the state of dhDNA/AuNPs/GCE before (a) and after (b) introduction of MUC1.

(dhDNA/AuNPs/GCE; Scheme 1B-a). The initial ratio responses of Fc-sP to MB-rP ((IFc-sP/IMB-rP)0) can be controlled at 1:1. Herein, MUC1 as a tumor-related biomarker is considered a model to analyze the practical application of the ratiometric probe. When the biosensor is treated with MUC1, the recognition and binding of the aptamer to MUC1 enforces the conformational change of Fc-sP, triggering the complementary segment dissociation and Fc-sP separation from the modified electrode (Scheme 1B-b). As a result, the electrochemical current responses of Fc-sP decrease. Meanwhile, MB-rP is kept stable and the electrochemical responses of MB-rP are kept constant. Accordingly, MUC1 concentrations can be monitored based on the ratio responses (IFc-sP/IMB-rP). Compared with conventional ratiometric electrochemical sensors, the proposed dhDNA sensor is anticipated to possess the following distinguishing features: (i) The integrated structure ensures an equal stoichiometric ratio and the same interface microenvironment for the modified Fc-sP and MB-rP, which provide the closest correlation between sP and rP, favoring the elimination of the disturbances from environmental changes. Even under different solution and interface conditions, the ratio responses are anticipated to be reproducible. (ii) The optimal (IFc-sP/IMB-rP)0 can be achieved by adjusting the distance between Fc molecules and electrode surface, which favors the increase in signal-to-noise ratio and sensitivity. (iii) The electrochemical sensor fabrication and target detection are simplified by one-step assembly of the ratiometric probe and one-step incubation procedure, respectively, which reduce the 3


Analytical Chemistry random errors. Therefore, the proposed dhDNA ratiometric probe is expected to exhibit high reproducibility, stability, accuracy, and sensitivity. Electrochemical Characterization of Modified Electrodes. The successive fabrication of the modified electrode was characterized by EIS and CV. EIS has been proven to be one of the most powerful tools for interfacial investigation. Electron transfer resistance (Ret) is the most directive and sensitive parameter that responds to changes on the electrode interface, and the variability of Ret is strongly related to different electrode modifications10,19. In Figure 1A, the bare GCE shows a small semicircle diameter and a long tail denoting diffusion of the redox probe [Fe(CN)6]3−/4− (curve a). When AuNPs were electrodeposited onto the surface of GCE, a straight line can be observed (curve b), which is due to the excellent conductivity of AuNPs and the large surface area of AuNPs/GCE20. However, the semicircle diameter increased dramatically (curve c) after assembling dhDNA on AuNPs/GCE, which is attributed to the presence of dhDNA blocking the interfacial electron transfer. The assembly of MCH also caused an obvious increase in Ret (curve d). The peak currents and the separation of peak potential in CV can reflect the electron transfer of the redox probe at the modified electrode21. As displayed in Figure 1B, a pair of well-defined redox peaks can be observed for the bare GCE (curve a), indicating the fast electron transfer of the redox probe. When AuNPs were electrodeposited onto the GCE, the redox peak currents were obviously enhanced (curve b), which can be attributed to the convenient electron transfer on the large surface area of AuNPs. Subsequently, the self-assembly

of dhDNA onto the AuNPs/GCE led to the decrease in redox peak currents (curve c) and the increase in peak-to-peak separation, which is due to the repulsion effect between the negatively charged phosphate backbone in dhDNA and negatively charged [Fe(CN)6]3−/4−. Afterward, when MCH was selfassembled onto dhDNA/AuNPs/GCE to block the remaining active sites, a further decrease in redox peak currents can be observed (curve d). All results prove that the stepwise fabrication of the modified electrode has been successfully achieved. Optimization of the Performance of the dhDNA Ratiometric Probe. The equal stoichiometric ratio between the modified MB-rP and Fc-sP is the key point for achieving highperformance ratiometric sensors. Given that the proposed dhDNA was achieved by hybridization reaction (Scheme 1A), the optimal reaction condition should be confirmed. In our work, 12 complementary sequences of MB-rP were designed to be complementary to Fc-sP. The free energy of the formed duplex between MB-rP and Fc-sP can be evaluated, and the binding constant (Ka) was calculated as 0.11 nM by referring to the literature22,23. This finding indicates that the hybridization reaction efficiency can be deemed as approximately 100% when the concentration ratio of Fc-sP and MB-rP (cFc-sP/cMB-rP) is 1:1 at µM degradation. In the experiment, given the uncertain effects in the practical environment, the value of cFc-sP/cMBrP was set as 2:1 in the mixture solution to ensure the complete hybridization of Fc-sP onto MB-rP. The excess Fc-sP can be easily rinsed out after the immobilization of dhDNA because of the absence of –SH. 250

A

2000

c

150

b a

c

I / nA

1500

Z'' / Ω

d

200

A

d

1000

b

100 50

500

a

0 -0.4

0

0

1000

2000

3000

4000

5000

40

6000

1.0

0.0

0.2

0.4

0.6

B

b

B

0.8

a

(Ι Fc-sP/Ι MB-rP)

0

c d

20

0

0.6 0.4

-20

0.2

-40

0.0

-0.1

-0.2

E/V

Z' / Ω

I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 0.0

0.1

0.2

0.3

0.4

0.5

3

6

Number of extened sequence (n)

E/V

Figure 2. (A) SWV curves of MCH/AuNPs/GCE (curve a), MCH/dhDNA1/AuNPs/GCE (curve b), MCH/dhDNA2/AuNPs/GC (curve c), and MCH/dhDNA/AuNPs/GCE (curve d) in PBS (10 mM, pH 7.4). (B) Effect of the number (n) of extended sequences in Fc-sP on (IFc-sP/IMB-rP)0.

Figure 1. Electrochemical impedance spectra (A) and cyclic voltammograms (B) of the different electrodes in 0.1 M KCl aqueous solution containing 5 mM [Fe(CN)6]3−/4−as redox probe. (a) Bare GCE, (b) AuNPs/GCE, (c) dhDNA/AuNPs/GCE, and (d) MCH/dhDNA/AuNPs/GCE.

1 4


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Table 2. The influence of fr on the performance of the dhDNA ratiometric probe and the conventional ratiometric probe, respectively.

Electrode area/cm2a

dhDNA Ratiometric Probe

Conventional ratiometric probe

fr (IFc-sP)0/µA

(IMB-rP)0/µA

((IFc-sP/IMB-rP)0

RSD

(IFc-csP)0/µA

(IMB-crP)0/µA

(IFc-csP/IMB-crP)0

RSD

0.093

3.0

0.173±0.027

0.186±0.026

0.937±0.048

5.1%

0.161±0.047

0.181±0.039

0.891±0.1578

17.7%

0.151

4.8

0.214±0.024

0.216±0.025

0.991±0.041

4.2%

0.186±0.035

0.193±0.034

0.959±0.1123

11.7%

0.174

5.5

0.228±0.025

0.236±0.028

0.975±0.057

5.8%

0.197±0.046

0.216±0.038

0.912±0.1356

14.9%

a

The real surface are of the AuNPs/GCE was estimated based on the amount of charge consumed during reduction of the Au surface oxide monolayer in 0.5 M H2SO4 and a reported value of 400 µC cm-2 was used for the calculation27.bThe electrode area is the average value for five independent sensors. The roughness factor (fr) of the AuNPs/GCE surface was defined as the ratio between the real surface and geometric surface area.

In conventional ratiometric electrochemical sensors, data normalization is necessary to achieve acceptable reproducibility because of the various initial response ratios of signal probe to inner reference probe. In the proposed design, (IMB0 0 rP) would be more obvious than (IFc-sP) because MB and Fc undergo two-electron and one-electron transfer25. The larger reference signal would significantly reduce the detection sensitivity. Therefore, if ((IFc-sP/IMB-rP)0) could be controlled at 1:1, then the detection process will be simplified, the error will be decreased, and the signal-to-noise ratio and sensitivity will be enhanced. Herein, according to the report that the electron transfer rate constant is related to the distance between redox labels and electrode surface26, distance d2 (MB to the electrode surface) was kept constant and distance d1 (Fc to the electrode) was regulated by changing the number of the extended sequence (n) in Fc-sP. Three ratiometric probes, i.e., dhDNA1, dhDNA2, and dhDNA, corresponding to the number of extended sequences, i.e., 0, 3, and 6, were designed and assembled onto the AuNPs/GCE. The corresponding SWV responses were investigated, as shown in Figure 2A. All three ratiometric probes exhibited two peak currents centered at −0.27 V and 0.38 V because of the electrochemical oxidation of MB and Fc, respectively20,24. The SWV responses of MB-rP (IMB0 rP) from three probes are constant, whereas the current responses of Fc-sP (IFc-sP)0 increased with n from 0 to 6. When the number of extended sequences increased from 0 to 3 and 6, (IFc-sP/IMB-rP)0 changed from 3/5 (n=3) to 4/5 (n=3) and 1/1 (n=6), respectively, as displayed in Figure 2B. These results reveal that the distance from the Fc molecules to the electrode surface can be effectively controlled by the number of extended sequences. When the number of extended sequences in FcsP is 6, (IFc-sP/IMB-rP)0 of 1:1 can be achieved. This finding obviously highlights the advantages of combining Fc-sP and MB-rP in one dhDNA as ratiometric probe. Feasibility of the dhDNA Sensor. The electrochemical signal from the smooth surface is too small to fulfill sensitive detection. AuNPs have been commonly used for signal amplification. This study investigated whether the dhDNA ratiometric probe is feasible for target detection with AuNP amplification. The SWV responses of the MCH/dhDNA/gold electrode and the MCH/dhDNA/AuNPs/GCE in PBS were investigated, as displayed in Figure 3. The peak currents of Fc and MB at MCH/dhDNA/AuNPs/GCE (curve b) are signifi-

cantly enlarged compared with those at the MCH/dhDNA/gold electrode (curve a). This finding indicates that AuNPs are relatively suitable for accelerating the electron transfer of the redox label and promoting the detection sensitivity of the dhDNA ratiometric sensor. With the introduction of the target (MUC1), the internal reference response at MCH/dhDNA/AuNPs/GCE is kept constant (curve c), indicating the unchanged state between the electrode and MB during the protein-binding process. However, the current response of Fc-sP significantly decreased, which is ascribed to the binding of MUC1 to Fc-sP (containing the MUC1-binding aptamer) and delivery of Fc-sP from the modified electrode. Thus, the concentrations of MUC1 can be monitored based on the ratio response (IFc-sP/IMB-rP). Notably, using the designed dhDNA ratiometric probe, the sensing interface can be easily fabricated by one-step assembly, the ratiometric detection can be simply achieved by one-step incubation, and the AuNPs play a crucial role in signal amplification. 250

b 200 150

I / nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

c

50

a

0 -0.4

-0.2

0.0

0.2

0.4

0.6

E/V

Figure 3. SWV curves of the different electrodes in PBS (0.1 M, pH 7.4). MCH/dhDNA/gold electrode (line a), MCH/dhDNA/AuNPs/GCE (line b), and MCH/dhDNA/AuNPs/GCE after incubation in 500 nM MUC1 (line c).

Influence of Surface Roughness. Nanomaterials are generally employed to increase the specific surface area of the working electrode and to improve the sensitivity of the ratiometric electrochemical detection. However, for the conventional ratiometric methods, the immobilizations of sP and rP

5


Analytical Chemistry Table 3. Comparison of specific values of non-ratiometric probe, conventional ratiometric probe, and dhDNA ratiometric probe before and after MUC1 detection.

Average Response RSD

Non-ratiometric probe


dhDNA Ratiometric Probe

(IFc-sP)0/(A)

IFc-sP/(A)

(IFc-csP/IMB-crP)0

IFc-csP/IMB-crP

(IFc-sP/IMB-rP)0

IFc-csP/IMB-crP

2.16e-7±0.34

1.43e-7±0.24

0.959±0.112

0.619±0.89

0.991±0.39

0.660±0.34

15.8%

17.1%

11.7%

14.5%

4.0%

5.2%

A 320

1.2

a

1.2

b

c 0.9

240

80

0

/Ι MB-rP)

0.6

0.3

dhDNA Ratiometric probe

Conventional ratiometric probe 1.2

320

1.2

e

d

f 0.9

Ι Fc-csP/Ι MB-crP

Ι Fc-sP/Ι MB-rP

0.9

240

80

0.6


0.6

0.3

0.3

0.0

0.0

0

0.3

0.0

0.0


160

0.6

Fc-sP

160

(Ι

( Ι Fc-csP /Ι MB-crP )

0

(Ι Fc-sP) / nA

0

0.9

0

B

Ι Fc-sP / nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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dhDNA Ratiometric probe

Figure 4. Reproducibility of the non-ratiometric probe, conventional ratiometric probe, and dhDNA ratiometric probe before (a, b, c) and after (d, e, f) MUC1 detection, respectively. The black histograms represent the background responses of 30 individual measurements over 10 electrodes. The red and black error bars represent the averages and standard deviations (SD) of 30 parallel experiments, respectively.

are random18. The effects of surface roughness and curvature on the immobilization of sP must be different from that on rP. Thus, the reproducibility and accuracy of these ratiometric sensors are significantly reduced. Herein, the influence of surface roughness on the conventional ratiometric probe and the proposed ratiometric probe has been evaluated. The electrode surface roughness was modulated by changing the deposition time for AuNPs. The roughness factor (fr), which is defined as the ratio between the real surface area and the geometric surface area (fr=Ar/Ag), was calculated. As expected, for the proposed dhDNA ratiometric probe, although fr affected the values of (IFc-sP)0 and (IMB-rP)0, the value of (IFc0 sP/IMB-rP) was almost kept constant at 1:1, and the relative standard deviation (RSD) for (IFc-sP/IMB-rP)0 exhibited a slight change, as displayed in Table 2. However, for the conventional ratiometric probe, fr significantly affected not only the individual values of (IFc-csP)0 and (IMB-crP)0 but also the ratio value (IFc-csP/IMB-crP)0, as shown in Table 227. The results confirm that combining sP and rP in one ratiometric probe, just as what we have designed, will ensure the synchronous immobilization and the completely same modification condition of sP and rP. Thus, the influence of the changes of surface

roughness and the microenvironment is significantly reduced and the detection reproducibility and accuracy are improved. Reproducibility of the Ratiometric Sensor. The ratiometric electrochemical probe has been expected to have higher reproducibility and accuracy. Therefore, the reproducibility of the dhDNA ratiometric sensor prior to (A) and after (B) target detection was compared with those of the nonratiometric and conventional ratiometric sensors. The corresponding results are displayed in Figure 4 and Table 3. A total of 30 individual measurements of the electrochemical responses before target detection were collected. For the nonratiometric sensor (Figure 4A-a), (IFc-sp)0 exhibited a wide variation with an average of 2.16 ×10−7 A and RSD of 15.8% (see Table 3). For the conventional ratiometric probe-based sensor (Figure 4A-b), (IFc-csP/IMB-crP)0 in 30 tests also show a wide variation with an average of 0.959 and RSD of 11.7% (see Table 3). However, the variation in the proposed dhDNA ratiometric electrochemical sensor was significantly reduced (Figure 4A-c) with an average background ratio response (IFc0 sP/IMB-rP) of 0.991 and RSD of 4.0% (see Table 3). When MUC1 was introduced, as shown in Figures 4B-d, 4B-e, and 4B-f and Table 3, the RSD of the dhDNA ratiometric electrochemical probe (5.2%) was smaller than those 6


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of the non-ratiometric probe (17.1%) and the conventional ratiometric probe (14.5%). For target detection, the higher reproducibility of the dhDNA ratiometric probe can also be achieved. All of the aforementioned data prove that the proposed ratiometric model is far more robust, reliable, and reproducible than the conventional ratiometric probe and the non-ratiometric probe. This finding also further illustrates the advantage of the combination of Fc-sP and MB-rP in one ratiometric probe, which can be expected to be a new class of ratiometric electrochemical probe. Ratiometric Detection of MUC1. The previously presented studies on different models revealed the advantages of the proposeddhDNA ratiometric probe. The prepared ratiometric sensor was employed to detect MUC1 in PBS (pH 7.4). Figure 5A shows the relationship between the SWV responses of the dhDNA ratiometric probe and the MUC1 concentrations. With the increase in MUC1 concentration, the SWV responses of Fc-sP decreased gradually, whereas the SWV responses of MB-rP did not change. Thus, IFc-sP/IMB-rP gradually decreased with the increase in MUC1 concentration. Figure 5B displays the calibration curve of the ratiometric sensor, which was obtained by monitoring IFc-sP/IMB-rP after the introduction of MUC1. The data were collected using different electrodes. IFc-sP/IMB-rP exhibited a good linear relationship with the logarithm of the MUC1 concentration ranging from 1 nM to 1 µM. The linear equation was obtained as I Fc-sP /I MB- rP = −0.2078logcMUC1+0.9921 (R=0.9955), which can be used for the quantitative detection of MUC1. The detection limit

0.5

∆ Ι Fc-sP / Ι MB-rP

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I / nA

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0.0

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B

Sample

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Ι Fc-sP / Ι MB-rP

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PBS

Added

Found (nM)

10 50 100

9.7 45.4 96.6

0.8

1 2 3

0.6

0.4 0.0

0.5

0.2

1.0

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2.0

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HSS

HSA

HIgG

was calculated to be 0.827 nM (S/N=3), which is more sensitive compared with other electrochemical biosensors for MUC128–30. Specificity of the Ratiometric Sensor. HSA and HIgG, which might coexist with MUC1 in human blood serum, were chosen as the possible interfering substances of target MUC1 to investigate the specificity of the developed ratiometric biosensor. As shown in Figure 6, the IFc-sP/IMB-rP value of the sensor in the presence of MUC1 (100 nM) is significantly obvious. However, the IFc-sP/IMB-rP values in diluted HSS and in PBS without or with other foreign proteins (HSA and HIgG) are almost negligible, despite their concentrations (1 µM) being larger than the target MUC1 concentration. This finding indicates that the specificity of the proposed ratiometric sensor is excellent for the detection of biomarkers, which is useful for the practical application of the ratiometric sensor. The ratiometric sensor was employed to measure MUC1 in real blood samples spiked with MUC1 to assess its analytical application in real blood samples further. The blood samples spiked with MUC1 at three different concentrations (10, 50, and 100 nM) were investigated, and the corresponding results are listed in Table 4. The recoveries for the added MUC1with the concentrations of 10, 50, and 100 nM are 97%, 90.8%, and 96.6%, respectively, which are acceptable for practical detection. Table 4. Recovery of MUC1 in 10 fold-diluted human serum samples.

g

-0.4

MUC1

Figure 6. Selectivity of the dhDNA sensor toward MUC1 at 100 nM against 10 mM PBS, 1 µM HSA, and 1 µM HIgG solution, and 10 times diluted HSS.

a

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RSD (%) 5.5 6.3 3.7

Recovery (%) 97.0 90.8 96.6

3.0

Log(cMUC1 / nM)

Stability of the dhDNA Ratiometric Sensor. By designing the sequence of rP and sP, this dhDNA ratiometric probe should have wide application in different fields, including the detection of biomarkers, mutated DNA/RNA, and even metal ions. In different application fields, the experimental conditions, particularly the pH value, changed significantly. Therefore, the effect of pH on the stability of dhDNAwas investigated. As shown in Figure 7A, although the current responses of Fc-sP and MB-rP were different, with the pH ranging from 5.0 to 9.0, the ratio values were nearly invariable (always maintaining 1:1). The ratiometric probe as a unity of Fc-sP and MB-rP is stable under different pH values. All of these

0.0 0

20

40

60

80

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cMUC1 / µM

Figure 5. (A) SWV curves for the detection of MUC1 under different concentrations (from a to g: 0 nM, 5 nM, 10 nM, 50 nM, 500 nM, 10 µM, and 100 µM). (B) Dependence of IFc-sP/IMB-rP on MUC1 concentration ranging from 1 nM to 100 µM. Inset: The linear relationship between IFc-sP/IMB-rP and the logarithm of MUC1 concentration. Error bars represent the SD of three parallel experiments.

7



This work was financially supported by the National High Technology Research and Development Program of China (2015AA020502), the National Key Basic Research Program of China (2014CB744502), the National Natural Science Foundation of China (21573290, 21273288, 21005090).

results indicate that the dhDNA ratiometric probe is reliable and stable for the detection of the target under different environments, which may be significant for the development of the ratiometric method in various fields. 250

A

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c d b

200 e

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a

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Figure 7. SWV responses (A) and (IFc-sP/IMB-rP)0 (B) of the dhDNA ratiometric probe under different pH values (from a to e: 5, 6, 7.4, 8 and 9).

CONCLUSIONS This study proposed a novel concept for the ratiometric probe. The concept achieved the combination of the inner reference probe (MB-rP) and the signal probe (Fc-sP) to form the integrated dual signal-tagged dhDNA used as the ratiometric probe. The obtained dhDNA ratiometric probe was self-assembled onto the AuNPs/GCE electrode to develop a ratiometric biosensor, in which the inner reference probe can provide the in situ information of the signal probe and sensing interface. The initial current ratio of Fc-sP to MB-rP is constant at 1:1. Moreover, the different surface roughness and pH values almost have no effect on the stability of the initial ratio responses. Therefore, compared with the non-ratiometric and conventional ratiometric sensors, the dhDNA ratiometric electrochemical sensor exhibits higher accuracy, robustness, sensitivity, and selectivity. The electrochemical strategy has been successfully used for the reliable detection of the biomarker (MUC1) by one-step incubation procedure. The simplicity in operation together with the excellent analytical performance of the biosensor should make it useful and powerful for applications in biochemical investigations. Therefore, this work provides a new class of ratiometric probe, which may lay the foundation for the development and application of the ratiometric method in clinical and other fields.

(16) Cheng, H.; Wang, X.; Wei, H. Anal. Chem. 2015, 87, 88898895. (17) Zhou, L.; Wang, J.; Li, D.; Li, Y. Food Chem. 2014, 162, 3440. (18) Yang, W.W.; Gerasimov, J. Y.; Lai, R. Y. Chem. Commun. 2009, 2902-2904. (19) Deng, C.; Chen, J.; Nie, Z.; Wang, M.; Chu, X.; Chen, X.; Xiao, X.; Lei, C.; Yao, S. Anal. Chem. 2009, 81, 739-745. (20) Guo, J.; Wang, J.; Zhao, J.; Guo, Z.; Zhang, Y. ACS Appl. Mat. Interfaces 2016, 8, 6898-6904. (21) Veloso, A. J.; Chow, A. M.; Ganesh, H. V. S.; Li, N.; Dhar, D.; Wu, D. C. H.; Mikhaylichenko, S.; Brown, I. R.; Kerman, K. Anal. Chem. 2014, 86, 4901-4909. (22) Breslauer, K. J.; Frank, R.; Blöcker, H.; Marky, L. A. Proc. Nat. Acad. Sci. 1986, 83, 3746-3750. (23) SantaLucia, J.; Allawi, H. T.; Seneviratne, P. A. Biochemistry 1996, 35, 3555-3562. (24) Ren, K.; Wu, J.; Yan, F.; Ju, H. Sci. Rep. 2014, 4, 4360.

AUTHOR INFORMATION

(25) Yang, W.; Lai, R. Y. Chem. Commun. 2012, 48, 8703-8705.

Corresponding Author

(26) Xiong, E.; Zhang, X.; Liu, Y.; Zhou, J.; Yu, P.; Li, X.; Chen, J. Anal. Chem. 2015, 87, 7291-7296.

* Tel.: +86-731-88876490; Fax: +86-731-88879616 E-mail: [email protected]

(27) Angerstein-Kozlowska, H.; Conway, B. E.; Hamelin, A.; Stoicoviciu, L. J. Electroanal. Chem. and Interf. Electrochem. 1987, 228, 429-453.

ACKNOWLEDGMENT 8


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(28) Guo, P.; Xiong, J.; Zheng, D.; Zhang, W.; Liu, L.; Wang, S.; Gu, H. RSC Advances 2015, 5, 66355-66359. (29) Ding, Y.; Ling, J.; Wang, H.; Zou, J.; Wang, K.; Xiao, X.; Yang, M. Anal. Methods 2015, 7, 7792-7798. (30) Hu, R.; Wen, W.; Wang, Q.; Xiong, H.; Zhang, X.; Gu, H.; Wang, S. Biosens. Bioelectron. 2014, 53, 384-389.

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