Polycytosine DNA Electric Current Generated Immunosensor for

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Polycytosine DNA Electric Current Generated Immunosensor for Electrochemical Detection of Human Epidermal Growth Factor Receptor 2 (HER2) Xiaoqing Li, Congcong Shen, Minghui Yang, and Avraham Rasooly Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00023 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Analytical Chemistry

Polycytosine DNA Electric Current Generated Immunosensor for Electrochemical Detection of Human Epidermal Growth Factor Receptor 2 (HER2)

Xiaoqing Li†, Congcong Shen†, Minghui Yang†* and Avraham Rasooly‡* †

College of Chemistry and Chemical Engineering, Central South University,

Changsha, China, 410083 ‡

National Cancer Institute, National Institutes of Health, Rockville, Maryland 20850,

United States

Email: [email protected] (M. Yang) [email protected] (A. Rasooly) Tel: (+86) 731 88836954

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ACS Paragon Plus Environment

Analytical Chemistry 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

Abstract: A polycytosine DNA based immunosensor for electrochemical detection was developed and tested for detection of human epidermal growth factor receptor 2 (HER2), a breast cancer biomarker. We utilized gold nanoparticles (AuNPs) as supporting matrix to immobilize polycytosine DNA sequence (dC20) for electrochemical current generation and anti-HER2 antibodies. In the presence of target HER2, a sandwiched immunocomplex forms between a peptide specific to HER2 immobilized on the gold electrode and the anti-HER2 antibodies on the AuNPs. The HER2 captured by the sensor is detected due to the reaction of the dC20 phosphate backbone with molybdate, forming redox molybdophosphate precipitate that generates an electrochemical current on the surface of the electrode. The assay is sensitive: the calculated limit of detection of HER2 was as low as 0.5pg/ml and the detection was linear to HER2 from 1 pg/mL to 1 ng/mL. The sensor’s specificity is high, and there is no cross reactivity with several potential interferents, such as human IgG, human IgA, p53, carcinoembryonic antigen (CEA) and protein kinase (PKA). The sensor’s performance with HER2 in clinical serum samples is similar to the performance of commercial ELISA assays. The configuration of polycytosine DNA as electrochemical current generating label and anti-HER2 antibodies on AuNPs is versatile and can be reconfigured to detect low levels of different analytes, or made more sensitive by amplifying the DNA to produce more phosphate to react with Na2MoO4.

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Successful design of a sensitive and precise immunosensor for detection of protein biomarkers requires careful selection of both binding ligands and signal amplification strategies.1-3 While immunoassays are widely used for detection, sensitivity is limited for many analytes because assay signal generating modes (e.g. enzymatic assays, colorimetric detection, chemiluminescence, fluorescence) are often difficult to amplify. Recently, DNA strand ligands such as aptamers were developed as alternatives to traditional antibodies for different analyte targets.4-6 While aptamers have the advantages of low cost, easy synthesis and potential for signal amplification, antibodies remain the most common ligands for biodetection applications7,8 even though DNA based signal strategies are versatile and easy to amplify.9,10 Immunoassays can be made orders of magnitude more sensitive by replacing the conventionally used enzyme probes with amplifiable DNA probes utilizing polymerase chain reaction (PCR)11,12, rolling circle amplification (RCA)13-16 or hybridization chain reaction (HCR).17-19 Previous efforts have combined the specificity of an immunoassay with DNA based signal amplification strategies such as immunoassays

with

PCR

(immuno-PCR),

and

immunoassays

with

RCA

(immuno-RCA).20-22 Although these methods are highly sensitive, they are not widely used because of their complexity. For example, for immuno-PCR, antibodies must be conjugated to the DNA strand, and very precise cycling temperatures are required along with special equipment to detect the amplicon. In addition, the utilization of such amplification strategies may lead to false signals such as those resulting from non-specific PCR amplification.23

Clearly, further efforts are needed to develop

immunoassays using DNA based signal amplification. 3



In previous work, our group studied electrochemical aptasensors for cancer biomarker detection based on a DNA generated electrochemical current.24,25 The reaction of the DNA phosphate backbone with molybdate can form redox molybdophosphate precipitate and generate electrochemical current.26,27 However, the use of DNA self-assembly for signal amplification complicates detection due to the additional step required for the protocol. It was reported that the use of the inherently conducting polycytosine DNA sequence improves electrochemical detection28 and that polycytosine DNA is a high-affinity ligand for inorganic nanomaterials.29 Despite this, the potential of polycytosine DNA sequences to improve biodetection has not been fully explored. With this in mind we present an alternative approach for DNA generated electrochemical current by forming redox-active molybdophosphate through the reaction between molybdate and the backbone phosphate of polycytosine DNA. HER2 is overexpressed in around 20-30% of breast cancer tumors and is associated with a more aggressive disease, higher recurrence rates, and increased mortality. It was demonstrated that elevated serum HER2/neu levels are associated with progressive metastatic cancer and poor response to chemotherapy and hormonal therapy. Serum HER2 testing is clinically important for monitoring metastatic breast cancer patients during treatment and it is widely used as biomarker for cancer diagnosis.30

We now demonstrate that HER2 can be detected with high

reproducibility and sensitivity using polycytosine to generate electrochemical current for electrochemical detection of this clinically important human growth factor receptor. For the new HER2 assay we combine the specificity of the immunoassay with the 4


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versatility of DNA signal generation. dC20 and anti-HER2 antibody (Ab) were bound to AuNPs for use as an electrochemical probe. The sensor was constructed using a sandwich structure between a peptide specific to HER2 immobilized on the gold electrode and the anti-HER2 antibodies on the AuNPs.

The subsequent reaction of

dC20 with molybdate generated electrochemical current. After validating the performance of the sensor it was used for detection of HER2 in serum samples. The configuration of polycytosine DNA and antibodies on nanoparticles is versatile and can be reconfigured using other antibodies, or integrated with other DNA amplification techniques to further enhance the sensitivity of immunosensors for a wide variety of biomedical applications.

EXPERIMENTAL SECTION Materials and Reagents. Thiol group modified peptide specific to HER2 (CKLRLEWNR) was purchased from GL Biochem Ltd. (Shanghai, China). Thiol group modified polycytosine (dC20) was synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Anti-HER2 Ab and recombinant human HER2 protein were obtained from Abcam Co., Ltd. (Cambridge, MA, USA). Sodium molybdate dihydrate (Na2MoO4·2H2O) and 6-mercapto-1-hexanol (MCH) were obtained from Sigma-Aldrich. The human HER2 ELISA kit was obtained from Jining Biotech Co., Ltd. (Shanghai, China). Human serum samples were collected from the Second Xiangya hospital. All other reagents were of analytical grade and used without further purification and all aqueous stock solutions were prepared with ultrapure water. Apparatus. Electrochemical measurements were carried out by a CHI-650D 5



electrochemical workstation (Shanghai CH Instruments Co., China) equipped with a conventional three-electrode configuration utilizing a working electrode (gold electrode, 2 mm in diameter), a saturated Ag/AgCl reference electrode and a platinum auxiliary electrode. A FEI Titan G2 60-300 transmission electron microscope (TEM) was used for synthesized AuNP characterization and a Shimadzu UV-2450 spectrophotometer was used for UV-Vis absorbance measurements. Electric Current Generated Immunosensor for Detection of HER2. Our detection methodology includes three steps: 1. Preparation of polycytosine and Ab modified AuNPs. 2. Preparation of the gold electrode and sandwich immunosensor for HER2 on the gold electrode utilizing the modified AuNPs and 3. Reaction of polycytosine with molybdate to generate electrochemical current. Preparation of polycytosine and antibody modified Gold Nanoparticles. Gold nanoparticles (AuNPs) were synthesized by reducing HAuCl4 with sodium citrate. Typically, to 100 mL of heated HAuCl4 (0.01%), 2 % sodium citrate (w/v) was quickly added after boiling. The mixture was refluxed for 15 min and stirred until cool. The AuNPs (~20 nm in diameter) were formed and stored at 4 oC. To modify the AuNPs, 50 µL of Ab solution (50 ng/mL) was added to 2 mL of gold colloids and incubated for 1 h at room temperature. Then the mixture was centrifuged and AuNPs were re-suspended into 2 mL of deionized water. Afterward, 200 µL of 10 mM polycytosine (dC20）solution was added to the mixture described above and incubated for 16 h. Sodium chloride (1M) was then added to the mixture every 8 hours until the final concentration of sodium chloride was 0.2 M. After incubation overnight at room temperature with gentle shaking, the mixture was centrifuged again to remove free 6


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DNA molecules. The obtained AuNPs were re-suspended in 2 mL of deionized water and stored at 4 oC until use. Preparation of the electrochemical sensor. The gold electrode was soaked in fresh Piranha solution (H2O2 (30%): concentrated sulfuric acid = 3:7 (v/v)) for 15 min and washed extensively with ethanol and deionized water. To bind peptide to the gold electrode, the gold electrode was immersed in 50 µg/mL of peptide solution overnight at 4 oC.

After thorough washing the electrode was

soaked in MCH (1 mM) solution for 1 h to block nonspecific sites. HER2 sandwich assay: Different concentrations of HER2 were added to the electrode surface for 2 h following washing to remove unbound HER2. Then 5 µL of the modified AuNP solution was added to the electrode surface for 1 h to bind the captured HER2 in a sandwich assay. Before detection an additional cycle of washing was performed to remove unbound AuNPs. Reaction of polycytosine with molybdate to generate electrochemical current: 5µL of 5 mM Na2MoO4 solution was dropped on the electrode surface and incubated for 20 min before electrochemical analysis in 0.5 M H2SO4 with the electrochemical detector. RESULTS AND DISCUSSION We utilized polycytosine (dC20) current generation as an electrochemical probe for immunodetection. Both Anti-HER2 Ab and polycytosine were immobilized onto the AuNP surface and used as new probe for electrochemical detection; this integrates the specificity of the traditional immunoassay with our DNA signal generation strategy. Scheme 1 is a representation of the sensor preparation and detection process. The 7



design of the electrochemical assay was straightforward, with the AuNP Ab prepared in stage I bound to target analyte (HER2) which was then captured by the peptide immobilized on the gold electrode (stage II). For signal generation the phosphate in the polycytosine DNA backbone on AuNP reacted with Na2MoO4 and the current generated was measured (stage III).

Scheme 1 Schematic representation of the sensor and detection process: I. Preparation of polycytosine and Ab modified AuNPs. II. Preparation of the sandwich immunosensor for HER2 on the gold electrode utilizing the modified AuNPs and III. Reaction of polycytosine with molybdate to generate electrochemical current. Characterization of polycytosine and antibody modified gold nanoparticles: The antibodies were physically adsorbed onto AuNPs, while the polycytosine was conjugated onto AuNPs via thiol groups. AuNPs were synthesized by reducing

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HAuCl4 with trisodium citrate. The size of the AuNPs was determined to be around 20

nm

(Figure

1A).

The

modification

of

the

AuNPs

was

measured

spectrophotometrically. As shown in Figure 1B, before modification (curve a) the UV-Vis spectra have two absorption peaks at around 280 and 520 nm for the synthesized AuNPs. After DNA conjugation onto AuNPs (curve b) the absorption peak at 280 nm decreased and became blurred, which proved the successful immobilization of DNA. Further adsorption of antibodies onto AuNPs (curve c) resulted in no obvious change of the spectrum.

This may be due to the relatively low

concentration of immobilized antibodies on the gold nanoparticles. After modification of the AuNPs with DNA molecules and antibodies, the size of the AuNPs characterized by dynamic light scattering was also increased (Figure 1C). The size of a base is ~0.43 nm and the size of dC20 is ~8.6 nm. If 3 dC20 molecules bind to each 100nm AuNP the size of the DNA- AuNP molecule will be about 126nm as suggested in Figure 1C. Besides the modification of the antibodies and DNA molecules onto AuNPs, partial aggregation of AuNPs during the process of the modification of AuNPs may also contributed to the increase of the size of AuNPs

A

25

B

1.0

20

0.8

C AuNPs

a Intensity (%)

Absorbance

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


0.6 0.4 0.2

c

AuNPs/DNA

15

AuNPs/DNA/Ab 10 5

0.0 200

300

400

500

600

700

W avelength/nm

0 0.1

1

10

100

1000

Size (d.nm )

Figure 1 Characterization of polycytosine and antibody modified gold nanoparticles: (A) TEM image of the synthesized AuNPs. (B) UV-Vis absorption of 9


10000


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AuNPs (curve a), AuNPs after conjugation of DNA molecules (curve b) and AuNPs after conjugation of DNA molecules and antibodies (curve c). (C) Size distribution of AuNPs before and after modification characterized by dynamic light scattering.

Electric current generated by AuNP probe: To demonstrate the ability of the modified AuNP electrochemical probe to generate current, the AuNP probe-induced generation of electrochemical current was studied. Two electrodes were prepared, one with immobilized unmodified AuNPs and a second having immobilized Ab as well as DNA modified AuNPs. After reaction with 5 mM molybdate the electrodes were tested in 0.5 M H2SO4 by cyclic voltammetry (CV). The testing of the sensor in 0.5 M H2SO4 does not affect the application of the sensor as before testing, the target HER2 was already captured onto the electrode and the sandwich structure was formed. The bare electrode in H2SO4 displays no current peaks (Figure 2, curve a). For the electrode immobilized with un-modified AuNPs, relatively weak redox currents are observed (Figure 2, curve b). On the other hand, the electrode with modified AuNPs shows two pairs of robust current peaks (Figure 2, curve c). This strong electrochemical current is due to the reaction of the dC20 DNA on the AuNP surface, specifically the reaction of the phosphate backbone of dC20 with molybdate to form the redox-active molybdophosphate precipitate as shown in our previous reports.31-34 This previous work probed in detail the reaction of DNA strands composed of different nucleotide bases with molybdate and leading to the possibility that the polycytosine sequence generate the highest current intensity.24

As in the description

above, electron transfer within the molybdophosphate lead to two pairs of redox peaks 10


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(0.22 and 0.35 V). In the absence of dC20 DNA on the AuNP surface, the small current generated was due to the reaction of molybdate itself on the electrode. These data demonstrate successful conjugation of dC20 with AuNPs, show that the AuNPs can generate current, and that they are useful as probes for electrochemical sensors.

Figure 2. Electric current generated by AuNP probe:

CV responses of different

electrodes in 0.5 M H2SO4, (a) bare electrode, (b) electrode immobilized with unmodified AuNPs and (c) electrode immobilized with Ab as well as DNA strand modified AuNPs.

Polycytosine DNA electric current generated immunosensor for detection of HER2: To test HER2 detection the modified AuNPs were used for detection of different concentrations of HER2. Two sensors were prepared, one as control for the detection of sample without HER2 and the second to measure 1 ng/mL of HER2. The corresponding square wave voltammetry (SWV) responses of the two sensors are

11



shown in Figure 3A. Two current peaks can be observed for both samples. For the control sample (a), the small current generated can be ascribed to nonspecific adsorption of AuNPs onto the electrode. The sensor for 1 ng/mL of HER2 generated much higher current intensity due to the specific capture of AuNPs by the electrode via Ab-antigen reaction. These results demonstrate the feasibility of the sensor for detection of HER2. An additional seven concentrations of HER2 in the range from 0 to 1 ng/mL were measured. As shown in Figure 3B, the response current of the sensor at 0.2 V increased with increasing concentrations of HER2. The relationship between current intensity and logarithm of HER2 concentration is linear in the range from 1 pg/mL to 1 ng/mL (inset of Figure 3B). Based on S/N of 3, the calculated limit of detection of the sensor is 0.5 pg/mL. The performance of the present sensor was compared with literature reported electrochemical sensor for HER detection. As shown in Table 1, it can be seen, the performance of the present sensor is better than literature reports.

Figure 3 Polycytosine DNA electric current generated immunosensor detection of HER2 (A) SWV responses of sensors in 0.5 M H2SO4: (a) sensor for blank sample 12


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and (b) sensor for 1 ng/mL of HER2; (B) Responses of the sensor to different concentrations of HER2, from a to g: 0, 1, 10, 50, 100, 500, 1000 pg/mL respectively. The inset is the calibration curve.

Table 1. Compare the performance of the present sensor with literature reported electrochemical sensors for HER2 detection Linear range

Detection limit

(ng/mL)

(pg/mL)

Aptamer

0.01 to 5

5

24

Horseradish peroxidase

5 to 200

4000

35

Horseradish peroxidase

10 to 150

4900

36

Alkaline phosphatase

9.8 to 50

2900

37

15 to 100

4400

38

0.001 to 1

0.5

This work

Signal probe

References

Alkaline phosphatase Polycytosine DNA (dC20)

Sensor selectivity and reproducibility:

To study the sensor selectivity we tested

several proteins that may coexist with HER2 in human serum samples, including human IgG, human IgA, p53, carcinoembryonic antigen (CEA) and protein kinase (PKA). As shown in Figure 4 the responses of the sensor to 1 ng/mL of the above proteins are rather low compared to 1 ng/mL of HER2. These results demonstrate good sensor selectivity resulting from the high specificity of the peptide and Ab towards HER2 binding. 13



Figure 4. Sensor selectivity: Responses of the sensor to proteins that may coexist with HER2 in human serum samples: p53, carcinoembryonic antigen (CEA), human IgG, human IgA, and protein kinase (PKA).

Sensor precision and reproducibility were evaluated by independent testing of each sample in triplicate. Experimental results indicate that the relative standard deviation of the sensor for 0.05 and 0.5 ng/mL of HER2 were 1.5 % and 2.6%, respectively, supporting high reliability of testing results. Sensor measurement of HER2 in serum of breast cancer patients: To demonstrate potential clinical utility of the sensor, HER2 levels in serum of six breast cancer patients were analyzed. Serum samples were diluted by phosphate buffer and measured with the sensor. The same samples were tested with a commercial ELISA kit as control. As shown in Figure 5, serum sample results are in agreement at all HER2 levels as determined by the two methods, with a correlation coefficient of 0.994.

This demonstrates that the performance of the new polycytosine DNA

immunosensor is similar to that of the commercial ELISA kit, and further supports 14


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application of the sensor in clinical testing protocols.

Figure 5 Sensor measurement of HER2 in serum of breast cancer patients: HER2 levels in breast cancer serum patients determined by the polycytosine DNA electric current generated immunosensor sensor vs. detection by commercial ELISA Kit.

The polycytosine DNA Ab/AuNPs technology is versatile and can be expanded beyond the electrode configuration shown in Scheme 1. For example, an additional potential application of the technology is detection of low level of analytes in large volume blood or serum samples. Reconfiguring the probe by using magnetic beads instead AuNP as supporting matrix for the polycytosine DNA and antibodies will generate a new magnetic electrochemical probe (stage I). It can utilize the Ab to capture analytes from large volumes of sample by using magnets to separate the captured analytes and detecting the captured analytes in the sandwich assay described in stages II and III. Another possible configuration integrates detection with PCR DNA amplification11,12, rolling circle amplification (RCA)13-16 or hybridization chain reaction (HCR)17-19 thereby enabling an increase in DNA phosphate concentration and resulting in increased electrical signal when reacted with Na2MoO4. Therefore our 15



approach can be used in wide array of applications for different bioassays to meet varying clinical and analytical needs.

CONCLUSIONS Gold nanoparticles were utilized as a supporting matrix for both polycytosine DNA sequence (dC20) and anti-HER2 antibodies. We have demonstrated that in the presence of target HER2 a sandwiched immunocomplex forms between a peptide specific to HER2 immobilized on the gold electrode and the anti-HER2 antibodies on the AuNPs. The immunocomplex was detected after reaction of the dC20 phosphate backbone with molybdate, forming redox-active molybdophosphate precipitate. In term of sensitivity, polycytosine generated current enables sensitive detection of HER2 with a limit of detection as low as 0.5 pg/ml. We have shown that our sensor’s specificity is high and that its performance with clinical samples is similar to that of a commercial ELISA assay. This approach is versatile, reconfiguring the polycytosine DNA/ antibodies supporting matrix may enable detection low levels of analytes in large volumes and

this system can also be made more sensitive by increasing

electrochemical signal strength through amplification of DNA to generate more phosphate to react with Na2MoO4.

Notes The authors declare no competing financial interest. The human samples used were obtained under the University human subject protection protocol.

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ACKNOWLEDGMENTS The authors thank the support of this work by the National Natural Science Foundation of China (Grant No.21575165), the National Key Basic Research Program of China (Grant 2014CB744502), and the support by Central South University (Grant No.2017gczd018).

REFERENCES (1) Tang, Z.; Ma, Z. Biosensor. Bioelectron. 2017, 98, 100-112. (2) Wang, Z.; Zong, S.; Wu, L.; Zhu, D.; Cui, Y. Chem. Rev. 2017, 117, 7910-7963. (3) Fu, X.; Chen, L.; Choo, J. Anal. Chem. 2017, 89, 124-137. (4) Dunn, M. R.; Jimenez, R. M.; Chaput, J. C. Nat. Rev. Chem. 2017, 1. (5) Yang, D.; Liu, X.; Zhou, Y.; Luo, L.; Zhang, J.; Huang, A.; Mao, Q.; Chen, X.; Tang, L. Anal. Methods 2017, 9, 1976-1990. (6) Park, K. S. Biosensor. Bioelectron. 2017, 102, 179-188. (7) Tian, S.; Zeng, K.; Yang, A.; Wang, Q.; Yang, M. J. Immunol. methods 2017, 451, 78-82. (8) Zeng, K.; Tian, S.; Wang, Z.; Shen, C.; Luo, J.; Yang, M.; Liu, Y.-N. Microchim. Acta 2017, 184, 2791-2796. (9) Zhang, H.; Dong, H.; Yang, G.; Chen, H.; Cai, C. Anal. Chem. 2016, 88, 11108-11114. (10) Zhang, B.; Liu, B.; Tang, D.; Niessner, R.; Chen, G.; Knopp, D. Anal. Chem. 2012, 84, 5392-5399. (11) Yu, T.; Zhao, W.; Xu, J.-J.; Chen, H.-Y. Talanta 2018, 178, 594-599. (12) Islam, M. N.; Gopalan, V.; Hague, M. H.; Masud, M. K.; Al Hossain, S.; Yamauchi, Y.; Nam-Trung, N.; Lam, A. K.-Y.; Shiddiky, M. J. A. Biosensor. Bioelectron. 2017, 98, 227-233. (13) Gao, F.; Zhou, F.; Chen, S.; Yao, Y.; Wu, J.; Yin, D.; Geng, D.; Wang, P. The Analyst 2017, 142, 4308-4316. (14) Jiang, W.; Liu, L.; Zhang, L.; Guo, Q.; Cui, Y.; Yang, M. Microchim. Acta 2017, 184, 4757-4763. (15) Li, Y.; Pu, Q.; Li, J.; Zhou, L.; Tao, Y.; Li, Y.; Yu, W.; Xie, G. Microchim. Acta 2017, 184, 4323-4330. (16) Zhou, Y.; Li, B.; Wang, M.; Wang, J.; Yin, H.; Ai, S. Microchim. Acta 2017, 184, 4359-4365. (17) Cai, W.; Xie, S.; Zhang, J.; Tang, D.; Tang, Y. Biosensor. Bioelectron. 2017, 98, 466-472. (18) Chen, Y.-X.; Huang, K.-J.; He, L.-L.; Wang, Y.-H. Biosensor.Bioelectron. 2018, 100, 274-281. (19) Bi, S.; Yue, S.; Zhang, S. Chem. Soc. Rev. 2017, 46, 4281-4298. 17



(20) Chang, L.; Li, J.; Wang, L. Anal. Chim. Acta 2016, 910, 12-24. (21) Ebai, T.; Souza de Oliveira, F. M.; Lof, L.; Wik, L.; Schweiger, C.; Larsson, A.; Keilholtz, U.; Haybaeck, J.; Landegren, U.; Kamali-Moghaddam, M. Clin. Chem. 2017, 63, 1497-1505. (22) Van Dessel, W.; Vandenbussche, F.; Staes, M.; Goris, N.; De Clercq, K. J Virol Methods 2008, 147, 151-156. (23) Malou, N.; Renvoise, A.; Nappez, C.; Raoult, D. Eur J Clin Microbiol Infect Dis 2012, 31, 1951-1960. (24) Hu, L.; Hu, S.; Guo, L.; Shen, C.; Yang, M.; Rasooly, A. Anal. Chem. 2017, 89, 2547-2552. (25) Shen, C.; Zeng, K.; Luo, J.; Li, X.; Yang, M.; Rasooly, A. Anal. Chem. 2017, 89, 10264-10269. (26) Shen, C.; Li, X.; Rasooly, A.; Guo, L.; Zhang, K.; Yang, M. Biosensor. Bioelectron. 2016, 85, 220-225. (27) Xie, B.; Zhou, N.; Ding, R.; Zhao, Y.; Zhang, B.; Li, T.; Yang, M. Anal. Methods 2017, 9, 6569-6573. (28) Wu, Y. Z.; Moulton, S. E.; Too, C. O.; Wallace, G. G.; Zhou, D. Z. Analyst 2004, 129, 585-588. (29) Lu, C.; Huang, Z.; Liu, B.; Liu, Y.; Ying, Y.; Liu, J. Angew Chem Int Ed Engl 2017, 56, 6208-6212. (30) Esteva, F. J.; Cheli, C. D.; Fritsche, H.; Fornier, M.; Slamon, D.; Thiel, R. P.; Luftner, D.; Ghani, F. Breast Cancer Res 2005, 7, R436-443. (31) Jiang, W.; Tian, D.; Zhang, L.; Guo, Q.; Cui, Y.; Yang, M. Microchim. Acta 2017, 184, 4375-4381. (32) Si, Z.; Xie, B.; Chen, Z.; Tang, C.; Li, T.; Yang, M. Microchim. Acta 2017, 184, 3215-3221. (33) Huang, Y.; Tang, C.; Liu, J.; Cheng, J.; Si, Z.; Li, T.; Yang, M. Microchim. Acta 2017, 184, 855-861. (34) Qu, F.; Yang, M.; Rasooly, A. Anal. Chem. 2016, 88, 10559-10565. (35) Tallapragada, S. D.; Layek, K.; Mukherjee, R.; Mistry, K. K.; Ghosh, M. Bioelectrochemistry 2017, 118, 25-30. (36) Yang, S.; You, M.; Zhang, F.; Wang, Q.; He, P. Sens. Actuators B: Chem. 2018, 258, 796-802. (37) Marques, R. C. B.; Costa-Rama, E.; Viswanathan, S.; Nouws, H. P. A.; Costa-García, A.; Delerue-Matos, C.; González-García, M. B. Sens. Actuators B: Chem. 2018, 2018, 918-925. (38) Marques, R. C. B.; Viswanathan, V.; Nouws, H. P. A.; Delerue-Matos, C.; González-García, M. B. Talanta 2014, 129, 594-599.

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Graphic presentation: Polycytosine DNA Electric Current Generated Immunosensor

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Polycytosine DNA Electric Current Generated Immunosensor for

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