Electrochemical Immunosensor for Detection of Epidermal Growth

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Analytical Chemistry

An Electrochemical Immunosensor for Detection of EGF Reaching Lower Detection Limit: Toward GSSG as a More Efficient Blocking Reagent for the Ab-AgNPs and Antigen Interaction Yuqing Lin,*,† Kangyu Liu,† Chao Wang, † Linbo Li, †,‡ Yuxin Liu, †

†

Department of Chemistry, Capital Normal University, Beijing 100048, China

‡

College of Resources Environment and Tourism, Capital Normal University, Beijing

100048, China

∗Corresponding author Tel.: +86 1068903047; Fax:+86 1068903047 E-mail address: [email protected](Y. Lin). 1

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ABSTRACT: Blocking reagent is of vital importance for immunosensor because it ensures the antifouling of the sensing interface and thus selective determination of target. This letter investigates a small inactive peptide, oxidized glutathione (GSSG) to replace the commonly used bovine serum albumin (BSA) as blocking reagent for immunosensor fabrication to lower detection limit of electrochemical immunosensors. The EGF (epidermal growth factor) detection as an example is used here to compare the blocking effects from GSSG and BSA, respectively. The relatively big size of BSA sterically hinders EGF and Ab-AgNPs binding. By comparison, GSSG cannot hinder EGF and Ab-AgNPs binding since it is much smaller than EGF, verified by scanning electron microscopy (SEM) results. The established GSSG blocking-based immunosensor for EGF reach a greatly low detection limit of 0.01 pM, exhibits wide linearity range between 0.1 pM and 0.1 µM and is more sensitive than the BSA blocking strategy. The proposed GSSG-blocking strategy in immunoassay paves an attractive platform for other biomolecules detection reaching a lower detection limit.

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Electrochemical immunosensors combine the advantages of electrochemical technology and immunoassays with the advantages of a short response time, high sensitivity, high specificity and easy manipulation, and they continue to attract enormous interest for monitoring the amounts of analytes, including small organic molecules, proteins, microorganisms or anything to which an antibody can be raised.1-4 Most electrochemical immunosensor works through electrochemical redox species being suppressed when an analytes such as proteins, bind to the sensing interface.In consequence, the resistant layer is vital and necessary to ensure a reliable biosensor since nonspecific adsorption of proteins or contaminants in complicated samples on the sensing interface could also suppress the electrochemistry of the redox probes, i.e. interference the electrochemical detection.5-10 Recently, almost all established electrochemical immunosensors as well as other immunochemical experiments such as enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunohistochemical studies utilize BSA, an inactive and easy obtained protein, as a blocking reagent to block uncoated surface sites and endow the electrode resistant to nonspecific adsorption of contaminants.11-14 These selective

strategies

have

then

been

used

for

developing

electrochemical

immunosensor for small molecules in complex samples. The fact that the immunosensor can reliably detect different species in real samples demonstrates that the BSA layer ensures the antibody specifically combine with the sensing interface. However, the major challenge with this design is the relatively large size of BSA, which weights 66 500 Da15, can sterically hinder antigen-antibody recognition, especially for smaller volume analytes such as peptide, hormone16, virus17,18 and other small molecules19-21.Consequently, this reduces the available specific binding sites and ultimately the detection sensitivity. It is expected that smaller blocking reagent in an immunosensor can form a relatively uniform blocking film that surrounds the analyte while not causing steric hindrance effects to prevent specific recognition of analytes and its antibody, and finally enables sensing at a lower detection limit. 3



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Nevertheless, blocking reagents other than BSA is still rarely studied, especially for the detection of smaller molecules in complex samples. This study investigates using a small inactive peptide, oxidized glutathione (GSSG) to replace BSA, as a more efficient blocking reagent for immunosensor fabrication to reach lower detection limit and applying such electrochemical immunosensor for EGF detection in real sample. The immune complex, i.e. anti-EGF antibody functionalized silver nanoparticles (Ab-AgNPs) combining with EGF on the polydopamine films (PDA) film inhibit redox probe [Fe(CN)6]3-/[Fe(CN)6]4-accessing to electrode and thus electrochemical signal attenuation. Thus, the specific recognition process between Ab-AgNPs and EGF can be monitored by the changes of peak currents of the redox ([Fe(CN)6]3-/[Fe(CN)6]4-) probe. The proposed effect for blocking the non-specific sites of GSSG and BSA is shown in Scheme 1. In the immunosensor, a non-specific adherent surface of PDA film is casted onto GCE, and EGF is captured by adhesive PDA layer. Then, GSSG and BSA are used to block the free sites of the PDA films. The exposed EGF on the PDA films can recognize and combine with Ab-AgNPs. In the blocking process using BSA as blocking reagent, a proportion of EGF on the PDA surface was blocked by BSA and prevented from specifically combining with the Ab-AgNPs because of its large size. By comparison, GSSG offers sufficient steric permission for EGF and Ab-AgNPs-specific binding because GSSG is a small peptide consisting 6 amino acids, and is much smaller than EGF. In such way, a clear electrochemical signal change can be observed in the EGF immunosensor with GSSG as blocking reagent and enables the electrochemical immunosensor to reach a lower detection limit. The morphology of Ab-AgNPs combining on EGF(GSSG)/PDA/GCE and EGF(BSA)/PDA/GCE, where GSSG and BSA are used as blocking reagent respectively, was investigated by using scanning electron microscopy (SEM). As shown

in

Figure

1A,

Ab-AgNPs

are

scattered

evenly

across

the

EGF(GSSG)/PDA/GCE surface, while on the EGF(BSA)/PDA/GCE surface, fewer 4


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particles were observed (Figure 1B), indicating the easier binding of EGF and Ab-AgNPs in the GSSG blocking strategy compared with BSA blocking. These features are consistent with the aforementioned mechanism proposed in Scheme 1, further verifying the steric hindrance effects of BSA to antigen and antibody binding.

Little Steric Hindrance of GSSG to Ab-Ag Binding

Easy Binding Ab-Ag Binding

GSSG Blocking Obvious Steric Hindrance of BSA to Ab-Ag Binding

Difficult

BSA Blocking

Binding Ab-Ag Binding

GSSG

EGF

Ab-AgNPs

BSA

GCE

Scheme 1. Schematic representation of two different blocking processes using GSSG and BSA as blocking reagent to design the electrochemical immunosensor, respectively.

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A

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B

5.0 µm

5.0 µm

10.0µm Figure 1. SEM images of Ab-AgNPs captured onto the surface of substrates through antigen and antibody binding after GSSG (A) and BSA (B) blocking, respectively.

In the designed immunosensor, the electron transfer between the redox probe and the electrode surface was used to characterize the electrode modification and antigen-antibody-specific recognition process. Therefore, the degree of current signals decrease depending on the density of Ab-AgNPs covering on the electrode surface in this designed sensor. We investigated the linear responses to EGF using two blocking reagent respectively, shown in Figure 2. Electrochemical signals were recorded before and after Ab-AgNPs recognizing EGF, i.e. I1 (µA) and I2 (µA). Here, we define ∆I= (I1-I2) and S = ∆I(µA)/I1(µA) to characterize the effect of two blocking reagent towards EGF detection. The linear equation of GSSG blocking model is S= 0.0977 lgCEGF(µM) + 0.946, with the correlation coefficient of 0.980 (Figure 2a). The linear calibration range of EGF in the GSSG blocking strategy is from 0.1 pM to 0.01 µM and the detection limit is 0.01 pM. On the other hand, the linear equation of BSA blocking model is S= 0.0754 lgCEGF(µM) + 0.309, with the correlation coefficient of 0.994 (Figure 3b). However, the linear calibration range in the BSA blocking strategy is narrower, i.e. from 1 nM to 5 µM, and detection limit is higher, i.e. 0.1 nM compared with that in GSSG blocking model. In the EGF concentration range between 1 nM and 10 nM, S of GSSG blocking model is higher than BSA blocking model, illustrating that GSSG blocking strategy amplifies signal changes and 6


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improves the sensitivity of the immunosensor. Consequently, in comparison with BSA blocking, GSSG blocking enable the immunosensor reach lower detection limit, wider linear range and higher sensitivity.

0.8 0.6

a b

S

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0.4 0.2 0.0 -8

-6

-4

-2

0

2

lgCEGF/µM

Figure 2.The linearity of the EGF immunosensor with GSSG (a) and BSA (b) as blocking reagent, respectively. Each data point represent mean ± SD (n=3).

In such designed immunosensor, the degree of current signals decreases depending on electrochemical redox species being suppressed when analytes bind to the sensing interface. However, before the Ab-AgNPs combining to designed sensor, the blocking film itself, especially for BSA which is a huge protein compared with GSSG, may already passivate the electrochemical signal and thus result in a lower sensitivity and higher detection limit. In order to further investigate the mechanism underlying the excellent analytical properties of the immunosensor with GSSG as blocking reagent, inhibition of current signals by the GSSG and BSA blocking effects were studied. Figure 3 shows the current response from GSSG blocking (Figure 3A) 7



and BSA blocking (Figure 3B). As expected, current signals of redox probe decreased after GSSG and BSA blocking, respectively. The corresponding inhibition ratio is shown (Figure 3C) and calculated by using the following expression: Inhibition (%) = [(I0-Iblocking)/I0]×100%, where I0 and Iblocking are the oxidation peak current before and after blocking, respectively. The calculation results demonstrate that BSA inhibition effect was more obvious than GSSG, which can be explained by the different size of these two reagents. 22,23 In this way, the comparison shows that GSSG benefits better to probe accessing to the electrode surface and enables the immunosensor to reach a lower detection limit and higher sensitivity.

20

20

1

A 10

B

1

10

2

2 0

-10

-10

-20

0

I/µA

I/µA

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-0.4

0.0

0.4

-20

0.8

-0.4

0.0

0.4

E/V vs. Ag/AgCl

E/V vs. Ag/AgCl

C Blocking I0 (µA)

Iblocking (µA)

I0- Iblocking(µA)

Inhibition (%)

GSSG

12.33 ± 2.01

9.86 ± 1.68

2.47 ± 0.33

20.0 ± 2.6

BSA

14.31 ± 2.89

11.12 ± 2.16

3.19 ± 0.73

22.3 ± 5.1

Reagent

Figure 3. Cyclic voltammograms (A and B) and current calculations (C) for GSSG (A) and BSA (B) blocking. The black curves (A-1 and B-1) represented CV response of PDA/GCE, and the red curve represented CV response of EGF(GSSG)/PDA/GCE 8


0.8

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(A-2) or EGF (BSA)/PDA/GCE (B-2). The following table (C) illustrates the corresponding oxidation peak current. Each data in table represent mean ± SD (n=3).

In addition, this GSSG blocking strategy was further verified in an electrochemical immunosensor for the detection of EGF. EGF is a 53-amino acid polypeptide that presents in mammalian tissues fluid, serum and urine, and mediates several important cellular metabolism processes such as signal events and cell cycle progression.24-26 PDA films can interact with and load EGF through π-π conjugation, p-π conjugation and hydrogen bonding27-29 as well as preserve the biocompatibility of the EGF, and thus significantly improve the biosensing performance. In addition, nanomaterials that were used for amplifying signal have been widely reported in previous work.30-33 In the present work, the signal amplifying effect of Ab-AgNPs was illustrated in Supporting Information. The fabrication process of this EGF immunosensor is depicted in Scheme S1 which demonstrated the sensing interface consists of three vital components: PDA films, GSSG, and Ab-AgNPs. Detailed electrode modification process, synthesis process and characterization of Ab-AgNPs (Figure S1, Figure S2 and Figure S3), the specific recognition of EGF by Ab-AgNPs (Figure S4), CV and EIS from different fabrication stages (Figure S5) and SEM images of different concentration of EGF captured by PDA film and linearity of the EGF immunosensor (Figure S6) are shown in the Supporting Information. These improved properties of the established EGF immunosensor are comparable or better than those of previous work for measuring EGF and are summarized in Table S1 in Supporting Information.34-37As far as we know, our method has the lowest detection limit and the widest linear range. Furthermore, this method was applied to detect the concentration of EGF in dialysates from submandibular gland tissue. The result of EGF concentration in this dialysate was approximately 8.5×10-14 M. Moreover, in order to validate the proposed method, the 9



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standard addition methodology was used to determine the recovery of EGF in the dialysate samples (Table S2 in the Supporting Information). The recoveries are ranged between 98.1% and 108.3%, indicating that our established method is promising for determining EGF in biological samples. In conclusion, this letter utilizes GSSG to replace BSA as blocking reagent for immunosensor fabrication reaching a lower detection limit, i.e. 0.01 pM. This result is ascribed to GSSG forming a relatively uniform blocking film around the EGF on the PDA surface without hindering EGF and Ab-AgNPs binding. This study establishes a promising alternative platform for immunoassays of disease-related biomolecules at extremely low concentration.

ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation (21375088), Scientific Research Project of Beijing Educational Committee (KM201410028006), Youth Talent Project of the Beijing Municipal Commission of Education (CIT&TCD201504072), and Scientific Research Base Development Program of the Beijing Municipal Commission of Education.

ASSOCIATED CONTENT Supporting Information Available Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES (1) Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.; Chen, X. ACS Nano 2012, 6, 6546-6561. (2) Johnson,A.; Song, Q. F.;Ferrigno, P. K.;Bueno, P. R.; Davis, J. J.Anal. Chem.2012, 84, 6553-6560. (3)Wang, Q.; Song, Y.; Chai, Y; Pan, G.; Li, T.; Yuan, Y.; Yuan, R.Biosens. Bioelectron.2014, 60, 118-123. (4) Liu, G.; Iyengar, S. G.; Gooding, J. J. Electroanalysis2013, 25, 881-887. (5) Makaraviciute, A.; Ramanaviciene, A. Biosens. Bioelectron. 2013, 50, 460-471. (6) Bogomolova, A.; Komarova, E.; Reber, K.; Gerasimov, T.; Yavuz, O.; Bhatt, S.; Aldissi, M. Anal. Chem. 2009, 81, 3944-3949. (7) Nassef, H. M.; Civit, L.; Fragoso, A.; O'Sullivan, C. K. Anal. Chem. 2009, 81, 5299-5307. (8) Liu,G.; Gooding,J.J.Langmuir2006, 22, 7421-7430. (9) Li, J.; Song, S. P.; Li, D.; Huang, Q.; Zhao, Y.; Fan, C. H. Biosens. Bioelectron.2009, 24, 3311-3315. (10) Le, N. C. H.; Gubala, V.; Gandhiraman, R. P.; Daniels, S.; Williams, D. E. Langmuir 2011, 27, 9043-9051. (11) Wang, J.; Han, H.; Jiang, X.; Huang, L.; Chen, L.; Li, N. Anal. Chem. 2012, 84, 4893-4899. (12) Jeong, B.; Akter, R.; Han, O. H.; Rhee, C. K.; Rahman, M. A. Anal. Chem. 2013, 85, 1784-1791. (13) Chen, L.; Zeng, X.; Si, P.; Chen, Y.; Chi, Y.; Kim, D.; Chen, G. Anal. Chem. 2014, 11



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

86, 4188-4195. (14) Fowler, J. M.; Stuart, M. C.; Wong, D. K. Y. Anal. Chem. 2007, 79, 350-354. (15) Chen, M.; Ling, M.; Lai, K.; Pramudityo, E. Biomacromolecules 2012, 13, 4022-4031. (16) Kaushik, A.; Vasudev, A.; Arya, S. K.; Bhansali, S. Biosens. Bioelectron. 2013, 50, 35-41. (17) Wang, X.; Wang, L.; Yang, W.; Ai, S. Sensor. Actuat. B-Chem. 2014, 194, 276-282. (18) Wang, R.; Wang, Y.; Lassiter, K.; Li, Y.; Hargis, B.; Tung, S.; Berghman, L.; Bottje, W. Talanta 2009, 79, 159-164. (19) Zhu, Y.; Cao, Y.; Sun, X.; Wang, X. Sensors 2013, 13, 5286-5301. (20) Miao, X.; Yuan, R.; Chai, Y.; Shi, Y.; Yuan, Y. Biochem. Eng. J. 2008, 38, 9-15. (21) Yuan, R.; Zhuo, Y.; Chai, Y.; Zhang, Y.; Sun, A. Sci. China, Ser. B 2007, 50, 97-104. (22) Furnus, C. C.; de Matos, D. G.; Picco, S.; Garcia, P. P.; Inda, A. M.; Mattioli, G.; Errecalde, A. L. Anim. Reprod. Sci. 2008, 109, 88-99. (23) Rose, S.; Melnyk, S.; Pavliv, O.; Bai, S.; Nick, T. G.; Frye, R. E.; James, S. J. Transl. Psychiat. 2012, 2, 1-8 (24)Hauner, H.; Rohrig, K.; Petruschke, T. Eur. J. Clin. Invest. 1995, 25, 90-96. (25) Thomas T. P.; Shukla, R.; Kotlyar, A.; Liang, B.; Ye, J.; Norris T. B.; Baker, J. R. Biomacromolecules 2008, 9, 603-609. (26) Bae, Y. S.; Kang, S. W.; Seo, M. S.; Baines, I. C.; Tekle, E.; Chock, P. B.; Rhee, S. G. J. Biol. Chem.1997, 272, 217-221. 12


Page 12 of 14

Page 13 of 14

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


(27) Liu, Y.; Ai. K.; Lu L. Chem. Rev. 2014, 114, 5057-5115. (28) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426-430. (29) Liu, Q.; Wang, N.; Caro, J.; Huang, A. J. Am. Chem. Soc. 2013, 135, 17679-17682. (30) Chang, T.; Wu, C.; Wang S.; Chau, L.; Hsieh, W. Anal. Chem. 2013, 85, 245-250. (31) Keighron, J. D.; Akesson, S.; Canns, A. Langmuir 2014, 30, 11348-11355. (32) Hu, C.; Yang, D.; Xu, K.; Cao, H.; Wu, B.; Cui, D.; Jia, N. Anal. Chem. 2012, 84, 10324-10331. (33)Gui, G.; Zhuo, Y.; Chai, Y.; Xiang, Y.; Yuan, R. Anal. Chem. 2014, 86, 5873-5880. (34) Carpenter, G.; Lembach, K. J.; Morrison, M. M.; Cohen, S. J. Biol. Chem. 1975, 250. 4297-304. (35) Vazquez, J.; Freyre, M.; Duarte, C.; Ferra, E.; Lopez, I.; Perez, E.; Gavilondo, J.V. Biotecnologia aplicada 1990, 7, 42-51. (36) Pesonen K.; Alfthan, H.; Stenman, U. H.; Viinikka, L.; Perheentupa, J. Anal. Biochem. 1986, 157. 208-211. (37) Lee, K. W.; Hwang, K. H.; Kim, C. S.; Han, K.; Chung, Y. B.; Park, J. S.; Lee, Y. M.; Moon, D. C. Arch. Pharm. Res. 2001, 24. 355-359.

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Electrochemical Immunosensor for Detection of Epidermal Growth

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