View Article Online View Journal
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. Guo, Y. Sha, Y. Hu and S. Wang, Chem. Commun., 2016, DOI: 10.1039/C6CC00787B.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 4
ChemComm
ChemComm
Dynamic Article Links► View Article Online
DOI: 10.1039/C6CC00787B
Cite this: DOI: 10.1039/c0xx00000x
COMMUNICATION
www.rsc.org/chemcomm
In-Electrode vs. On-Electrode: Ultrasensitive Electrochemiluminescence Immunoassay
Faradaycage-Type
5
10
15
20
25
30
35
40
45
50
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A new-concept "in-electrode" Faradaycage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement on sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively. Immunoassays are analytical techniques based on the avidity and specificity of the antigen-antibody reaction.1 They have been successfully exploited for screening the presence, progression and therapeutic outcome of disease or infection with molecular markers,2 and monitoring the presence and variation of drugs, toxic substances and environmental contaminants.2b, 3 Up to now, mainstream immunoassays mainly include enzyme-linked immunoabsorbent assay (ELISA),4a radioimmunoassay (RIA),4b electrophoretic immunoassay (EIA),4c fluorescence immunoassay (FIA),4d chemiluminescence immunoassay (CLIA),4e 4f electrochemical immunoassay (ECIA) and electrochemiluminescence immunoassay (ECLIA),4g etc. Among them, ECLIA has attracted particular attention recently due to its high selectivity and sensitivity, simple setup, low background signal and absence of radioactive or toxic markers.5 ECLIA has been commercialized and been extensively applied in clinical settings to detect tens kinds of analytes using 50-150 µL of serum with detection limits at the picomolar level.6 However, many blood serum or plasma concentrations of analytes associated with early stage cancers and infectious diseases range from 10−16 to 10−12 mol/L.7 In addition, analysis of tissues is greatly restricted by the sample amount, and can be achieved only by increasing detection sensitivity.8 These urge us to develop an ultrasensitive ECLIA to selectively detect low-abundance analytes in trace amount of real samples. Currently, sandwich-type immunoassay is the commonly used mode of ECLIA, in which high level of sensitivity and specificity is achieved due to the use of a couple of matching antibodies.9 Generally, the detection mechanism relies on three methods presented in Figure 1. A capture antibody is first immobilized on the electrode surface (Figure 1A and Figure 1B) or magnetic beads with the diameters of several micrometers (Figure 1C). After the binding of antigen in the sample, a detector antibody labeled with electrochemiluminophores (Figure 1A and Figure 1C) or a nanoparticle immobilized simultaneously by detector antibody and electrochemiluminophore (Figure 1B) is added to obtain an immunocomplex. The quantity of the labeled detector antibody is directly proportional to the amount of antigen present This journal is © The Royal Society of Chemistry [year]
55
60
65
70
75
80
85
90
in the sample which increases the detectable signal with increasing target analyte. However, the electrochemiluminous efficiency and the labeling amount of electrochemiluminophores limit the enhancement of sensitivity of the proposed assay to some extent.10 The former is very difficult to improve theoretically as well as practically,11 while the latter is limited due to following reasons: (1) the surface area of the detector antibody or the nanoparticle is small; (2) usually no more than 20 electrochemiluminophores could be labeled on a detector antibody molecule to maintain its immunocompetence; and (3) the diameter of nanoparticle should usually be smaller than 100 nm to ensure reliable capture.
Figure 1. Factors restricting the sensitivity of the conventional “onelectrode” sandwich-type ECLIA.
As a matter of fact, the critical factor that restricts the improvement of the detection sensitivity of ECLIA is neglected, i.e., the sandwich-type of assay format itself. In sandwich-type ELISA, RIA, EIA, FIA and CLIA, the distance between the markers labeled on the detector antibody and the solid plate on which the formed immunocomplex immobilized is entirely irrelevant with the generation of detection signals. However, in the case of ECLIA, the distance between electrochemiluminophores labeled on the detector antibody and the electrode surface directly influences the generation of signals, which ranges from ~0 to several micrometers decided by the size of the immunocomplex and the labeling sites. It means that most of the electrochemiluminophores labeled are positioned beyond the outer Helmholtz plane (OHP), the thickness of which is from several angstroms to several nanometers normally.12 In electrode kinetics, OHP is defined as the plane where solvated ions can approach the electrode only nearest to.13 When proper electrode potential is applied, the potential at the OHP is correspondingly modulated to activate the electrons of electrochemiluminophores near the OHP with the same energy as the electrons on the electrode. Then, radiationless electronic rearrangements will occur followed by isoenergetic electron transfer between electrochemiluminophores near the OHP and the electrode, i.e., electrochemical reaction is launched. On the other hand, for electrochemiluminophores labeled on the detector antibody at a fixed distance x from the electrode, the act of electron transfer is [journal], [year], [vol], 00–00 |1
ChemComm Accepted Manuscript
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
Zhiyong Guo,* Yuhong Sha, Yufang Hu, Sui Wang
ChemComm
Page 2 of 4 View Article Online
5
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
10
15
20
usually considered as tunneling of the electron between states in the electrode and those on the electrochemiluminophores. Typically, the probability of electron tunneling is proportional to exp(–βx), where β is a factor that depends upon the height of the energy barrier and the nature of the medium between the states. For proteins such as antigen and antibody, β could be roughly estimated as 0.1 nm–1 considering the typical value for saturated carbon chains. Thus, it is easy to understand that electrochemiluminophores near the OHP are "effective" in participating in the electrochemical reactions and emitting electrochemiluminescence (ECL) signals, while electrochemiluminophores far away from the OHP are "ineffective".12, 13 To be simple, once the distance between labeled electrochemiluminophores and the electrode is 10 times higher than that between the OHP and the electrode, the probability of electron tunneling will be only 5 ppm, indicating ineffective electrochemical reactions. In reality, this is just the case in current sandwich-type assay format of the ECLIA in which the immunocomplex is immobilized "on-electrode", resulting in the restriction for improving the detection sensitivity. If the immunocomplex is immobilized "in-electrode", then all electrochemiluminophores labeled would be electrochemically "effective", and thus a higher detection sensitivity of ECLIA could be achieved.
45
50
55
60
65
70
75
80
85
25
Figure 2. (A) Structure of the capture and detector units. (B) The detection protocol and sensitivity improvement mechanism of “inelectrode” Faradaycage-type ECLIA.
30
35
40
As a proof of concept for ECLIA based on "in-electrode", we designed a novel biosensing platform using two kinds of multifunctionalized graphene oxide (GO) materials, capture unit (antibody(Ab1)-Fe3O4 nanoparticles@GO, referred as Ab1nanoFe3O4@GO) and detector unit (antibody(Ab2)&N-(4aminobutyl)-N-ethylisoluminol)@GO, referred as (Ab2&ABEI)@GO) (Figure 2A); the preparation process of which are provided in supporting information. GO, which is a single atom thick and two dimensional carbon nanomaterial,14 can contribute greatly to this process because of its unique properties, such as large surface area for high loading and good electric conductivity for favoring the energy and electron transfer.
90
95
100
105
2| Journal Name, [year], [vol], 00–00
The capture unit, which was prepared by immobilizing capture antibody Ab1 on GO coated by Fe3O4 nanoparticles (nanoFe3O4@GO), had three functions: Ab1 acted as the capture device for antigen through antigen-antibody immunoreaction; nanoFe3O4@GO provided the feasibility of one-step preparation; and the size of nanoFe3O4@GO ensured the construct of a nanoscale immune complex system to achieve "in-electrode" mode. And the detector unit, which was prepared by simultaneously immobilizing detector antibody Ab2 and electrochemiluminophore ABEI on GO sheet, also had three functions: ABEI acted as the electrochemiluminophore; Ab2 acted as the recognition device for antigen through antigenantibody immunoreaction; GO could load a great amount of ABEI molecules and enabled all of them effective in emitting ECL signals and thus improved the detection sensitivity greatly. The detection protocol is shown in Figure 2B and described detailedly in supporting information. Firstly, a simple one-step preparation of ECL immunosensor was achieved through dropping and attracting certain amount of capture unit on the surface of a magnetic glass carbon electrode in a few minutes, and the ECL immunosensor was ready for assay. Secondly, the sample containing the antigen was added and incubated. Thirdly, detector unit was added and incubated. Finally, the potential was applied to launch the electrochemical reaction, the ECL signal was generated and recorded, and the antigen was quantified through the relationship between the antigen concentration and the ECL intensity. Though this protocol seems roughly same as that of the sandwich-type ECLIA, the assay scheme is significantly different. GO in the detector unit extends the OHP of the electrode, making all the electrochemiluminophores labeled effective as such they are immobilized directly on the surface of the electrode or even "in the electrode" (Figure 2B). In this case of "in-electrode" state, detector unit lapped on the electrode surface directly, so electrons could flow freely between the detector unit and the electrode. In other words, detector unit had become de facto part of the electrode. Considering the skin effect of the electric field, the whole immune complex system seemed like a Faraday cage with some small windows because some Ab1 or Ab2 molecules were immobilized on the edge of the GO in capture unit and detector unit. The Ab1-antigen-Ab2 immunocomplex was sealed in the Faraday cage. All electrochemiluminophores labeled on detector unit were on the surface of the Faraday cage, so all of them could take part in the electrode reaction to emit ECL signal, profiting from the free flow of electrons resulting from the isopotential character of the Faraday cage. Thus, the two bottlenecks which restrict the sensitivity improvement of sandwich-type ECLIA were substantially eliminated, i.e., the labeling amount of electrochemiluminophores increased and all electrochemiluminophores labeled were ensured to be "effective". To demonstrate the feasibility and sensitivity of this proposed "in-electrode" Faradaycage-type ECLIA, neurotensin (NT) was analyzed as a model target analyte. NT is a 13 amino acid neuropeptide, involved in a number of important biological processes including dopamine transmission, analgesia, hypothermia, etc.15 Recent reports suggest that the detection of very low concentration of NT peptides, which ranges from several to tens of pg/mL and even lower levels in some diseases, is helpful to identify sub-healthy humans or early-stage patients.16 Scanning electron microscope (SEM) was used to characterize the stepwise fabrication and detection process of the immunosensor. The SEM image of nanoFe3O4@GO (Figure 3A) exhibits that numerous Fe3O4 nanoparticles were well-dispersed onto the uniform GO film (Figure S1) with a typical twodimensional structure. In the case of capture unit (Figure 3B) and This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
DOI: 10.1039/C6CC00787B
Page 3 of 4
ChemComm View Article Online
5
NT-capture unit (Figure 3C), the surface became much rougher and richer in texture due to the successive immobilization of the capture unit and the antigen NT. When immobilized finally (Figure 3D), detection unit covered the antigen and the capture unit with some bulges, demonstrating the existence of the immunocomplex detector unit-NT-capture unit.
45
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
50
55
60
electrode" Faradaycage-type ECLIA. Afterwards, electrochemical impedance spectroscopy (EIS) was used to monitor the interface properties of the Faradaycagetype ECLIA in the assembly process (Figure S3). Compared with electron transfer resistance, Ret, of bare MGCE (curve a), the Ret of nanoFe3O4@GO/MGCE (curve b) was lower due to the good electronic conductivity of nanoFe3O4@GO. In the case of capture unit/MGCE (curve c) and NT–capture unit/MGCE (curve d), Ret increased markedly because Ab1, bovine serum protein (BSA) and NT are proteins which would hinder the electron transfer on the electrode interface. As to the detector unit–NT–capture unit/MGCE (curve e), Ret decreased greatly, demonstrating the accomplishment of the "in-electrode" Faradaycage-type ECLIA. When the detector unit was immobilized to form the immunocomplex, GO in the detector unit lapped on the electrode surface directly as if becoming a part of the electrode and thus building the as-designed Faradaycage-type ECLIA. Therefore, electron could transfer freely between the electrode and the electrochemical probe without the hindrance of Ab1, Ab2, BSA and NT, thus Ret decreased near to zero.
Figure 3. Representative SEM images of (A) nanoFe3O4@GO, (B) capture unit, (C) NT-capture unit, and (D) detector unit-NT-capture unit.
10
15
20
25
30
35
40
Figure 4. (A) ECL signals of (a) magnetic glassy carbon electrode (MGCE), (b) capture unit/MGCE, (c) NT–capture unit/MGCE and (d) detector unit–NT–capture unit/MGCE. (B) ECL signals of (a) detector unit–NT–capture unit/MGCE, (b) the same process as (a) without NT, (c) the same process as (a) without ABEI, (d) the same process as (a) without Ab2 and (e) the same process as (a) using g-C3N4 instead of GO in the detector unit. The concentration of NT is 5 pg/mL.
ECL signals at each immobilization step were recorded to monitor the fabrication of the immunosensor. As shown in Figure 4A, the ECL intensities of bare magnetic glassy carbon electrode (MGCE), capture unit/MGCE and NT–capture unit/MGCE were all near to zero, while that of detector unit–NT–capture unit/MGCE was strong and stable at several thousand arbitrary unit, indicating that the ECL signal was originated from the electrochemiluminophores ABEI immobilized on the detector unit. To obtain further convincing support on the signal amplification capability of the developed Faradaycage-type ECLIA strategy, the ECL intensities of the sensors incubated with/without NT, ABEI or Ab2 were compared. As depicted in Figure 4B, the absence of ABEI resulted in negligible ECL signal. It is evident that the high ECL signal could be ascribed to ABEI in detector unit. While the absence of NT or Ab2 led to a sharp decrease in ECL intensity, implying that NT or Ab2 is also important for loading detector unit and supporting it to achieve the proposed efficient ECL "in-electrode" strategy. If graphitelike carbon nitride (g-C3N4) with similar size as GO (Figure S2) was used to build the detector unit instead of GO, the electric conductivity of which was much poorer than that of GO, the ECL intensity decreased down to about one tenth (Figure 4B, curve e), demonstrating that the good electric conductivity of GO help increase the ECL intensity well and the validity of the "inThis journal is © The Royal Society of Chemistry [year]
65
70
75
80
85
Figure 5. (A) ECL intensity in the presence of NT at different concentrations; (B) Calibration curve for NT determination; (C) Selectivity of the ECL immunosensor toward other proteins; and (D) ECL intensity of the proposed immunosensor to 0.005 fg/mL NT in the presence of 1 µg/mL of other proteins, individually, or in a mixture with all these proteins.
Under the optimal detection conditions of pH of the electrolyte solution (9.74), concentration of H2O2 in electrolyte solution (1 mmol/L), incubation temperature (37 °C) and incubation time (1 h) (Figure S4), the ECL intensity of the proposed Faradaycagetype immunosensor increased with the increasing concentration of NT (Figure 5A). The calibration plot showed a good linear relationship between the ECL intensity (y) and the logarithmic value of NT concentration (x) ranging from 5×10-3 fg/mL to 5×104 fg/mL with a correlation coefficient r of 0.9989 (Figure 5B). The detection limit was 2×10-3 fg/mL at a signal-to-noise ratio of 3, which is much better than those detection limits from other methods reported in literature (Table S1). To demonstrate the sensitivity enhancement of this emerging "in-electrode" Faradaycage-type ECLIA further, we also measured NT with conventional "on-electrode" sandwich-type for comparison, using magnetic beads immobilized by Ab1 as capture unit and Ab2 labeled with ABEI as detector unit. The linear relationship ranged from 10 fg/mL to 1×105 fg/mL, with the detection limit of 5 fg/mL. Results showed that the detection sensitivity increased about three orders of magnitude when using "in-electrode" Faradaycage-type than that of "on-electrode" sandwich-type Journal Name, [year], [vol], 00–00 |3
ChemComm Accepted Manuscript
DOI: 10.1039/C6CC00787B
ChemComm
Page 4 of 4 View Article Online
DOI: 10.1039/C6CC00787B
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
10
15
20
25
30
35
40
45
50
55
65
2 (a) R. Ekin, Nucl. Med. Biol., 1994, 21, 495-521; (b) E. N. Brown, T. J. 3
70
4
75
80
85
5
90
6 7 95
McDermott, K. J. Bloch and A. D. McCollom, Clin. Chem., 1996, 42, 893903. (a) J. M. Hicks, Hum. Pathol., 1984, 15, 112-116; (b) J. Sherry, Chemosphere, 1997, 34, 1011-1025; (c) A. Hatch, A. E. Kamholz, K. R. Hawkins, M. S. Munson, E. A. Schilling, B. H. Weigl and P. Yager, Nat. Biotechnol., 2001, 19, 461-465. (a) K. D. Chandra, K. V. Sandeep, T. O. Feidhlim, O. Brian, D. M. Brian and O. Richard, Anal. Chem., 2010, 82, 7049-7052; (b) D. H. Charles, Anal. Chem., 1973, 45, 878-890; (c) N. M. Victor and Y. M. Tamara, Anal. Chem., 2003, 75, 6813-6819; (d) S. I. Stoeva, J. S. Lee, J. E. Smith, S. T. Rosen and C. A. Mirkin, J. Am. Chem. Soc., 2006, 128, 8378-8379; (e) A. T. Hashem, G. B. Dean, J. B. John, Y. Chen, S. C. Richard, D. S. Renuka, A. E. Robert, E. G. Jose, S. H. Richard, P. K. Kenneth, K. L. Senia, E. M. Michael, L. M. Terri, D. M. James, H. O. Bruce, G. R. Sarada, S. Michael, A. S. Barry, S. Nir, R. S. Katherine, C. T. Jeff, G. P. Wang and W. H. Xie, J. Am. Chem. Soc., 2013, 135, 4191-4194; (f) R. H. William and H. B. Halsall, Anal. Chem., 1985, 57, 1321A-1331A; (g) Y. H. Sha, Z. Y. Guo, B. B. Chen, S. Wang, G. P. Ge, B. Qiu and X. H. Jiang, Biosens. Bioelectron., 2015, 66, 468-473. (a) W. Miao, Chem. Rev., 2008, 108, 2506-2553; (b) M. S. Wu, H. W. Shi, L. J. He, J. J. Xu and H. Y. Chen, Anal. Chem., 2012, 84, 4207-4213; (c) Y. Zhuo, N. Liao, Y. Q. Chai, G. F. Gui, M. Zhao, J. Han, Y. Xiang and R. Yuan, Anal. Chem., 2014, 86, 1053-1060; (d) M. M. Richter, Chem. Rev., 2004, 104, 3003-3036; (e) Z. Y. Liu, W. J. Qi and G. B. Xu, Chem. Soc. Rev., 2015, 44, 3117-3142. (a) X. Yu, B. Munge, V. Patel, G. Jensen, A. Bhirde, J. D. Gong, S. N. Kim, J. Gillespie, J. S. Gutkind, F. Papadimitrakopoulos and J. F. Rusling, J. Am. Chem. Soc., 2006, 128, 11199-11205. D. M. Rissin, C. W. Kan, T. G. Campbell, S. C. Howes, D. R. Fournier, L. Song, T. Piech, P. P. Patel, L. Chang, A. J. Rivnak, E. P. Ferrell, J. D. Randall, G. K. Provuncher, D. R. Walt and D. C. Duffy, Nat. Biotechnol., 2010, 28, 595-599.
8 (a) S. Krishnan, V. Mani, D. Wasalathanthri, C. V. Kumar and J. F. Rusling, Angew. Chem. Int. Ed., 2011, 50, 1175-1178; (b) K. D. Wegner, 100
105
110
115
120
125
This study was financially supported by National Natural Science Foundation of China (Nos. 41576098 and 81273130) and K. C. Wong Magna Fund in Ningbo University.
Z. W. Jin, S. Lindén, T. L. Jennings and N. Hildebrandt, ACS Nano, 2013, 7, 7411-7419. 9 (a) P. Vicennati, N. Bensel, A. Wagner, C. Créminon and F. Taran, Angew. Chem. Int. Ed., 2005, 44, 6863-6866; (b) J. Quinton, S. Kolodych, M. Chaumonet, V. Bevilacqua, M. Nevers, H. Volland, S. Gabillet, P. Thuery, C. Creminon and F. Taran, Angew. Chem. Int. Ed., 2012, 51, 6144-6148. 10 (a) J. I. Kim, I. S. Shin, H. Kim and J. K. Lee, J. Am. Chem. Soc., 2005, 127, 1614-1615; (b) X. Liu, L. Shi, W. Niu, H. Li and G. Xu, Angew. Chem., 2007, 119, 425-428; (c) X. Q. Liu, L. H. Shi, W. X. Niu, H. J. Li and G. B. Xu, Angew. Chem. Int. Ed., 2007, 46, 421-424. 11 (a) S. Rashidnadimi, T. H. Hung, K. T. Wong and A. J. Bard, J. Am. Chem. Soc., 2008, 130, 634-639; (b) J. W. Oh, Y. O. Lee, T. H. Kim, K, C. Ko, J. Y. Lee, H. Kim and J. S. Kim, Angew. Chem. Int. Ed., 2009, 48, 2522-2524. 12 S. P. Du, Z. Y. Guo, B. B. Chen, Y. H. Sha, X. H. Jiang, X. Li, N. Gan and S. Wang, Biosens. Bioelectron., 2014, 53, 135-141. 13 (a) A. J. Bard and L. R. Faulkner, Electrochemical Methods, Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2001; (b) M. Nakamura, H. Kaminaga, O. Endo, H. Tajiri, O. Sakata and Nagahiro Hoshi, J. Phys. Chem. C, 2014, 118, 22136-22140. 14 (a) K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I. V. Grigorieva and A. A. Firsov, Science, 2004, 306, 666-669; (b) R. F. Service, Science, 2009, 324, 875-877; (c) S. Park and R. S. Ruoff, Nat. Nanotechnol., 2009, 4, 217-224. 15 (a) W. H. Rostène and M. J. Alexander, Front. Neuroendocrinol., 1997, 18, 115-173; (b) J. Gobom, K. O. Kraeuter, R. Persson, H. Steen, P. Roepstorff and R. Ekman, Anal. Chem., 2000, 72, 3320-3326. 16 (a) A. Mascarin, I. E. Valverde, S. Vomstein, and T. L. Mindt, Bioconjugate Chem., 2015, 26, 2143-2152; (b) Z. Y. Guo, S. P. Du, B. B. Chen, Y. H. Sha, B. Qiu, X. H. Jiang, S. Wang, and X. Li, Electrochim. Acta, 2014, 135, 519-525.
130
Notes and references
60
School of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, China. Tel: 86-574-87600798 E-mail: [email protected] † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. 1 (a) S. Brakmann, Angew. Chem. Int. Ed., 2004, 43, 5730-5734; (b) R. Batrla and B. W. Jordan, Ann. N. Y. Acad. Sci., 2015, 1346, 71-80; (c) J. Durner, Angew. Chem. Int. Ed., 2010, 49, 1026-1051.
4| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
5
format. In order to illustrate the possible interference of non-specific antigens, various species of interfering proteins including alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), neuronspecific enolase (NSE), bovine serum albumin (BSA) and squamous cell carcinoma antigen (SCCA), at the concentration of 1 µg/mL, were measured using the proposed immunoassay. The ECL intensities of them were all around 150, nearly similar to that of the blank sample, and much lower than that of 0.005 fg/mL NT (Figure 5C), demonstrating that the immunosensor developed has an excellent specificity for the determination of target protein NT. Meanwhile, the interference of these proteins tested in presence of NT either individually or in a mix (Figure 5D) exhibited unchanged coexistence ECL intensity in the presence of other interference proteins. These results demonstrate that the developed sensing method is highly selective toward NT against other interference molecules, due to the highly specific binding capability of antibody to NT. To assess the potential application of this method for real biological samples, a NTtrapping test was performed using human serum or urine samples spiked with different concentrations of NT. The observed recoveries were between 94.4% and 112.6% (Table S2), providing a new insight into NT-relevant determination in biological fluids sample. The stability of the developed immunosensor was investigated by assessing its relative activity after storage at 4 °C in dark for one month. The ECL intensity for the detection of 5 fg/mL NT was 92.5 ± 7.4% (n = 5) of the initial value which was obtained when the immunosensor was constructed freshly, indicating that the developed immunosensor has acceptable stability during storage. The relative standard deviations (RSD) of an intra-assay and an inter-assay were used to evaluate the reproducibility of the proposed immunosensor. The intra-assay precision was 7.3%, which was evaluated from the response to 5 fg/mL NT at five different immunosensors fabricated in the same batch, while an inter-assay precision of 7.8% was obtained by assaying 5 fg/mL NT with five different immunosensors prepared with different batches (Figure S5). Results indicated the excellent precision and reproducibility. In addition, the reversibility of this immunosensor could be easily achieved in the absence and presence of the external magnetic field. To conclude, we demonstrated a new-concept of "In-Electrode" "Faradaycage-Type" immunoassay mode using multifunctionalized GO composite materials, aiming at eliminating the key bottlenecks restricting the improvement of the sensitivity of ECLIA based on conventional "On-Electrode" "Sandwich-Type" immunoassay mode. The results confirmed that the detection sensitivity of the model protein reached the level of ag/mL and showed high specificity, stability and reproducibility, providing a great application prospect for the early diagnosis of NT-related diseases.
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. Guo, Y. Sha, Y. Hu and S. Wang, Chem. Commun., 2016, DOI: 10.1039/C6CC00787B.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 4
ChemComm
ChemComm
Dynamic Article Links► View Article Online
DOI: 10.1039/C6CC00787B
Cite this: DOI: 10.1039/c0xx00000x
COMMUNICATION
www.rsc.org/chemcomm
In-Electrode vs. On-Electrode: Ultrasensitive Electrochemiluminescence Immunoassay
Faradaycage-Type
5
10
15
20
25
30
35
40
45
50
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A new-concept "in-electrode" Faradaycage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement on sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively. Immunoassays are analytical techniques based on the avidity and specificity of the antigen-antibody reaction.1 They have been successfully exploited for screening the presence, progression and therapeutic outcome of disease or infection with molecular markers,2 and monitoring the presence and variation of drugs, toxic substances and environmental contaminants.2b, 3 Up to now, mainstream immunoassays mainly include enzyme-linked immunoabsorbent assay (ELISA),4a radioimmunoassay (RIA),4b electrophoretic immunoassay (EIA),4c fluorescence immunoassay (FIA),4d chemiluminescence immunoassay (CLIA),4e 4f electrochemical immunoassay (ECIA) and electrochemiluminescence immunoassay (ECLIA),4g etc. Among them, ECLIA has attracted particular attention recently due to its high selectivity and sensitivity, simple setup, low background signal and absence of radioactive or toxic markers.5 ECLIA has been commercialized and been extensively applied in clinical settings to detect tens kinds of analytes using 50-150 µL of serum with detection limits at the picomolar level.6 However, many blood serum or plasma concentrations of analytes associated with early stage cancers and infectious diseases range from 10−16 to 10−12 mol/L.7 In addition, analysis of tissues is greatly restricted by the sample amount, and can be achieved only by increasing detection sensitivity.8 These urge us to develop an ultrasensitive ECLIA to selectively detect low-abundance analytes in trace amount of real samples. Currently, sandwich-type immunoassay is the commonly used mode of ECLIA, in which high level of sensitivity and specificity is achieved due to the use of a couple of matching antibodies.9 Generally, the detection mechanism relies on three methods presented in Figure 1. A capture antibody is first immobilized on the electrode surface (Figure 1A and Figure 1B) or magnetic beads with the diameters of several micrometers (Figure 1C). After the binding of antigen in the sample, a detector antibody labeled with electrochemiluminophores (Figure 1A and Figure 1C) or a nanoparticle immobilized simultaneously by detector antibody and electrochemiluminophore (Figure 1B) is added to obtain an immunocomplex. The quantity of the labeled detector antibody is directly proportional to the amount of antigen present This journal is © The Royal Society of Chemistry [year]
55
60
65
70
75
80
85
90
in the sample which increases the detectable signal with increasing target analyte. However, the electrochemiluminous efficiency and the labeling amount of electrochemiluminophores limit the enhancement of sensitivity of the proposed assay to some extent.10 The former is very difficult to improve theoretically as well as practically,11 while the latter is limited due to following reasons: (1) the surface area of the detector antibody or the nanoparticle is small; (2) usually no more than 20 electrochemiluminophores could be labeled on a detector antibody molecule to maintain its immunocompetence; and (3) the diameter of nanoparticle should usually be smaller than 100 nm to ensure reliable capture.
Figure 1. Factors restricting the sensitivity of the conventional “onelectrode” sandwich-type ECLIA.
As a matter of fact, the critical factor that restricts the improvement of the detection sensitivity of ECLIA is neglected, i.e., the sandwich-type of assay format itself. In sandwich-type ELISA, RIA, EIA, FIA and CLIA, the distance between the markers labeled on the detector antibody and the solid plate on which the formed immunocomplex immobilized is entirely irrelevant with the generation of detection signals. However, in the case of ECLIA, the distance between electrochemiluminophores labeled on the detector antibody and the electrode surface directly influences the generation of signals, which ranges from ~0 to several micrometers decided by the size of the immunocomplex and the labeling sites. It means that most of the electrochemiluminophores labeled are positioned beyond the outer Helmholtz plane (OHP), the thickness of which is from several angstroms to several nanometers normally.12 In electrode kinetics, OHP is defined as the plane where solvated ions can approach the electrode only nearest to.13 When proper electrode potential is applied, the potential at the OHP is correspondingly modulated to activate the electrons of electrochemiluminophores near the OHP with the same energy as the electrons on the electrode. Then, radiationless electronic rearrangements will occur followed by isoenergetic electron transfer between electrochemiluminophores near the OHP and the electrode, i.e., electrochemical reaction is launched. On the other hand, for electrochemiluminophores labeled on the detector antibody at a fixed distance x from the electrode, the act of electron transfer is [journal], [year], [vol], 00–00 |1
ChemComm Accepted Manuscript
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
Zhiyong Guo,* Yuhong Sha, Yufang Hu, Sui Wang
ChemComm
Page 2 of 4 View Article Online
5
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
10
15
20
usually considered as tunneling of the electron between states in the electrode and those on the electrochemiluminophores. Typically, the probability of electron tunneling is proportional to exp(–βx), where β is a factor that depends upon the height of the energy barrier and the nature of the medium between the states. For proteins such as antigen and antibody, β could be roughly estimated as 0.1 nm–1 considering the typical value for saturated carbon chains. Thus, it is easy to understand that electrochemiluminophores near the OHP are "effective" in participating in the electrochemical reactions and emitting electrochemiluminescence (ECL) signals, while electrochemiluminophores far away from the OHP are "ineffective".12, 13 To be simple, once the distance between labeled electrochemiluminophores and the electrode is 10 times higher than that between the OHP and the electrode, the probability of electron tunneling will be only 5 ppm, indicating ineffective electrochemical reactions. In reality, this is just the case in current sandwich-type assay format of the ECLIA in which the immunocomplex is immobilized "on-electrode", resulting in the restriction for improving the detection sensitivity. If the immunocomplex is immobilized "in-electrode", then all electrochemiluminophores labeled would be electrochemically "effective", and thus a higher detection sensitivity of ECLIA could be achieved.
45
50
55
60
65
70
75
80
85
25
Figure 2. (A) Structure of the capture and detector units. (B) The detection protocol and sensitivity improvement mechanism of “inelectrode” Faradaycage-type ECLIA.
30
35
40
As a proof of concept for ECLIA based on "in-electrode", we designed a novel biosensing platform using two kinds of multifunctionalized graphene oxide (GO) materials, capture unit (antibody(Ab1)-Fe3O4 nanoparticles@GO, referred as Ab1nanoFe3O4@GO) and detector unit (antibody(Ab2)&N-(4aminobutyl)-N-ethylisoluminol)@GO, referred as (Ab2&ABEI)@GO) (Figure 2A); the preparation process of which are provided in supporting information. GO, which is a single atom thick and two dimensional carbon nanomaterial,14 can contribute greatly to this process because of its unique properties, such as large surface area for high loading and good electric conductivity for favoring the energy and electron transfer.
90
95
100
105
2| Journal Name, [year], [vol], 00–00
The capture unit, which was prepared by immobilizing capture antibody Ab1 on GO coated by Fe3O4 nanoparticles (nanoFe3O4@GO), had three functions: Ab1 acted as the capture device for antigen through antigen-antibody immunoreaction; nanoFe3O4@GO provided the feasibility of one-step preparation; and the size of nanoFe3O4@GO ensured the construct of a nanoscale immune complex system to achieve "in-electrode" mode. And the detector unit, which was prepared by simultaneously immobilizing detector antibody Ab2 and electrochemiluminophore ABEI on GO sheet, also had three functions: ABEI acted as the electrochemiluminophore; Ab2 acted as the recognition device for antigen through antigenantibody immunoreaction; GO could load a great amount of ABEI molecules and enabled all of them effective in emitting ECL signals and thus improved the detection sensitivity greatly. The detection protocol is shown in Figure 2B and described detailedly in supporting information. Firstly, a simple one-step preparation of ECL immunosensor was achieved through dropping and attracting certain amount of capture unit on the surface of a magnetic glass carbon electrode in a few minutes, and the ECL immunosensor was ready for assay. Secondly, the sample containing the antigen was added and incubated. Thirdly, detector unit was added and incubated. Finally, the potential was applied to launch the electrochemical reaction, the ECL signal was generated and recorded, and the antigen was quantified through the relationship between the antigen concentration and the ECL intensity. Though this protocol seems roughly same as that of the sandwich-type ECLIA, the assay scheme is significantly different. GO in the detector unit extends the OHP of the electrode, making all the electrochemiluminophores labeled effective as such they are immobilized directly on the surface of the electrode or even "in the electrode" (Figure 2B). In this case of "in-electrode" state, detector unit lapped on the electrode surface directly, so electrons could flow freely between the detector unit and the electrode. In other words, detector unit had become de facto part of the electrode. Considering the skin effect of the electric field, the whole immune complex system seemed like a Faraday cage with some small windows because some Ab1 or Ab2 molecules were immobilized on the edge of the GO in capture unit and detector unit. The Ab1-antigen-Ab2 immunocomplex was sealed in the Faraday cage. All electrochemiluminophores labeled on detector unit were on the surface of the Faraday cage, so all of them could take part in the electrode reaction to emit ECL signal, profiting from the free flow of electrons resulting from the isopotential character of the Faraday cage. Thus, the two bottlenecks which restrict the sensitivity improvement of sandwich-type ECLIA were substantially eliminated, i.e., the labeling amount of electrochemiluminophores increased and all electrochemiluminophores labeled were ensured to be "effective". To demonstrate the feasibility and sensitivity of this proposed "in-electrode" Faradaycage-type ECLIA, neurotensin (NT) was analyzed as a model target analyte. NT is a 13 amino acid neuropeptide, involved in a number of important biological processes including dopamine transmission, analgesia, hypothermia, etc.15 Recent reports suggest that the detection of very low concentration of NT peptides, which ranges from several to tens of pg/mL and even lower levels in some diseases, is helpful to identify sub-healthy humans or early-stage patients.16 Scanning electron microscope (SEM) was used to characterize the stepwise fabrication and detection process of the immunosensor. The SEM image of nanoFe3O4@GO (Figure 3A) exhibits that numerous Fe3O4 nanoparticles were well-dispersed onto the uniform GO film (Figure S1) with a typical twodimensional structure. In the case of capture unit (Figure 3B) and This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
DOI: 10.1039/C6CC00787B
Page 3 of 4
ChemComm View Article Online
5
NT-capture unit (Figure 3C), the surface became much rougher and richer in texture due to the successive immobilization of the capture unit and the antigen NT. When immobilized finally (Figure 3D), detection unit covered the antigen and the capture unit with some bulges, demonstrating the existence of the immunocomplex detector unit-NT-capture unit.
45
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
50
55
60
electrode" Faradaycage-type ECLIA. Afterwards, electrochemical impedance spectroscopy (EIS) was used to monitor the interface properties of the Faradaycagetype ECLIA in the assembly process (Figure S3). Compared with electron transfer resistance, Ret, of bare MGCE (curve a), the Ret of nanoFe3O4@GO/MGCE (curve b) was lower due to the good electronic conductivity of nanoFe3O4@GO. In the case of capture unit/MGCE (curve c) and NT–capture unit/MGCE (curve d), Ret increased markedly because Ab1, bovine serum protein (BSA) and NT are proteins which would hinder the electron transfer on the electrode interface. As to the detector unit–NT–capture unit/MGCE (curve e), Ret decreased greatly, demonstrating the accomplishment of the "in-electrode" Faradaycage-type ECLIA. When the detector unit was immobilized to form the immunocomplex, GO in the detector unit lapped on the electrode surface directly as if becoming a part of the electrode and thus building the as-designed Faradaycage-type ECLIA. Therefore, electron could transfer freely between the electrode and the electrochemical probe without the hindrance of Ab1, Ab2, BSA and NT, thus Ret decreased near to zero.
Figure 3. Representative SEM images of (A) nanoFe3O4@GO, (B) capture unit, (C) NT-capture unit, and (D) detector unit-NT-capture unit.
10
15
20
25
30
35
40
Figure 4. (A) ECL signals of (a) magnetic glassy carbon electrode (MGCE), (b) capture unit/MGCE, (c) NT–capture unit/MGCE and (d) detector unit–NT–capture unit/MGCE. (B) ECL signals of (a) detector unit–NT–capture unit/MGCE, (b) the same process as (a) without NT, (c) the same process as (a) without ABEI, (d) the same process as (a) without Ab2 and (e) the same process as (a) using g-C3N4 instead of GO in the detector unit. The concentration of NT is 5 pg/mL.
ECL signals at each immobilization step were recorded to monitor the fabrication of the immunosensor. As shown in Figure 4A, the ECL intensities of bare magnetic glassy carbon electrode (MGCE), capture unit/MGCE and NT–capture unit/MGCE were all near to zero, while that of detector unit–NT–capture unit/MGCE was strong and stable at several thousand arbitrary unit, indicating that the ECL signal was originated from the electrochemiluminophores ABEI immobilized on the detector unit. To obtain further convincing support on the signal amplification capability of the developed Faradaycage-type ECLIA strategy, the ECL intensities of the sensors incubated with/without NT, ABEI or Ab2 were compared. As depicted in Figure 4B, the absence of ABEI resulted in negligible ECL signal. It is evident that the high ECL signal could be ascribed to ABEI in detector unit. While the absence of NT or Ab2 led to a sharp decrease in ECL intensity, implying that NT or Ab2 is also important for loading detector unit and supporting it to achieve the proposed efficient ECL "in-electrode" strategy. If graphitelike carbon nitride (g-C3N4) with similar size as GO (Figure S2) was used to build the detector unit instead of GO, the electric conductivity of which was much poorer than that of GO, the ECL intensity decreased down to about one tenth (Figure 4B, curve e), demonstrating that the good electric conductivity of GO help increase the ECL intensity well and the validity of the "inThis journal is © The Royal Society of Chemistry [year]
65
70
75
80
85
Figure 5. (A) ECL intensity in the presence of NT at different concentrations; (B) Calibration curve for NT determination; (C) Selectivity of the ECL immunosensor toward other proteins; and (D) ECL intensity of the proposed immunosensor to 0.005 fg/mL NT in the presence of 1 µg/mL of other proteins, individually, or in a mixture with all these proteins.
Under the optimal detection conditions of pH of the electrolyte solution (9.74), concentration of H2O2 in electrolyte solution (1 mmol/L), incubation temperature (37 °C) and incubation time (1 h) (Figure S4), the ECL intensity of the proposed Faradaycagetype immunosensor increased with the increasing concentration of NT (Figure 5A). The calibration plot showed a good linear relationship between the ECL intensity (y) and the logarithmic value of NT concentration (x) ranging from 5×10-3 fg/mL to 5×104 fg/mL with a correlation coefficient r of 0.9989 (Figure 5B). The detection limit was 2×10-3 fg/mL at a signal-to-noise ratio of 3, which is much better than those detection limits from other methods reported in literature (Table S1). To demonstrate the sensitivity enhancement of this emerging "in-electrode" Faradaycage-type ECLIA further, we also measured NT with conventional "on-electrode" sandwich-type for comparison, using magnetic beads immobilized by Ab1 as capture unit and Ab2 labeled with ABEI as detector unit. The linear relationship ranged from 10 fg/mL to 1×105 fg/mL, with the detection limit of 5 fg/mL. Results showed that the detection sensitivity increased about three orders of magnitude when using "in-electrode" Faradaycage-type than that of "on-electrode" sandwich-type Journal Name, [year], [vol], 00–00 |3
ChemComm Accepted Manuscript
DOI: 10.1039/C6CC00787B
ChemComm
Page 4 of 4 View Article Online
DOI: 10.1039/C6CC00787B
Published on 29 January 2016. Downloaded by UNSW Library on 04/02/2016 10:27:20.
10
15
20
25
30
35
40
45
50
55
65
2 (a) R. Ekin, Nucl. Med. Biol., 1994, 21, 495-521; (b) E. N. Brown, T. J. 3
70
4
75
80
85
5
90
6 7 95
McDermott, K. J. Bloch and A. D. McCollom, Clin. Chem., 1996, 42, 893903. (a) J. M. Hicks, Hum. Pathol., 1984, 15, 112-116; (b) J. Sherry, Chemosphere, 1997, 34, 1011-1025; (c) A. Hatch, A. E. Kamholz, K. R. Hawkins, M. S. Munson, E. A. Schilling, B. H. Weigl and P. Yager, Nat. Biotechnol., 2001, 19, 461-465. (a) K. D. Chandra, K. V. Sandeep, T. O. Feidhlim, O. Brian, D. M. Brian and O. Richard, Anal. Chem., 2010, 82, 7049-7052; (b) D. H. Charles, Anal. Chem., 1973, 45, 878-890; (c) N. M. Victor and Y. M. Tamara, Anal. Chem., 2003, 75, 6813-6819; (d) S. I. Stoeva, J. S. Lee, J. E. Smith, S. T. Rosen and C. A. Mirkin, J. Am. Chem. Soc., 2006, 128, 8378-8379; (e) A. T. Hashem, G. B. Dean, J. B. John, Y. Chen, S. C. Richard, D. S. Renuka, A. E. Robert, E. G. Jose, S. H. Richard, P. K. Kenneth, K. L. Senia, E. M. Michael, L. M. Terri, D. M. James, H. O. Bruce, G. R. Sarada, S. Michael, A. S. Barry, S. Nir, R. S. Katherine, C. T. Jeff, G. P. Wang and W. H. Xie, J. Am. Chem. Soc., 2013, 135, 4191-4194; (f) R. H. William and H. B. Halsall, Anal. Chem., 1985, 57, 1321A-1331A; (g) Y. H. Sha, Z. Y. Guo, B. B. Chen, S. Wang, G. P. Ge, B. Qiu and X. H. Jiang, Biosens. Bioelectron., 2015, 66, 468-473. (a) W. Miao, Chem. Rev., 2008, 108, 2506-2553; (b) M. S. Wu, H. W. Shi, L. J. He, J. J. Xu and H. Y. Chen, Anal. Chem., 2012, 84, 4207-4213; (c) Y. Zhuo, N. Liao, Y. Q. Chai, G. F. Gui, M. Zhao, J. Han, Y. Xiang and R. Yuan, Anal. Chem., 2014, 86, 1053-1060; (d) M. M. Richter, Chem. Rev., 2004, 104, 3003-3036; (e) Z. Y. Liu, W. J. Qi and G. B. Xu, Chem. Soc. Rev., 2015, 44, 3117-3142. (a) X. Yu, B. Munge, V. Patel, G. Jensen, A. Bhirde, J. D. Gong, S. N. Kim, J. Gillespie, J. S. Gutkind, F. Papadimitrakopoulos and J. F. Rusling, J. Am. Chem. Soc., 2006, 128, 11199-11205. D. M. Rissin, C. W. Kan, T. G. Campbell, S. C. Howes, D. R. Fournier, L. Song, T. Piech, P. P. Patel, L. Chang, A. J. Rivnak, E. P. Ferrell, J. D. Randall, G. K. Provuncher, D. R. Walt and D. C. Duffy, Nat. Biotechnol., 2010, 28, 595-599.
8 (a) S. Krishnan, V. Mani, D. Wasalathanthri, C. V. Kumar and J. F. Rusling, Angew. Chem. Int. Ed., 2011, 50, 1175-1178; (b) K. D. Wegner, 100
105
110
115
120
125
This study was financially supported by National Natural Science Foundation of China (Nos. 41576098 and 81273130) and K. C. Wong Magna Fund in Ningbo University.
Z. W. Jin, S. Lindén, T. L. Jennings and N. Hildebrandt, ACS Nano, 2013, 7, 7411-7419. 9 (a) P. Vicennati, N. Bensel, A. Wagner, C. Créminon and F. Taran, Angew. Chem. Int. Ed., 2005, 44, 6863-6866; (b) J. Quinton, S. Kolodych, M. Chaumonet, V. Bevilacqua, M. Nevers, H. Volland, S. Gabillet, P. Thuery, C. Creminon and F. Taran, Angew. Chem. Int. Ed., 2012, 51, 6144-6148. 10 (a) J. I. Kim, I. S. Shin, H. Kim and J. K. Lee, J. Am. Chem. Soc., 2005, 127, 1614-1615; (b) X. Liu, L. Shi, W. Niu, H. Li and G. Xu, Angew. Chem., 2007, 119, 425-428; (c) X. Q. Liu, L. H. Shi, W. X. Niu, H. J. Li and G. B. Xu, Angew. Chem. Int. Ed., 2007, 46, 421-424. 11 (a) S. Rashidnadimi, T. H. Hung, K. T. Wong and A. J. Bard, J. Am. Chem. Soc., 2008, 130, 634-639; (b) J. W. Oh, Y. O. Lee, T. H. Kim, K, C. Ko, J. Y. Lee, H. Kim and J. S. Kim, Angew. Chem. Int. Ed., 2009, 48, 2522-2524. 12 S. P. Du, Z. Y. Guo, B. B. Chen, Y. H. Sha, X. H. Jiang, X. Li, N. Gan and S. Wang, Biosens. Bioelectron., 2014, 53, 135-141. 13 (a) A. J. Bard and L. R. Faulkner, Electrochemical Methods, Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2001; (b) M. Nakamura, H. Kaminaga, O. Endo, H. Tajiri, O. Sakata and Nagahiro Hoshi, J. Phys. Chem. C, 2014, 118, 22136-22140. 14 (a) K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I. V. Grigorieva and A. A. Firsov, Science, 2004, 306, 666-669; (b) R. F. Service, Science, 2009, 324, 875-877; (c) S. Park and R. S. Ruoff, Nat. Nanotechnol., 2009, 4, 217-224. 15 (a) W. H. Rostène and M. J. Alexander, Front. Neuroendocrinol., 1997, 18, 115-173; (b) J. Gobom, K. O. Kraeuter, R. Persson, H. Steen, P. Roepstorff and R. Ekman, Anal. Chem., 2000, 72, 3320-3326. 16 (a) A. Mascarin, I. E. Valverde, S. Vomstein, and T. L. Mindt, Bioconjugate Chem., 2015, 26, 2143-2152; (b) Z. Y. Guo, S. P. Du, B. B. Chen, Y. H. Sha, B. Qiu, X. H. Jiang, S. Wang, and X. Li, Electrochim. Acta, 2014, 135, 519-525.
130
Notes and references
60
School of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, China. Tel: 86-574-87600798 E-mail: [email protected] † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. 1 (a) S. Brakmann, Angew. Chem. Int. Ed., 2004, 43, 5730-5734; (b) R. Batrla and B. W. Jordan, Ann. N. Y. Acad. Sci., 2015, 1346, 71-80; (c) J. Durner, Angew. Chem. Int. Ed., 2010, 49, 1026-1051.
4| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
5
format. In order to illustrate the possible interference of non-specific antigens, various species of interfering proteins including alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), neuronspecific enolase (NSE), bovine serum albumin (BSA) and squamous cell carcinoma antigen (SCCA), at the concentration of 1 µg/mL, were measured using the proposed immunoassay. The ECL intensities of them were all around 150, nearly similar to that of the blank sample, and much lower than that of 0.005 fg/mL NT (Figure 5C), demonstrating that the immunosensor developed has an excellent specificity for the determination of target protein NT. Meanwhile, the interference of these proteins tested in presence of NT either individually or in a mix (Figure 5D) exhibited unchanged coexistence ECL intensity in the presence of other interference proteins. These results demonstrate that the developed sensing method is highly selective toward NT against other interference molecules, due to the highly specific binding capability of antibody to NT. To assess the potential application of this method for real biological samples, a NTtrapping test was performed using human serum or urine samples spiked with different concentrations of NT. The observed recoveries were between 94.4% and 112.6% (Table S2), providing a new insight into NT-relevant determination in biological fluids sample. The stability of the developed immunosensor was investigated by assessing its relative activity after storage at 4 °C in dark for one month. The ECL intensity for the detection of 5 fg/mL NT was 92.5 ± 7.4% (n = 5) of the initial value which was obtained when the immunosensor was constructed freshly, indicating that the developed immunosensor has acceptable stability during storage. The relative standard deviations (RSD) of an intra-assay and an inter-assay were used to evaluate the reproducibility of the proposed immunosensor. The intra-assay precision was 7.3%, which was evaluated from the response to 5 fg/mL NT at five different immunosensors fabricated in the same batch, while an inter-assay precision of 7.8% was obtained by assaying 5 fg/mL NT with five different immunosensors prepared with different batches (Figure S5). Results indicated the excellent precision and reproducibility. In addition, the reversibility of this immunosensor could be easily achieved in the absence and presence of the external magnetic field. To conclude, we demonstrated a new-concept of "In-Electrode" "Faradaycage-Type" immunoassay mode using multifunctionalized GO composite materials, aiming at eliminating the key bottlenecks restricting the improvement of the sensitivity of ECLIA based on conventional "On-Electrode" "Sandwich-Type" immunoassay mode. The results confirmed that the detection sensitivity of the model protein reached the level of ag/mL and showed high specificity, stability and reproducibility, providing a great application prospect for the early diagnosis of NT-related diseases.
