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Received 00th January 2012, Accepted 00th January 2012
Ultrasensitive electrochemical immunoassay of proteins based on in-situ duple amplification of gold nanoparticles biolabel signal Xiaoli Qin,a Aigui Xu,a Ling Liu,a Wenfang Deng,a Chao Chen,a Yueming Tan,a Yingchun Fu,a Qingji Xie,a* and Shouzhuo Yaoa,b
DOI: 10.1039/x0xx00000x www.rsc.org/
An electrochemical sandwich immunoassay method that can be sensitive to a few protein molecules (human immunoglobulin G or human prostate-specific antigen) is reported, based on HAuCl4-NH2OH redox reaction to enlarge the size of second antibody labeled gold nanoparticles and insitu microliter-droplet anodic stripping voltammetry analysis with enhanced cathodic preconcentration of the gold. Early warning of serious diseases such as cancers is crucially important for their diagnosis, therapy and prognosis.1-3 Ultrasensitive quantitative analysis or even single-molecule-level detection of molecular biomarkers is a useful approach for this purpose.4-7 Various optical and electrochemical analysis techniques and their combination with chromatographic or magnetic separation have been frequently used for bioanalysis.8, 9 Electroanalytical methods have the advantages of high sensitivity and selectivity, low limits of detection (LODs), facile operation, simple instrumentation as well as ease of automation and device miniaturization, thus they are promising for rapid and sensitive bioanalysis and biosensing.10-14 The nanomaterials of metals and their compounds have been used as biolabels for bioelectroanalysis, because the metal components can be sensitively determined by anodic stripping voltammetry (ASV) in a very convenient way. The metal-labeled amperometric immunoassay (MLAI) involving a sandwich immuno-interface has been proven a highly promising bioanalysis method.15-18 From the microcosmic viewpoint, the valence-electron-movement issue should be one of the core scientific issues in chemistry and its downstream disciplines such as molecules/atoms-based biology and material sciences, since the valence-electron movement including partial electron transfer, electron sharing and complete electron gain/loss is directly related to various chemical reactions and intermolecular interactions. The electrode in electrochemistry is an excellent platform to study gains and losses of valence electrons of atoms, ions and/or molecules in electrolyte phases. Electrochemical studies have revealed the fact that the efficient distance for valence electron transfer/communication is only ~3 nm even with the electron-conducting nanomaterial relay.19-21 Hence, driving the electroactive species to move as close as possible to the electrode surface is very important in fulfilling an analytical task occurring on
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the electrode with enhanced sensitivity.22 In the sandwich MLAI, it is thus favorable to perform simultaneous chemical-dissolution of the metal biolabels and cathodic-preconcentration of atomic metal for in-situ ASV analysis on the bioelectrode, which can maximally make use of both the electrically contacted and noncontacted metal biolables in the sandwich biostructure on the bioelectrode to achieve greatly enhanced ASV signaling. However, such attempts are not reported to date. Moreover, the metal staining methods are capable of enhancing the readout,23-32 including gold label/silver staining,23-27 and gold label/gold staining.28-32 The gold staining method is more sensitive than the silver staining one probably due to the unique Augrowth mechanism.29, 32 Such metal staining methods belong to nanomaterial-based signal-amplification ones and are simpler than many bioamplification methods in principle and operation, which are thus adaptable to robust, low-cost and ultrasensitive bioanalysis and biosensing. Herein, we report a new protocol for sandwich MLAI with signal amplification (MLAIsa) of the metal biolabels, based on simultaneous chemical-dissolution and cathodic-preconcentration of the gold-stained AuNPs biolabels as well as the microliter-droplet ASV analysis directly on the immunoelectrode. As demonstrationof-concept examples here, our MLAIsa method shows limits of detection (LODs, S/N=3) of 0.3 fg mL-1 (7 molecules in 6 L sample) for human immunoglobulin G (hIgG) and 0.1 fg mL-1 (11 molecules in 6 L sample) for human prostate-specific antigen (hPSA) under optimized conditions, which are much better than the reported results for the two proteins (Table S1). Our MLAIsa protocol is depicted in Scheme 1. Briefly, the first antibody (Ab1) was attached on a carboxylated multiwalled carbon nanotubes (MWCNTs) cast-coated glassy carbon electrode (GCE) by the 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry, bovine serum albumin (BSA) was used to block the nonspecific binding sites, and then the target antigen was immobilized by the immunological reaction. The second antibody (Ab2) labeled with AuNPs (Ab2AuNPs) was immunogically captured onto the electrode to form a sandwich-type immuno-interface. Afterwards, gold was stained selectively on the AuNPs surfaces through the AuNPs-catalyzed HAuCl4-NH2OH redox reaction.28-32 As shown in Scheme 1(b), a cathodic potential (0 V here) was first applied on the
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Journal Name dissolution gave =42.5% after 600-s preconcentration. The maximum was only 8.1% for the conventional solutionView Article Online the replacement protocol,34 which was performed by dissolving DOI: 10.1039/C5CC01439E AuNPs with 8 L of 1.0 M HBr-Br2 and transferring it into a mixed solution of 2 L 3-phenoxypropionic acid and 990-L 1.0 M HCl for preconcentration at 0 V for 600 s and then LSV stripping. A direct anodic stripping of the cast-coated AuNPs in 1.0 M aqueous HCl yielded an experimental of 96.1% (Fig. S1a). Another reported electroanalysis protocol involving an electrooxidation step at 1.25 V for 150 s in 1.0 M aqueous HCl, followed by a cathodic scan from 1.25 to 0 V and measurement of the cathodic peak current at 0.49 V,35, 36 yielded an experimental of only 2.1% (Fig. S1b). It is expected that, at the sandwich immunoelectrodes rather than the bare electrode used here, the direct anodic stripping protocol without simultaneous chemical-dissolution and cathodic-preconcentration will give a signaling efficiency notably lower than our MLAIsa protocol, due to the obvious blocking of electron communication by the insulating protein layers between the metal labels and the electrode surface, as confirmed in Fig. S2. Hence, this work provides an alternative method with high sensitivity for MLAI.
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Scheme 1. Illustration of immunoelectrode preparation (a) and key electrochemical steps (b) of our MLAIsa protocol. To compare the signaling efficiency (, Eq. (1)) of our MLAIsa protocol with those of the protocol similar to MLAIsa but the cathodic potential was applied soon after AuNPs dissolution (potential control only in liquid), the conventional solutionreplacement protocol,33, 34 and the direct anodic stripping protocol, we conducted simulation experiments simply by cast-coating an appropriate amount of AuNPs (nAuNPs-cast in mol) on a GCE and then detecting the recovered amount of the cast-coated AuNPs (nAuNPs-LSV in mol) by linear sweep voltammetry (LSV) and the Faraday law.
=nAuNPs-LSV /nAuNPs-cast =Qp /( zFnAuNPs-cast )
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where Qp is the charge under the LSV stripping peak of cast-coated AuNPs, z is the number of electrons transferred (z=3 here), and F is the Faraday constant (96485.3 C mol-1). The anodic stripping LSV curves and corresponding values as functions of the cathodic-preconcentration time are shown in Fig. 1. After simultaneous dissolution of the AuNPs and cathodic preconcentration for 600 s and LSV stripping in 8 L of 1.0 M HBrBr2, the for our MLAIsa protocol was 82.6%. The protocol similar to MLAIsa but the cathodic potential was applied soon after AuNPs
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immunoelectrode in air, and 8 L of 1.0 M aqueous HBr-Br2 was then added to connect the glassy carbon working electrode (WE), the KCl-saturated calomel reference electrode (RE) with a salt bridge of the test solution, and the platinum wire counter electrode (CE), as well as to dissolve the AuNPs and stained gold for diffusioncontrolled cathodic preconcentration of atomic gold onto the immunoelectrode substrate. Finally, differential pulse ASV analysis was conducted for immunoassay signaling. Here, the beforehand open-circuit exertion of a cathodic potential in air and the use of a small-volume solution can minimize the diffusion-layer thickness to rapidly electrodeposit the metal label onto the bioelectrode surface as entirely as possible from the lysate, which can greatly enhance the subsequent ASV signal for bioanalysis. It should be noted that the open-circuit exertion of a constant potential in air is always safe for an electrochemical instrument working in its potentiostatic mode but may damage the instrument running in its galvanostatic mode. The open-circuit potentiostatic operation (i.e., potential control in air) is rarely used in analytical chemistry and electrochemistry, as compared with potential control in liquid solutions, and we prove here that this special procedure can be used to effectively amplify the bioelectroanalysis signal.
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Fig. 1. versus preconcentration-time curves (A, and Insets of C and D, n=3) and anodic stripping LSV curves of the AuNPs cast-coated on GCE (B, C and D, 100 mV s-1) for our MLAIsa protocol (B, a), the protocol similar to MLAIsa but the cathodic preconcentration potential was applied soon after AuNPs dissolution (C, b) and conventional solution-replacement protocol (D, c). See ESI for experimental details. The volume of 1.0 M HBr-Br2 used to dissolve AuNPs was optimized. As shown in Fig. S3, decreased with the increase of HBr-Br2 volume, since a smaller solution volume can decrease the diffusion-layer thickness for enhanced preconcentration of atomic gold on the WE and can thus give a larger ASV peak signal. To maximize the signal, we will use 8-L HBr-Br2 below.
Fig. 2. UV-Vis spectra and photographs (Inset) of 0.50 mL NH2OH (1), gold-staining solution (2), BSA/anti-hIgG/AuNPs (3) and after its gold-staining for 20 min (4). Final concentrations: 10 g mL-1 anti-hIgG, 1.0% (w/v) BSA, 18 mM NH2OH and 3 mM HAuCl4.
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Under optimum conditions, the ASV peak current is linear with the common logarithm of hIgG concentration from 0.4 fg mL-1 to Articleof Online 400 ng mL-1, with a sensitivity of 0.337 mA dec-1 andView a LOD 0.3 -6 10.1039/C5CC01439E fg mL-1 (7 molecules in 6 L sample, 610DOI: 0.3 10-151036.02 1023/1.5105=7.27, S/N=3) by our MLAIsa method (Fig. S7). The semi-derivative treatment of these ASV curves can notably improve the peak-signal resolution and decrease the background,38,39 as also shown in Fig. S7. The reproducibility of our MLAIsa method was evaluated using hIgG at three concentration levels (0.500, 5.00 and 50.0 ng mL-1), with relative standard deviations (RSDs) of (61)%, indicating acceptable reproducibility. The LOD obtained here is much better than those reported for hIgG to date (Table S1). hPSA is the most widely used tumor marker worldwide for early warning, screening, diagnosing and monitoring of prostate cancer that is usually localized and has no obvious symptoms in its early stage. hPSA in blood is the most sensitive biomarker for this purpose, which is commonly determined by immunoassay methods.40, 41 We used our MLAIsa method to detect hPSA (Fig. 4). Under the optimum conditions, we obtained linearity from 0.18 fg mL-1 to 450 ng mL-1 with a sensitivity of 0.609 mA dec-1 and a LOD of 0.1 fg mL-1 (11 molecules in 6 L sample, 610-60.110-151036.02 1023/3.4104=10.611, S/N=3). The LOD of our method is also much better the literature-reported values for hPSA (Table S1). The Au-stripping anodic peaks of improved resolution after the semiderivative treatment of the voltammetric curves in Fig. 4 and the corresponding calibration curve are shown in Fig. S8. We also examined the applicability of our MLAIsa method to hPSA assay in seven clinical human-serum samples (only 6 L serum sample was required for each assay). Our results agreed well with the hospital results from chemiluminescence assay (within 7% RSD), as listed in Table S2, validating our MLAIsa method for analysis in clinical human sera. From the above two immunoassay examples, our MLAIsa method with several-molecules-level sensibility to proteins may be promising for challenging trace analysis of proteins (e.g., some ultratrace proteins in single circulating tumor cells,42, 43 and interleukin-2 with a normal pM~fM concentration in human serum44) and may find wide applications in the early warning, diagnosis and treatment of the serious diseases including cancers. 8
450 ng mL-1 -1 180 ng mL 18 ng mL-1 -1 1.8 ng mL 180 pg mL-1 -1 18 pg mL 1.8 pg mL-1 -1 180 fg mL 18 fg mL-1 -1 1.8 fg mL 0.9 fg mL-1 0.18 fg mL-1 0
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In this work, we employed performed AuNPs to label Ab2 for AuNPs-catalyzed gold-staining. As shown in Fig. 2, NH2OH solution was colorless and showed no obvious absorption peak. After 3 mM HAuCl4 was added, the solution turned into light yellow. The BSA/anti-hIgG/AuNPs suspension showed a wine red color and an absorption peak at 537 nm. After adding the gold staining solution, the suspension showed a blue-gray color and an absorption peak at 579 nm (sample 4) due to seeded-growth of AuNPs here. Taking hIgG immunoassay as an example, electrode modifications in our MLAIsa protocol were examined by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM), as shown in Figs. S4 and S5 with discussion details. We also used scanning electron microscopy (SEM) to characterize the immunoelectrode fabrication, as shown in Fig. 3. The surface of bare GCE is smooth (Fig. 3A). On the antigen/BSA/Ab1/MWCNTs/GCE, nanotubes are seen (Fig. 3B). After further immobilization of Ab2-AuNPs (Figs. 3C and 3E), some anchored Ab2-AuNPs granules were observed, proving the successful immuno-recognition. After the AuNPs-catalyzed gold staining reaction (Fig. 3D and F, gold/Ab2-AuNPs/antigen/BSA/Ab1/ MWCNTs/GCE), the Ab2-AuNPs granules become larger and denser. As a control, similar gold staining operation on an Ab2-AuNPs-free antigen/BSA/Ab1/MWCNTs/GCE hardly changed the electrode surface (Fig. S6). This observation supports that gold staining occurs only on AuNPs due to seed-mediated nucleation growth.28, 37 The energy-dispersive X-ray spectroscopy (EDX) results for Ab2AuNPs/antigen/BSA/Ab1/MWCNTs/GCE (Fig. 3G) and gold/Ab2AuNPs/antigen/BSA/Ab1/MWCNTs/GCE (Fig. 3H) confirm the increased mass and atomic fractions of Au after the AuNPscatalyzed gold staining reaction. The CV, EIS, QCM, SEM, and EDX characterizations here have proven the successful fabrication of the immunoelectrode.
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Fig. 3. SEM images and EDX spectra of bare GCE (A), antigen/BSA/Ab1/MWCNTs/GCE (B), Ab2-AuNPs/antigen/BSA/ Ab1/MWCNTs/GCE (C, E and G), and gold/Ab2-AuNPs/antigen/ BSA/Ab1/MWCNTs/GCE (D, F and H). Concentration of hIgG: 4 ng mL-1 (C and D) and 400 ng mL-1 (E, F, G and H).
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Fig. 4. Linear sweep ASV curves for hPSA immunoassay by our MLAIsa protocol (A) and the corresponding calibration curves (B) (n=3). Scan rate: 100 mV s-1. In summary, we have reported that a new and ultrasensitive MLAIsa method, based on simultaneous microliter-droplet chemicaldissolution and cathodic-preconcentration of the AuNPs biolabel after gold staining and then in-situ ASV analysis directly on the immunoelectrode, can be sensitive to a few hIgG or hPSA molecules. Virtually, amperometric detection of a single bioelectrode-supported AuNP after sufficient size-enlargement may be possible, thus the principle here may be promising even for quantitative singlemolecule analysis of proteins. Our method has been used for hPSA analysis in clinical samples with satisfactory results. Our MLAIsa protocol has the advantages of ultrahigh sensitivity, wide linear detection range, good accuracy/precision/stability, easy operation, small consumption of reagents/samples, and application potential for
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Journal Name 17 V. Pavlov, Y. Xiao, B. Shlyahovsky and I. Willner, J. Am. Chem. Soc., 2004, 126, 11768.
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Anal. Chem., 2011, 18 G. S. Lai, F. Yan, J. Wu, C. Leng and H. X. Ju,10.1039/C5CC01439E DOI: 83, 2726.
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This work was supported by the National Natural Science Foundation of China (21475041, 21175042, 21305041, 21405042), Hunan Lotus Scholars Program, Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, and Hunan Provincial Innovation Foundation For Postgraduate (CX2014B169).
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine
Research (MOE of China), National & Local Joint Engineering Laboratory for New Petro-chemical Materials and Fine Utilization of Resources, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China. State Key Laboratory of Chemo/Biosensing and Chemometrics, College
of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P.R. China. †
Electronic Supplementary Information (ESI) available: Experimental
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DOI: 10.1039/c000000x/ 1
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This article can be cited before page numbers have been issued, to do this please use: X. Qin, A. Xu, L. Liu, W. Deng, C. Chen, Y. Tan, Y. Fu, Q. Xie and S. Yao, Chem. Commun., 2015, DOI: 10.1039/C5CC01439E.
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.
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Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012, Accepted 00th January 2012
Ultrasensitive electrochemical immunoassay of proteins based on in-situ duple amplification of gold nanoparticles biolabel signal Xiaoli Qin,a Aigui Xu,a Ling Liu,a Wenfang Deng,a Chao Chen,a Yueming Tan,a Yingchun Fu,a Qingji Xie,a* and Shouzhuo Yaoa,b
DOI: 10.1039/x0xx00000x www.rsc.org/
An electrochemical sandwich immunoassay method that can be sensitive to a few protein molecules (human immunoglobulin G or human prostate-specific antigen) is reported, based on HAuCl4-NH2OH redox reaction to enlarge the size of second antibody labeled gold nanoparticles and insitu microliter-droplet anodic stripping voltammetry analysis with enhanced cathodic preconcentration of the gold. Early warning of serious diseases such as cancers is crucially important for their diagnosis, therapy and prognosis.1-3 Ultrasensitive quantitative analysis or even single-molecule-level detection of molecular biomarkers is a useful approach for this purpose.4-7 Various optical and electrochemical analysis techniques and their combination with chromatographic or magnetic separation have been frequently used for bioanalysis.8, 9 Electroanalytical methods have the advantages of high sensitivity and selectivity, low limits of detection (LODs), facile operation, simple instrumentation as well as ease of automation and device miniaturization, thus they are promising for rapid and sensitive bioanalysis and biosensing.10-14 The nanomaterials of metals and their compounds have been used as biolabels for bioelectroanalysis, because the metal components can be sensitively determined by anodic stripping voltammetry (ASV) in a very convenient way. The metal-labeled amperometric immunoassay (MLAI) involving a sandwich immuno-interface has been proven a highly promising bioanalysis method.15-18 From the microcosmic viewpoint, the valence-electron-movement issue should be one of the core scientific issues in chemistry and its downstream disciplines such as molecules/atoms-based biology and material sciences, since the valence-electron movement including partial electron transfer, electron sharing and complete electron gain/loss is directly related to various chemical reactions and intermolecular interactions. The electrode in electrochemistry is an excellent platform to study gains and losses of valence electrons of atoms, ions and/or molecules in electrolyte phases. Electrochemical studies have revealed the fact that the efficient distance for valence electron transfer/communication is only ~3 nm even with the electron-conducting nanomaterial relay.19-21 Hence, driving the electroactive species to move as close as possible to the electrode surface is very important in fulfilling an analytical task occurring on
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the electrode with enhanced sensitivity.22 In the sandwich MLAI, it is thus favorable to perform simultaneous chemical-dissolution of the metal biolabels and cathodic-preconcentration of atomic metal for in-situ ASV analysis on the bioelectrode, which can maximally make use of both the electrically contacted and noncontacted metal biolables in the sandwich biostructure on the bioelectrode to achieve greatly enhanced ASV signaling. However, such attempts are not reported to date. Moreover, the metal staining methods are capable of enhancing the readout,23-32 including gold label/silver staining,23-27 and gold label/gold staining.28-32 The gold staining method is more sensitive than the silver staining one probably due to the unique Augrowth mechanism.29, 32 Such metal staining methods belong to nanomaterial-based signal-amplification ones and are simpler than many bioamplification methods in principle and operation, which are thus adaptable to robust, low-cost and ultrasensitive bioanalysis and biosensing. Herein, we report a new protocol for sandwich MLAI with signal amplification (MLAIsa) of the metal biolabels, based on simultaneous chemical-dissolution and cathodic-preconcentration of the gold-stained AuNPs biolabels as well as the microliter-droplet ASV analysis directly on the immunoelectrode. As demonstrationof-concept examples here, our MLAIsa method shows limits of detection (LODs, S/N=3) of 0.3 fg mL-1 (7 molecules in 6 L sample) for human immunoglobulin G (hIgG) and 0.1 fg mL-1 (11 molecules in 6 L sample) for human prostate-specific antigen (hPSA) under optimized conditions, which are much better than the reported results for the two proteins (Table S1). Our MLAIsa protocol is depicted in Scheme 1. Briefly, the first antibody (Ab1) was attached on a carboxylated multiwalled carbon nanotubes (MWCNTs) cast-coated glassy carbon electrode (GCE) by the 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry, bovine serum albumin (BSA) was used to block the nonspecific binding sites, and then the target antigen was immobilized by the immunological reaction. The second antibody (Ab2) labeled with AuNPs (Ab2AuNPs) was immunogically captured onto the electrode to form a sandwich-type immuno-interface. Afterwards, gold was stained selectively on the AuNPs surfaces through the AuNPs-catalyzed HAuCl4-NH2OH redox reaction.28-32 As shown in Scheme 1(b), a cathodic potential (0 V here) was first applied on the
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Journal Name dissolution gave =42.5% after 600-s preconcentration. The maximum was only 8.1% for the conventional solutionView Article Online the replacement protocol,34 which was performed by dissolving DOI: 10.1039/C5CC01439E AuNPs with 8 L of 1.0 M HBr-Br2 and transferring it into a mixed solution of 2 L 3-phenoxypropionic acid and 990-L 1.0 M HCl for preconcentration at 0 V for 600 s and then LSV stripping. A direct anodic stripping of the cast-coated AuNPs in 1.0 M aqueous HCl yielded an experimental of 96.1% (Fig. S1a). Another reported electroanalysis protocol involving an electrooxidation step at 1.25 V for 150 s in 1.0 M aqueous HCl, followed by a cathodic scan from 1.25 to 0 V and measurement of the cathodic peak current at 0.49 V,35, 36 yielded an experimental of only 2.1% (Fig. S1b). It is expected that, at the sandwich immunoelectrodes rather than the bare electrode used here, the direct anodic stripping protocol without simultaneous chemical-dissolution and cathodic-preconcentration will give a signaling efficiency notably lower than our MLAIsa protocol, due to the obvious blocking of electron communication by the insulating protein layers between the metal labels and the electrode surface, as confirmed in Fig. S2. Hence, this work provides an alternative method with high sensitivity for MLAI.
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Scheme 1. Illustration of immunoelectrode preparation (a) and key electrochemical steps (b) of our MLAIsa protocol. To compare the signaling efficiency (, Eq. (1)) of our MLAIsa protocol with those of the protocol similar to MLAIsa but the cathodic potential was applied soon after AuNPs dissolution (potential control only in liquid), the conventional solutionreplacement protocol,33, 34 and the direct anodic stripping protocol, we conducted simulation experiments simply by cast-coating an appropriate amount of AuNPs (nAuNPs-cast in mol) on a GCE and then detecting the recovered amount of the cast-coated AuNPs (nAuNPs-LSV in mol) by linear sweep voltammetry (LSV) and the Faraday law.
=nAuNPs-LSV /nAuNPs-cast =Qp /( zFnAuNPs-cast )
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where Qp is the charge under the LSV stripping peak of cast-coated AuNPs, z is the number of electrons transferred (z=3 here), and F is the Faraday constant (96485.3 C mol-1). The anodic stripping LSV curves and corresponding values as functions of the cathodic-preconcentration time are shown in Fig. 1. After simultaneous dissolution of the AuNPs and cathodic preconcentration for 600 s and LSV stripping in 8 L of 1.0 M HBrBr2, the for our MLAIsa protocol was 82.6%. The protocol similar to MLAIsa but the cathodic potential was applied soon after AuNPs
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immunoelectrode in air, and 8 L of 1.0 M aqueous HBr-Br2 was then added to connect the glassy carbon working electrode (WE), the KCl-saturated calomel reference electrode (RE) with a salt bridge of the test solution, and the platinum wire counter electrode (CE), as well as to dissolve the AuNPs and stained gold for diffusioncontrolled cathodic preconcentration of atomic gold onto the immunoelectrode substrate. Finally, differential pulse ASV analysis was conducted for immunoassay signaling. Here, the beforehand open-circuit exertion of a cathodic potential in air and the use of a small-volume solution can minimize the diffusion-layer thickness to rapidly electrodeposit the metal label onto the bioelectrode surface as entirely as possible from the lysate, which can greatly enhance the subsequent ASV signal for bioanalysis. It should be noted that the open-circuit exertion of a constant potential in air is always safe for an electrochemical instrument working in its potentiostatic mode but may damage the instrument running in its galvanostatic mode. The open-circuit potentiostatic operation (i.e., potential control in air) is rarely used in analytical chemistry and electrochemistry, as compared with potential control in liquid solutions, and we prove here that this special procedure can be used to effectively amplify the bioelectroanalysis signal.
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Fig. 1. versus preconcentration-time curves (A, and Insets of C and D, n=3) and anodic stripping LSV curves of the AuNPs cast-coated on GCE (B, C and D, 100 mV s-1) for our MLAIsa protocol (B, a), the protocol similar to MLAIsa but the cathodic preconcentration potential was applied soon after AuNPs dissolution (C, b) and conventional solution-replacement protocol (D, c). See ESI for experimental details. The volume of 1.0 M HBr-Br2 used to dissolve AuNPs was optimized. As shown in Fig. S3, decreased with the increase of HBr-Br2 volume, since a smaller solution volume can decrease the diffusion-layer thickness for enhanced preconcentration of atomic gold on the WE and can thus give a larger ASV peak signal. To maximize the signal, we will use 8-L HBr-Br2 below.
Fig. 2. UV-Vis spectra and photographs (Inset) of 0.50 mL NH2OH (1), gold-staining solution (2), BSA/anti-hIgG/AuNPs (3) and after its gold-staining for 20 min (4). Final concentrations: 10 g mL-1 anti-hIgG, 1.0% (w/v) BSA, 18 mM NH2OH and 3 mM HAuCl4.
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Under optimum conditions, the ASV peak current is linear with the common logarithm of hIgG concentration from 0.4 fg mL-1 to Articleof Online 400 ng mL-1, with a sensitivity of 0.337 mA dec-1 andView a LOD 0.3 -6 10.1039/C5CC01439E fg mL-1 (7 molecules in 6 L sample, 610DOI: 0.3 10-151036.02 1023/1.5105=7.27, S/N=3) by our MLAIsa method (Fig. S7). The semi-derivative treatment of these ASV curves can notably improve the peak-signal resolution and decrease the background,38,39 as also shown in Fig. S7. The reproducibility of our MLAIsa method was evaluated using hIgG at three concentration levels (0.500, 5.00 and 50.0 ng mL-1), with relative standard deviations (RSDs) of (61)%, indicating acceptable reproducibility. The LOD obtained here is much better than those reported for hIgG to date (Table S1). hPSA is the most widely used tumor marker worldwide for early warning, screening, diagnosing and monitoring of prostate cancer that is usually localized and has no obvious symptoms in its early stage. hPSA in blood is the most sensitive biomarker for this purpose, which is commonly determined by immunoassay methods.40, 41 We used our MLAIsa method to detect hPSA (Fig. 4). Under the optimum conditions, we obtained linearity from 0.18 fg mL-1 to 450 ng mL-1 with a sensitivity of 0.609 mA dec-1 and a LOD of 0.1 fg mL-1 (11 molecules in 6 L sample, 610-60.110-151036.02 1023/3.4104=10.611, S/N=3). The LOD of our method is also much better the literature-reported values for hPSA (Table S1). The Au-stripping anodic peaks of improved resolution after the semiderivative treatment of the voltammetric curves in Fig. 4 and the corresponding calibration curve are shown in Fig. S8. We also examined the applicability of our MLAIsa method to hPSA assay in seven clinical human-serum samples (only 6 L serum sample was required for each assay). Our results agreed well with the hospital results from chemiluminescence assay (within 7% RSD), as listed in Table S2, validating our MLAIsa method for analysis in clinical human sera. From the above two immunoassay examples, our MLAIsa method with several-molecules-level sensibility to proteins may be promising for challenging trace analysis of proteins (e.g., some ultratrace proteins in single circulating tumor cells,42, 43 and interleukin-2 with a normal pM~fM concentration in human serum44) and may find wide applications in the early warning, diagnosis and treatment of the serious diseases including cancers. 8
450 ng mL-1 -1 180 ng mL 18 ng mL-1 -1 1.8 ng mL 180 pg mL-1 -1 18 pg mL 1.8 pg mL-1 -1 180 fg mL 18 fg mL-1 -1 1.8 fg mL 0.9 fg mL-1 0.18 fg mL-1 0
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In this work, we employed performed AuNPs to label Ab2 for AuNPs-catalyzed gold-staining. As shown in Fig. 2, NH2OH solution was colorless and showed no obvious absorption peak. After 3 mM HAuCl4 was added, the solution turned into light yellow. The BSA/anti-hIgG/AuNPs suspension showed a wine red color and an absorption peak at 537 nm. After adding the gold staining solution, the suspension showed a blue-gray color and an absorption peak at 579 nm (sample 4) due to seeded-growth of AuNPs here. Taking hIgG immunoassay as an example, electrode modifications in our MLAIsa protocol were examined by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM), as shown in Figs. S4 and S5 with discussion details. We also used scanning electron microscopy (SEM) to characterize the immunoelectrode fabrication, as shown in Fig. 3. The surface of bare GCE is smooth (Fig. 3A). On the antigen/BSA/Ab1/MWCNTs/GCE, nanotubes are seen (Fig. 3B). After further immobilization of Ab2-AuNPs (Figs. 3C and 3E), some anchored Ab2-AuNPs granules were observed, proving the successful immuno-recognition. After the AuNPs-catalyzed gold staining reaction (Fig. 3D and F, gold/Ab2-AuNPs/antigen/BSA/Ab1/ MWCNTs/GCE), the Ab2-AuNPs granules become larger and denser. As a control, similar gold staining operation on an Ab2-AuNPs-free antigen/BSA/Ab1/MWCNTs/GCE hardly changed the electrode surface (Fig. S6). This observation supports that gold staining occurs only on AuNPs due to seed-mediated nucleation growth.28, 37 The energy-dispersive X-ray spectroscopy (EDX) results for Ab2AuNPs/antigen/BSA/Ab1/MWCNTs/GCE (Fig. 3G) and gold/Ab2AuNPs/antigen/BSA/Ab1/MWCNTs/GCE (Fig. 3H) confirm the increased mass and atomic fractions of Au after the AuNPscatalyzed gold staining reaction. The CV, EIS, QCM, SEM, and EDX characterizations here have proven the successful fabrication of the immunoelectrode.
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Fig. 3. SEM images and EDX spectra of bare GCE (A), antigen/BSA/Ab1/MWCNTs/GCE (B), Ab2-AuNPs/antigen/BSA/ Ab1/MWCNTs/GCE (C, E and G), and gold/Ab2-AuNPs/antigen/ BSA/Ab1/MWCNTs/GCE (D, F and H). Concentration of hIgG: 4 ng mL-1 (C and D) and 400 ng mL-1 (E, F, G and H).
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Fig. 4. Linear sweep ASV curves for hPSA immunoassay by our MLAIsa protocol (A) and the corresponding calibration curves (B) (n=3). Scan rate: 100 mV s-1. In summary, we have reported that a new and ultrasensitive MLAIsa method, based on simultaneous microliter-droplet chemicaldissolution and cathodic-preconcentration of the AuNPs biolabel after gold staining and then in-situ ASV analysis directly on the immunoelectrode, can be sensitive to a few hIgG or hPSA molecules. Virtually, amperometric detection of a single bioelectrode-supported AuNP after sufficient size-enlargement may be possible, thus the principle here may be promising even for quantitative singlemolecule analysis of proteins. Our method has been used for hPSA analysis in clinical samples with satisfactory results. Our MLAIsa protocol has the advantages of ultrahigh sensitivity, wide linear detection range, good accuracy/precision/stability, easy operation, small consumption of reagents/samples, and application potential for
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Journal Name 17 V. Pavlov, Y. Xiao, B. Shlyahovsky and I. Willner, J. Am. Chem. Soc., 2004, 126, 11768.
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Anal. Chem., 2011, 18 G. S. Lai, F. Yan, J. Wu, C. Leng and H. X. Ju,10.1039/C5CC01439E DOI: 83, 2726.
19 C. R. Bradbury, J. J. Zhao and D. J. Fermı´n, J. Phys. Chem. C, 2008, 112, 10153.
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This work was supported by the National Natural Science Foundation of China (21475041, 21175042, 21305041, 21405042), Hunan Lotus Scholars Program, Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, and Hunan Provincial Innovation Foundation For Postgraduate (CX2014B169).
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine
Research (MOE of China), National & Local Joint Engineering Laboratory for New Petro-chemical Materials and Fine Utilization of Resources, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China. State Key Laboratory of Chemo/Biosensing and Chemometrics, College
of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P.R. China. †
Electronic Supplementary Information (ESI) available: Experimental
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DOI: 10.1039/c000000x/ 1
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