Electrostatic Force Triggering Elastic Condensation of Double

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Electrostatic Force Triggering Elastic Condensation of DoubleStranded DNA for High-Performance One-Step Immunoassay Chunyan Deng, Manman Zhang, Chunyan Liu, Honghua Deng, Yan Huang, Minghui Yang, Juan Xiang, and Bin Ren Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02556 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Electrostatic Force Triggering Elastic Condensation of Double-Stranded DNA for High-Performance One-Step Immunoassay

Chunyan Deng1, Manman Zhang1, Chunyan Liu1, Honghua Deng3, Yan Huang3, Minghui Yang1, Juan Xiang*1, Bin Ren*2

1

College of Chemistry and Chemical Engineering, Central South University,

Changsha 410083, China 2

Collaborative Innovation Center of Chemistry for Energy Materials, State Key

Laboratory of Physical Chemistry of Solid Surfaces, and The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China 3

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry

and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China

*

Corresponding Author

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

1

ACS Paragon Plus Environment

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

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ABSTRACT Current strategies for high-performance immunoassay generally require a sandwich structure for signal amplification. This strategy is limited to multivalent antigens and complicates the detection scheme. Herein we demonstrate a class of simple one-step ultrasensitive immunoassay with the adoption of double-stranded DNA

(dsDNA)

as

“conductive

spring”

to

bridge

the

electrode

and

redox-reporter/antibody-receptor co-modified gold nanoparticles (AbFc@AuNPs). Upon bio-recognition between antigen and antibody, the charge of the AuNPs changes, enhancing the electrostatic interaction between the AuNPs and Au electrode surface, and condensing the dsDNA chain. For the first time, the sensitive response of the electrochemical redox current to the DNA chain length is utilized to achieve an ultrahigh sensitivity down to fM level. Only the primary antibody needed in the recognition interface ensures the one-step immunoreaction works well with monovalent antigens, which ensure this method a promising general alternative means for fast, high-throughput or point-of-care clinical applications even for very challenging clinically relevant samples.

Keywords: Electrostatic force; Elastic condensation; Conductive spring; One-step immunoassay

2


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INTRODUCTION In the past several decades, immunoassays, based on specific antigen-antibody recognition, have been a working horse in early diagnosis and clinic analysis of diseases, food safety control, environmental monitoring, public security, and home-health care.1-5 Traditional one-step immunoassays are based on the direct binding between a biomarker and its primary antibody.6-8 Being simple, fast and useful for high-throughput and point-of-care detection, its low sensitivity (µM~nM) made its application difficult for low abundant biomarkers in real samples. In the past ten years, various signal amplification strategies, including nanomaterial-enhanced, enzyme-based, or DNA-based methods, have been proposed and developed to improve the sensitivity of immunoassays.9-14 These strategies usually rely on a sandwich structure to quantify antigens between two layers of antibodies or other specific molecules.15,16 The requirement of at least two antigenic sites limits the sandwich assays to multivalent antigens, such as proteins or polysaccharides. The use of antibody pairs also makes the assay more expensive. In addition, sandwich immunoassays require multiple washing steps, which are time consuming and hard for high-throughput applications. Therefore, it will be highly appreciated if a more general method for monovalent antigens with high sensitivity can be developed. Double-stranded DNA (dsDNA) is a privileged class of biomolecules, whose conductance is highly sensitive to the chain length and structure. Mechanical condensing or stretching may lead to an exponential change of the charge transport through dsDNA.17-21 We developed here a general strategy for high-performance 3



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one-step immunoassay by using elastic condensation of dsDNA induced by the increased electrostatic force between the electrode surface and the immunoreacted Au nanoparticles (AuNPs). The ultrahigh sensitivity was achieved by the synergetic amplification of dsDNA and AuNPs. This scheme only requires one-step immunoreaction between the antigen and its primary antibody, makes the immunoassay applicable for not only multivalent antigens but also monovalent antigens such as short peptides or small molecules. More importantly, the one-step detection simplifies the operation and reduces the cost, which renders the immunoassay a promising tool for fast and high-throughput, or point-of-care clinical applications. EXPERIMENTAL SECTION Reagents and Chemicals. Tris-(hydroxymethyl) aminomethane (Tris), sodium citrate, chloroauric acid (HAuCl4·4H2O, ≥99.9%), KCl, MgCl2, CaCl2, NaCl 6-Mercaptohexanol (MCH), streptavidin (SA), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP),

human

serum

N-2-hydroxyethylpiperazine-N-ethane-sulphonic

albumin acid

(HEPES),

(HAS), glucose

and

n-hexane were obtained from Sigma-Aldrich (St Louis, MO). 6-(Ferrocenyl) hexanethiol (Fc-SH) was obtained from J&K Technology Co., Ltd (Beijing, China). Human immunoglobulin G (IgG), α-synuclein (α-syn), insulin, mucin 1 (MUC1) and carcinoembryonic antigen (CEA) were from Shanghai Apeptide Co., Ltd (Shanghai, China). Biotin-conjugated CEA, IgG and Aβ40 monomer (Aβ40M) antibody were purchased from Beijing KEY Biological Technology Co., Ltd. (Beijing, China), 4


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Shanghai Sangon biotechnology (Shanghai, China), and Biolegend (San Diego, CA.), respectively. Aβ40 and Aβ42 were purchased from Xinghao medicine Co., Ltd (Wuhan, China). Antigens and antibodies were dissolved in 10 mM phosphate buffer saline (PBS, pH 7.4). All oligonucleotides listed in Table S1 were purchased from Shanghai Sangon biotechnology (Shanghai, China). They were diluted to 10 µM in 34 mM Tris-HCl buffer (pH 7.4, 233 mM NaCl, 8.5 mM KCl, 1.7 mM MgCl2, 1.7 mM CaCl2) for use. Artificial cerebrospinal fluid (ACSF) was prepared according to our previous works22. All other chemicals were of analytical-grade purity and used without further purification. Deionized water (18.2 MΩ·cm; Millipore M-Q System Inc., Milford, MA) was used for all experiments. All of the obtained solutions were kept at 4 °C. Instruments. The measurements of cyclic voltammetry (CV) and square wave voltammetry (SWV) were performed on an electrochemical workstation (CHI Instruments 832B, Shanghai Chenhua Equipment, China). The characterization of electrochemical impedance spectroscopy (EIS) was performed on Gamry Reference 600 electrochemical workstation (Gamry Instruments Co., Ltd., Warminster, PA, USA). A conventional three-electrode system with a platinum wire as the auxiliary electrode and an Ag/AgCl electrode as the reference electrode (inner filled solution: saturated KCl solution) was employed for the electrochemical measurements. All the potentials were referred to the Ag/AgCl electrode. The SWV parameters were referred to our previous work22. UV-visible-light

(UV-vis)

spectra

were

monitored

using

a

UV-2450

spectrophotometer (Shimadzu, Kyoto, Japan). Dynamic-light-scattering (DLS) 5



measurements were conducted using a Zetasizer Nano ZS Instrument (Marlvern Instruments, Southborough, U.K.). Transmission-electron-microscope (TEM) images were conducted on a JEM 2010 (JEOL Company Ltd., Japan). The hydrophobicity of interfaces was characterized with contact angle changes on a JC2000D1 (Shanghai Zhong Chen Digital Technology Equipment Co., Ltd., Shanghai). Preparation of AuNPs and AbFc@AuNPs. AuNPs (14 nm in diameter) were prepared by citrate reduction of HAuCl4 according to the literature.23 A 1.5 mL AuNP solution (2.5 nM) was incubated with a 15 µL antibody solution (5 mM) for 4 h at room temperature to obtain the Ab@AuNP solution.24 Afterward, Fc-SH dissolved in n-hexane solution with a final concentration of 3 mM was added into the Ab@AuNP solution and kept for 24 h, resulting in the self-assembly of Fc onto the unoccupied Au sites of Ab@AuNP surface to obtain the AbFc@AuNP solution. The prepared AuNP and AbFc@AuNP solutions were kept at 4 °C. Construction of the Electrochemical Biosensor. The bare Au electrode (2 mm in diameter) was mechanically polished and electrochemically cleaned according to our previous works25,26. Various DNAs were designed for experiments, as shown in Table S1. 10 µM DNA1s were mixed with equal volume of 10 µM DNA2s, respectively, and incubated in a water bath at 37 °C for 2 h to achieve the hybridization between DNA1s and DNA2s and obtain different dsDNAs. Then, the obtained dsDNA solution (15 µL) was dropped on the Au electrode, and incubated in the dark for 15 h. After rinsed with PBS thoroughly, 2 mM MCH was used to block the unmodified Au spots and displace nonspeciﬁcally bound dsDNA. Afterwards, a 6


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0.25 mg·mL-1 SA solution (10 µL) was dropped on the dsDNA modified Au electrode and incubated for 1 h to achieve the reaction between SA and biotin-modified DNA2. Finally, a 10 µL AbFc@AuNP solution was placed on the resulted electrode for 1 h to form the sensing interface (AbFc@AuNPs-dsDNA/Au) via the interaction between SA and biotin-conjugated antibodies. Preparation of Aβ40 Oligomer and Fibril. Aβ40 oligomer and fibril (Aβ40O, Aβ40F) were prepared according to reports in the literature.27 In brief, the purchased Aβ40M was directly dissolved in HFIP at a final concentration of 1 mg·mL-1 and incubated overnight at 4 °C to dissolve any pre-existing oligomers22. Then, the solution was sonicated for 30 min and lyophilized on freeze-dryer (Virtis Benchtop K, SP Scientific, Gardiner, NY). The Aβ40 solution was then prepared by dissolving the obtained lyophilized powder in PBS (10 mM, pH 7.4) at a concentration of 80 µM and incubating it in a thermostatic water bath at 37 °C for 1 day and 7 days to obtain the Aβ40O and Aβ40F, respectively. RESULTS AND DISCUSSION Sensing Principle. Scheme 1 shows the scheme of the immunoassay. Antibody and ferrocene (Fc), acted respectively as specific receptor and electrochemical signal reporter, were loaded onto AuNPs to form antibody/Fc@Au nanoparticles (AbFc@AuNPs). dsDNA was pre-modified on the Au electrode and used as a conductive spring bridging the AbFc@AuNPs and the electrode. The dsDNA had 20 bases, and thus the distance between AuNP and Au electrode was 6~7 nm. In the absence of a target, due to the high loading of Fc on AuNPs, the faradaic current from 7



Fc can be detected at sub-µA level. After the introduction of an antigen (usually negative charged in neutral solution), the specific antigen-antibody binding made the surface of the AuNPs more negative. If the electrode surface is controlled at a positive charged state, electrostatic force will pull the negative charged AuNPs to the positively charged electrode surface (at potentials corresponding to the Fc redox transformation). As a result, the dsDNA is condensed and its conductivity is significantly increased to promote electron transfer from Fc to electrode. Then, the current changes with the addition of antigen (∆I) could be employed for quantitative detections. For example, a typical antigen, carcino-embryonic antigen (CEA), with an isoelectric point (pI) of 3.4, could be conveniently detected by this method. As expected, the introduction of 50 pM CEA induced over 10 µA current increase, indicating the high sensitivity of this type of one-step immunoassay. Characterization of AuNPs and AbFc@AuNPs. The prepared AuNP and AbFc@AuNP were characterized with UV-vis spectra and TEM. As shown in Figure S1, the AuNP solution displayed a maximal absorbance at ∼520 nm (Figure S1-A, curve a). The TEM image showed that the prepared AuNPs were nearly monodisperse spheres with an average size of about 14 nm (Figure S1-B) and an average hydrodynamic diameter of ~21 nm (Figure S1-C). However, the absorption peak of AbFc@AuNPs appeared at 528 nm (Figure S1-A, curve b), which most likely resulted from a change in the dielectric property of the media surrounding the AuNPs,28,29 demonstrating the successful co-modification of Fc and antibody on the AuNP surface. 8


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Characterization of the Electrochemical Biosensor. The modification process of the Au electrodes was characterized by EIS and CV. The semicircle diameter in EIS, which corresponds to the electron transfer resistance (Ret), is strongly dependent on the electrode modification25. As seen from Figure S2-A, the bare Au electrode (curve a) displays the smallest Ret value (ca. 200 Ω). With the immobilization of dsDNA (curve b) and MCH (curve c), the Ret increases to 4090 and 7345 Ω, respectively. The significant increases are attributed to the presence of dsDNA and MCH blocking the interfacial electron transfer. Similarly, the binding of SA to biotin-modified DNA2 causes a great increase in Ret (curve d). However, when incubated

with

the

AbFc@AuNPs

solution,

the

Ret

value

of

the

AbFc@AuNP-dsDNA/Au electrode significantly decreases (curve e), which is ascribed to the excellent electron-transfer of AuNPs,25,30 also implying the successful immobilization of AbFc@AuNPs onto the modified Au electrode. The electrode modification process was also characterized by the peak currents and the peak-to-peak potential separation in CV31. As shown in Figure S2-B, due to the fast electron transfer on the bare Au electrode, [Fe(CN)6]3-/4- displays a pair of reversible redox peaks (curve a). Once the electrode is modified with dsDNA, the redox peak currents decrease and the peak-to-peak separation increases obviously (curve b), which is consistent with the reports in the literature25,31. Similarly, the respectively immobilization of MCH and SA on the dsDNA modified-electrode, furtherly decreases the redox peak currents (curves c, d). Upon AbFc@AuNPs bounded to the modified Au electrode, the redox peak currents greatly increase (curve 9



e), which results from the excellent electron-transfer of AuNPs. These electrochemical results are consistent with EIS results, further confirming the successful fabrication of the sensing interface. Mechanism for Signal Amplification of the Immunoassay. Both AuNPs and dsDNA may play critical roles in the signal amplification of the immunoassay. AuNPs were employed as supports to increase the loading of Fc and antibodies to amplify the electrochemical signal and the recognition event, respectively, as a result of high surface-to-volume ratio. dsDNA acts as a conductive spring to mediate the electron transfer from Fc to electrode, and a slight condensing of the DNA chain may dramatically change the electron transfer current. In fact, there are various possible electron transfer pathways at the interface, including electron transfer mediated by the stacked-bases of dsDNA (Figure 1a I), or those unmediated by the stacked-bases such as direct electron transfer while dsDNA rod is lying flat or elastically bending (Figure 1a II), and free rotational motion of the dsDNA rod (Figure 1a III).32 In our design, the application of AuNPs can effectively restrict the free rotational motion of the dsDNA. So, model I may be the main pathway. To verify the exact electron transfer pathway, the relationship between the scan rate and redox current was studied. The anodic and cathodic peak currents (Ia and Ic) scaled linearly with the scan rate, confirming the surface-confined electron transfer (Figure S3 in supporting info). If model I is the key factor, we would expect to see an obvious current change while we change the mismatched base pairs or the length of the DNA chain. Indeed, we found the redox current exponentially decays with the increased number of mismatched base 10


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pairs in dsDNA (Figure 1b).33 The mismatch changes the π-π stacking interaction and the stacking integrity, which sensitively changes the electron transfer efficiency through the stacked bases.25,30 Given the whole DNA has more than 20 base pairs, even three mismatches did not affect the overall duplex structure (melting temperature: Tm=69.5). Therefore, this result strongly confirms that the DNA mediated electron transfer in our system is mainly through the π-π stacking interactions between neighboring base pairs (model I).34,35 The electron transfer proceeds through the base pairs in the interior of the duplex, which also ensures the stability of the current response even under complicated solution conditions. We also found a very sensitive current response to the number of complementary base pairs (Figure 1c). A short dsDNA (below 10 base pairs) favors direct electron transfer from Fc on AuNPs to Au electrode. As a result, the current response is insensitive to antigen binding. On the other hand, a long dsDNA has a large flexibility, which favors its random structural distortion (ie. elastic bending) and sharply attenuates the long-rang electron transfer. Furthermore, a longer chain tends to accumulate more negative charge (Table 1), which may shield the electrostatic interaction between AbFc@AuNPs and the electrode, disfavors its signal amplification. Therefore, there is an optimal length of 20-30 base pairs to achieve the optimal signal response, including large current changes and small deviations. Electrostatic Effect and Ionic Strength Effect on the Immunoassay. Immunoreaction usually induces a change in the surface charge (shown in Table S2). To compare with the result shown in Scheme 1, we replaced the electrochemical 11



reporter molecule from Fc to Mb (methyl blue). Different from Fc, Mb reacts at potentials when the surface is negatively charged. Figure 2a shows the SWV responses of AbMb@AuNPs with or without the introduction of 50 pM CEA. The current is decreased after immunoreaction. The negatively charged Au surface repels the negatively charged nanoparticles away from the electrode surface as a result of electrostatic interaction. Thus, dsDNA was slightly stretched and its conductivity was significantly decreased, which reduced the electron transfer efficiency from Mb to electrode, as also observed by Plaxco group.36,37 The opposite effect of immunoreaction on the current response in the Fc and Mb reactions strongly confirms the dominant effect of the electrostatic force between the AuNPs and the electrode surface for the sensitive current response for such a one-step immunoassay. These experimental results provide us an interesting strategy to further improve the sensitivity and specificity for immunoassays. For example, if the immunoreaction induces a decrease of the surface charge, we can use an electrochemical reporter with its redox potential corresponding to positively charged electrode surface, such as Fc. In this way, it ensures the condensation of dsDNA and the increase of ∆I to improve detection sensitivity and linear range. On the other hand, if antigen binding results in an increase of the surface charge, we should use an electrochemical reporter with its redox potential corresponding to negatively charged electrode surface, such as Mb. The ionic strength can determine the conformation of DNA and affect electrostatic interactions between AuNPs and electrode. Therefore, we investigated the effect of ionic strength. To ensure an effective hybridization of DNA strands,38 the 12


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ionic strength was above 5 mM. As shown in Figure 2b, both the blank current and the current change decreased with the increase of the ionic strength (Figure 2b). Usually, ionic strength has a minor effect on the electron transfer in dsDNA, since the electron transfer proceeds through the base pairs in the interior of the duplex. Therefore, the significant change in the current response at different ionic strengths most probably results from the change of potential drops in the electrical double-layer at different ionic strengths. According to the Guoy–Chapman–Stern (GCS) model,39,40 a double layer at the electrode/solution surface consists of a compact layer (CL) with a sharp potential drop and a diffuse layer (DL) with a slow potential drop (Figure 2c). The ionic strength can critically influence the double-layer structure according to equation κ=(3.29×107)zc1/2,38 in which κ is the reciprocal of characteristic thickness of the diffuse layer, c is the bulk z:z electrolyte concentration. At a low ionic strength, the potential drop is gradual and the diffuse layer can be reach ~10 nm when the electrolyte concentration is about 1 mM. It ensures the effective electrostatic interaction between AuNPs and electrode, showing an electrostatic force on the single AuNP to be 10-9~10-12 N (taking the electric field intensity of ~107 V·m-1, and the surface charge of AuNPs at 10-16~10-19 C). Since the spring constants of dsDNA molecules with 20 base pairs is about 0.1 nN·nm-1,41 the electrostatic force on AuNPs is capable of inducing the elastic condensation of dsDNA from several pm to nm scale. On the other hand, at a high ionic strength, the potential drop is precipitous and the diffuse layer is relatively compact. For example, when the electrolyte concentration is above 0.1 M, the potential drops down to zero within 1 nm, and essentially no 13



potential drop occurs on the AuNPs. The solution with such a high ionic strength can provide sufficient screening of the electrostatic interactions between AuNPs and electrode. Therefore, to match the potential distribution with the length of AbFc@AuNP-dsDNA and to ensure the stability of the dsDNA strand, the solution should be controlled at a low ionic strength, e.g., 10 mM NaCl. Feasibility of the Biosensor. To test the feasibility of the immunoassay for different biomarkers, three typical antigens with different isoelectric point (pI), amyloid β peptide (Aβ, pI: 5.5), immunoglobulin G (IgG, pI: 4.5), and CEA (pI: 3.4), were detected with the corresponding sensor (Figure 3a). Also, the relationship between ∆I and the surface charge changes of AbFc@AuNPs with the addition of antigen (∆Z) is shown as inset in Figure 3a. As anticipated, the antigen which can induce higher surface charge changes (such as CEA) has more significant current change. Upon changing the concentration of biomarkers, we obtained the quantitative relationships for three biomarkers (Figure 3b). Good linear relationships between ∆I and the logarithm of biomarker’s concentration were obtained, in the range from 0.5 fM to 50 pM for cCEA, from 6.7 fM to 67 pM for cIgG, and from 50 fM to 50 pM for cAβ40M. The more negatively charged biomarkers show a lower detection limit, which agrees well with the electrostatic model of the immunoassay. Selectivity. We further evaluated the selectivity of such a one-step immunoassay using Aβ40 monomer as the target, while Aβ40 oligomer, Aβ40 fiber and Aβ42 monomer were chosen as interfering species. As shown in Figure 4a, these interfering species with 106 higher concentrations didn’t result in a significant current change. Similar 14


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results were obtained for IgG (Figure 4b) and CEA (Figure 4c). The high selectivity of proposed immunoassays toward corresponding biomarkers should be attributed to the high specific recognition between biomarker and its primary antibody, and the simple composition of the immunoassay interface to greatly reduce its cross-interactions with those interfering species. The recovery rate is above 90% (Table S3-6) for all the three immunoassays in artificial cerebrospinal fluid (ACSF) or 100-fold diluted human serum samples, which confirms the applicability of the immunoassay for real sample detections. CONCLUSIONS In summary, taking dsDNA as the conductive spring, we provided a new and previously unattainable general strategy for effective amplification of the signal and simplification of the interface, enabling high-performance detection for not only multivalent antigens but also monovalent antigens such as short peptides or small molecules. This fast one-step immunoassay strategy has the advantages of high sensitivity and selectivity of both traditional and sandwich immunoassays. This method was used successfully to detect three typical biomarkers. In principle, it is applicable to any target molecules with positive or negative charges enough to alter the surface charge of nanoparticles. It provides a promising alternative means for fast, convenient, high sensitive, high-throughput, point-of-care clinical applications even for very challenging clinically relevant samples. ASSOCIATED CONTENT Supporting Information: 15



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Supplementary figures showed schematic representation of the fabrication of the one-step immunoassay interface, TEM image of AuNPs, UV-Vis spectra of AuNPs and AbFc@AuNPs, electrochemical characterization of the modified electrodes, and the dependence of the peak currents of Fc redox (Ia and Ic) on the potential scan rate υ. Supplementary tables listed names and sequences of all the oligonucleotides used in this work, pI and Zeta potentials of different solutions in 10 mM PBS, recoveries of Aβ40M and IgG in ACSF, and recoveries of Aβ40M and CEA in 100-fold diluted human serum. Additional information

is available free of

charge

via

the Internet at

http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors: Tel.: +86-731-88876490; Fax: +86-731-88879616 E-mail: [email protected] Notes The authors declare no competing financial interest. Conflict of Interest No financial conflict of interest was reported by the authors of this paper.

Acknowledgements

The work was financially supported by the National Key Basic Research Program of China (2014CB744502), the National Natural Science Foundation of 16


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China (21573290, 21727810, 21633005, 21790354, 21711530704, 21505056 and 21621091), and the Hunan Provincial Natural Science Foundation of China (13JJ3004).

17



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(24) Lee, I. H.; Lee, J. M.; Jung, Y. ACS Appl. Mat. Interfaces. 2014, 6, 7659-7664. (25) Deng, C. Y.; Pi, X. M.; Qian, P.; Chen, X. Q.; Wu, W. M.; Xiang, J. Anal. Chem. 2016, 89, 966-973. (26) Kelf, T. A.; Sugawara, Y.; Cole, R. M.; Baumberg, J. J.; Abdelsalam, M. E.; Cintra, S.; Mahajan, S.; Russell, A. E.; Bartlett, P. N. Phys. Rev. B. 2006, 74, 4070-4079. (27) Hatai, J.; Motiei, L.; Margulies, D. J. Am. Chem. Soc. 2017, 139, 2136-2139. (28) Petrizza, L.; Genovese, D.; Valenti, G.; Iurlo, M.; Fiorani, A.; Paolucci, F.; Rapino, S.; Marcaccio, M.; Electroanal. 2016, 28, 2777-2784. (29) Wan, L.; Qin, Y.; Xiang, J. Electrochim. Acta 2017, 238, 220-226. (30) Larguinho, M.; Baptista, P. V. J. Proteomics, 2012, 75, 2811-2823. (31) Veloso, A. J.; Chow, A. M.; Ganesh, H. V. S.; Li, N.; Dhar, D.; Wu, D. C. H.; Mikhaylichenko, S.; Brown, I. R.; Kerman, K. Anal. Chem. 2014, 86, 4901-4909. (32) Anne, A.; Demaille, C. J. Am. Chem. Soc. 2008, 130, 9812-9823. (33) Bhattacharya, P. K.; Cha, J.; Barton, J. K. Nucleic Acids Res. 2002, 30, 4740-4750. (34) Lewis, F. D.; Liu, X.; Liu, J.; Miller, S. E.; Hayes, R. T.; Wasielewski, M. R. Nature 2000, 406, 51-53. (35) Giese, B.; Amaudrut, J.; Kohler, A.; Spormann, M.; Wessely, S. Nature 2001, 412, 318-320. (36) Kang, D.; Sun, S.; Kurnik, M; Morales, D.; Dahlquist, F. W.; Plaxco, K. W. J. Am. Chem. Soc. 2017, 139, 12113-12116. (37) Cash, K. J.; Ricci, F.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131, 6955-6957. (38) Kaiser, W.; Rant, U. J. Am. Chem. Soc. 2010, 132, 7935-7945. (39) Electrochemical Methods: Funda-mentals and Applications,2nd ed, volume 6, (A. J. Bard, L. R. Faulkner, A. Bard, L. Faulkner), Wiley 2001, pp 534-579. (40) Oldham, K. B. J. Electroanal. Chem. 2008, 613, 131-138. (41) Noy, A.; Vezenov, D. V.; Kayyem, J. F.; Meade, T. J.; Lieber, C. M. Chem. Biol. 1997, 4, 519-527.

19



Page 20 of 26

Table 1. Zeta Potentials of dsDNAs with Different Complementary Bases in 10 mM PBS.

Numbers of complementary bases

Zeta potential (mV)

RSD (n=3)

20

-3.60±0.34

9.44%

30

-5.04±0.33

6.54%

40

-9.84±0.31

4.11%

50

-13.83±0.32

2.31%

60

-25.73±0.44

1.71%

20


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


Scheme 1. Schematic representation of the detection principle. Inset is the electrochemical responses of AbFc@AuNPs in 10 mM PBS (pH 7.4) before (dash line) and after the introduction of 50 pM CEA (solid line).

21



a II

I

12

b

10

III

c

8

∆I / µA

9

∆I / µA

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

Page 22 of 26

6

6 4 2

3

0

0

10 20 30 40 50 Number of complimentary base pairs

0 1 2 3 Number of mismatched base pairs

Figure 1. Electron transfer mechanism studies. (a) Possible types of electron transfer mechanisms. I: electron transfer through the stacked-bases of dsDNA; II: direct electron transfer while dsDNA rod is lying flat or elastically bending; III: free rotational motion of the dsDNA rod. (b) Effect of mismatched dsDNA on the SWV responses of 50 pM CEA on the corresponding CEA AbFc@AuNP-dsDNA/Au. (c) Dependence of ∆I on the number of complementary base pairs ranging from 10 to 50.

22


Page 23 of 26

1.4 1.2

a

3.0

Current / µ A

1.0

Current / µΑ

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


0.8 0.6 0.4 0.2 0.0 -0.2

b

2.5 2.0 1.5 1.0 0.5 0.0

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0

20

40

60

80

100

cPBS / mM

Potential / V

c I

II

Figure 2. Electrostatic effect on the immunoassay. (a) SWV responses of AbMb@AuNPs in 10 mM PBS (pH 7.4) before (dash line) and after the introduction of 50 pM CEA (solid line). (b) Effect of ionic strength on the SWV blank current of AbFc@AuNPs-dsDNA/Au interface (circle symbol) and the current change ∆I with the addition of CEA (square symbol). (c) Illumination of the potential drops at the AbFc@AuNPs-dsDNA/Au interface under high (left panel) and low (right panel) ionic strength, respectively.

23



Current / µA

10

b

8

12 CEA 10 8 IgG 6 4 Aβ40 M 2 -12 -10 -8 -6 -4 -2 ∆

12

6 4

a

2

8 6 4 2

0 -2

CEA IgG Aβ40M

b

10

∆I / µA

c

a

∆I / µA

12

V m / Z

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

Page 24 of 26

def 0.1

0.2

0.3

0.4

0

0.5

0.6

0.7

0.8

-4

-3

-2

-1

0

1

2

log c / pM

Potential / V

Figure 3. Feasibility of the immunoassay for different biomarkers. (a) SWV responses of 50 pM Aβ40M (blue curve), IgG (red curve), and CEA (black curve) on the corresponding AbFc@AuNP-dsDNA/Au in 10 mM PBS (pH 7.4). Green, yellow and

dark

blue

curves

are

the

blank

responses

on

the

corresponding

AbFc@AuNPs-dsDNA/Au. Inset is the relationship between ∆I and the corresponding surface potential changes of AbFc@AuNPs with the addition of antigen. (b) Linear relationship of ∆I and the logarithm of analyte concentration for three typical biomarkers.

24


Page 25 of 26

3.0

a

∆I / µA

1.5 1.0 0.5

3.0

b

2.5

2.5

2.0

2.0

∆I / µA

2.0

∆I / µA

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


1.5 1.0 0.5

0.0 Aβ40M

Aβ40F

Aβ42M

Aβ42O

0.0

c

1.5 1.0 0.5

IgG

MUC1

a-syn

CEA

0.0

CEA

IgG

Insulin

MUC1

Figure 4. (a) Selectivity of the proposed immunosensor toward Aβ40M at 0.01 nM against Aβ40F, Aβ40O, and Aβ42M at 80 µM; (b) Selectivity of the proposed immunosensor toward IgG at 0.05 pM against 10 nM MUC1, 70 nM α-syn, and 5 pM CEA; (c) Selectivity of the proposed immunosensor toward CEA at 0.005 pM against 0.5 pM IgG, 10 nM insulin, and 10 nM MUC1.

25



For TOC only

26


Page 26 of 26

Electrostatic Force Triggering Elastic Condensation of Double

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